Sample records for targeting insulin-degrading enzyme

  1. Inhibition of Insulin Degrading Enzyme and Insulin Degradation by UV-Killed Lactobacillus acidophilus.

    PubMed

    Neyazi, Nadia; Motevaseli, Elahe; Khorramizadeh, Mohammad Reza; Mohammadi Farsani, Taiebeh; Nouri, Zahra; Nasli Esfahani, Ensieh; Ghahremani, Mohammad Hossein

    2018-05-11

    Probiotics have beneficial effects on management of type 2 diabetes (T2D). The major hallmarks of T2D are insulin deficiency and insulin resistance which emphasize insulin therapy in onset of disease. Lactobacilli such as Lactobacillus acidophilus ( L. acidophilus ) have well known properties on prevention of T2D and insulin resistance but not on insulin degradation. Insulin-degrading enzyme (IDE) degrades insulin in the human body. We studied the effects of cell-free supernatant (CFS) and ultraviolet (UV)-killed L. acidophilus (ATCC 314) on IDE activity and insulin degradation in vitro. Cell growth inhibition by CFS and UV-killed L. acidophilus (ATCC 314) was studied and Western blotting and a fluoregenic assay was performed to determine IDE expression and its activity, respectively. Insulin degradation was evaluated by sandwich enzyme-linked immunosorbent assay(ELISA). IDE expression and activity was reduced by CFS and UV-killed L. acidophilus (ATCC 314). Although, decreased enzyme expression and activity was not significant for CFS in contrast to MRL (MRS with same pH as CFS). Also, reduction in IDE activity was not statistically considerable when compared to IDE expression. Insulin degradation was increased by CFS but decreased by UV-killed L. acidophilus (ATCC 314).

  2. Targeting Insulin-Degrading Enzyme to Treat Type 2 Diabetes Mellitus.

    PubMed

    Tang, Wei-Jen

    2016-01-01

    Insulin-degrading enzyme (IDE) selectively degrades peptides, such as insulin, amylin, and amyloid β (Aβ) that form toxic aggregates, to maintain proteostasis. IDE defects are linked to the development of type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD). Structural and biochemical analyses revealed the molecular basis for IDE-mediated destruction of amyloidogenic peptides and this information has been exploited to develop promising inhibitors of IDE to improve glucose homeostasis. However, the inhibition of IDE can also lead to glucose intolerance. In this review, I focus on recent advances regarding our understanding of the structure and function of IDE and the discovery of IDE inhibitors, as well as challenges in developing IDE-based therapy for human diseases, particularly T2DM. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. A highly sensitive peptide substrate for detecting two Aß-degrading enzymes: neprilysin and insulin-degrading enzyme.

    PubMed

    Chen, Po-Ting; Liao, Tai-Yan; Hu, Chaur-Jong; Wu, Shu-Ting; Wang, Steven S-S; Chen, Rita P-Y

    2010-06-30

    Neprilysin has been singled out as the most promising candidate for use in the degradation of Abeta as a therapy for Alzheimer's disease. In this study, a quenched fluorogenic peptide substrate containing the first seven residues of the Abeta peptide plus a C-terminal Cysteine residue was synthesized to detect neprilysin activity. A fluorophore was attached to the C-terminal Cysteine and its fluorescence was quenched by a quencher linked to the N-terminus of the peptide. When this peptide substrate was degraded by an endopeptidase, fluorescence was produced and proved to be a sensitive detection system for endopeptidase activity. Our results showed that this assay system was extremely sensitive to neprilysin and insulin-degrading enzyme, but insensitive, or much less sensitive, to other Abeta-degrading enzymes. As low as 0.1 nM of neprilysin and 0.2 nM of insulin-degrading enzyme can be detected. Copyright 2010 Elsevier B.V. All rights reserved.

  4. Ensemble cryoEM elucidates the mechanism of insulin capture and degradation by human insulin degrading enzyme

    PubMed Central

    Bailey, Lucas J; Tan, Yong Zi; Wei, Hui; Wang, Andrew; Farcasanu, Mara; Woods, Virgil A; McCord, Lauren A; Lee, David; Shang, Weifeng; Deprez-Poulain, Rebecca; Deprez, Benoit; Liu, David R; Koide, Akiko; Koide, Shohei; Kossiakoff, Anthony A

    2018-01-01

    Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies. PMID:29596046

  5. Catalytic site inhibition of insulin-degrading enzyme by a small molecule induces glucose intolerance in mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Deprez-Poulain, Rebecca; Hennuyer, Nathalie; Bosc, Damien

    Insulin-degrading enzyme (IDE) is a protease that cleaves insulin and other bioactive peptides such as amyloid-β. Knockout and genetic studies have linked IDE to Alzheimer’s disease and type-2 diabetes. As the major insulin-degrading protease, IDE is a candidate drug target in diabetes. Here we have used kinetic target-guided synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and small angle X-ray scattering analyses show that it locks IDE in a closed conformation. Among a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signalling and surprisinglymore » impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes.« less

  6. Catalytic site inhibition of insulin-degrading enzyme by a small molecule induces glucose intolerance in mice

    DOE PAGES

    Deprez-Poulain, Rebecca; Hennuyer, Nathalie; Bosc, Damien; ...

    2015-09-23

    Insulin-degrading enzyme (IDE) is a protease that cleaves insulin and other bioactive peptides such as amyloid-β. Knockout and genetic studies have linked IDE to Alzheimer’s disease and type-2 diabetes. As the major insulin-degrading protease, IDE is a candidate drug target in diabetes. Here we have used kinetic target-guided synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and small angle X-ray scattering analyses show that it locks IDE in a closed conformation. Among a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signalling and surprisinglymore » impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes.« less

  7. Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones

    PubMed Central

    Maianti, Juan Pablo; McFedries, Amanda; Foda, Zachariah H.; Kleiner, Ralph E.; Du, Xiu Quan; Leissring, Malcolm A.; Tang, Wei-Jen; Charron, Maureen J.; Seeliger, Markus A.; Saghatelian, Alan; Liu, David R.

    2014-01-01

    Despite decades of speculation that inhibiting endogenous insulin degradation might treat type-2 diabetes1, 2, and the identification of IDE (insulin-degrading enzyme) as a diabetes susceptibility gene3, 4, the relationship between the activity of the zinc metalloprotein IDE and glucose homeostasis remains unclear. Although Ide−/− mice have elevated insulin levels, they exhibit impaired, rather than improved, glucose tolerance that may arise from compensatory insulin signalling dysfunction5, 6. IDE inhibitors that are active in vivo are therefore needed to elucidate IDE’s physiological roles and to determine its potential to serve as a target for the treatment of diabetes. Here we report the discovery of a physiologically active IDE inhibitor identified from a DNA-templated macrocycle library. An X-ray structure of the macrocycle bound to IDE reveals that it engages a binding pocket away from the catalytic site, which explains its remarkable selectivity. Treatment of lean and obese mice with this inhibitor shows that IDE regulates the abundance and signalling of glucagon and amylin, in addition to that of insulin. Under physiological conditions that augment insulin and amylin levels, such as oral glucose administration, acute IDE inhibition leads to substantially improved glucose tolerance and slower gastric emptying. These findings demonstrate the feasibility of modulating IDE activity as a new therapeutic strategy to treat type-2 diabetes and expand our understanding of the roles of IDE in glucose and hormone regulation. PMID:24847884

  8. Characterization of Insulin Degrading Enzyme and other Aβ Degrading Proteases in Human Serum: a Role in Alzheimer’s disease?

    PubMed Central

    Liu, Zhiheng; Zhu, Haihao; Fang, Guang Guang; Walsh, Kathryn; Mwamburi, Maya; Wolozin, Benjamin; Abdul-Hay, Same O.; Ikezu, Tsuneya; Lessring, Malcolm A.; Qiu, Wei Qiao

    2013-01-01

    Sporadic Alzheimer’s disease (AD) patients have low amyloid-β peptide (Aβ) clearance in the central nervous system (CNS). The peripheral Aβ clearance may also be important but its role in AD remains unclear. We aimed to study the Aβ degrading proteases including insulin degrading enzyme (IDE), angiotensin converting enzyme (ACE) and others in blood. Using the fluorogenic substrate V—a substrate of IDE and other metalloproteases, we showed that human serum degraded the substrate V, and the activity was inhibited by adding increasing dose of Aβ. The existence of IDE activity was demonstrated by the inhibition of insulin, amylin or EDTA, and further confirmed by immunocapture of IDE using monoclonal antibodies. The involvement of ACE was indicated by the ability of the ACE inhibitor, lisinopril, to inhibit the substrate V degradation. To test the variations of substrate V degradation in humans, we used serum samples from a homebound elderly population with cognitive diagnoses. Compared with the elderly who had normal cognition, those with probable AD and amnestic mild cognitive impairment (amnestic MCI) had lower peptidase activities. Probable AD or amnestic MCI as an outcome remained negatively associated with serum substrate V degradation activity after adjusting for the confounders. The elderly with probable AD had lower serum substrate V degradation activity compared with those who had vascular dementia. The blood proteases mediating Aβ degradation may be important for the AD pathogenesis. More studies are needed to specify each Aβ degrading protease in blood as a useful biomarker and a possible treatment target for AD. PMID:22232014

  9. Eicosapentaenoic acid and docosahexaenoic acid increase the degradation of amyloid-β by affecting insulin-degrading enzyme.

    PubMed

    Grimm, Marcus O W; Mett, Janine; Stahlmann, Christoph P; Haupenthal, Viola J; Blümel, Tamara; Stötzel, Hannah; Grimm, Heike S; Hartmann, Tobias

    2016-12-01

    Omega-3 polyunsaturated fatty acids (PUFAs) have been proposed to be highly beneficial in Alzheimer's disease (AD). AD pathology is closely linked to an overproduction and accumulation of amyloid-β (Aβ) peptides as extracellular senile plaques in the brain. Total Aβ levels are not only dependent on its production by proteolytic processing of the amyloid precursor protein (APP), but also on Aβ-clearance mechanisms, including Aβ-degrading enzymes. Here we show that the omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increase Aβ-degradation by affecting insulin-degrading enzyme (IDE), the major Aβ-degrading enzyme secreted into the extracellular space of neuronal and microglial cells. The identification of the molecular mechanisms revealed that EPA directly increases IDE enzyme activity and elevates gene expression of IDE. DHA also directly stimulates IDE enzyme activity and affects IDE sorting by increasing exosome release of IDE, resulting in enhanced Aβ-degradation in the extracellular milieu. Apart from the known positive effect of DHA in reducing Aβ production, EPA and DHA might ameliorate AD pathology by increasing Aβ turnover.

  10. Identification of residues in the insulin molecule important for binding to insulin-degrading enzyme.

    PubMed

    Affholter, J A; Cascieri, M A; Bayne, M L; Brange, J; Casaretto, M; Roth, R A

    1990-08-21

    Insulin-degrading enzyme (IDE) hydrolyzes insulin at a limited number of sites. Although the positions of these cleavages are known, the residues of insulin important in its binding to IDE have not been defined. To this end, we have studied the binding of a variety of insulin analogues to the protease in a solid-phase binding assay using immunoimmobilized IDE. Since IDE binds insulin with 600-fold greater affinity than it does insulin-like growth factor I (25 nM and approximately 16,000 nM, respectively), the first set of analogues studied were hybrid molecules of insulin and IGF I. IGF I mutants [insB1-17,17-70]IGF I, [Tyr55,Gln56]IGF I, and [Phe23,Phe24,Tyr25]IGF I have been synthesized and share the property of having insulin-like amino acids at positions corresponding to primary sites of cleavage of insulin by IDE. Whereas the first two exhibit affinities for IDE similar to that of wild type IGF I, the [Phe23,Phe24,Tyr25]IGF I analogue has a 32-fold greater affinity for the immobilized enzyme. Replacement of Phe-23 by Ser eliminates this increase. Removal of the eight amino acid D-chain region of IGF I (which has been predicted to interfere with binding to the 23-25 region) results in a 25-fold increase in affinity for IDE, confirming the importance of residues 23-25 in the high-affinity recognition of IDE. A similar role for the corresponding (B24-26) residues of insulin is supported by the use of site-directed mutant and semisynthetic insulin analogues. Insulin mutants [B25-Asp]insulin and [B25-His]insulin display 16- and 20-fold decreases in IDE affinity versus wild-type insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Expression of metalloprotease insulin-degrading enzyme insulysin in normal and malignant human tissues.

    PubMed

    Yfanti, Christina; Mengele, Karin; Gkazepis, Apostolos; Weirich, Gregor; Giersig, Cecylia; Kuo, Wen-Liang; Tang, Wei-Jen; Rosner, Marsha; Schmitt, Manfred

    2008-10-01

    Insulin-degrading enzyme (IDE, insulysin, insulinase; EC 3.4.22.11), a thiol metalloendopeptidase, is involved in intracellular degradation of insulin, thereby inhibiting its translocation and accumulation to the nucleus. Recently, protein expression of IDE has been demonstrated in the epithelial ducts of normal breast and breast cancer tissue. Utilizing four different antibodies generated against different epitopes of the IDE molecule, we performed Western blot analysis and immunohistochemical staining on several normal human tissues, on a plethora of tumor cell lines of different tissue origin, and on malignant breast and ovarian tissue. Applying the four IDE-directed antibodies, we demonstrated IDE expression at the protein level, by means of immunoblotting and immunocytochemistry, in each of the tumor cell lines analyzed. Insulin-degrading enzyme protein expression was found in normal tissues of the kidney, liver, lung, brain, breast and skeletal muscle, as well as in breast and ovarian cancer tissues. Immunohistochemical visualization of IDE indicated cytoplasmic localization of IDE in each of the cell lines and tissues assessed. In conclusion, we performed for the first time a wide-ranging survey on IDE protein expression in normal and malignant tissues and cells thus extending our knowledge on the cellular and tissue distribution of IDE, an enzyme which to date has mainly been studied in connection with Alzheimer's disease and diabetes but not in cancer.

  12. [Effect of tongluo xingnao effervescent tablet on learning and memory of AD rats and expression of insulin-degrading enzyme in hippocampus].

    PubMed

    Zhang, Yin-Jie; Dai, Yuan; Hu, Yong; Ma, Yun-Tong; Xu, Shi-Jun; Wang, Yong-Yan

    2013-09-01

    To study the effect of Tongluo Xingnao effervescent tablet on learning and memory of dementia rats induced by injection of Abeta25-35 in hippocampus and expression of insulin-degrading enzyme in hippocampus, in order to provide basis for preventing and treating senile dementia. The dementia rat model was established by injecting Abeta25-35 in hippocampus. The rats were divided into the model control group, the Aricept (1.4 mg x kg(-1)) group, and Tongluo Xingnao effervescent tablet high dose (7.56 g x kg(-1)), middle dose (3.78 g x kg(-1)) and low dose (1.59 g x kg(-1)) groups. A sham operation group was established by injecting normal saline in hippocampus. The rats were orally given drugs for 90 days, once a day. Their learning and memory were tested by using Morris water maze. Immunohistochemistry and image analysis were utilized for a quantitative analysis on the expression of insulin-degrading enzyme in hippocampus. Tongluo Xingnao effervescent tablet could significantly shorten the escape latency of rats in the directional navigation test, prolong the retention time in the first quadrant dwell, decrease the retention time in the third quadrant dwell, increase the frequency of crossing the platform, show a more notable statistical significance than the model control group (P < 0.05). Additionally, it could also remarkably increase the average optical density of insulin-degrading enzyme in hippocampus, promote the expression of insulin-degrading enzyme in hippocampus, and show a more notable statistical significance than the model control group (P < 0.05). Tongluo Xingnao effervescent tablet has the effects of improving learning and memory capacity of AD rats and promoting the expression of insulin-degrading enzyme in hippocampus. Its effect in promoting intelligence will be related to increased insulin-degrading enzyme in hippocampus.

  13. Aβ-degrading enzymes: potential for treatment of Alzheimer disease.

    PubMed

    Miners, James Scott; Barua, Neil; Kehoe, Patrick Gavin; Gill, Steven; Love, Seth

    2011-11-01

    There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme, and endothelin-converting enzyme reduce Aβ levels and protect against cognitive impairment in mouse models of AD. The activity of several Aβ-degrading enzymes rises with age and increases still further in AD, perhaps as a physiological response to minimize the buildup of Aβ. The age- and disease-related changes in expression of more recently recognized Aβ-degrading enzymes (e.g. NEP-2 and cathepsin B) remain to be investigated, and there is strong evidence that reduced NEP activity contributes to the development of cerebral amyloid angiopathy. Regardless of the role of Aβ-degrading enzymes in the development of AD, experimental data indicate that increasing the activity of these enzymes (NEP in particular) has therapeutic potential in AD, although targeting their delivery to the brain remains a major challenge. The most promising current approaches include the peripheral administration of agents that enhance the activity of Aβ-degrading enzymes and the direct intracerebral delivery of NEP by convection-enhanced delivery. In the longer term, genetic approaches to increasing the intracerebral expression of NEP or other Aβ-degrading enzymes may offer advantages.

  14. Somatostatin Modulates Insulin-Degrading-Enzyme Metabolism: Implications for the Regulation of Microglia Activity in AD

    PubMed Central

    Tundo, Grazia; Ciaccio, Chiara; Sbardella, Diego; Boraso, Mariaserena; Viviani, Barbara; Coletta, Massimiliano; Marini, Stefano

    2012-01-01

    The deposition of β-amyloid (Aβ) into senile plaques and the impairment of somatostatin-mediated neurotransmission are key pathological events in the onset of Alzheimer's disease (AD). Insulin-degrading-enzyme (IDE) is one of the main extracellular protease targeting Aβ, and thus it represents an interesting pharmacological target for AD therapy. We show that the active form of somatostatin-14 regulates IDE activity by affecting its expression and secretion in microglia cells. A similar effect can also be observed when adding octreotide. Following a previous observation where somatostatin directly interacts with IDE, here we demonstrate that somatostatin regulates Aβ catabolism by modulating IDE proteolytic activity in IDE gene-silencing experiments. As a whole, these data indicate the relevant role played by somatostatin and, potentially, by analogue octreotide, in preventing Aβ accumulation by partially restoring IDE activity. PMID:22509294

  15. Somatostatin modulates insulin-degrading-enzyme metabolism: implications for the regulation of microglia activity in AD.

    PubMed

    Tundo, Grazia; Ciaccio, Chiara; Sbardella, Diego; Boraso, Mariaserena; Viviani, Barbara; Coletta, Massimiliano; Marini, Stefano

    2012-01-01

    The deposition of β-amyloid (Aβ) into senile plaques and the impairment of somatostatin-mediated neurotransmission are key pathological events in the onset of Alzheimer's disease (AD). Insulin-degrading-enzyme (IDE) is one of the main extracellular protease targeting Aβ, and thus it represents an interesting pharmacological target for AD therapy. We show that the active form of somatostatin-14 regulates IDE activity by affecting its expression and secretion in microglia cells. A similar effect can also be observed when adding octreotide. Following a previous observation where somatostatin directly interacts with IDE, here we demonstrate that somatostatin regulates Aβ catabolism by modulating IDE proteolytic activity in IDE gene-silencing experiments. As a whole, these data indicate the relevant role played by somatostatin and, potentially, by analogue octreotide, in preventing Aβ accumulation by partially restoring IDE activity.

  16. Impact of Insulin Degrading Enzyme and Neprilysin in Alzheimer's Disease Biology: Characterization of Putative Cognates for Therapeutic Applications.

    PubMed

    Jha, Niraj Kumar; Jha, Saurabh Kumar; Kumar, Dhiraj; Kejriwal, Noopur; Sharma, Renu; Ambasta, Rashmi K; Kumar, Pravir

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative process primarily characterized by amyloid-β (Aβ) agglomeration, neuroinflammation, and cognitive dysfunction. The prominent cause for dementia is the deposition of Aβ plaques and tau-neurofibrillary tangles that hamper the neuronal organization and function. Aβ pathology further affects numerous signaling cascades that disturb the neuronal homeostasis. For instance, Aβ deposition is responsible for altered expression of insulin encoding genes that lead to insulin resistance, and thereby affecting insulin signaling pathway and glucose metabolism in the brain. As a result, the common pathology of insulin resistance between Type-2 diabetes mellitus and AD has led AD to be proposed as a form of diabetes and termed 'Type-3 diabetes'. Since accumulation of Aβ is the prominent cause of neuronal toxicity in AD, its clearance is the prime requisite for therapeutic prospects. This purpose is expertly fulfilled by the potential role of Aβ degrading enzymes such as insulin degrading enzyme (IDE) and Neprilysin (NEP). Therefore, their molecular study is important to uncover the proteolytic and regulatory mechanism of Aβ degradation. Herein, (i) In silico sequential and structural analysis of IDE and NEP has been performed to identify the molecular entities for proteolytic degradation of Aβ in the AD brain, (ii) to analyze their catalytic site to demonstrate the enzymatic action played by IDE and NEP, (iii) to identify their structural homologues that could behave as putative partners of IDE and NEP with similar catalytic action and (iv) to illustrate various IDE- and NEP-mediated therapeutic approaches and factors for clearing Aβ in AD.

  17. Dual Exosite-binding Inhibitors of Insulin-degrading Enzyme Challenge Its Role as the Primary Mediator of Insulin Clearance in Vivo*

    PubMed Central

    Durham, Timothy B.; Toth, James L.; Klimkowski, Valentine J.; Cao, Julia X. C.; Siesky, Angela M.; Alexander-Chacko, Jesline; Wu, Ginger Y.; Dixon, Jeffrey T.; McGee, James E.; Wang, Yong; Guo, Sherry Y.; Cavitt, Rachel Nicole; Schindler, John; Thibodeaux, Stefan J.; Calvert, Nathan A.; Coghlan, Michael J.; Sindelar, Dana K.; Christe, Michael; Kiselyov, Vladislav V.; Michael, M. Dodson; Sloop, Kyle W.

    2015-01-01

    Insulin-degrading enzyme (IDE, insulysin) is the best characterized catabolic enzyme implicated in proteolysis of insulin. Recently, a peptide inhibitor of IDE has been shown to affect levels of insulin, amylin, and glucagon in vivo. However, IDE−/− mice display variable phenotypes relating to fasting plasma insulin levels, glucose tolerance, and insulin sensitivity depending on the cohort and age of animals. Here, we interrogated the importance of IDE-mediated catabolism on insulin clearance in vivo. Using a structure-based design, we linked two newly identified ligands binding at unique IDE exosites together to construct a potent series of novel inhibitors. These compounds do not interact with the catalytic zinc of the protease. Because one of these inhibitors (NTE-1) was determined to have pharmacokinetic properties sufficient to sustain plasma levels >50 times its IDE IC50 value, studies in rodents were conducted. In oral glucose tolerance tests with diet-induced obese mice, NTE-1 treatment improved the glucose excursion. Yet in insulin tolerance tests and euglycemic clamp experiments, NTE-1 did not enhance insulin action or increase plasma insulin levels. Importantly, IDE inhibition with NTE-1 did result in elevated plasma amylin levels, suggesting the in vivo role of IDE action on amylin may be more significant than an effect on insulin. Furthermore, using the inhibitors described in this report, we demonstrate that in HEK cells IDE has little impact on insulin clearance. In total, evidence from our studies supports a minimal role for IDE in insulin metabolism in vivo and suggests IDE may be more important in helping regulate amylin clearance. PMID:26085101

  18. Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme.

    PubMed

    McCord, Lauren A; Liang, Wenguang G; Dowdell, Evan; Kalas, Vasilios; Hoey, Robert J; Koide, Akiko; Koide, Shohei; Tang, Wei-Jen

    2013-08-20

    Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer's disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ~18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N- and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides.

  19. Expression of metalloprotease insulin-degrading enzyme (insulysin) in normal and malignant human tissues

    PubMed Central

    Yfanti, Christina; Mengele, Karin; Gkazepis, Apostolos; Weirich, Gregor; Giersig, Cecylia; Kuo, Wen-Liang; Tang, Wei-Jen; Rosner, Marsha; Schmitt, Manfred

    2013-01-01

    Background Insulin-degrading enzyme (IDE, insulysin, insulinase; EC 3.4.22.11), a thiol metalloendopeptidase, is involved in intracellular degradation of insulin, thereby inhibiting its translocation and accumulation to the nucleus. Recently, protein expression of IDE has been demonstrated in the epithelial ducts of normal breast and in breast cancer tissue (Radulescu et al., Int J Oncol 30:73; 2007). Materials and Methods Utilizing four different antibodies generated against different epitopes of the IDE molecule, we performed western blot analysis and immunohistochemical staining on several normal human tissues, on a plethora of tumor cell lines of different tissue origin, and on malignant breast and ovarian tissue. Results Applying the four IDE-directed antibodies, we demonstrate IDE expression at the protein level, both by means of immunoblotting and immunocytochemistry, in all of the tumor cell lines analyzed. Besides, IDE protein expression was found in normal tissues of the kidney, liver, lung, brain, breast and skeletal muscle, as well as in breast and ovarian cancer tissues. Immunohistochemical visualization of IDE indicated cytoplasmic localization of IDE in all of the cell lines and tissues assessed. Conclusions We performed for the first time a wide-ranging survey on IDE protein expression in normal and malignant tissues and cells and thus extend knowledge about cellular and tissue distribution of IDE, an enzyme which so far has mainly been studied in connection with Alzheimer’s disease and diabetes but not in cancer. PMID:18813847

  20. Nigella sativa Oil and Chromium Picolinate Ameliorate Fructose-Induced Hyperinsulinemia by Enhancing Insulin Signaling and Suppressing Insulin-Degrading Enzyme in Male Rats.

    PubMed

    Elseweidy, Mohamed Mahmoud; Amin, Rawia Sarhan; Atteia, Hebatallah Husseini; Aly, Maha Abdo

    2017-10-04

    In vivo and in vitro studies suggested that chromium enhances insulin sensitivity by promoting insulin receptor signaling. However, its effect on insulin clearance has not been yet identified. Nigella sativa, a widely used spice, possesses an antidiabetic activity. We, therefore, hypothesized that chromium picolinate may alter insulin clearance by modulating insulin-degrading enzyme (IDE) in insulin-resistant rats. We evaluated also the effect of Nigella sativa oil on insulin signaling and degradation with respect to chromium picolinate. To assess these hypotheses, insulin resistance was induced in 30 male Wistar albino rats through daily oral administration of high-fructose water (HFW, 20% w/v) for 45 days. These rats were then divided into three groups (n = 10/group). They were given either no treatment (control group) or Nigella sativa oil (500 mg/kg bw/day) or chromium picoloinate (200 μg/kg bw/day) orally along with HFW (20% w/v) for 45 days. Nigella sativa oil or chromium picolinate concurrent administration with HFW significantly decreased body weight, serum lipids, glucagon, insulin resistance, and hepatic IDE level but increased its mRNA expression and insulin receptor phosphorlyation as well as high-density lipoprotein cholesterol (HDL-C) level as compared to control group values, suggesting their potential as modulators for insulin signaling and clearance. However, Nigella sativa oil exerted better improvement in feeding efficacy ratio as well as the levels of glucagon, insulin, insulin resistance, hepatic IDE level and insulin receptor phosphorylation than chromium picolinate, suggesting its greater insulin sensitizing capacity. Our data, for the first time, prove that Nigella sativa oil and chromium picolinate monotherapy can reduce fructose-induced insulin resistance by reduction of hepatic IDE protein and activation of insulin receptor signaling.

  1. Structure–activity relationships of imidazole-derived 2-[N-carbamoylmethyl-alkylamino]acetic acids, dual binders of human insulin-degrading enzyme

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Charton, Julie; Gauriot, Marion; Totobenazara, Jane

    Insulin degrading enzyme (IDE) is a zinc metalloprotease that degrades small amyloid peptides such as amyloid-â and insulin. So far the dearth of IDE-specific pharmacological inhibitors impacts the understanding of its role in the physiopathology of Alzheimer's disease, amyloid-â clearance, and its validation as a potential therapeutic target. Hit 1 was previously discovered by high-throughput screening. Here we describe the structure-activity study, that required the synthesis of 48 analogues. We found that while the carboxylic acid, the imidazole and the tertiary amine were critical for activity, the methyl ester was successfully optimized to an amide or a 1,2,4-oxadiazole. Along withmore » improving their activity, compounds were optimized for solubility, lipophilicity and stability in plasma and microsomes. The docking or co-crystallization of some compounds at the exosite or the catalytic site of IDE provided the structural basis for IDE inhibition. The pharmacokinetic properties of best compounds 44 and 46 were measured in vivo. As a result, 44 (BDM43079) and its methyl ester precursor 48 (BDM43124) are useful chemical probes for the exploration of IDE's role.« less

  2. Structure-activity relationships of Imidazole-derived 2-[N-carbamoylmethyl-alkylamino]acetic acids, dual binders of human Insulin-Degrading Enzyme

    PubMed Central

    Charton, Julie; Gauriot, Marion; Totobenazara, Jane; Hennuyer, Nathalie; Dumont, Julie; Bosc, Damien; Marechal, Xavier; Elbakali, Jamal; Herledan, Adrien; Wen, Xiaoan; Ronco, Cyril; Gras-Masse, Helene; Heninot, Antoine; Pottiez, Virginie; Landry, Valerie; Staels, Bart; Liang, Wenguang G.; Leroux, Florence; Tang, Wei-Jen; Deprez, Benoit; Deprez-Poulain, Rebecca

    2015-01-01

    Insulin degrading enzyme (IDE) is a zinc metalloprotease that degrades small amyloid peptides such as amyloid-β and insulin. So far the dearth of IDE-specific pharmacological inhibitors impacts the understanding of its role in the physiopathology of Alzheimer’s disease, amyloid-β clearance, and its validation as a potential therapeutic target. Hit 1 was previously discovered by high-throughput screening. Here we describe the structure-activity study, that required the synthesis of 48 analogues. We found that while the carboxylic acid, the imidazole and the tertiary amine were critical for activity, the methyl ester was successfully optimized to an amide or a 1,2,4-oxadiazole. Along with improving their activity, compounds were optimized for solubility, lipophilicity and stability in plasma and microsomes. The docking or co-crystallization of some compounds at the exosite or the catalytic site of IDE provided the structural basis for IDE inhibition. The pharmacokinetic properties of best compounds 44 and 46 were measured in vivo. As a result, 44 (BDM43079) and its methyl ester precursor 48 (BDM43124) are useful chemical probes for the exploration of IDE’s role. PMID:25489670

  3. Metabolism of Cryptic Peptides Derived from Neuropeptide FF Precursors: The Involvement of Insulin-Degrading Enzyme

    PubMed Central

    Grasso, Giuseppe; Mielczarek, Przemyslaw; Niedziolka, Magdalena; Silberring, Jerzy

    2014-01-01

    The term “cryptome” refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes. PMID:25247577

  4. Somatostatin: a novel substrate and a modulator of insulin-degrading enzyme activity.

    PubMed

    Ciaccio, Chiara; Tundo, Grazia R; Grasso, Giuseppe; Spoto, Giuseppe; Marasco, Daniela; Ruvo, Menotti; Gioia, Magda; Rizzarelli, Enrico; Coletta, Massimo

    2009-02-06

    Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes beta-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward beta-amyloid. In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k(cat) (s(-1))=0.38 (+/-0.05); K(m) (M)=7.5 (+/-0.9) x 10(-6)] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic beta-amyloid through a decrease of the K(m) toward this substrate, which corresponds to the 10-25 amino acid sequence of the Abeta(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn(2+) ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic beta-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.

  5. Multiple allosteric sites are involved in the modulation of insulin-degrading-enzyme activity by somatostatin.

    PubMed

    Tundo, Grazia R; Di Muzio, Elena; Ciaccio, Chiara; Sbardella, Diego; Di Pierro, Donato; Polticelli, Fabio; Coletta, Massimo; Marini, Stefano

    2016-10-01

    Somatostatin is a cyclic peptide, released in the gastrointestinal system and the central nervous system, where it is involved in the regulation of cognitive and sensory functions, motor activity and sleep. It is a substrate of insulin-degrading enzyme (IDE), as well as a modulator of its activity and expression. In the present study, we have investigated the modulatory role of somatostatin on IDE activity at 37 °C and pH 7.3 for various substrates [i.e. insulin, β-amyloid (Aβ) 1-40 and bradykinin], aiming to quantitatively characterize the correlation between the specific features of the substrates and the regulatory mechanism. Functional data indicate that somatostatin, in addition to the catalytic site of IDE (being a substrate), is also able to bind to two additional exosites, which play different roles according to the size of the substrate and its binding mode to the IDE catalytic cleft. In particular, one exosite, which displays high affinity for somatostatin, regulates only the interaction of IDE with larger substrates (such as insulin and Aβ 1-40 ) in a differing fashion according to their various modes of binding to the enzyme. A second exosite, which is involved in the regulation of enzymatic processing by IDE of all substrates investigated (including a 10-25 amino acid long amyloid-like peptide, bradykinin and somatostatin itself, which had been studied previously), probably acts through the alteration of an 'open-closed' equilibrium. © 2016 Federation of European Biochemical Societies.

  6. Abeta-degrading enzymes in Alzheimer's disease.

    PubMed

    Miners, James Scott; Baig, Shabnam; Palmer, Jennifer; Palmer, Laura E; Kehoe, Patrick G; Love, Seth

    2008-04-01

    In Alzheimer's disease (AD) Abeta accumulates because of imbalance between the production of Abeta and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Abeta is partly, if not in some cases solely, because of defects in its removal--mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Abeta. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Abeta-degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin-degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Abetain vitro or in animal studies include plasmin; endothelin-converting enzymes ECE-1 and -2; matrix metalloproteinases MMP-2, -3 and -9; and angiotensin-converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE-2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEepsilon4. We found no change in the level or activity of MMP-2, -3 or -9 in AD. The level and activity of ACE are increased, the level being directly related to Abeta plaque load. Up-regulation of some Abeta-degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Abeta-clearance pathways, reductions in the activity of particular Abeta-degrading enzymes may become critical, leading to the development of AD and CAA.

  7. C allele of the rs2209972 single nucleotide polymorphism of the insulin degrading enzyme gene and Alzheimer's disease in type 2 diabetes, a case control study.

    PubMed

    Gutiérrez-Hermosillo, Hugo; Díaz De León-González, Enrique; Palacios-Corona, Rebeca; Cedillo-Rodríguez, Javier Armando; Camacho-Luis, Abelardo; Reyes-Romero, Miguel Arturo; Medina-Chávez, Juan Humberto; Blandón, Pedro A

    2015-02-20

    In the last few decades we have witnessed an interesting transformation of the population pyramids throughout the world. As the population's life expectancy increases, there are more chronic diseases such as diabetes mellitus and dementias, and both of them have shown an association. To determine the association between Alzheimer's disease in diabetic patients and the insulin degrading enzyme in outpatients of a second level Hospital in Monterrey, Mexico. This was a case control study in which we included outpatients from the Geriatrics Clinic of a Hospital in Northeastern Mexico. Cases were patients with a Mini Mental Score Exam (MMSE) below 24 and DSM-IV criteria for Dementia. Controls were patients who had MMSE scores greater than 24. Data from 97 patients were analyzed. Regarding physical examination and the results of laboratory tests, there were no differences between the two groups (p>0.05). A 98% prevalence of the insulin degrading enzyme was documented in the sample studied. We found an association between a homozygous status for the CC genotype and Dementia with an estimated Odds Ratio (OR) of 2.5 (CI 95% 1.6-3.3) on the bivariate test, while, on the multivariate analysis, the OR was estimated 3.3 (CI 95% 1.3-8.2). Evidence shows that cognitive impairment is more frequent among those exposed to the C allele of the rs2209972 SNP of the insulin degrading enzyme gene. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

  8. Molecular Basis for the Recognition and Cleavages of IGF-II, TGF-[alpha], and Amylin by Human Insulin-Degrading Enzyme

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Guo, Qing; Manolopoulou, Marika; Bian, Yao

    2010-02-11

    Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-{beta}, peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulin-like growth factor (IGF)-II and transforming growth factor-{alpha} (TGF-{alpha}) over IGF-I and epidermal growth factor, respectively. Here, we used high-accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-{alpha}, amylin, reduced amylin, and amyloid-{beta} bymore » human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-{alpha} at 2.3 {angstrom} and IDE-amylin at 2.9 {angstrom}. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (amino acids 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and epidermal growth factor families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their N-terminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.« less

  9. Central insulin signaling is attenuated by long-term insulin exposure via insulin receptor substrate-1 serine phosphorylation, proteasomal degradation, and lysosomal insulin receptor degradation.

    PubMed

    Mayer, Christopher M; Belsham, Denise D

    2010-01-01

    Central insulin signaling is critical for the prevention of insulin resistance. Hyperinsulinemia contributes to insulin resistance, but it is not yet clear whether neurons are subject to cellular insulin resistance. We used an immortalized, hypothalamic, clonal cell line, mHypoE-46, which exemplifies neuronal function and expresses the components of the insulin signaling pathway, to determine how hyperinsulinemia modifies neuronal function. Western blot analysis indicated that prolonged insulin treatment of mHypoE-46 cells attenuated insulin signaling through phospho-Akt. To understand the mechanisms involved, time-course analysis was performed. Insulin exposure for 4 and 8 h phosphorylated Akt and p70-S6 kinase (S6K1), whereas 8 and 24 h treatment decreased insulin receptor (IR) and IR substrate 1 (IRS-1) protein levels. Insulin phosphorylation of S6K1 correlated with IRS-1 ser1101 phosphorylation and the mTOR-S6K1 pathway inhibitor rapamycin prevented IRS-1 serine phosphorylation. The proteasomal inhibitor epoxomicin and the lysosomal pathway inhibitor 3-methyladenine prevented the degradation of IRS-1 and IR by insulin, respectively, and pretreatment with rapamycin, epoxomicin, or 3-methyladenine prevented attenuation of insulin signaling by long-term insulin exposure. Thus, a sustained elevation of insulin levels diminishes neuronal insulin signaling through mTOR-S6K1-mediated IRS-1 serine phosphorylation, proteasomal degradation of IRS-1 and lysosomal degradation of the IR.

  10. Experimental strategy to discover microbes with gluten-degrading enzyme activities

    NASA Astrophysics Data System (ADS)

    Helmerhorst, Eva J.; Wei, Guoxian

    2014-06-01

    Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

  11. Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities.

    PubMed

    Helmerhorst, Eva J; Wei, Guoxian

    2014-05-05

    Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

  12. Effect of Tong Luo Jiu Nao on Aβ-degrading enzymes in AD rat brains.

    PubMed

    Liu, Yuan; Hua, Qian; Lei, Hongtao; Li, Pengtao

    2011-09-02

    Tong Luo Jiu Nao (TLJN) is a modern Chinese formula based on Traditional Chinese Medicine theory that has been used to treat ischemic cerebral stroke and vascular dementia. TLJN belongs to the ethnopharmacological family of medicines. In this study, we investigated the mechanism of the TLJN effect on Alzheimer's disease (AD). To investigate the effect of TLJN on β-amyloid-degrading enzymes and learning and memory in the AD rat brain. AD rats whose disease was induced by Aβ(25-35) injection into the bilateral hippocampus CA1 region were subjected to intragastric administration of various preparations. The experimental animals were healthy male Sprague-Dawley rats which were randomly divided into normal, sham, model, TLJN min, TLJN max and donepezil hydrochloride groups. Spontaneous alternation and passive avoidance behavior, which are regarded as measures of spatial learning and memory, were investigated using Y-maze testing. Western blotting and immunohistochemistry were used to observe the therapeutic effect of TLJN on the deposits of amyloid plaque and on the expression of synaptophysin, insulin-degrading enzyme and neprilysin. Y-maze results showed that the AD model group presented with spatial learning and memory impairments. Hematoxylin-eosin and Congo red staining indicated neuronal impairment and deposits of amyloid plaque in the model group and these results were consistent with their learning and memory deficits in the Y-maze. The TLJN-treated groups exhibited prolonged a cavity delitescence, decreased arm entries and improvement in learning and memory. Moreover, the structure of the neurons of the treated groups was restored and the expression of synaptophysin increased in both the hippocampus and cortex. In addition, their levels of insulin-degrading enzyme and neprilysin in the cortex and hippocampus were upregulated and the amyloid plaque was decreased. TLJN can improve learning and memory, up-regulate insulin-degrading enzyme and neprilysin levels

  13. Immunohistochemical evidence for ubiquitous distribution of metalloendoprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cell lines

    PubMed Central

    Weirich, Gregor; Mengele, Karin; Yfanti, Christina; Gkazepis, Apostolos; Hellmann, Daniela; Welk, Anita; Giersig, Cecylia; Kuo, Wen-Liang; Rosner, Marsha Rich; Tang, Wei-Jen; Schmitt, Manfred

    2013-01-01

    Immunohistochemical evidence for ubiquitous distribution of metalloprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cells is presented. Immunohistochemical staining was performed on a multi-organ tissue microarray (pancreas, lung, kidney, central/peripheral nervous system, liver, breast, placenta, myocardium, striated muscle, bone marrow, thymus, spleen) and on a cell microarray encompassing 31 tumor cell lines of different origin plus trophoblast cells, and normal blood lymphocytes and granulocytes. IDE protein is expressed by all of the tissues assessed and in all of the tumor cell lines except Raji and HL-60; trophoblast cells and granulocytes but not normal lymphocytes are also IDE-positive. PMID:18783335

  14. Targeted protein degradation by PROTACs.

    PubMed

    Neklesa, Taavi K; Winkler, James D; Crews, Craig M

    2017-06-01

    Targeted protein degradation using the PROTAC technology is emerging as a novel therapeutic method to address diseases driven by the aberrant expression of a disease-causing protein. PROTAC molecules are bifunctional small molecules that simultaneously bind a target protein and an E3-ubiquitin ligase, thus causing ubiquitination and degradation of the target protein by the proteasome. Like small molecules, PROTAC molecules possess good tissue distribution and the ability to target intracellular proteins. Herein, we highlight the advantages of protein degradation using PROTACs, and provide specific examples where degradation offers therapeutic benefit over classical enzyme inhibition. Foremost, PROTACs can degrade proteins regardless of their function. This includes the currently "undruggable" proteome, which comprises approximately 85% of all human proteins. Other beneficial aspects of protein degradation include the ability to target overexpressed and mutated proteins, as well as the potential to demonstrate prolonged pharmacodynamics effect beyond drug exposure. Lastly, due to their catalytic nature and the pre-requisite ubiquitination step, an exquisitely potent molecules with a high degree of degradation selectivity can be designed. Impressive preclinical in vitro and in vivo PROTAC data have been published, and these data have propelled the development of clinically viable PROTACs. With the molecular weight falling in the 700-1000Da range, the delivery and bioavailability of PROTACs remain the largest hurdles on the way to the clinic. Solving these issues and demonstrating proof of concept clinical data will be the focus of many labs over the next few years. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Immunohistochemical evidence of ubiquitous distribution of the metalloendoprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cell lines.

    PubMed

    Weirich, Gregor; Mengele, Karin; Yfanti, Christina; Gkazepis, Apostolos; Hellmann, Daniela; Welk, Anita; Giersig, Cecylia; Kuo, Wen-Liang; Rosner, Marsha Rich; Tang, Wei-Jen; Schmitt, Manfred

    2008-11-01

    Immunohistochemical evidence of ubiquitous distribution of the metalloprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cells is presented. Immunohistochemical staining was performed on a multi-organ tissue microarray (pancreas, lung, kidney, central/peripheral nervous system, liver, breast, placenta, myocardium, striated muscle, bone marrow, thymus, and spleen) and on a cell microarray of 31 tumor cell lines of different origin, as well as trophoblast cells and normal blood lymphocytes and granulocytes. IDE protein was expressed in all the tissues assessed and all the tumor cell lines except for Raji and HL-60. Trophoblast cells and granulocytes, but not normal lymphocytes, were also IDE-positive.

  16. Elucidation of interactions of Alzheimer amyloid beta peptides (Abeta40 and Abeta42) with insulin degrading enzyme: a molecular dynamics study.

    PubMed

    Bora, Ram Prasad; Prabhakar, Rajeev

    2010-05-11

    In this study, interactions of the two full-length Alzheimer amyloid beta peptides (Abeta40 and Abeta42) with the fully active form of insulin degrading enzyme (IDE) through unrestrained, all-atom MD simulations have been investigated. This enzyme is a Zn-containing metallopeptidase that catalyzes the degradation of the monomeric forms of these peptides, and this process is critical for preventing the progression of Alzheimer's disease (AD). The available X-ray structures of the free and small fragment-bound (Asp1-Glu3 and Lys16-Asp23 of Abeta40 and Asp1-Glu3 and Lys16-Glu22 of Abeta42) mutated forms of IDE and NMR structures of the full-length Abeta40 and Abeta42 have been used to build the starting structures for these simulations. The most representative structures derived from the Abeta40-IDE and Abeta42-IDE simulations accurately reproduced the locations of the active site Zn(2+) metal and small fragments of the substrates and their interactions with the enzyme from the X-ray structures. The remaining fragments of both the substrates were found to interact with IDE through several hydrogen bonding, pi-pi, CH-pi, and NH-pi interactions. In comparison to Abeta40, Abeta42 is more flexible and interacts through a smaller number (17-22) of hydrogen bonds in the catalytic chamber of IDE. Both the substrates adopted more beta-sheet character in the IDE environment, an observation that is in line with experiments. Their structural characteristics inside IDE are significantly different than the ones observed in aqueous solution. The atomistic level details provided by these simulations can help in the elucidation of binding and degrading mechanisms of the Abeta peptides by IDE.

  17. Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner.

    PubMed

    Hahn, Friedrich; Schmalen, Adrian; Setz, Christian; Friedrich, Melanie; Schlößer, Stefan; Kölle, Julia; Spranger, Robert; Rauch, Pia; Fraedrich, Kirsten; Reif, Tatjana; Karius-Fischer, Julia; Balasubramanyam, Ashok; Henklein, Petra; Fossen, Torgils; Schubert, Ulrich

    2017-01-01

    There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The 52 amino acid HIV-1 p6 Gag protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two late (L-) domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet. We found that the mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metalloendopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE-insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition. Our cumulative data support a model in which removal of p6 during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1.

  18. Association between polymorphisms of the insulin-degrading enzyme gene and late-onset Alzheimer disease.

    PubMed

    Wang, Shitao; He, Feiyan; Wang, Ying

    2015-06-01

    The insulin-degrading enzyme (IDE) gene is a strong positional and biological candidate for late-onset Alzheimer disease (LOAD) susceptibility, with recent studies independently demonstrating an association between IDE gene variants and LOAD. However, previous data have been controversial. To investigate the relationship between IDE gene polymorphisms and LOAD risk, a case-control association study of 406 Han Chinese participants in Xinjiang, China, was undertaken. The LOAD and control groups consisted of 202 and 204 participants, respectively. The single-nucleotide polymorphisms rs1887922 and rs1999764 of the IDE gene were linked to LOAD incidence. The presence of the CT+CC genotype of rs1999764 had a protective effect compared to the TT genotype (adjusted P=.0001; odds ratio [OR]=0.226; 95% confidence interval [CI]=0.116-0.441), while the CT+CC genotype of rs1887922 was associated with increased LOAD risk (adjusted P=.0001; OR=3.640; 95% CI=1.889-7.016). Moreover, the effects of rs1887922 and rs1999764 were associated with LOAD risk independent of the apolipoprotein E ∊4 polymorphism and were more significant in men and women, respectively. These results demonstrate that the polymorphisms rs1887922 and rs1999764 of the IDE gene are associated with LOAD susceptibility in the Xinjiang Han population. © The Author(s) 2014.

  19. Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

    PubMed Central

    Yui, Daishi; Nishida, Yoichiro; Nishina, Tomoko; Mogushi, Kaoru; Tajiri, Mio; Ishibashi, Satoru; Ajioka, Itsuki; Ishikawa, Kinya; Mizusawa, Hidehiro; Murayama, Shigeo; Yokota, Takanori

    2015-01-01

    Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa -/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa -/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa -/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD. PMID:26637123

  20. Glycation & Insulin Resistance: Novel Mechanisms and Unique Targets?

    PubMed Central

    Song, Fei; Schmidt, Ann Marie

    2012-01-01

    Objectives Multiple biochemical, metabolic and signal transduction pathways contribute to insulin resistance. In this review, we present the evidence that the post-translational process of protein glycation may play role in insulin resistance. The post-translational modifications, the advanced glycation endproducts (AGEs), are formed and accumulate by endogenous and exogenous mechanisms. Methods and Results AGEs may contribute to insulin resistance by a variety of mechanisms, including generation of tumor necrosis factor-alpha, direct modification of the insulin molecule thereby leading to its impaired action, generation of oxidative stress, and impairment of mitochondrial function, as examples. AGEs may stimulate signal transduction via engagement of cellular receptors, such as RAGE, or receptor for AGE. AGE-RAGE interaction perpetuates AGE formation and cellular stress via induction of inflammation, oxidative stress and reduction in the expression and activity of the enzyme, glyoxalase I that detoxifies the AGE precursor, methylglyoxal, or MG. Conclusions Once set in motion, glycation-promoting mechanisms may stimulate ongoing AGE production and target tissue stresses that reduce insulin responsiveness. Strategies to limit AGE accumulation and action may contribute to prevention of insulin resistance and its consequences. PMID:22815341

  1. Knockdown of Both Mitochondrial Isocitrate Dehydrogenase Enzymes In Pancreatic Beta Cells Inhibits Insulin Secretion

    PubMed Central

    MacDonald, Michael J.; Brown, Laura J.; Longacre, Melissa J.; Stoker, Scott W.; Kendrick, Mindy A.; Hasan, Noaman M.

    2013-01-01

    Background There are three isocitrate dehydrogenases (IDHs) in the pancreatic insulin cell; IDH1 (cytosolic) and IDH2 (mitochondrial) use NADP(H). IDH3 is mitochondrial, uses NAD(H) and was believed to be the IDH that supports the citric acid cycle. Methods With shRNAs targeting mRNAs for these enzymes we generated cell lines from INS-1 832/13 cells with severe (80%–90%) knockdown of the mitochondrial IDHs separately and together in the same cell line. Results With knockdown of both mitochondrial IDH’s mRNA, enzyme activity and protein level, but not with knockdown of one mitochondrial IDH, glucose- and BCH (an allosteric activator of glutamate dehydrogenase)-plus-glutamine-stimulated insulin release were inhibited. Cellular levels of citrate, α-ketoglutarate, malate and ATP were altered in patterns consistent with blockage at the mitochondrial IDH reactions. We were able to generate only 50% knockdown of Idh1 mRNA in multiple cell lines (without inhibition of insulin release) possibly because greater knockdown of IDH1 was not compatible with cell line survival. Conclusions The mitochondrial IDHs are redundant for insulin secretion. When both enzymes are severely knocked down, their low activities (possibly assisted by transport of IDH products and other metabolic intermediates from the cytosol into mitochondria) are sufficient for cell growth, but inadequate for insulin secretion when the requirement for intermediates is certainly more rapid. The results also indicate that IDH2 can support the citric acid cycle. General Significance As almost all mammalian cells possess substantial amounts of all three IDH enzymes, the biological principles suggested by these results are probably extrapolatable to many tissues. PMID:23876293

  2. Bacterial enzymes involved in lignin degradation.

    PubMed

    de Gonzalo, Gonzalo; Colpa, Dana I; Habib, Mohamed H M; Fraaije, Marco W

    2016-10-20

    Lignin forms a large part of plant biomass. It is a highly heterogeneous polymer of 4-hydroxyphenylpropanoid units and is embedded within polysaccharide polymers forming lignocellulose. Lignin provides strength and rigidity to plants and is rather resilient towards degradation. To improve the (bio)processing of lignocellulosic feedstocks, more effective degradation methods of lignin are in demand. Nature has found ways to fully degrade lignin through the production of dedicated ligninolytic enzyme systems. While such enzymes have been well thoroughly studied for ligninolytic fungi, only in recent years biochemical studies on bacterial enzymes capable of lignin modification have intensified. This has revealed several types of enzymes available to bacteria that enable them to act on lignin. Two major classes of bacterial lignin-modifying enzymes are DyP-type peroxidases and laccases. Yet, recently also several other bacterial enzymes have been discovered that seem to play a role in lignin modifications. In the present review, we provide an overview of recent advances in the identification and use of bacterial enzymes acting on lignin or lignin-derived products. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

  3. Maternal Lead Exposure Induces Down-regulation of Hippocampal Insulin-degrading Enzyme and Nerve Growth Factor Expression in Mouse Pups.

    PubMed

    Li, Xing; Li, Ning; Sun, Hua Lei; Yin, Jun; Tao, Yu Chang; Mao, Zhen Xing; Yu, Zeng Li; Li, Wen Jie; Bogden, John D

    2017-03-01

    Lead exposure is a known potential risk factor for neurodegenerative diseases such as Alzheimer's disease (AD). Exposure to lead during the critical phase of brain development has been linked with mental retardation and hypophrenia in later life. This study was aimed to investigate the effects of lead exposure of pregnant mice on the expressions of insulin-degrading enzyme (IDE) and nerve growth factor (NGF) in the hippocampus of their offspring. Blood samples were collected from the tail vein, and after anesthetizing the pups, the brain was excised on postnatal day 21. Lead concentrations were determined by graphite furnace atomic absorption spectrophotometry, and the expressions of IDE and NGF were determined by immunohistochemistry and Western blotting. Results showed that the reduction in IDE and NGF expression in the hippocampus of pups might be associated with impairment of learning and memory and dementia induced by maternal lead exposure during pregnancy and lactation. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  4. Synthesis and Testing of Polymers Susceptible to Degradation by Proteolytic Enzymes

    DTIC Science & Technology

    1975-05-01

    diisocyanatohexane, was biodegraded by the enzymes urease and rennin and also by two fungi. The tensile strength was greater than 10,000 psi, with high...Copolymer Degradation by Urease Enzyme Copolymer Degradation by Rennin Enzyme Degradation of Modified Gelatins: Undrawn Bulk Material Degradation of...bacteria. Results with urease enzyme did indicate significant degradation, as shown by the following tables: Table 1. Copolymer Degradation by

  5. Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity.

    PubMed

    Kooistra, T; Lloyd, J B

    1985-01-01

    The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation.

  6. Liposomes containing glycocholate as potential oral insulin delivery systems: preparation, in vitro characterization, and improved protection against enzymatic degradation

    PubMed Central

    Niu, Mengmeng; Lu, Yi; Hovgaard, Lars; Wu, Wei

    2011-01-01

    Background: Oral delivery of insulin is challenging and must overcome the barriers of gastric and enzymatic degradation as well as low permeation across the intestinal epithelium. The present study aimed to develop a liposomal delivery system containing glycocholate as an enzyme inhibitor and permeation enhancer for oral insulin delivery. Methods: Liposomes containing sodium glycocholate were prepared by a reversed-phase evaporation method followed by homogenization. The particle size and entrapment efficiency of recombinant human insulin (rhINS)-loaded sodium glycocholate liposomes can be easily adjusted by tuning the homogenization parameters, phospholipid:sodium glycocholate ratio, insulin:phospholipid ratio, water:ether volume ratio, interior water phase pH, and the hydration buffer pH. Results: The optimal formulation showed an insulin entrapment efficiency of 30% ± 2% and a particle size of 154 ± 18 nm. A conformational study by circular dichroism spectroscopy and a bioactivity study confirmed the preserved integrity of rhINS against preparative stress. Transmission electron micrographs revealed a nearly spherical and deformed structure with discernable lamella for sodium glycocholate liposomes. Sodium glycocholate liposomes showed better protection of insulin against enzymatic degradation by pepsin, trypsin, and α-chymotrypsin than liposomes containing the bile salt counterparts of sodium taurocholate and sodium deoxycholate. Conclusion: Sodium glycocholate liposomes showed promising in vitro characteristics and have the potential to be able to deliver insulin orally. PMID:21822379

  7. Proteomic researches for lignocellulose-degrading enzymes: A mini-review.

    PubMed

    Guo, Hongliang; Wang, Xiao-Dong; Lee, Duu-Jong

    2018-05-31

    Protective action of lignin/hemicellulose networks and crystalline structures of embedded cellulose render lignocellulose material resistant to external enzymatic attack. To eliminate this bottleneck, research has been conducted in which advanced proteomic techniques are applied to identify effective commercial hydrolytic enzymes. This mini-review summarizes researches on lignocellulose-degrading enzymes, the mechanisms of the responses of various lignocellulose-degrading strains and microbial communities to various carbon sources and various biomass substrates, post-translational modifications of lignocellulose-degrading enzymes, new lignocellulose-degrading strains, new lignocellulose-degrading enzymes and a new method of secretome analysis. The challenges in the practical use of enzymatic hydrolysis process to realize lignocellulose biorefineries are discussed, along with the prospects for the same. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Targeted polypeptide degradation

    DOEpatents

    Church, George M [Brookline, MA; Janse, Daniel M [Brookline, MA

    2008-05-13

    This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.

  9. Glucose-responsive microgels integrated with enzyme nanocapsules for closed-loop insulin delivery.

    PubMed

    Gu, Zhen; Dang, Tram T; Ma, Minglin; Tang, Benjamin C; Cheng, Hao; Jiang, Shan; Dong, Yizhou; Zhang, Yunlong; Anderson, Daniel G

    2013-08-27

    A glucose-responsive closed-loop insulin delivery system represents the ideal treatment of type 1 diabetes mellitus. In this study, we develop uniform injectable microgels for controlled glucose-responsive release of insulin. Monodisperse microgels (256 ± 18 μm), consisting of a pH-responsive chitosan matrix, enzyme nanocapsules, and recombinant human insulin, were fabricated through a one-step electrospray procedure. Glucose-specific enzymes were covalently encapsulated into the nanocapsules to improve enzymatic stability by protecting from denaturation and immunogenicity as well as to minimize loss due to diffusion from the matrix. The microgel system swelled when subjected to hyperglycemic conditions, as a result of the enzymatic conversion of glucose into gluconic acid and protonation of the chitosan network. Acting as a self-regulating valve system, microgels were adjusted to release insulin at basal release rates under normoglycemic conditions and at higher rates under hyperglycemic conditions. Finally, we demonstrated that these microgels with enzyme nanocapsules facilitate insulin release and result in a reduction of blood glucose levels in a mouse model of type 1 diabetes.

  10. ENZYME DEGRADATION OF CHIRAL ORGANIC PHOSPHORUS INSECTICIDES

    EPA Science Inventory

    Chiral organic phosphorus pesticides (OPs) are expected to be biologically degraded enantioselectively by endogenous enzymes. Various chiral Ops were treated with the enzyme phosphotriesterase (PTE) obtained from partially purified extracts of Escherichia coli strain DH-5- carryi...

  11. Targeted enzyme prodrug therapies.

    PubMed

    Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

    2010-09-01

    The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

  12. Monooxygenase, a Novel Beta-Cypermethrin Degrading Enzyme from Streptomyces sp

    PubMed Central

    Xiao, Ying; Deng, Yinyue; Chang, Changqing; Zhong, Guohua; Hu, Meiying; Zhang, Lian-Hui

    2013-01-01

    The widely used insecticide beta-cypermethrin has become a public concern because of its environmental contamination and toxic effects on mammals. In this study, a novel beta-cypermethrin degrading enzyme designated as CMO was purified to apparent homogeneity from a Streptomyces sp. isolate capable of utilizing beta-cypermethrin as a growth substrate. The native enzyme showed a monomeric structure with a molecular mass of 41 kDa and pI of 5.4. The enzyme exhibited the maximal activity at pH 7.5 and 30°C. It was fairly stable in the pH range from 6.5–8.5 and at temperatures below 10°C. The enzyme activity was significantly stimulated by Fe2+, but strongly inhibited by Ag+, Al3+, and Cu2+. The enzyme catalyzed the degradation of beta-cypermethrin to form five products via hydroxylation and diaryl cleavage. A novel beta-cypermethrin detoxification pathway was proposed based on analysis of these products. The purified enzyme was identified as a monooxygenase by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis (MALDI-TOF-MS) and N-terminal protein sequencing. Given that all the characterized pyrethroid-degrading enzymes are the members of hydrolase family, CMO represents the first pyrethroid-degrading monooxygenase identified from environmental microorganisms. Taken together, our findings depict a novel pyrethroid degradation mechanism and indicate that the purified enzyme may be a promising candidate for detoxification of beta-cypermethrin and environmental protection. PMID:24098697

  13. Structures of Human CCL18, CCL3, and CCL4 Reveal Molecular Determinants for Quaternary Structures and Sensitivity to Insulin-Degrading Enzyme

    DOE PAGES

    Liang, Wenguang G.; Ren, Min; Zhao, Fan; ...

    2015-01-27

    CC chemokine ligands (CCL) are 8-14 kDa signaling proteins involved in diverse immune functions. While CCLs share similar tertiary structures, oligomerization produces highly diverse quaternary structures that protect chemokines from proteolytic degradation and modulate their functions. CCL18 is closely related to CCL3 and CCL4 with respect to both protein sequence and genomic location, yet CCL18 has distinct biochemical and biophysical properties. Here in this paper, we report a crystal structure of human CCL18 and its oligomerization states in solution based on crystallographic and small angle X-ray scattering (SAXS) analyses. Our data shows that CCL18 adopts an α-helical conformation at itsmore » N-terminus that weakens its dimerization, explaining CCL18’s preference for the monomeric state. Multiple contacts between monomers allow CCL18 to reversibly form a unique open-ended oligomer different from those of CCL3, CCL4, and CCL5. Furthermore, these differences hinge on proline 8, which is conserved in CCL3 and CCL4, but is replaced by lysine in human CCL18. Our structural analyses suggest that a proline 8 to alanine mutation stabilizes a type I β-turn at the N-terminus of CCL4 to prevent dimerization but prevents dimers from making key contacts with each other in CCL3. Thus, the P8A mutation induces depolymerization of CCL3 and CCL4 by distinct mechanisms. Finally, we used structural, biochemical, and functional analyses to unravel why insulin-degrading enzyme (IDE) degrades CCL3 and CCL4 but not CCL18. Lastly, our results elucidate the molecular basis for the oligomerization of three closely related CC chemokines and suggest how oligomerization shapes CCL chemokine function.« less

  14. Structures of human CCL18, CCL3, and CCL4 reveal molecular determinants for quaternary structures and sensitivity to insulin-degrading enzyme.

    PubMed

    Liang, Wenguang G; Ren, Min; Zhao, Fan; Tang, Wei-Jen

    2015-03-27

    CC chemokine ligands (CCLs) are 8- to 14-kDa signaling proteins involved in diverse immune functions. While CCLs share similar tertiary structures, oligomerization produces highly diverse quaternary structures that protect chemokines from proteolytic degradation and modulate their functions. CCL18 is closely related to CCL3 and CCL4 with respect to both protein sequence and genomic location, yet CCL18 has distinct biochemical and biophysical properties. Here, we report a crystal structure of human CCL18 and its oligomerization states in solution based on crystallographic and small-angle X-ray scattering analyses. Our data show that CCL18 adopts an α-helical conformation at its N-terminus that weakens its dimerization, explaining CCL18's preference for the monomeric state. Multiple contacts between monomers allow CCL18 to reversibly form a unique open-ended oligomer different from those of CCL3, CCL4, and CCL5. Furthermore, these differences hinge on proline 8, which is conserved in CCL3 and CCL4 but is replaced by lysine in human CCL18. Our structural analyses suggest that a mutation of proline 8 to alanine stabilizes a type 1 β-turn at the N-terminus of CCL4 to prevent dimerization but prevents dimers from making key contacts with each other in CCL3. Thus, the P8A mutation induces depolymerization of CCL3 and CCL4 by distinct mechanisms. Finally, we used structural, biochemical, and functional analyses to unravel why insulin-degrading enzyme degrades CCL3 and CCL4 but not CCL18. Our results elucidate the molecular basis for the oligomerization of three closely related CC chemokines and suggest how oligomerization shapes CCL chemokine function. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Novel N-substituted aminobenzamide scaffold derivatives targeting the dipeptidyl peptidase-IV enzyme.

    PubMed

    Al-Balas, Qosay A; Sowaileh, Munia F; Hassan, Mohammad A; Qandil, Amjad M; Alzoubi, Karem H; Mhaidat, Nizar M; Almaaytah, Ammar M; Khabour, Omar F

    2014-01-01

    The dipeptidyl peptidase-IV (DPP-IV) enzyme is considered a pivotal target for controlling normal blood sugar levels in the body. Incretins secreted in response to ingestion of meals enhance insulin release to the blood, and DPP-IV inactivates these incretins within a short period and stops their action. Inhibition of this enzyme escalates the action of incretins and induces more insulin to achieve better glucose control in diabetic patients. Thus, inhibition of this enzyme will lead to better control of blood sugar levels. In this study, computer-aided drug design was used to help establish a novel N-substituted aminobenzamide scaffold as a potential inhibitor of DPP-IV. CDOCKER software available from Discovery Studio 3.5 was used to evaluate a series of designed compounds and assess their mode of binding to the active site of the DPP-IV enzyme. The designed compounds were synthesized and tested against a DPP-IV enzyme kit provided by Enzo Life Sciences. The synthesized compounds were characterized using proton and carbon nuclear magnetic resonance, mass spectrometry, infrared spectroscopy, and determination of melting point. Sixty-nine novel compounds having an N-aminobenzamide scaffold were prepared, with full characterization. Ten of these compounds showed more in vitro activity against DPP-IV than the reference compounds, with the most active compounds scoring 38% activity at 100 μM concentration. The N-aminobenzamide scaffold was shown in this study to be a valid scaffold for inhibiting the DPP-IV enzyme. Continuing work could unravel more active compounds possessing the same scaffold.

  16. Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

    PubMed Central

    Janssen, George; Christis, Chantal; Kooy-Winkelaar, Yvonne; Edens, Luppo; Smith, Drew

    2015-01-01

    Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme

  17. Effect of ethylenediamine on chemical degradation of insulin aspart in pharmaceutical solutions.

    PubMed

    Poulsen, Christian; Jacobsen, Dorte; Palm, Lisbeth

    2008-11-01

    To examine the effect of different amine compounds on the chemical degradation of insulin aspart at pharmaceutical formulation conditions. Insulin aspart preparations containing amine compounds or phosphate (reference) were prepared and the chemical degradation was assessed following storage at 37 degrees C using chromatographic techniques. Ethylenediamine was examined at multiple concentrations and the resulting insulin-ethylenediamine derivates were structurally characterized using matrix assisted laser desorption ionization time-of-flight mass spectroscopy. The effects on ethylenediamine when omitting glycerol or phenolic compounds from the formulations were investigated. Ethylenediamine was superior in terms of reducing formation of high molecular weight protein and insulin aspart related impurities compared to the other amine compounds and phosphate. Monotransamidation of insulin aspart in the presence of ethylenediamine was observed at all of the six possible Asn/Gln residues with Asn(A21) having the highest propensity to react with ethylenediamine. Data from formulations studies suggests a dual mechanism of ethylenediamine and a mandatory presence of phenolic compounds to obtain the effect. The formation of high molecular weight protein and insulin aspart related impurities was reduced by ethylenediamine in a concentration dependant manner.

  18. Autophagy and Mis-targeting of Therapeutic Enzyme in Skeletal Muscle in Pompe Disease

    PubMed Central

    Fukuda, Tokiko; Ahearn, Meghan; Roberts, Ashley; Mattaliano, Robert J.; Zaal, Kristien; Ralston, Evelyn; Plotz, Paul H.; Raben, Nina

    2009-01-01

    Enzyme replacement therapy (ERT) became a reality for patients with Pompe disease, a fatal cardiomyopathy and skeletal muscle myopathy caused by a deficiency of glycogen-degrading lysosomal enzyme acid alpha-glucosidase (GAA). The therapy, which relies on receptor-mediated endocytosis of recombinant human GAA (rhGAA), appears to be effective in cardiac muscle, but less so in skeletal muscle. We have previously shown a profound disturbance of the lysosomal degradative pathway (autophagy) in therapy-resistant muscle of GAA knockout mice (KO). Our findings here demonstrate a progressive age-dependent autophagic build-up in addition to enlargement of glycogen-filled lysosomes in multiple muscle groups in the KO. Trafficking and processing of the therapeutic enzyme along the endocytic pathway appear to be affected by the autophagy. Confocal microscopy of live single muscle fibers exposed to fluorescently labeled rhGAA indicates that a significant portion of the endocytosed enzyme in the KO was trapped as a partially processed form in the autophagic areas instead of reaching its target – the lysosomes. A fluid-phase endocytic marker was similarly mis-targeted and accumulated in vesicular structures within the autophagic areas. These findings may explain why ERT often falls short of reversing the disease process, and point to new avenues for the development of pharmacological intervention. PMID:17008131

  19. Lignocellulose-degrading enzymes from termites and their symbiotic microbiota.

    PubMed

    Ni, Jinfeng; Tokuda, Gaku

    2013-11-01

    Lignocellulose-the dry matter of plants, or "plant biomass"-digestion is of increasing interest in organismal metabolism research, specifically the conversion of biomass into biofuels. Termites efficiently decompose lignocelluloses, and studies on lignocellulolytic systems may elucidate mechanisms of efficient lignocellulose degradation in termites as well as offer novel enzyme sources, findings which have significant potential industrial applications. Recent progress in metagenomic and metatranscriptomic research has illuminated the diversity of lignocellulolytic enzymes within the termite gut. Here, we review state-of-the-art research on lignocellulose-degrading systems in termites, specifically cellulases, xylanases, and lignin modification enzymes produced by termites and their symbiotic microbiota. We also discuss recent investigations into heterologous overexpression of lignocellulolytic enzymes from termites and their symbionts. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Specific uptake, dissociation, and degradation of /sup 125/I-labeled insulin in isolated turtle (Chrysemys dorbigni) thyroid glands

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Marques, M.; da Silva, R.S.; Turyn, D.

    1985-11-01

    Thyroid glands from turtles (Chrysemys dorbigni) pretreated with potassium iodide were incubated with /sup 125/I-insulin in the presence or absence of unlabeled insulin, in order to study its specific uptake. At 24 degrees, the specific uptake reached a plateau at 180 min of incubation. The dose of bovine insulin that inhibited 50% of the /sup 125/I-insulin uptake was 2 micrograms/ml of incubation medium. Most of the radioactive material (71%) extracted from the gland, after 30 min incubation with /sup 125/I-insulin, eluted in the same position as labeled insulin on Sephadex G-50. Only 24% eluted in the salt position. After 240more » min incubation, increased amount of radioactivity appeared in the Na/sup 125/I position. When bovine insulin was added together with the labeled hormone, a substantial reduction of radioactivity was observed in the insulin and Na/sup 125/I elution positions. Dissociation studies were performed at 6 degrees in glands preincubated with /sup 125/I-insulin either at 24 or 6 degrees. The percentage of trichloroacetic acid (TCA)-soluble radioactive material in the dissociation medium increased with incubation time at both temperatures. However, the degradation activity was lower at 6 than at 24 degrees. The addition of bovine insulin to the incubation buffer containing /sup 125/I-insulin reduced the radioactive degradation products in the dissociated medium. Chloroquine or bacitracin inhibited the degradation activity. Incubation of thyroid glands with /sup 125/I-hGH or /sup 125/I-BSA showed values of uptake, dissociation, and degradation similar to those experiments in which an excess of bovine insulin was added together with the labeled hormone. Thus, by multiple criteria, such as specific uptake, dissociation, and degradation, the presence of insulin-binding sites in the turtle thyroid gland may be suggested.« less

  1. Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice.

    PubMed

    Robinson, Matthew M; Dasari, Surendra; Karakelides, Helen; Bergen, H Robert; Nair, K Sreekumaran

    2016-09-01

    Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that ∼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria. Copyright © 2016 the American Physiological Society.

  2. Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice

    PubMed Central

    Robinson, Matthew M.; Dasari, Surendra; Karakelides, Helen; Bergen, H. Robert

    2016-01-01

    Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that ∼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria. PMID:27436610

  3. Integrative computational approach for genome-based study of microbial lipid-degrading enzymes.

    PubMed

    Vorapreeda, Tayvich; Thammarongtham, Chinae; Laoteng, Kobkul

    2016-07-01

    Lipid-degrading or lipolytic enzymes have gained enormous attention in academic and industrial sectors. Several efforts are underway to discover new lipase enzymes from a variety of microorganisms with particular catalytic properties to be used for extensive applications. In addition, various tools and strategies have been implemented to unravel the functional relevance of the versatile lipid-degrading enzymes for special purposes. This review highlights the study of microbial lipid-degrading enzymes through an integrative computational approach. The identification of putative lipase genes from microbial genomes and metagenomic libraries using homology-based mining is discussed, with an emphasis on sequence analysis of conserved motifs and enzyme topology. Molecular modelling of three-dimensional structure on the basis of sequence similarity is shown to be a potential approach for exploring the structural and functional relationships of candidate lipase enzymes. The perspectives on a discriminative framework of cutting-edge tools and technologies, including bioinformatics, computational biology, functional genomics and functional proteomics, intended to facilitate rapid progress in understanding lipolysis mechanism and to discover novel lipid-degrading enzymes of microorganisms are discussed.

  4. A Simple Endpoint Assay for Starch-Degrading Enzymes.

    ERIC Educational Resources Information Center

    Kroen, William K.

    1998-01-01

    Since many of the important energy-transferring pathways involve synthesis or degradation of biological macromolecules, observations of the enzymes responsible for starch breakdown provide a useful case study. Provides a short, one-step assay for the enzymes amylase and amyloglucosidase. Topics covered include goals, preparation, assay procedure,…

  5. degradation or cerebral perfusion? Divergent effects of multifunctional enzymes.

    PubMed

    Miners, J Scott; Palmer, Jennifer C; Tayler, Hannah; Palmer, Laura E; Ashby, Emma; Kehoe, Patrick G; Love, Seth

    2014-01-01

    There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), endothelin-converting enzyme (ECE), and angiotensin-converting enzyme (ACE) reduce Aβ levels and protect against cognitive impairment in mouse models of AD. In post-mortem human brain tissue we have found that the activity of these Aβ-degrading enzymes rise with age and increases still further in AD, perhaps as a physiological response that helps to minimize the build-up of Aβ. ECE-1/-2 and ACE are also rate-limiting enzymes in the production of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors, increases in the levels of which are likely to contribute to reduced blood flow in AD. This review considers the possible interdependence between Aβ-degrading enzymes, ischemia and Aβ in AD: ischemia has been shown to increase Aβ production both in vitro and in vivo, whereas increased Aβ probably enhances ischemia by vasoconstriction, mediated at least in part by increased ECE and ACE activity. In contrast, NEP activity may help to maintain cerebral perfusion, by reducing the accumulation of Aβ in cerebral blood vessels and lessening its toxicity to vascular smooth muscle cells. In assessing the role of Aβ-degrading proteases in the pathogenesis of AD and, particularly, their potential as therapeutic agents, it is important to bear in mind the multifunctional nature of these enzymes and to consider their effects on other substrates and pathways.

  6. Insulin-egg yolk dispersions in self microemulsifying system.

    PubMed

    Singnurkar, P S; Gidwani, S K

    2008-11-01

    Formulation of insulin into a microemulsion very often presents a physicochemical instability during their preparation and storage. In order to overcome this lack of stability and facilitate the handling of these colloidal systems, stabilization of insulin in presence of hydrophobic components of a microemulsion appears as the most promising strategy. The present paper reports the use of egg yolk for stabilization of insulin in self microemulsifying dispersions. Insulin loaded egg yolk self microemulsifying dispersions were prepared by lyophilization followed by dispersion into self microemulsifying vehicle. The physicochemical characterization of selfmicroemulsifying dispersions includes such as insulin encapsulation efficiency, in vitro stability of insulin in presence of proteolytic enzymes and in vitro release. The biological activity of insulin from the dispersion was estimated by enzyme-linked immunosorbant assay and in vivo using Wistar diabetic rats. The particle size ranged 1.023±0.316 μm in diameter and insulin encapsulation efficiency was 98.2±0.9 %. Insulin hydrophobic self microemulsifying dispersions suppressed insulin release in pH 7.4 phosphate buffer and shown to protect insulin from enzymatic degradation in vitro in presence of chymotripsin. Egg yolk encapsulated insulin was bioactive, demonstrated through both in vivo and in vitro.

  7. Targeting Insulin Signaling for the Treatment of Alzheimer's Disease.

    PubMed

    Chen, Yanxing; Zhang, Jianfang; Zhang, Baorong; Gong, Cheng-Xin

    2016-01-01

    Sporadic Alzheimer's disease (AD) is caused by multiple etiological factors, among which impaired brain insulin signaling and decreased brain glucose metabolism are important metabolic factors. Contrary to previous belief that insulin would not act in the brain, studies in the last three decades have proven important roles of insulin and insulin signaling in various biological functions in the brain. Impaired brain insulin signaling or brain insulin resistance and its role in the molecular pathogenesis of sporadic AD have been demonstrated. Thus, targeting brain insulin signaling for the treatment of cognitive impairment and AD has now attracted much attention in the field of AD drug discovery. This article reviews recent studies that target brain insulin signaling, especially those investigations on intranasal insulin administration and drugs that improve insulin sensitivity, including incretins, dipeptidyl peptidase IV inhibitors, thiazolidinediones, and metformin. These drugs have been previously approved for the treatment of diabetes mellitus, which could expedite their development for the treatment of AD. Although larger clinical trials are needed for validating their efficacy for the treatment of cognitive impairment and AD, results of animal studies and clinical trials available to date are encouraging.

  8. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

    PubMed

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-07-22

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation*

    PubMed Central

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-01-01

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

  10. An ethanolic extract of Artemisia dracunculus L. regulates gene expression of ubiquitin-proteasome system enzymes in skeletal muscle: potential role in the treatment of sarcopenic obesity.

    PubMed

    Kirk-Ballard, Heather; Kilroy, Gail; Day, Britton C; Wang, Zhong Q; Ribnicky, David M; Cefalu, William T; Floyd, Z Elizabeth

    2014-01-01

    Obesity is linked to insulin resistance, a primary component of metabolic syndrome and type 2 diabetes. The problem of obesity-related insulin resistance is compounded when age-related skeletal muscle loss, called sarcopenia, occurs with obesity. Skeletal muscle loss results from elevated levels of protein degradation and prevention of obesity-related sarcopenic muscle loss will depend on strategies that target pathways involved in protein degradation. An extract from Artemisia dracunculus, termed PMI 5011, improves insulin signaling and increases skeletal muscle myofiber size in a rodent model of obesity-related insulin resistance. The aim of this study was to examine the effect of PMI 5011 on the ubiquitin-proteasome system, a central regulator of muscle protein degradation. Gastrocnemius and vastus lateralis skeletal muscle was obtained from KK-A(y) obese diabetic mice fed a control or 1% (w/w) PMI 5011-supplemented diet. Regulation of genes encoding enzymes of the ubiquitin-proteasome system was determined using real-time quantitative reverse transcriptase polymerase chain reaction. Although MuRF-1 ubiquitin ligase gene expression is consistently down-regulated in skeletal muscle, atrogin-1, Fbxo40, and Traf6 expression is differentially regulated by PMI 5011. Genes encoding other enzymes of the ubiquitin-proteasome system ranging from ubiquitin to ubiquitin-specific proteases are also regulated by PMI 5011. Additionally, expression of the gene encoding the microtubule-associated protein-1 light chain 3 (LC3), a ubiquitin-like protein pivotal to autophagy-mediated protein degradation, is down-regulated by PMI 5011 in the vastus lateralis. PMI 5011 alters the gene expression of ubiquitin-proteasome system enzymes that are essential regulators of skeletal muscle mass. This suggests that PMI 5011 has therapeutic potential in the treatment of obesity-linked sarcopenia by regulating ubiquitin-proteasome-mediated protein degradation. Copyright © 2014 Elsevier Inc

  11. [Ligninolytic enzyme production by white rot fungi during paraquat (herbicide) degradation].

    PubMed

    Camacho-Morales, Reyna L; Gerardo-Gerardo, José Luis; Guillén Navarro, Karina; Sánchez, José E

    Paraquat is a widely used herbicide in agriculture. Its inappropriate use and wide distribution represents a serious pollution problem for soil and water. White rot fungi are capable of degrading pollutants having a similar structure to that of lignin, such as paraquat. This study evaluated the degradation effect of paraquat on the production of ligninolytic enzymes by white rot fungi isolated from the South of Mexico. Six fungal strains showed tolerance to the herbicide in solid culture. Three of the six evaluated strains showed levels of degradation of 32, 26 and 47% (Polyporus tricholoma, Cilindrobasidium laeve and Deconica citrispora, respectively) after twelve days of cultivation in the presence of the xenobiotic. An increase in laccase and manganese peroxidase (MnP) activities was detected in the strains showing the highest percentage of degradation. Experiments were done with enzyme extracts from the extracellular medium with the two strains showing more degradation potential and enzyme production. After 24hours of incubation, a degradation of 49% of the initial paraquat concentration was observed for D. citrispora. These results suggest that paraquat degradation can be attributed to the presence of extracellular enzymes from white rot fungi. In this work the first evidence of the biodegradation potential of D. citrispora and Cilindrobasidium leave is shown. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  12. Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme

    PubMed Central

    Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; Holton, James M.; Charles, Emeric; O’Connell, Mitchell R.; Doudna, Jennifer A.

    2018-01-01

    CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR-Cas systems include the Cas13a enzyme, an RNA-activated ribonuclease (RNase) capable of crRNA processing and single-stranded RNA degradation upon target transcript binding. Here we present the 2.0 Å resolution crystal structure of a crRNA-bound L. bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define for the first time the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked prior to target RNA recognition, with implications for both bacterial immunity and diagnostic applications. PMID:28892041

  13. Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.

    CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

  14. Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

    DOE PAGES

    Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; ...

    2017-09-11

    CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

  15. Lignin degradation: microorganisms, enzymes involved, genomes analysis and evolution.

    PubMed

    Janusz, Grzegorz; Pawlik, Anna; Sulej, Justyna; Swiderska-Burek, Urszula; Jarosz-Wilkolazka, Anna; Paszczynski, Andrzej

    2017-11-01

    Extensive research efforts have been dedicated to describing degradation of wood, which is a complex process; hence, microorganisms have evolved different enzymatic and non-enzymatic strategies to utilize this plentiful plant material. This review describes a number of fungal and bacterial organisms which have developed both competitive and mutualistic strategies for the decomposition of wood and to thrive in different ecological niches. Through the analysis of the enzymatic machinery engaged in wood degradation, it was possible to elucidate different strategies of wood decomposition which often depend on ecological niches inhabited by given organism. Moreover, a detailed description of low molecular weight compounds is presented, which gives these organisms not only an advantage in wood degradation processes, but seems rather to be a new evolutionatory alternative to enzymatic combustion. Through analysis of genomics and secretomic data, it was possible to underline the probable importance of certain wood-degrading enzymes produced by different fungal organisms, potentially giving them advantage in their ecological niches. The paper highlights different fungal strategies of wood degradation, which possibly correlates to the number of genes coding for secretory enzymes. Furthermore, investigation of the evolution of wood-degrading organisms has been described. © FEMS 2017.

  16. Lignin degradation: microorganisms, enzymes involved, genomes analysis and evolution

    PubMed Central

    Pawlik, Anna; Sulej, Justyna; Świderska-Burek, Urszula; Jarosz-Wilkołazka, Anna; Paszczyński, Andrzej

    2017-01-01

    Abstract Extensive research efforts have been dedicated to describing degradation of wood, which is a complex process; hence, microorganisms have evolved different enzymatic and non-enzymatic strategies to utilize this plentiful plant material. This review describes a number of fungal and bacterial organisms which have developed both competitive and mutualistic strategies for the decomposition of wood and to thrive in different ecological niches. Through the analysis of the enzymatic machinery engaged in wood degradation, it was possible to elucidate different strategies of wood decomposition which often depend on ecological niches inhabited by given organism. Moreover, a detailed description of low molecular weight compounds is presented, which gives these organisms not only an advantage in wood degradation processes, but seems rather to be a new evolutionatory alternative to enzymatic combustion. Through analysis of genomics and secretomic data, it was possible to underline the probable importance of certain wood-degrading enzymes produced by different fungal organisms, potentially giving them advantage in their ecological niches. The paper highlights different fungal strategies of wood degradation, which possibly correlates to the number of genes coding for secretory enzymes. Furthermore, investigation of the evolution of wood-degrading organisms has been described. PMID:29088355

  17. Insulin attenuates atrophy of unweighted soleus muscle by amplified inhibition of protein degradation

    NASA Technical Reports Server (NTRS)

    Tischler, M. E.; Satarug, S.; Aannestad, A.; Munoz, K. A.; Henriksen, E. J.

    1997-01-01

    Unweighting atrophy of immature soleus muscle occurs rapidly over the first several days, followed by slower atrophy coinciding with increased sensitivity to insulin of in vitro protein metabolism. This study determined whether this increased sensitivity might account for the diminution of atrophy after 3 days of tall-cast hindlimb suspension. The physiological significance of the increased response to insulin in unweighted muscle was evaluated by analyzing in vivo protein metabolism for day 3 (48 to 72 hours) and day 4 (72 to 96 hours) of unweighting in diabetic animals either injected with insulin or not treated. Soleus from nontreated diabetic animals showed a similar loss of protein during day 3 (-16.2%) and day 4 (-14.5%) of unweighting, whereas muscle from insulin-treated animals showed rapid atrophy (-14.5%) during day 3 only, declining to just -3.1% the next day. Since fractional protein synthesis was similar for both day 3 (8.6%/d) and day 4 (7.0%/d) of unweighting in insulin-treated animals, the reduction in protein loss must be accounted for by a slowing of protein degradation due to circulating insulin. Intramuscular (IM) injection of insulin (600 nmol/L) stimulated in situ protein synthesis similarly in 4-day unweighted (+56%) and weight-bearing (+90%) soleus, even though unweighted muscle showed a greater in situ response of 2-deoxy-[3H]glucose uptake to IM injection of either insulin (133 nmol/L) or insulin-like growth factor-I (IGF-I) (200 nmol/L) than control muscle. These findings suggest that unweighted muscle is selectively more responsive in vivo to insulin, and that the slower atrophy after 3 days of unweighting was due to an increased effect of insulin on inhibiting protein degradation.

  18. Phage lytic enzymes targeting streptococci

    USDA-ARS?s Scientific Manuscript database

    Streptococcal pathogens contribute to a wide variety of human and livestock diseases. There is a need for new antimicrobials to replace over-used conventional antibiotics. Bacteriophage (viruses that infect bacteria) endolysins (enzymes that help degrade the bacterial cell wall) are ideal candidat...

  19. Immobilization of an enzyme from a Fusarium fungus WZ-I for chlorpyrifos degradation.

    PubMed

    Xie, Hui; Zhu, Lusheng; Ma, Tingting; Wang, Jun; Wang, Jinhua; Su, Jun; Shao, Bo

    2010-01-01

    The free enzyme extracted from WZ-I, which was identified as Fusarium LK. ex Fx, could effectively degrade chlorpyrifos, an organophosphate insecticide. The methods of immobilizing this free enzyme and determined its degradation-related characteristics were investigated. The properties of the immobilized enzyme were compared with those of the free enzyme. The optimal immobilization of the enzyme was achieved in a solution of 30 g/L sodium alginate at 4 degrees C for 4-12 hr. The immobilized enzyme showed the maximal activity at pH 8.0, 45 degrees C. The maximum initial rate and the substrate concentration of the immobilized enzyme were less than that of the free enzyme. The immobilized enzyme, therefore, had a higher capacity to withstand a broader range of temperatures and pH conditions than the free enzyme. With varying pH and temperatures, the immobilized enzyme was more active than the free enzyme in the degradation reaction. In addition, the immobilized enzyme exhibited only a slight loss in its initial activity, even after three repeated uses. The results showed that the immobilized enzyme was more resistant to different environmental conditions, suggesting that it was viable for future practical use.

  20. Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger

    PubMed Central

    van Munster, Jolanda M.; Thomas, Baptiste; Riese, Michel; Davis, Adrienne L.; Gray, Christopher J.; Archer, David B.; Flitsch, Sabine L.

    2017-01-01

    Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry. PMID:28220903

  1. Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.

    PubMed

    van Munster, Jolanda M; Thomas, Baptiste; Riese, Michel; Davis, Adrienne L; Gray, Christopher J; Archer, David B; Flitsch, Sabine L

    2017-02-21

    Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.

  2. Enzymes Involved in Naproxen Degradation by Planococcus sp. S5.

    PubMed

    Wojcieszyńska, Danuta; Domaradzka, Dorota; Hupert-Kocurek, Katarzyna; Guzik, Urszula

    2016-01-01

    Naproxen is a one of the most popular non-steroidal anti-inflammatory drugs (NSAIDs) entering the environment as a result of high consumption. For this reason, there is an emerging need to recognize mechanisms of its degradation and enzymes engaged in this process. Planococcus sp. S5 is a gram positive strain able to degrade naproxen in monosubstrate culture (27%). However, naproxen is not a sufficient growth substrate for this strain. In the presence of benzoate, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid or vanillic acid as growth substrates, the degradation of 21.5%, 71.71%, 14.75% and 8.16% of naproxen was observed respectively. It was shown that the activity of monooxygenase, hydroxyquinol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxyegnase in strain S5 was induced after growth of the strain with naproxen and 4-hydroxybenzoate. Moreover, in the presence of naproxen activity of gentisate 1,2-dioxygenase, enzyme engaged in 4-hydroxybenzoate metabolism, was completely inhibited. The obtained results suggest that monooxygenase and hydroxyquinol 1,2-dioxygenase are the main enzymes in naproxen degradation by Planococcus sp. S5.

  3. Inhibition and kinetic studies of lignin degrading enzymes of Ganoderma boninense by naturally occurring phenolic compounds.

    PubMed

    Surendran, Arthy; Siddiqui, Yasmeen; Saud, Halimi Mohd; Ali, Nusaibah Syd; Manickam, Sivakumar

    2018-05-22

    Lignolytic (Lignin degrading) enzyme, from oil palm pathogen Ganoderma boninense Pat. (Syn G. orbiforme (Ryvarden), is involved in the detoxification and the degradation of lignin in the oil palm and is the rate-limiting step in the infection process of this fungus. Active inhibition of lignin degrading enzymes secreted by G. boninense by various naturally occurring phenolic compounds and estimation of efficiency on pathogen suppression was aimed at. In our work, ten naturally occurring phenolic compounds were evaluated for their inhibitory potential towards the lignolytic enzymes of G.boninense. Additionally, the lignin degrading enzymes were characterised. Most of the peholic compounds exhibited an uncompetitive inhibition towards the lignin degrading enzymes. Benzoic acid was the superior inhibitor to the production of lignin degrading enzymes, when compared between the ten phenolic compounds. The inhibitory potential of the phenolic compounds toward the lignin degrading enzymes are higher than that of the conventional metal ion inhibitor. The lignin degrading enzymes were stable in a wide range of pH but were sensitive to higher to temperature. The study demonstrated the inhibitor potential of ten naturally occurring phenolic compounds toward the lignin degrading enzymes of G. boninense with different efficacies. The study has shed a light towards a new management strategy to control BSR in oil palm. It serves as replacement for the existing chemical control. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  4. Plant-Polysaccharide-Degrading Enzymes from Basidiomycetes

    PubMed Central

    Rytioja, Johanna; Hildén, Kristiina; Yuzon, Jennifer; Hatakka, Annele; de Vries, Ronald P.

    2014-01-01

    SUMMARY Basidiomycete fungi subsist on various types of plant material in diverse environments, from living and dead trees and forest litter to crops and grasses and to decaying plant matter in soils. Due to the variation in their natural carbon sources, basidiomycetes have highly varied plant-polysaccharide-degrading capabilities. This topic is not as well studied for basidiomycetes as for ascomycete fungi, which are the main sources of knowledge on fungal plant polysaccharide degradation. Research on plant-biomass-decaying fungi has focused on isolating enzymes for current and future applications, such as for the production of fuels, the food industry, and waste treatment. More recently, genomic studies of basidiomycete fungi have provided a profound view of the plant-biomass-degrading potential of wood-rotting, litter-decomposing, plant-pathogenic, and ectomycorrhizal (ECM) basidiomycetes. This review summarizes the current knowledge on plant polysaccharide depolymerization by basidiomycete species from diverse habitats. In addition, these data are compared to those for the most broadly studied ascomycete genus, Aspergillus, to provide insight into specific features of basidiomycetes with respect to plant polysaccharide degradation. PMID:25428937

  5. Micropollutant degradation via extracted native enzymes from activated sludge.

    PubMed

    Krah, Daniel; Ghattas, Ann-Kathrin; Wick, Arne; Bröder, Kathrin; Ternes, Thomas A

    2016-05-15

    A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the

  6. Association and haplotype analysis of the insulin-degrading enzyme (IDE) gene, a strong positional and biological candidate for type 2 diabetes susceptibility.

    PubMed

    Groves, Christopher J; Wiltshire, Steven; Smedley, Damian; Owen, Katherine R; Frayling, Timothy M; Walker, Mark; Hitman, Graham A; Levy, Jonathan C; O'Rahilly, Stephen; Menzel, Stephan; Hattersley, Andrew T; McCarthy, Mark I

    2003-05-01

    The gene for insulin-degrading enzyme (IDE) represents a strong positional and biological candidate for type 2 diabetes susceptibility. IDE maps to chromosome 10q23.3, a region linked to diabetes in several populations; the rat homolog has been directly implicated in diabetes susceptibility; and known functions of IDE support an important role in glucose homeostasis. We sought evidence for association between IDE variation and diabetes by mutation screening, defining local haplotype structure, and genotyping variants delineating common haplotypic diversity. An initial case-control analysis (628 diabetic probands from multiplex sibships and 604 control subjects) found no haplotypic associations, although one variant (IDE2, -179T-->C) showed modest association with diabetes (odds ratio [OR]1.25, P = 0.03). Linkage partitioning analyses failed to support this association, but provided borderline evidence for a different variant (IDE10, IVS20-405A-->G) (P = 0.06). Neither variant was associated with diabetes when replication was sought in 377 early onset diabetic subjects and 825 control subjects, though combined analysis of all typed cohorts indicated a nominally significant effect at IDE2 (OR 1.21 [1.04-1.40], P = 0.013). In the absence of convincing support for this association from linkage partitioning or analyses of continuous measures of glycemia, we conclude that analysis of over 2,400 samples provides no compelling evidence that variation in IDE contributes to diabetes susceptibility in humans.

  7. Injectable nano-network for glucose-mediated insulin delivery.

    PubMed

    Gu, Zhen; Aimetti, Alex A; Wang, Qun; Dang, Tram T; Zhang, Yunlong; Veiseh, Omid; Cheng, Hao; Langer, Robert S; Anderson, Daniel G

    2013-05-28

    Diabetes mellitus, a disorder of glucose regulation, is a global burden affecting 366 million people across the world. An artificial "closed-loop" system able to mimic pancreas activity and release insulin in response to glucose level changes has the potential to improve patient compliance and health. Herein we develop a glucose-mediated release strategy for the self-regulated delivery of insulin using an injectable and acid-degradable polymeric network. Formed by electrostatic interaction between oppositely charged dextran nanoparticles loaded with insulin and glucose-specific enzymes, the nanocomposite-based porous architecture can be dissociated and subsequently release insulin in a hyperglycemic state through the catalytic conversion of glucose into gluconic acid. In vitro insulin release can be modulated in a pulsatile profile in response to glucose concentrations. In vivo studies validated that these formulations provided improved glucose control in type 1 diabetic mice subcutaneously administered with a degradable nano-network. A single injection of the developed nano-network facilitated stabilization of the blood glucose levels in the normoglycemic state (<200 mg/dL) for up to 10 days.

  8. Understanding Lignin-Degrading Reactions of Ligninolytic Enzymes: Binding Affinity and Interactional Profile

    PubMed Central

    Chen, Ming; Zeng, Guangming; Tan, Zhongyang; Jiang, Min; Li, Hui; Liu, Lifeng; Zhu, Yi; Yu, Zhen; Wei, Zhen; Liu, Yuanyuan; Xie, Gengxin

    2011-01-01

    Previous works have demonstrated that ligninolytic enzymes mediated effective degradation of lignin wastes. The degrading ability greatly relied on the interactions of ligninolytic enzymes with lignin. Ligninolytic enzymes mainly contain laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP). In the present study, the binding modes of lignin to Lac, LiP and MnP were systematically determined, respectively. Robustness of these modes was further verified by molecular dynamics (MD) simulations. Residues GLU460, PRO346 and SER113 in Lac, residues ARG43, ALA180 and ASP183 in LiP and residues ARG42, HIS173 and ARG177 in MnP were most crucial in binding of lignin, respectively. Interactional analyses showed hydrophobic contacts were most abundant, playing an important role in the determination of substrate specificity. This information is an important contribution to the details of enzyme-catalyzed reactions in the process of lignin biodegradation, which can be used as references for designing enzyme mutants with a better lignin-degrading activity. PMID:21980516

  9. Potential of extracellular enzymes from Trametes versicolor F21a in Microcystis spp. degradation.

    PubMed

    Du, Jingjing; Pu, Gaozhong; Shao, Chen; Cheng, Shujun; Cai, Ji; Zhou, Liang; Jia, Yong; Tian, Xingjun

    2015-03-01

    Studies have shown that microorganisms may be used to eliminate cyanobacteria in aquatic environments. The present study showed that the white-rot fungus Trametes versicolor F21a could degrade Microcystis aeruginosa. After T. versicolor F21a and Microcystis spp. were co-incubated for 60h, >96% of Microcystis spp. cells were degraded by T. versicolor F21a. The activities of extracellular enzymes showed that cellulase, β-glucosidase, protease, and laccase were vital to Microcystis spp. degradation in the early stage (0h to 24h), while β-glucosidase, protease, laccase, and manganese peroxidase in the late stage (24h to 60h). The positive and significant correlation of the degradation rate with these enzyme activities indicated that these enzymes were involved in the degradation rate of Microcystis spp. cells at different phases. It suggested that the extracellular enzymes released by T. versicolor F21a might be vital to Microcystis spp. degradation. The results of this study may be used to develop alternative microbial control agents for cyanobacterial control. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Early-branching Gut Fungi Possess A Large, And Comprehensive Array Of Biomass-Degrading Enzymes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Solomon, Kevin V.; Haitjema, Charles; Henske, John K.

    The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. Its more primitive members, however, remain relatively unexploited. We developed a systems-level approach that integrates RNA-Seq, proteomics, phenotype and biochemical studies of relatively unexplored early-branching free-living fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, unpretreated plant biomass, and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite repressed,more » and are further regulated by a rich landscape of noncoding regulatory RNAs. Furthermore, we identified several promising sequence divergent enzyme candidates for lignocellulosic bioprocessing.« less

  11. Insulin alleviates degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system in septic rats.

    PubMed

    Chen, Qiyi; Li, Ning; Zhu, Weiming; Li, Weiqin; Tang, Shaoqiu; Yu, Wenkui; Gao, Tao; Zhang, Juanjuan; Li, Jieshou

    2011-06-03

    Hypercatabolism is common under septic conditions. Skeletal muscle is the main target organ for hypercatabolism, and this phenomenon is a vital factor in the deterioration of recovery in septic patients. In skeletal muscle, activation of the ubiquitin-proteasome system plays an important role in hypercatabolism under septic status. Insulin is a vital anticatabolic hormone and previous evidence suggests that insulin administration inhibits various steps in the ubiquitin-proteasome system. However, whether insulin can alleviate the degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system under septic condition is unclear. This paper confirmed that mRNA and protein levels of the ubiquitin-proteasome system were upregulated and molecular markers of skeletal muscle proteolysis (tyrosine and 3-methylhistidine) simultaneously increased in the skeletal muscle of septic rats. Septic rats were infused with insulin at a constant rate of 2.4 mU.kg-1.min-1 for 8 hours. Concentrations of mRNA and proteins of the ubiquitin-proteasome system and molecular markers of skeletal muscle proteolysis were mildly affected. When the insulin infusion dose increased to 4.8 mU.kg-1.min-1, mRNA for ubiquitin, E2-14 KDa, and the C2 subunit were all sharply downregulated. At the same time, the levels of ubiquitinated proteins, E2-14KDa, and the C2 subunit protein were significantly reduced. Tyrosine and 3-methylhistidine decreased significantly. We concluded that the ubiquitin-proteasome system is important skeletal muscle hypercatabolism in septic rats. Infusion of insulin can reverse the detrimental metabolism of skeletal muscle by inhibiting the ubiquitin-proteasome system, and the effect is proportional to the insulin infusion dose.

  12. Enzyme-driven metabolomic screening: a proof-of-principle method for discovery of plant defence compounds targeted by pathogens.

    PubMed

    Carere, Jason; Colgrave, Michelle L; Stiller, Jiri; Liu, Chunji; Manners, John M; Kazan, Kemal; Gardiner, Donald M

    2016-11-01

    Plants produce a variety of secondary metabolites to defend themselves from pathogen attack, while pathogens have evolved to overcome plant defences by producing enzymes that degrade or modify these defence compounds. However, many compounds targeted by pathogen enzymes currently remain enigmatic. Identifying host compounds targeted by pathogen enzymes would enable us to understand the potential importance of such compounds in plant defence and modify them to make them insensitive to pathogen enzymes. Here, a proof of concept metabolomics-based method was developed to discover plant defence compounds modified by pathogens using two pathogen enzymes with known targets in wheat and tomato. Plant extracts treated with purified pathogen enzymes were subjected to LC-MS, and the relative abundance of metabolites before and after treatment were comparatively analysed. Using two enzymes from different pathogens the in planta targets could be found by combining relatively simple enzymology with the power of untargeted metabolomics. Key to the method is dataset simplification based on natural isotope occurrence and statistical filtering, which can be scripted. The method presented here will aid in our understanding of plant-pathogen interactions and may lead to the development of new plant protection strategies. © 2016 CSIRO. New Phytologist © 2016 New Phytologist Trust.

  13. Notch signaling proteins HES-1 and Hey-1 bind to insulin degrading enzyme (IDE) proximal promoter and repress its transcription and activity: Implications for cellular Aβ metabolism

    PubMed Central

    Leal, María C.; Surace, Ezequiel I.; Holgado, María P.; Ferrari, Carina C.; Tarelli, Rodolfo; Pitossi, Fernando; Wisniewski, Thomas; Castaño, Eduardo M.; Morelli, Laura

    2012-01-01

    Cerebral amyloid β (Aβ) accumulation is pathogenically associated with sporadic Alzheimer’s disease (SAD). BACE-1 is involved in Aβ generation while insulin-degrading enzyme (IDE) partakes in Aβ proteolytic clearance. Vulnerable regions in AD brains show increased BACE-1 protein levels and enzymatic activity while the opposite occurs with IDE. Another common feature in SAD brains is Notch1 overexpression. Here we demonstrate an increase in mRNA levels of Hey-1, a Notch target gene, and a decrease of IDE transcripts in the hippocampus of SAD brains as compared to controls. Transient transfection of Notch intracellular domain (NICD) in N2aSW cells, mouse neuroblastoma cells (N2a) stably expressing human amyloid precursor protein (APP) Swedish mutation, reduce IDE mRNA levels, promoting extracellular Aβ accumulation. Also, NICD, HES-1 and Hey-1 overexpression result in decreased IDE proximal promoter activity. This effect was mediated by 2 functional sites located at −379/−372 and −310 −303 from the first translation start site in the −575/−19 (556 bp) fragment of IDE proximal promoter. By site-directed mutagenesis of the IDE promoter region we reverted the inhibitory effect mediated by NICD transfection suggesting that these sites are indeed responsible for the Notch-mediated inhibition of the IDE gene expression. Intracranial injection of the Notch ligand JAG-1 in Tg2576 mice, expressing the Swedish mutation in human APP, induced overexpression of HES-1 and Hey-1 and reduction of IDE mRNA levels, respectively. Our results support our theory that a Notch-dependent IDE transcriptional modulation may impact on Aβ metabolism providing a functional link between Notch signaling and the amyloidogenic pathway in SAD. PMID:22036964

  14. Metabolic adaptation via regulated enzyme degradation in the pathogenic yeast Candida albicans.

    PubMed

    Ting, S Y; Ishola, O A; Ahmed, M A; Tabana, Y M; Dahham, S; Agha, M T; Musa, S F; Muhammed, R; Than, L T L; Sandai, D

    2017-03-01

    The virulence of Candida albicans is dependent upon fitness attributes as well as virulence factors. These attributes include robust stress responses and metabolic flexibility. The assimilation of carbon sources is important for growth and essential for the establishment of infections by C. albicans. Previous studies showed that the C. albicans ICL1 genes, which encode the glyoxylate cycle enzymes isocitratelyase are required for growth on non-fermentable carbon sources such as lactate and oleic acid and were repressed by 2% glucose. In contrast to S. cerevsiae, the enzyme CaIcl1 was not destabilised by glucose, resulting with its metabolite remaining at high levels. Further glucose addition has caused CaIcl1 to lose its signal and mechanisms that trigger destabilization in response to glucose. Another purpose of this study was to test the stability of the Icl1 enzyme in response to the dietary sugars, fructose, and galactose. In the present study, the ICL1 mRNAs expression was quantified using Quantitative Real Time PCR, whereby the stability of protein was measured and quantified using Western blot and phosphoimager, and the replacing and cloning of ICL1 ORF by gene recombination and ubiquitin binding was conducted via co-immuno-precipitation. Following an analogous experimental approach, the analysis was repeated using S. cerevisiaeas a control. Both galactose and fructose were found to trigger the degradation of the ICL1 transcript in C. albicans. The Icl1 enzyme was stable following galactose addition but was degraded in response to fructose. C. albicans Icl1 (CaIcl1) was also subjected to fructose-accelerated degradation when expressed in S. cerevisiae, indicating that, although it lacks a ubiquitination site, CaIcl1 is sensitive to fructose-accelerated protein degradation. The addition of an ubiquitination site to CaIcl1 resulted in this enzyme becoming sensitive to galactose-accelerated degradation and increases its rate of degradation in the

  15. The denaturation and degradation of stable enzymes at high temperatures.

    PubMed Central

    Daniel, R M; Dines, M; Petach, H H

    1996-01-01

    Now that enzymes are available that are stable above 100 degrees C it is possible to investigate conformational stability at this temperature, and also the effect of high-temperature degradative reactions in functioning enzymes and the inter-relationship between degradation and denaturation. The conformational stability of proteins depends upon stabilizing forces arising from a large number of weak interactions, which are opposed by an almost equally large destabilizing force due mostly to conformational entropy. The difference between these, the net free energy of stabilization, is relatively small, equivalent to a few interactions. The enhanced stability of very stable proteins can be achieved by an additional stabilizing force which is again equivalent to only a few stabilizing interactions. There is currently no strong evidence that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in proteins that are stable at 100 degrees C than in those stable at 50 degrees C, or that the structures of very stable proteins are systematically different from those of less stable proteins. The major degradative mechanisms are deamidation of asparagine and glutamine, and succinamide formation at aspartate and glutamate leading to peptide bond hydrolysis. In addition to being temperature-dependent, these reactions are strongly dependent upon the conformational freedom of the susceptible amino acid residues. Evidence is accumulating which suggests that even at 100 degrees C deamidation and succinamide formation proceed slowly or not at all in conformationally intact (native) enzymes. Whether this is the case at higher temperatures is not yet clear, so it is not known whether denaturation of degradation will set the upper limit of stability for enzymes. PMID:8694749

  16. Ectonucleotidase NTPDase3 is abundant in pancreatic β-cells and regulates glucose-induced insulin secretion.

    PubMed

    Syed, Samreen K; Kauffman, Audra L; Beavers, Lisa S; Alston, James T; Farb, Thomas B; Ficorilli, James; Marcelo, Marialuisa C; Brenner, Martin B; Bokvist, Krister; Barrett, David G; Efanov, Alexander M

    2013-11-15

    Extracellular ATP released from pancreatic β-cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide-metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. Therefore, we investigated the expression and role of ectonucleotidases in pancreatic β-cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding the NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulin-secreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose-dependent insulin secretion. Downregulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated the effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic β-cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors.

  17. Industrially Important Carbohydrate Degrading Enzymes from Yeasts: Pectinases, Chitinases, and β-1,3-Glucanases

    NASA Astrophysics Data System (ADS)

    Gummadi, Sathyanarayana N.; Kumar, D. Sunil; Dash, Swati S.; Sahu, Santosh Kumar

    Polysaccharide degrading enzymes are hydrolytic enzymes, which have a lot of industrial potential and also play a crucial role in carbon recycling. Pectinases, chitinases and glucanases are the three major polysaccharide degrading enzymes found abundantly in nature and these enzymes are mainly produced by fungal strains. Production of these enzymes by yeasts is advantageous over fungi, because the former are easily amenable to genetic manipulations and time required for growth and production is less than that of the latter. Several yeasts belonging to Saccharomyces, Pichia, Rhodotorula and Cryptococcus produce extracellular pectinases, glucanases and chitinases. This chapter emphasizes on the biological significance of these enzymes, their production and their industrial applications.

  18. Degradation of phenolic compounds with hydrogen peroxide catalyzed by enzyme from Serratia marcescens AB 90027.

    PubMed

    Yao, Ri-Sheng; Sun, Min; Wang, Chun-Ling; Deng, Sheng-Song

    2006-09-01

    In this paper, the degradation of phenolic compounds using hydrogen peroxide as oxidizer and the enzyme extract from Serratia marcescens AB 90027 as catalyst was reported. With such an enzyme/H2O2 combination treatment, a high chemical oxygen demand (COD) removal efficiency was achieved, e.g., degradation of hydroquinone exceeded 96%. From UV-visible and IR spectra, the degradation mechanisms were judged as a process of phenyl ring cleavage. HPLC analysis shows that in the degradation p-benzoquinone, maleic acid and oxalic acid were formed as intermediates and that they were ultimately converted to CO2 and H2O. With the enzyme/H2O2 treatment, vanillin, hydroquinone, catechol, o-aminophenol, p-aminophenol, phloroglucinol and p-hydroxybenzaldehyde were readily degraded, whereas the degradation of phenol, salicylic acid, resorcinol, p-cholorophenol and p-nitrophenol were limited. Their degradability was closely related to the properties and positions of their side chain groups. Electron-donating groups, such as -OH, -NH2 and -OCH3 enhanced the degradation, whereas electron-withdrawing groups, such as -NO2, -Cl and -COOH, had a negative effect on the degradation of these compounds in the presence of enzyme/H2O2. Compounds with -OH at ortho and para positions were more readily degraded than those with -OH at meta positions.

  19. Isoquercetin ameliorates hyperglycemia and regulates key enzymes of glucose metabolism via insulin signaling pathway in streptozotocin-induced diabetic rats.

    PubMed

    Jayachandran, Muthukumaran; Zhang, Tongze; Ganesan, Kumar; Xu, Baojun; Chung, Stephen Sum Man

    2018-06-15

    Among the foremost common flavonoids within the human diet, quercetin glycosides possess neuroprotective, cardioprotective, anti-oxidative, chemopreventive, and anti-allergic properties. Isoquercetin is one such promising candidate with anti-diabetic potential. However, complete studies of its molecular action on insulin signaling pathway and carbohydrate metabolizing enzymes remain unclear. Hence, we have designed this study to accumulate the experimental evidence in support of anti-diabetic effects of isoquercetin. Male albino Wistar rats were divided into seven groups. Rats (Groups 3-7) were administered a single intraperitoneal injection of streptozotocin (STZ; 40 mg/kg b.w) to induce diabetes mellitus. As an extension, STZ rats received isoquercetin at three different doses (20, 40 and 80 mg/kg b.w), and Group 7 rats received glibenclamide (standard drug) (600 μg/kg b.w). The results showed that STZ exaggerated blood sugar, decreased insulin, altered metabolizing enzymes, and impaired the mRNA expression of insulin signaling genes and carbohydrate metabolizing enzyme genes. Supplementation with isoquercetin significantly normalized blood sugar levels, insulin and regulated the mRNA expression of insulin signaling genes and carbohydrate metabolizing enzyme genes. The results achieved with isoquercetin are similar to that of standard drug glibenclamide. The findings suggest isoquercetin could be a possible therapeutic agent for treating diabetes mellitus in the near future. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Outer membrane vesicles from Fibrobacter succinogenes S85 contain an array of carbohydrate-active enzymes with versatile polysaccharide-degrading capacity.

    PubMed

    Arntzen, Magnus Ø; Várnai, Anikó; Mackie, Roderick I; Eijsink, Vincent G H; Pope, Phillip B

    2017-07-01

    Fibrobacter succinogenes is an anaerobic bacterium naturally colonising the rumen and cecum of herbivores where it utilizes an enigmatic mechanism to deconstruct cellulose into cellobiose and glucose, which serve as carbon sources for growth. Here, we illustrate that outer membrane vesicles (OMVs) released by F. succinogenes are enriched with carbohydrate-active enzymes and that intact OMVs were able to depolymerize a broad range of linear and branched hemicelluloses and pectin, despite the inability of F. succinogenes to utilize non-cellulosic (pentose) sugars for growth. We hypothesize that the degradative versatility of F. succinogenes OMVs is used to prime hydrolysis by destabilising the tight networks of polysaccharides intertwining cellulose in the plant cell wall, thus increasing accessibility of the target substrate for the host cell. This is supported by observations that OMV-pretreatment of the natural complex substrate switchgrass increased the catalytic efficiency of a commercial cellulose-degrading enzyme cocktail by 2.4-fold. We also show that the OMVs contain a putative multiprotein complex, including the fibro-slime protein previously found to be important in binding to crystalline cellulose. We hypothesize that this complex has a function in plant cell wall degradation, either by catalysing polysaccharide degradation itself, or by targeting the vesicles to plant biomass. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. The Epoxyeicosatrienoic Acid Pathway Enhances Hepatic Insulin Signaling and is Repressed in Insulin-Resistant Mouse Liver*

    PubMed Central

    Schäfer, Alexander; Neschen, Susanne; Kahle, Melanie; Sarioglu, Hakan; Gaisbauer, Tobias; Imhof, Axel; Adamski, Jerzy; Hauck, Stefanie M.; Ueffing, Marius

    2015-01-01

    Although it is widely accepted that ectopic lipid accumulation in the liver is associated with hepatic insulin resistance, the underlying molecular mechanisms have not been well characterized. Here we employed time resolved quantitative proteomic profiling of mice fed a high fat diet to determine which pathways were affected during the transition of the liver to an insulin-resistant state. We identified several metabolic pathways underlying altered protein expression. In order to test the functional impact of a critical subset of these alterations, we focused on the epoxyeicosatrienoic acid (EET) eicosanoid pathway, whose deregulation coincided with the onset of hepatic insulin resistance. These results suggested that EETs may be positive modulators of hepatic insulin signaling. Analyzing EET activity in primary hepatocytes, we found that EETs enhance insulin signaling on the level of Akt. In contrast, EETs did not influence insulin receptor or insulin receptor substrate-1 phosphorylation. This effect was mediated through the eicosanoids, as overexpression of the deregulated enzymes in absence of arachidonic acid had no impact on insulin signaling. The stimulation of insulin signaling by EETs and depression of the pathway in insulin resistant liver suggest a likely role in hepatic insulin resistance. Our findings support therapeutic potential for inhibiting EET degradation. PMID:26070664

  2. Winery biomass waste degradation by sequential sonication and mixed fungal enzyme treatments.

    PubMed

    Karpe, Avinash V; Dhamale, Vijay V; Morrison, Paul D; Beale, David J; Harding, Ian H; Palombo, Enzo A

    2017-05-01

    To increase the efficiency of winery-derived biomass biodegradation, grape pomace was ultrasonicated for 20min in the presence of 0.25M, 0.5Mand1.0MKOH and 1.0MNaOH. This was followed by treatment with a 1:1 (v/v) mix of crude enzyme preparation derived from Phanerochaete chrysosporium and Trametes versicolor for 18h and a further 18h treatment with a 60:14:4:2 percent ratio combination of enzymes derived from Aspergillus niger: Penicillium chrysogenum: Trichoderma harzianum: P. citrinum, repsectively. Process efficiency was evaluated by its comparison to biological only mixed fungal degradation over 16days. Ultrasonication treatment with 0.5MKOH followed by mixed enzyme treatment yielded the highest lignin degradation of about 13%. Cellulase, β-glucosidase, xylanase, laccase and lignin peroxidase activities of 77.9, 476, 5,390.5, 66.7 and 29,230.7U/mL, respectively, were observed during biomass degradation. Gas chromatography-mass spectrometry (GC-MS) analysis of the degraded material identified commercially important compounds such as gallic acid, lithocholic acid, glycolic acid and lactic acid which were generated in considerable quantities. Thus, the combination of sonication pre-treatment and enzymatic degradation has the potential to considerably improve the breakdown of agricultural biomass and produce commercially useful compounds in markedly less time (<40h) with respect to biological only degradation (16days). Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Dietary Anthocyanins and Insulin Resistance: When Food Becomes a Medicine.

    PubMed

    Belwal, Tarun; Nabavi, Seyed Fazel; Nabavi, Seyed Mohammad; Habtemariam, Solomon

    2017-10-12

    Insulin resistance is an abnormal physiological state that occurs when insulin from pancreatic β-cells is unable to trigger a signal transduction pathway in target organs such as the liver, muscles and adipose tissues. The loss of insulin sensitivity is generally associated with persistent hyperglycemia (diabetes), hyperinsulinemia, fatty acids and/or lipid dysregulation which are often prevalent under obesity conditions. Hence, insulin sensitizers are one class of drugs currently employed to treat diabetes and associated metabolic disorders. A number of natural products that act through multiple mechanisms have also been identified to enhance insulin sensitivity in target organs. One group of such compounds that gained interest in recent years are the dietary anthocyanins. Data from their in vitro, in vivo and clinical studies are scrutinized in this communication to show their potential health benefit through ameliorating insulin resistance. Specific mechanism of action ranging from targeting specific signal transduction receptors/enzymes to the general antioxidant and anti-inflammatory mechanisms of insulin resistance are presented.

  4. Expression of neuropeptides and their degrading enzymes in ACD.

    PubMed

    Bak, H; Lee, W J; Lee, Y W; Chang, S-E; Choi, J-H; Kim, M N; Kim, B J; Choi, Y S; Suh, H S

    2010-04-01

    Sensory neuropeptides such as neurokinin A or substance P modulate skin and immune cells the functions of neurokinin receptor activation during neurogenic inflammation. Zinc metalloproteases, such as neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), effectively control the bioavailability of these neuropeptide mediators, which are released from sensory nerves, immune and skin cells during cutaneous responses to endogenous or exogenous noxious stimuli. Recently, studies have suggested that neuropeptides are one of the major pathogenetic fact in many dermatoses, such as allergic contact dermatitis (ACD), atopic dermatitis and psoriasis. To investigate the expression of major neuropeptides, SP and its degrading enzymes such as NEP and ACE, in the lesions of ACD. A skin biopsy was obtained from 10 patients with ACD. We analysed the expression of these molecules by immunohistochemical staining, confocal laser scanning microscopy, western blotting and reverse transcription PCR. There was a significant increase in expression of SP in keratinocytes from ACD lesions compared with those in control skin. There was also increased expression of ACE but not NEP in ACD. Neuropeptides and their degrading enzymes, particularly SP and ACE, have a significant role in the pathogenesis of ACD.

  5. Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin-cadmium induced diabetic nephrotoxic rats.

    PubMed

    Kandasamy, Neelamegam; Ashokkumar, Natarajan

    2014-09-01

    Diabetic nephropathy is the kidney disease that occurs as a result of diabetes. The present study was aimed to evaluate the therapeutic potential of myricetin by assaying the activities of key enzymes of carbohydrate metabolism, insulin signaling molecules and renal function markers in streptozotocin (STZ)-cadmium (Cd) induced diabetic nephrotoxic rats. After myricetin treatment schedule, blood and tissue samples were collected to determine plasma glucose, insulin, hemoglobin, glycosylated hemoglobin and renal function markers, carbohydrate metabolic enzymes in the liver and insulin signaling molecules in the pancreas and skeletal muscle. A significant increase of plasma glucose, glycosylated hemoglobin, urea, uric acid, creatinine, blood urea nitrogen (BUN), urinary albumin, glycogen phosphorylase, glucose-6-phosphatase, and fructose-1,6-bisphosphatase and a significant decrease of plasma insulin, hemoglobin, hexokinase, glucose-6-phosphate dehydrogenase, glycogen and glycogen synthase with insulin signaling molecule expression were found in the STZ-Cd induced diabetic nephrotoxic rats. The administration of myricetin significantly normalizes the carbohydrate metabolic products like glucose, glycated hemoglobin, glycogen phosphorylase and gluconeogenic enzymes and renal function markers with increase insulin, glycogen, glycogen synthase and insulin signaling molecule expression like glucose transporter-2 (GLUT-2), glucose transporter-4 (GLUT-4), insulin receptor-1 (IRS-1), insulin receptor-2 (IRS-2) and protein kinase B (PKB). Based on the data, the protective effect of myricetin was confirmed by its histological annotation of the pancreas, liver and kidney tissues. These findings suggest that myricetin improved carbohydrate metabolism which subsequently enhances glucose utilization and renal function in STZ-Cd induced diabetic nephrotoxic rats. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Application of residual polysaccharide-degrading enzymes in dried shiitake mushrooms as an enzyme preparation in food processing.

    PubMed

    Tatsumi, E; Konishi, Y; Tsujiyama, S

    2016-11-01

    To examine the activities of residual enzymes in dried shiitake mushrooms, which are a traditional foodstuff in Japanese cuisine, for possible applications in food processing. Polysaccharide-degrading enzymes remained intact in dried shiitake mushrooms and the activities of amylase, β-glucosidase and pectinase were high. A potato digestion was tested using dried shiitake powder. The enzymes reacted with potato tuber specimens to solubilize sugars even under a heterogeneous solid-state condition and that their reaction modes were different at 38 and 50 °C. Dried shiitake mushrooms have a potential use in food processing as an enzyme preparation.

  7. RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica

    PubMed Central

    Blackman, Leila M.; Cullerne, Darren P.; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R.

    2015-01-01

    RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1

  8. RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica.

    PubMed

    Blackman, Leila M; Cullerne, Darren P; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R

    2015-01-01

    RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1

  9. Mechanisms linking brain insulin resistance to Alzheimer's disease

    PubMed Central

    Matioli, Maria Niures P.S.; Nitrini, Ricardo

    2015-01-01

    Several studies have indicated that Diabetes Mellitus (DM) can increase the risk of developing Alzheimer's disease (AD). This review briefly describes current concepts in mechanisms linking DM and insulin resistance/deficiency to AD. Insulin/insulin-like growth factor (IGF) resistance can contribute to neurodegeneration by several mechanisms which involve: energy and metabolism deficits, impairment of Glucose transporter-4 function, oxidative and endoplasmic reticulum stress, mitochondrial dysfunction, accumulation of AGEs, ROS and RNS with increased production of neuro-inflammation and activation of pro-apoptosis cascade. Impairment in insulin receptor function and increased expression and activation of insulin-degrading enzyme (IDE) have also been described. These processes compromise neuronal and glial function, with a reduction in neurotransmitter homeostasis. Insulin/IGF resistance causes the accumulation of AβPP-Aβ oligomeric fibrils or insoluble larger aggregated fibrils in the form of plaques that are neurotoxic. Additionally, there is production and accumulation of hyper-phosphorylated insoluble fibrillar tau which can exacerbate cytoskeletal collapse and synaptic disconnection. PMID:29213950

  10. Co-operative actions and degradation analysis of purified xylan-degrading enzymes from Thermomonospora fusca BD25 on oat-spelt xylan.

    PubMed

    Tuncer, M; Ball, A S

    2003-01-01

    To determine and quantify the products from the degradation of xylan by a range of purified xylan-degrading enzymes, endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase produced extracellularly by Thermomonospora fusca BD25. The amounts of reducing sugars released from oat-spelt xylan by the actions of endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase were equal to 28.1, 4.6 and 7% hydrolysis (as xylose equivalents) of the substrate used, respectively. However, addition of beta-xylosidase and alpha-l-arabinofuranosidase preparation to endoxylanase significantly enhanced (70 and 20% respectively) the action of endoxylanase on the substrate. The combination of purified endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase preparations produced a greater sugar yield (58.6% hydrolysis) and enhanced the total reducing sugar yield by around 50%. The main xylooligosaccharide products released using the action of endoxylanase alone on oat-spelt xylan were identified as xylobiose and xylopentose. alpha-l-Arabinofuranosidase was able to release arabinose and xylobiose from oat-spelt xylan. In the presence of all three purified enzymes the hydrolysis products of oat-spelt xylan were mainly xylose, arabinose and substituted xylotetrose with lesser amount of substituted xylotriose. The addition of the beta-xylosidase and alpha-l-arabinofuranosidase enzymes to purified xylanases more than doubled the degradation of xylan from 28 to 58% of the total substrate with xylose and arabinose being the major sugars produced. The results highlight the role of xylan de-branching enzymes in the degradation of xylan and suggest that the use of enzyme cocktails may significantly improve the hydrolysis of xylan in industrial processes.

  11. Enzymes involved in a novel anaerobic cyclohexane carboxylic acid degradation pathway.

    PubMed

    Kung, Johannes W; Meier, Anne-Katrin; Mergelsberg, Mario; Boll, Matthias

    2014-10-01

    The anaerobic degradation of cyclohexane carboxylic acid (CHC) has so far been studied only in Rhodopseudomonas palustris, in which CHC is activated to cyclohexanoyl coenzyme A (cyclohexanoyl-CoA [CHCoA]) and then dehydrogenated to cyclohex-1-ene-1-carboxyl-CoA (CHeneCoA). This intermediate is further degraded by reactions of the R. palustris-specific benzoyl-CoA degradation pathway of aromatic compounds. However, CHeneCoA is not an intermediate in the degradation of aromatic compounds in all other known anaerobic bacteria; consequently, degradation of CHC was mostly unknown in anaerobic bacteria. We identified a previously unknown CHC degradation pathway in the Fe(III)-reducing Geobacter metallireducens by determining the following CHC-induced in vitro activities: (i) the activation of CHC to CHCoA by a succinyl-CoA:CHC CoA transferase, (ii) the 1,2-dehydrogenation of CHCoA to CHeneCoA by CHCoA dehydrogenase, and (iii) the unusual 1,4-dehydrogenation of CHeneCoA to cyclohex-1,5-diene-1-carboxyl-CoA. This last represents a previously unknown joint intermediate of the CHC and aromatic compound degradation pathway in bacteria other than R. palustris. The enzymes catalyzing the three reactions were purified and characterized as specific enzymes after heterologous expression of the encoding genes. Quantitative reverse transcription-PCR revealed that expression of these genes was highly induced during growth with CHC but not with benzoate. The newly identified CHC degradation pathway is suggested to be present in nearly all CHC-degrading anaerobic bacteria, including denitrifying, Fe(III)-reducing, sulfate-reducing, and fermenting bacteria. Remarkably, all three CHC degradation pathways always link CHC catabolism to the catabolic pathways of aromatic compounds. We propose that the capacity to use CHC as a carbon source evolved from already-existing aromatic compound degradation pathways. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  12. Enzymes Involved in a Novel Anaerobic Cyclohexane Carboxylic Acid Degradation Pathway

    PubMed Central

    Kung, Johannes W.; Meier, Anne-Katrin; Mergelsberg, Mario

    2014-01-01

    The anaerobic degradation of cyclohexane carboxylic acid (CHC) has so far been studied only in Rhodopseudomonas palustris, in which CHC is activated to cyclohexanoyl coenzyme A (cyclohexanoyl-CoA [CHCoA]) and then dehydrogenated to cyclohex-1-ene-1-carboxyl-CoA (CHeneCoA). This intermediate is further degraded by reactions of the R. palustris-specific benzoyl-CoA degradation pathway of aromatic compounds. However, CHeneCoA is not an intermediate in the degradation of aromatic compounds in all other known anaerobic bacteria; consequently, degradation of CHC was mostly unknown in anaerobic bacteria. We identified a previously unknown CHC degradation pathway in the Fe(III)-reducing Geobacter metallireducens by determining the following CHC-induced in vitro activities: (i) the activation of CHC to CHCoA by a succinyl-CoA:CHC CoA transferase, (ii) the 1,2-dehydrogenation of CHCoA to CHeneCoA by CHCoA dehydrogenase, and (iii) the unusual 1,4-dehydrogenation of CHeneCoA to cyclohex-1,5-diene-1-carboxyl-CoA. This last represents a previously unknown joint intermediate of the CHC and aromatic compound degradation pathway in bacteria other than R. palustris. The enzymes catalyzing the three reactions were purified and characterized as specific enzymes after heterologous expression of the encoding genes. Quantitative reverse transcription-PCR revealed that expression of these genes was highly induced during growth with CHC but not with benzoate. The newly identified CHC degradation pathway is suggested to be present in nearly all CHC-degrading anaerobic bacteria, including denitrifying, Fe(III)-reducing, sulfate-reducing, and fermenting bacteria. Remarkably, all three CHC degradation pathways always link CHC catabolism to the catabolic pathways of aromatic compounds. We propose that the capacity to use CHC as a carbon source evolved from already-existing aromatic compound degradation pathways. PMID:25112478

  13. Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP).

    PubMed

    Ma, Hongyan; Delafield, Daniel G; Wang, Zhe; You, Jianlan; Wu, Si

    2017-04-01

    The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

  14. Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

    NASA Astrophysics Data System (ADS)

    Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

    2017-04-01

    The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

  15. Heparanase augments insulin receptor signaling in breast carcinoma

    PubMed Central

    Goldberg, Rachel; Sonnenblick, Amir; Hermano, Esther; Hamburger, Tamar; Meirovitz, Amichay; Peretz, Tamar; Elkin, Michael

    2017-01-01

    Recently, growing interest in the potential link between metabolic disorders (i.e., diabetes, obesity, metabolic syndrome) and breast cancer has mounted, including studies which indicate that diabetic/hyperinsulinemic women have a significantly higher risk of bearing breast tumors that are more aggressive and associated with higher death rates. Insulin signaling is regarded as a major contributor to this phenomenon; much less is known about the role of heparan sulfate-degrading enzyme heparanase in the link between metabolic disorders and cancer. In the present study we analyzed clinical samples of breast carcinoma derived from diabetic/non-diabetic patients, and investigated effects of heparanase on insulin signaling in breast carcinoma cell lines, as well as insulin-driven growth of breast tumor cells. We demonstrate that heparanase activity leads to enhanced insulin signaling and activation of downstream tumor-promoting pathways in breast carcinoma cells. In agreement, heparanase enhances insulin-induced proliferation of breast tumor cells in vitro. Moreover, analyzing clinical data from diabetic breast carcinoma patients, we found that concurrent presence of both diabetic state and heparanase in tumor tissue (as opposed to either condition alone) was associated with more aggressive phenotype of breast tumors in the patient cohort analyzed in our study (two-sided Fisher's exact test; p=0.04). Our findings highlight the emerging role of heparanase in powering effect of hyperinsulinemic state on breast tumorigenesis and imply that heparanase targeting, which is now under intensive development/clinical testing, could be particularly efficient in a growing fraction of breast carcinoma patients suffering from metabolic disorders. PMID:28038446

  16. Expression, functional analysis and mutation of a novel neutral zearalenone-degrading enzyme.

    PubMed

    Wang, Meixing; Yin, Lifeng; Hu, Huizhen; Selvaraj, Jonathan Nimal; Zhou, Yuling; Zhang, Guimin

    2018-06-24

    The crops and grains were often contaminated by high level of mycotoxin zearalenone (ZEN). In order to remove ZEN and keep food safe, ZEN-degrading or detoxifying enzymes are urgently needed. Here, a newly identified lactonohydrolase responsible for the detoxification of ZEN, annotated as Zhd518, was expressed and characterized. Zhd518 showed 65% amino acid identity with Zhd101, which was widely studied for its ZEN-degrading ability. A detailed activity measurement method of ZEN-degrading enzyme was provided. Biochemical analysis indicated that the purified recombinant Zhd518 from E. coli exhibited a high activity against ZEN (207.0 U/mg), with the optimal temperature and pH of 40 °C and 8.0, respectively. The Zhd518 can degrade ZEN derivatives, and the specific activities against α-Zearalenol, β-Zearalenol, α-Zearalanol and β-Zearalanol were 23.0 U/mg, 64.7 U/mg, 119.8 U/mg and 66.5 U/mg, respectively. The active sites of Zhd518 were predicted by structure modeling and determined by mutation analysis. A point mutant N156H exhibited 3.3-fold activity against α-Zearalenol comparing to Zhd518. Zhd518 is the first reported neutral and the second characterized ZEN-degrading enzyme, which provides a new and more excellent candidate for ZEN detoxifying in food and feed industry. Copyright © 2018. Published by Elsevier B.V.

  17. It’s War Out There: Fighting for life with xenobiotic degrading enzymes

    USDA-ARS?s Scientific Manuscript database

    It’s War Out There: Fighting for life with xenobiotic degrading enzymes Beta-lactamase enzymes are well studied because of their tremendous impact on medicine. Their prominent role is in resistance to beta-lactam (four membered lactam ring) antibiotics including the first and most famous fungally d...

  18. Protein kinases: mechanisms and downstream targets in inflammation-mediated obesity and insulin resistance.

    PubMed

    Nandipati, Kalyana C; Subramanian, Saravanan; Agrawal, Devendra K

    2017-02-01

    Obesity-induced low-grade inflammation (metaflammation) impairs insulin receptor signaling. This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, inhibitor of NF-kB kinase complex β (IKKβ), AMP-activated protein kinase, protein kinase C, Rho-associated coiled-coil containing protein kinase, and RNA-activated protein kinase. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in type 2 diabetes mellitus (T2-DM). Identifying the specific protein kinases involved in obesity-induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity-induced T2-DM.

  19. Methods to identify enzymes that degrade the main extracellular polysaccharide component of Staphylococcus epidermidis biofilms

    PubMed Central

    Gökçen, Anke; Vilcinskas, Andreas; Wiesner, Jochen

    2013-01-01

    The production of extracellular poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by Staphylococcus epidermidis is the principal determinant of biofilm formation on indwelling medical devices. Enzymes that degrade PNAG therefore provide an attractive strategy for biofilm removal and for the manufacture of biofilm-resistant coatings. Here we present methods that allow the identification of PNAG-degrading enzymes with the ability to detach biofilms. Our protocol includes the preparation of soluble PNAG from S. epidermidis cultures, the incubation of soluble PNAG with candidate enzymes and assays that detect the release of N-acetyl-d-glucosamine using high-pH anion-exchange chromatography (HPAEC) followed in parallel by pulsed amperometric detection (PAD) and online electrospray ionization mass spectrometry (ESI-MS). We validated our procedures using dispersin B, which is currently the only known PNAG-degrading enzyme. PMID:23357872

  20. Hidden targets of ubiquitin proteasome system: To prevent diabetic nephropathy.

    PubMed

    Goru, Santosh Kumar; Kadakol, Almesh; Gaikwad, Anil Bhanudas

    2017-06-01

    Diabetic nephropathy (DN) is the major cause of end stage renal failure. Although, several therapeutic targets have emerged to prevent the progression of DN, the number of people with DN still continues to rise worldwide, suggesting an urgent need of novel targets to prevent DN completely. Currently, the role of ubiquitin proteasome system (UPS) has been highlighted in the pathogenesis and progression of various diseases like obesity, insulin resistance, atherosclerosis, cancers, neurodegerative disorders and including secondary complications of diabetes. UPS mainly involves in protein homeostatis through ubiquitination (post translational modification) and proteasomal degradation of various proteins. Ubiquitination, not only involves in proteasomal degradation, but also directs the substrate proteins to participate in multitude of cell signalling pathways. However, very little is known about ubiquitination and UPS in the progression of DN. This review mainly focuses on UPS and its components including E2 conjugating enzymes, E3 ligases and deubiquitinases (DUBs) in the development of DN and thus may help us to find novel therapeutic targets with in UPS to prevent DN completely in future. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Autophagy Differentially Regulates Insulin Production and Insulin Sensitivity.

    PubMed

    Yamamoto, Soh; Kuramoto, Kenta; Wang, Nan; Situ, Xiaolei; Priyadarshini, Medha; Zhang, Weiran; Cordoba-Chacon, Jose; Layden, Brian T; He, Congcong

    2018-06-12

    Autophagy, a stress-induced lysosomal degradative pathway, has been assumed to exert similar metabolic effects in different organs. Here, we establish a model where autophagy plays different roles in insulin-producing β cells versus insulin-responsive cells, utilizing knockin (Becn1 F121A ) mice manifesting constitutively active autophagy. With a high-fat-diet challenge, the autophagy-hyperactive mice unexpectedly show impaired glucose tolerance, but improved insulin sensitivity, compared to mice with normal autophagy. Autophagy hyperactivation enhances insulin signaling, via suppressing ER stress in insulin-responsive cells, but decreases insulin secretion by selectively sequestrating and degrading insulin granule vesicles in β cells, a process we term "vesicophagy." The reduction in insulin storage, insulin secretion, and glucose tolerance is reversed by transient treatment of autophagy inhibitors. Thus, β cells and insulin-responsive tissues require different autophagy levels for optimal function. To improve insulin sensitivity without hampering secretion, acute or intermittent, rather than chronic, activation of autophagy should be considered in diabetic therapy development. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. [Piperine regulates glucose metabolism disorder in HepG2 cells of insulin resistance models via targeting upstream target of AMPK signaling pathway].

    PubMed

    Wan, Chun-Ping; Wei, Ya-Gai; Li, Xiao-Xue; Zhang, Li-Jun; Yang, Rui; Bao, Zhao-Ri-Ge-Tu

    2017-02-01

    To investigate the effect of piperine on the disorder of glucose metabolism in the cell model with insulin resistance (IR) and explore the molecules mechanism on intervening the upstream target of AMPK signaling pathway. The insulin resistance models in HepG2 cells were established by fat emulsion stimulation. Then glucose consumption in culture supernatant was detected by GOD-POD method. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of leptin(LEP) and adiponectin(APN) in culture supernatant; Real-time quantitative PCR was used to assess the mRNA expression of APN and LEP; and the protein expression levels of LepR, AdipoR1, AdipoR2 and the activation of AMPK signaling pathway were detected by Western blot analysis. The results showed that piperine, rosiglitazone and AMPK agonist AICAR could significantly elevate the glucose consumption in insulin resistance cell models, enhance the level of APN, promote APN mRNA transcripts and increase the protein expression of Adipo receptor. Meanwhile,AMPKα mRNA and р-AMPKα protein expressions were also increased in piperine treated cells, but both LEP mRNA expression and LepR protein expressions were decreased in piperine treated group. The results indicated that piperine could significantly ameliorate the glucose metabolism disorder in insulin resistance cell models through regulating upstream molecules (APN and LEP) of AMPK signaling pathway, and thus activate the AMPK signaling pathway. Copyright© by the Chinese Pharmaceutical Association.

  3. Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin–cadmium induced diabetic nephrotoxic rats

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kandasamy, Neelamegam; Ashokkumar, Natarajan, E-mail: npashokkumar1@gmail.com

    Diabetic nephropathy is the kidney disease that occurs as a result of diabetes. The present study was aimed to evaluate the therapeutic potential of myricetin by assaying the activities of key enzymes of carbohydrate metabolism, insulin signaling molecules and renal function markers in streptozotocin (STZ)–cadmium (Cd) induced diabetic nephrotoxic rats. After myricetin treatment schedule, blood and tissue samples were collected to determine plasma glucose, insulin, hemoglobin, glycosylated hemoglobin and renal function markers, carbohydrate metabolic enzymes in the liver and insulin signaling molecules in the pancreas and skeletal muscle. A significant increase of plasma glucose, glycosylated hemoglobin, urea, uric acid, creatinine,more » blood urea nitrogen (BUN), urinary albumin, glycogen phosphorylase, glucose-6-phosphatase, and fructose-1,6-bisphosphatase and a significant decrease of plasma insulin, hemoglobin, hexokinase, glucose-6-phosphate dehydrogenase, glycogen and glycogen synthase with insulin signaling molecule expression were found in the STZ–Cd induced diabetic nephrotoxic rats. The administration of myricetin significantly normalizes the carbohydrate metabolic products like glucose, glycated hemoglobin, glycogen phosphorylase and gluconeogenic enzymes and renal function markers with increase insulin, glycogen, glycogen synthase and insulin signaling molecule expression like glucose transporter-2 (GLUT-2), glucose transporter-4 (GLUT-4), insulin receptor-1 (IRS-1), insulin receptor-2 (IRS-2) and protein kinase B (PKB). Based on the data, the protective effect of myricetin was confirmed by its histological annotation of the pancreas, liver and kidney tissues. These findings suggest that myricetin improved carbohydrate metabolism which subsequently enhances glucose utilization and renal function in STZ–Cd induced diabetic nephrotoxic rats. - Highlights: • Diabetic rats are more susceptible to cadmium nephrotoxicity. • Cadmium plays as a

  4. A novel regulation of IRS1 (insulin receptor substrate-1) expression following short term insulin administration

    PubMed Central

    2005-01-01

    Reduced insulin-mediated glucose transport in skeletal muscle is a hallmark of the pathophysiology of T2DM (Type II diabetes mellitus). Impaired intracellular insulin signalling is implicated as a key underlying mechanism. Attention has focused on early signalling events such as defective tyrosine phosphorylation of IRS1 (insulin receptor substrate-1), a major target for the insulin receptor tyrosine kinase. This is required for normal induction of signalling pathways key to many of the metabolic actions of insulin. Conversely, increased serine/threonine phosphorylation of IRS1 following prolonged insulin exposure (or in obesity) reduces signalling capacity, partly by stimulating IRS1 degradation. We now show that IRS1 levels in human muscle are actually increased 3-fold following 1 h of hyperinsulinaemic euglycaemia. Similarly, transient induction of IRS1 (3-fold) in the liver or muscle of rodents occurs following feeding or insulin injection respectively. The induction by insulin is also observed in cell culture systems, although to a lesser degree, and is not due to reduced proteasomal targeting, increased protein synthesis or gene transcription. Elucidation of the mechanism by which insulin promotes IRS1 stability will permit characterization of the importance of this novel signalling event in insulin regulation of liver and muscle function. Impairment of this process would reduce IRS1 signalling capacity, thereby contributing to the development of hyperinsulinaemia/insulin resistance prior to the appearance of T2DM. PMID:16128672

  5. Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs.

    PubMed

    Jabłońska-Trypuć, Agata; Matejczyk, Marzena; Rosochacki, Stanisław

    2016-01-01

    The main group of enzymes responsible for the collagen and other protein degradation in extracellular matrix (ECM) are matrix metalloproteinases (MMPs). Collagen is the main structural component of connective tissue and its degradation is a very important process in the development, morphogenesis, tissue remodeling, and repair. Typical structure of MMPs consists of several distinct domains. MMP family can be divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other non-classified MMPs. MMPs and their inhibitors have multiple biological functions in all stages of cancer development: from initiation to outgrowth of clinically relevant metastases and likewise in apoptosis and angiogenesis. MMPs and their inhibitors are extensively examined as potential anticancer drugs. MMP inhibitors can be divided into two main groups: synthetic and natural inhibitors. Selected synthetic inhibitors are in clinical trials on humans, e.g. synthetic peptides, non-peptidic molecules, chemically modified tetracyclines, and bisphosphonates. Natural MMP inhibitors are mainly isoflavonoids and shark cartilage.

  6. Effect of solvents on the enzyme mediated degradation of copolymers

    NASA Astrophysics Data System (ADS)

    Banerjee, Aditi; Chatterjee, Kaushik; Madras, Giridhar

    2015-09-01

    The biodegradation of polycaprolactone (PCL), polylactic acid (PLA), polyglycolide (PGA) and their copolymers, poly (lactide-co-glycolide) and poly (D, L-lactide-co-caprolactone) (PLCL) was investigated. The influence of different solvents on the degradation of these polymers at 37 °C in the presence of two different lipases namely Novozym 435 and the free lipase of porcine pancreas was investigated. The rate coefficients for the polymer degradation and enzyme deactivation were determined using continuous distribution kinetics. Among the homopolymers, the degradation of PGA was nearly an order of magnitude lower than that for PCL and PLA. The overall rate coefficients of the copolymers were higher than their respective homopolymers. Thus, PLCL degraded faster than either PCL or PLA. The degradation was highly dependent on the viscosity of the solvent used with the highest degradation observed in acetone. The degradation of the polymers in acetone was nearly twice that observed in dimethyl sulfoxide indicating that the degradation decreases with increase in the solvent viscosity. The degradation of the polymers in water-solvent mixtures indicated an optimal water content of 2.5 wt% of water.

  7. A Novel Enzyme Portfolio for Red Algal Polysaccharide Degradation in the Marine Bacterium Paraglaciecola hydrolytica S66T Encoded in a Sizeable Polysaccharide Utilization Locus.

    PubMed

    Schultz-Johansen, Mikkel; Bech, Pernille K; Hennessy, Rosanna C; Glaring, Mikkel A; Barbeyron, Tristan; Czjzek, Mirjam; Stougaard, Peter

    2018-01-01

    Marine microbes are a rich source of enzymes for the degradation of diverse polysaccharides. Paraglaciecola hydrolytica S66 T is a marine bacterium capable of hydrolyzing polysaccharides found in the cell wall of red macroalgae. In this study, we applied an approach combining genomic mining with functional analysis to uncover the potential of this bacterium to produce enzymes for the hydrolysis of complex marine polysaccharides. A special feature of P. hydrolytica S66 T is the presence of a large genomic region harboring an array of carbohydrate-active enzymes (CAZymes) notably agarases and carrageenases. Based on a first functional characterization combined with a comparative sequence analysis, we confirmed the enzymatic activities of several enzymes required for red algal polysaccharide degradation by the bacterium. In particular, we report for the first time, the discovery of novel enzyme activities targeting furcellaran, a hybrid carrageenan containing both β-carrageenan and κ/β-carrageenan motifs. Some of these enzymes represent a new subfamily within the CAZy classification. From the combined analyses, we propose models for the complete degradation of agar and κ/β-type carrageenan by P. hydrolytica S66 T . The novel enzymes described here may find value in new bio-based industries and advance our understanding of the mechanisms responsible for recycling of red algal polysaccharides in marine ecosystems.

  8. Structural and Functional Analysis of Phytotoxin Toxoflavin-Degrading Enzyme

    PubMed Central

    Kim, Myung-Il; Ma, Jun; Nagamatsu, Tomohisa; Goo, Eunhye; Kim, Hongsup; Hwang, Ingyu; Han, Jaehong; Rhee, Sangkee

    2011-01-01

    Pathogenic bacteria synthesize and secrete toxic low molecular weight compounds as virulence factors. These microbial toxins play essential roles in the pathogenicity of bacteria in various hosts, and are emerging as targets for antivirulence strategies. Toxoflavin, a phytotoxin produced by Burkholderia glumae BGR1, has been known to be the key factor in rice grain rot and wilt in many field crops. Recently, toxoflavin-degrading enzyme (TxDE) was identified from Paenibacillus polymyxa JH2, thereby providing a possible antivirulence strategy for toxoflavin-mediated plant diseases. Here, we report the crystal structure of TxDE in the substrate-free form and in complex with toxoflavin, along with the results of a functional analysis. The overall structure of TxDE is similar to those of the vicinal oxygen chelate superfamily of metalloenzymes, despite the lack of apparent sequence identity. The active site is located at the end of the hydrophobic channel, 9 Å in length, and contains a Mn(II) ion interacting with one histidine residue, two glutamate residues, and three water molecules in an octahedral coordination. In the complex, toxoflavin binds in the hydrophobic active site, specifically the Mn(II)-coordination shell by replacing a ligating water molecule. A functional analysis indicated that TxDE catalyzes the degradation of toxoflavin in a manner dependent on oxygen, Mn(II), and the reducing agent dithiothreitol. These results provide the structural features of TxDE and the early events in catalysis. PMID:21799856

  9. Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.

    PubMed

    Mecklenburg, M; Lindbladh, C; Li, H; Mosbach, K; Danielsson, B

    1993-08-01

    A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.

  10. Cellulose and hemicellulose-degrading enzymes in Fusarium commune transcriptome and functional characterization of three identified xylanases.

    PubMed

    Huang, Yuhong; Busk, Peter Kamp; Lange, Lene

    2015-06-01

    Specific enzymes from plant-pathogenic microbes demonstrate high effectiveness for natural lignocellulosic biomass degradation and utilization. The secreted lignocellulolytic enzymes of Fusarium species have not been investigated comprehensively, however. In this study we compared cellulose and hemicellulose-degrading enzymes of classical fungal enzyme producers with those of Fusarium species. The results indicated that Fusarium species are robust cellulose and hemicellulose degraders. Wheat bran, carboxymethylcellulose and xylan-based growth media induced a broad spectrum of lignocellulolytic enzymes in Fusarium commune. Prediction of the cellulose and hemicellulose-degrading enzymes in the F. commune transcriptome using peptide pattern recognition revealed 147 genes encoding glycoside hydrolases and six genes encoding lytic polysaccharide monooxygenases (AA9 and AA11), including all relevant cellulose decomposing enzymes (GH3, GH5, GH6, GH7, GH9, GH45 and AA9), and abundant hemicellulases. We further applied peptide pattern recognition to reveal nine and seven subfamilies of GH10 and GH11 family enzymes, respectively. The uncharacterized XYL10A, XYL10B and XYL11 enzymes of F. commune were classified, respectively, into GH10 subfamily 1, subfamily 3 and GH11 subfamily 1. These xylanases were successfully expressed in the PichiaPink™ system with the following properties: the purified recombinant XYL10A had interesting high specific activity; XYL10B was active at alkaline conditions with both endo-1,4-β-d-xylanase and β-xylosidase activities; and XYL11 was a true xylanase characterized by high substrate specificity. These results indicate that F. commune with genetic modification is a promising source of enzymes for the decomposition of lignocellulosic biomass. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Protein kinases: mechanisms and downstream targets in inflammation mediated obesity and insulin resistance

    PubMed Central

    Nandipati, Kalyana C; Subramanian, Saravanan; Agrawal, Devendra K

    2016-01-01

    Obesity induced low-grade inflammation (metaflammation) impairs insulin receptor signaling (IRS). This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), inhibitor of NF-kB kinase complex beta (IKKβ), AMP activated protein kinase (AMPK), protein kinase C (PKC), Rho associated coiled-coil containing protein kinase (ROCK) and RNA-activated protein kinase (PKR), etc. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor (IR) and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in Type II Diabetes Mellitus (T2-DM). Identifying the specific protein kinases involved in obesity induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity induced T2-DM. PMID:27868170

  12. Oral Insulin Delivery: How Far Are We?

    PubMed Central

    Fonte, Pedro; Araújo, Francisca; Reis, Salette; Sarmento, Bruno

    2013-01-01

    Oral delivery of insulin may significantly improve the quality of life of diabetes patients who routinely receive insulin by the subcutaneous route. In fact, compared with this administration route, oral delivery of insulin in diabetes treatment offers many advantages: higher patient compliance, rapid hepatic insulinization, and avoidance of peripheral hyperinsulinemia and other adverse effects such as possible hypoglycemia and weight gain. However, the oral delivery of insulin remains a challenge because its oral absorption is limited. The main barriers faced by insulin in the gastrointestinal tract are degradation by proteolytic enzymes and lack of transport across the intestinal epithelium. Several strategies to deliver insulin orally have been proposed, but without much clinical or commercial success. Protein encapsulation into nanoparticles is regarded as a promising alternative to administer insulin orally because they have the ability to promote insulin paracellular or transcellular transport across the intestinal mucosa. In this review, different delivery systems intended to increase the oral bioavailability of insulin will be discussed, with a special focus on nanoparticulate carrier systems, as well as the efforts that pharmaceutical companies are making to bring to the market the first oral delivery system of insulin. The toxicological and safety data of delivery systems, the clinical value and progress of oral insulin delivery, and the future prospects in this research field will be also scrutinized. PMID:23567010

  13. The Insulin Receptor: A New Target for Cancer Therapy

    PubMed Central

    Malaguarnera, Roberta; Belfiore, Antonino

    2011-01-01

    A large body of evidences have shown that both the IGF-I receptor (IGF-IR) and the insulin receptor (IR) play a role in cancer development and progression. In particular, IR overactivation by IGF-II is common in cancer cells, especially in dedifferentiated/stem-like cells. In spite of these findings, until very recently, only IGF-IR but not IR has been considered a target in cancer therapy. Although several preclinical studies have showed a good anti-cancer activity of selective anti-IGF-IR drugs, the results of the clinical first trials have been disappointing. In fact, only a small subset of malignant tumors has shown an objective response to these therapies. Development of resistance to anti-IGF-IR drugs may include upregulation of IR isoform A (IR-A) in cancer cells and its overactivation by increased secretion of autocrine IGF-II. These findings have led to the concept that co-targeting IR together with IGF-IR may increase therapy efficacy and prevent adaptive resistance to selective anti-IGF-IR drugs. IR blockade should be especially considered in tumors with high IR-A:IGF-IR ratio and high levels of autocrine IGF-II. Conversely, insulin sensitizers, which ameliorate insulin resistance associated with metabolic disorders and cancer treatments, may have important implications for cancer prevention and management. Only few drugs co-targeting the IR and IGF-IR are currently available. Ideally, future IR targeting strategies should be able to selectively inhibit the tumor promoting effects of IR without impairing its metabolic effects. PMID:22654833

  14. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

    PubMed

    Schumacher, Marc M; Elsabrouty, Rania; Seemann, Joachim; Jo, Youngah; DeBose-Boyd, Russell A

    2015-03-05

    Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

  15. The influence of short-term endurance training on the insulin blood level, binding, and degradation of 125I-insulin by erythrocyte receptors in patients after myocardial infarction.

    PubMed

    Dylewicz, P; Przywarska, I; Szcześniak, L; Rychlewski, T; Bieńkowska, S; Długiewicz, I; Wilk, M

    1999-01-01

    This study was directed toward establishing whether and to what extent, short-term endurance training influences the insulin blood level, and the binding and degradation of 125I-insulin by erythrocyte receptors in patients undergoing rehabilitation after myocardial infarction. The study was conducted in a group of 60 patients who had had myocardial infarction within the past 1.5 to 3 months and who did not have arterial hypertension and diabetes mellitus. All the patients took a symptom-limited cardiopulmonary exercise test. Before and after the test, venous blood was collected to determine lactic acid and insulin blood levels as well as the binding and degradation of 125I-insulin. The study group was randomized into two subgroups. One subgroup entered into a 3-week in-patient rehabilitation course. The control group was discharged from the hospital and was given no recommendations for physical exercise. The same investigation was repeated 3 weeks later. In the patients (50%) with hyperinsulinemia (insulin resistance index, > 10 microIU/mL), which was detected during the first investigation, insulin blood level decreased from 23.9 +/- 4.4 to 15.0 +/- 1.9 microIU/mL (P < 0.05) after rehabilitation, whereas insulin binding increased from 0.67 +/- 0.05 to 0.85 +/- 0.08 pg 125I/10(11) erythrocytes (P < 0.05). In the control group, which included normal subjects and those with hyperinsulinemia, the results obtained during the first and second investigations showed no statistically significant changes when compared. The results suggest that a 3-week endurance training period during rehabilitation after myocardial infarction reduces insulin resistance in patients with hyperinsulinemia.

  16. Bioinformatics Analysis and Characterization of Highly Efficient Polyvinyl Alcohol (PVA)-Degrading Enzymes from the Novel PVA Degrader Stenotrophomonas rhizophila QL-P4.

    PubMed

    Wei, Yahong; Fu, Jing; Wu, Jianying; Jia, Xinmiao; Zhou, Yunheng; Li, Cuidan; Dong, Mengxing; Wang, Shanshan; Zhang, Ju; Chen, Fei

    2018-01-01

    Polyvinyl alcohol (PVA) is used widely in industry, and associated environmental pollution is a serious problem. Herein, we report a novel, efficient PVA degrader, Stenotrophomonas rhizophila QL-P4, isolated from fallen leaves from a virgin forest in the Qinling Mountains. The complete genome was obtained using single-molecule real-time (SMRT) technology and corrected using Illumina sequencing. Bioinformatics analysis revealed eight PVA/vinyl alcohol oligomer (OVA)-degrading genes. Of these, seven genes were predicted to be involved in the classic intracellular PVA/OVA degradation pathway, and one (BAY15_3292) was identified as a novel PVA oxidase. Five PVA/OVA-degrading enzymes were purified and characterized. One of these, BAY15_1712, a PVA dehydrogenase (PVADH), displayed high catalytic efficiency toward PVA and OVA substrate. All reported PVADHs only have PVA-degrading ability. Most importantly, we discovered a novel PVA oxidase (BAY15_3292) that exhibited higher PVA-degrading efficiency than the reported PVADHs. Further investigation indicated that BAY15_3292 plays a crucial role in PVA degradation in S. rhizophila QL-P4. Knocking out BAY15_3292 resulted in a significant decline in PVA-degrading activity in S. rhizophila QL-P4. Interestingly, we found that BAY15_3292 possesses exocrine activity, which distinguishes it from classic PVADHs. Transparent circle experiments further proved that BAY15_3292 greatly affects extracellular PVA degradation in S. rhizophila QL-P4. The exocrine characteristics of BAY15_3292 facilitate its potential application to PVA bioremediation. In addition, we report three new efficient secondary alcohol dehydrogenases (SADHs) with OVA-degrading ability in S. rhizophila QL-P4; in contrast, only one OVA-degrading SADH was reported previously. IMPORTANCE With the widespread application of PVA in industry, PVA-related environmental pollution is an increasingly serious issue. Because PVA is difficult to degrade, it accumulates in aquatic

  17. Combined effect of enzyme inducers and nitrate on selective lignin degradation in wheat straw by Ganoderma lobatum.

    PubMed

    Hermosilla, Edward; Schalchli, Heidi; Mutis, Ana; Diez, María Cristina

    2017-09-01

    Lignin is one of the main barriers to obtaining added-value products from cellulosic fraction of lignocellulosic biomass due to its random aromatic structure and strong association with cellulose and hemicellulose. Inorganic and organic compounds have been used as enzyme inducers to increase the ligninolytic potential of white-rot fungi, without considering their effect on the selectivity of degradation. In this study, the selective lignin degradation in wheat straw by Ganoderma lobatum was optimized using a central composite design to evaluate the combined effect of Fe 2+ and Mn 2+ as inducers of ligninolytic enzymes and NO 3 - as an additional nitrogen source. Selective lignin degradation was promoted to maximize lignin degradation and minimize weight losses. The optimal conditions were 0.18 M NO 3 - , 0.73 mM Fe 2+ , and 1 mM Mn 2+ , which resulted in 50.0% lignin degradation and 18.5% weight loss after 40 days of fungal treatment. A decrease in absorbance at 1505 and 900 cm -1 in fungal-treated samples was observed in the FTIR spectra, indicating lignin and cellulose degradation in fungal-treated wheat straw, respectively. The main ligninolytic enzymes detected during lignin degradation were manganese-dependent and manganese-independent peroxidases. Additionally, confocal laser scanning microscopy revealed that lignin degradation in wheat straw by G. lobatum resulted in higher cellulose accessibility. We concluded that the addition of enzyme inducers and NO 3 - promotes selective lignin degradation in wheat straw by G. lobatum.

  18. Insulin-coated gold nanoparticles as a new concept for personalized and adjustable glucose regulation.

    PubMed

    Shilo, Malka; Berenstein, Peter; Dreifuss, Tamar; Nash, Yuval; Goldsmith, Guy; Kazimirsky, Gila; Motiei, Menachem; Frenkel, Dan; Brodie, Chaya; Popovtzer, Rachela

    2015-12-28

    Diabetes mellitus is a chronic metabolic disease, characterized by high blood glucose levels, affecting millions of people around the world. Currently, the main treatment for diabetes requires multiple daily injections of insulin and self-monitoring of blood glucose levels, which markedly affect patients' quality of life. In this study we present a novel strategy for controlled and prolonged glucose regulation, based on the administration of insulin-coated gold nanoparticles (INS-GNPs). We show that both intravenous and subcutaneous injection of INS-GNPs into a mouse model of type 1 diabetes decreases blood glucose levels for periods over 3 times longer than free insulin. We further showed that conjugation of insulin to GNPs prevented its rapid degradation by the insulin-degrading-enzyme, and thus allows controlled and adjustable bio-activity. Moreover, we assessed different sizes and concentrations of INS-GNPs, and found that both parameters have a critical effect in vivo, enabling specific adjustment of blood glucose levels. These findings have the potential to improve patient compliance in diabetes mellitus.

  19. Insulin-coated gold nanoparticles as a new concept for personalized and adjustable glucose regulation

    NASA Astrophysics Data System (ADS)

    Shilo, Malka; Berenstein, Peter; Dreifuss, Tamar; Nash, Yuval; Goldsmith, Guy; Kazimirsky, Gila; Motiei, Menachem; Frenkel, Dan; Brodie, Chaya; Popovtzer, Rachela

    2015-12-01

    Diabetes mellitus is a chronic metabolic disease, characterized by high blood glucose levels, affecting millions of people around the world. Currently, the main treatment for diabetes requires multiple daily injections of insulin and self-monitoring of blood glucose levels, which markedly affect patients' quality of life. In this study we present a novel strategy for controlled and prolonged glucose regulation, based on the administration of insulin-coated gold nanoparticles (INS-GNPs). We show that both intravenous and subcutaneous injection of INS-GNPs into a mouse model of type 1 diabetes decreases blood glucose levels for periods over 3 times longer than free insulin. We further showed that conjugation of insulin to GNPs prevented its rapid degradation by the insulin-degrading-enzyme, and thus allows controlled and adjustable bio-activity. Moreover, we assessed different sizes and concentrations of INS-GNPs, and found that both parameters have a critical effect in vivo, enabling specific adjustment of blood glucose levels. These findings have the potential to improve patient compliance in diabetes mellitus.

  20. Human β-glucuronidase: structure, function, and application in enzyme replacement therapy.

    PubMed

    Naz, Huma; Islam, Asimul; Waheed, Abdul; Sly, William S; Ahmad, Faizan; Hassan, Imtaiyaz

    2013-10-01

    Lysosomal storage diseases occur due to incomplete metabolic degradation of macromolecules by various hydrolytic enzymes in the lysosome. Despite structural differences, most of the lysosomal enzymes share many common features including a lysosomal targeting motif and phosphotransferase recognition sites. β-Glucuronidase (GUSB) is an important lysosomal enzyme involved in the degradation of glucuronate-containing glycosaminoglycan. The deficiency of GUSB causes mucopolysaccharidosis type VII (MPSVII), leading to lysosomal storage in the brain. GUSB is a well-studied protein for its expression, sequence, structure, and function. The purpose of this review is to summarize our current understanding of sequence, structure, function, and evolution of GUSB and its lysosomal enzyme targeting. Enzyme replacement therapy reported for this protein is also discussed.

  1. The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study.

    PubMed

    Burslem, George M; Smith, Blake E; Lai, Ashton C; Jaime-Figueroa, Saul; McQuaid, Daniel C; Bondeson, Daniel P; Toure, Momar; Dong, Hanqing; Qian, Yimin; Wang, Jing; Crew, Andrew P; Hines, John; Crews, Craig M

    2018-01-18

    Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. The role of proteolytic enzymes in degradation of plant tissues: Summary report

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lewosz, J.; Kelman, A.; Sequeira, L.

    1989-01-01

    The proteolytic enzymes produced by Erwinia carotovora subsp. carotovora (Ecc-strain SR 394) grown on various media were examined by isoelectrofocusing in polyacrylamide gels over a pH range of 3-10. In addition to the main protease present in culture filtrates, low concentrations of several other proteases were present in extracts from potato tubers infected by Ecc. Proteases from all these sources were similar and had the following properties: pH optimum near 8.0, calcium dependent, insensitive to serine proteinase and SH-proteinase inhibitors, inhibited by EDTA, and highly thermostable. These enzymes degraded gelatin, soluble collagen and Hide Powder Azure, and showed weak activitymore » on casein, but did not degrade insoluble collagen or elastin.« less

  3. Endothelial Targeting of Semi-permeable Polymer Nanocarriers for Enzyme Therapies

    PubMed Central

    Dziubla, Thomas D; Shuvaev, Vladimir V.; Hong, Nan Kang; Hawkins, Brian; Muniswamy, Madesh; Takano, Hajime; Simone, Eric; Nakada, Marian T.; Fisher, Aron; Albelda, Steven M.; Muzykantov, Vladimir R.

    2007-01-01

    The medical utility of proteins, e.g. therapeutic enzymes, is greatly restricted by their liable nature and inadequate delivery. Most therapeutic enzymes do not accumulate in their targets and are inactivated by proteases. Targeting of enzymes encapsulated into substrate-permeable Polymeric Nano-Carriers (PNC) impermeable for proteases might overcome these limitations. To test this hypothesis, we designed endothelial targeted PNC loaded with catalase, the H2O2-detoxifying enzyme, and tested if this approach protects against vascular oxidative stress, a pathological process implicated in ischemia-reperfusion and other disease conditions. Encapsulation of catalase (MW 240KD), peroxidase (MW 42kD) and xanthine oxidase (XO, MW 300 kD) into ~300nm diameter PNC composed of co-polymers of PEG-PLGA (polyethylene glycol and poly-lactic/poly-glycolic acid) was in the range ~10% for all enzymes. PNC/catalase and PNC/peroxidase were protected from external proteolysis and exerted the enzymatic activity on their PNC diffusible substrates, H2O2 and ortho-phenylendiamine, whereas activity of encapsulated XO was negligible due to polymer impermeability to the substrate. PNC targeted to platelet-endothelial cell adhesion molecule-1 delivered active encapsulated catalase to endothelial cells and protected the endothelium against oxidative stress in cell culture and animal studies. Vascular targeting of PNC-loaded detoxifying enzymes may find wide medical applications including management of oxidative stress and other toxicities. PMID:17950837

  4. Defective insulin signaling pathway and increased glycogen synthase kinase-3 activity in the brain of diabetic mice: parallels with Alzheimer's disease and correction by insulin.

    PubMed

    Jolivalt, C G; Lee, C A; Beiswenger, K K; Smith, J L; Orlov, M; Torrance, M A; Masliah, E

    2008-11-15

    We have evaluated the effect of peripheral insulin deficiency on brain insulin pathway activity in a mouse model of type 1 diabetes, the parallels with Alzheimer's disease (AD), and the effect of treatment with insulin. Nine weeks of insulin-deficient diabetes significantly impaired the learning capacity of mice, significantly reduced insulin-degrading enzyme protein expression, and significantly reduced phosphorylation of the insulin-receptor and AKT. Phosphorylation of glycogen synthase kinase-3 (GSK3) was also significantly decreased, indicating increased GSK3 activity. This evidence of reduced insulin signaling was associated with a concomitant increase in tau phosphorylation and amyloid beta protein levels. Changes in phosphorylation levels of insulin receptor, GSK3, and tau were not observed in the brain of db/db mice, a model of type 2 diabetes, after a similar duration (8 weeks) of diabetes. Treatment with insulin from onset of diabetes partially restored the phosphorylation of insulin receptor and of GSK3, partially reduced the level of phosphorylated tau in the brain, and partially improved learning ability in insulin-deficient diabetic mice. Our data indicate that mice with systemic insulin deficiency display evidence of reduced insulin signaling pathway activity in the brain that is associated with biochemical and behavioral features of AD and that it can be corrected by insulin treatment.

  5. Effects of soybean oligosaccharides on antioxidant enzyme activities and insulin resistance in pregnant women with gestational diabetes mellitus.

    PubMed

    Fei, Bei-bei; Ling, Li; Hua, Chen; Ren, Shu-yan

    2014-09-01

    The effects of soybean oligosaccharides (SBOS) on antioxidant enzyme activities and insulin resistance in pregnant women with gestational diabetes mellitus (GDM) were investigated. Ninety-seven pregnant women with GDM were randomly divided into two groups, the control group (51 cases) and the SBOS group (46 cases). Before the group separation, the blood sugar level in patients was maintained stable by regular diet and insulin treatment. The control group was continued with the insulin treatment, while the SBOS group was treated with the combination of insulin and SBOS. Results showed that SBOS were able to reduce oxidative stress and alleviate insulin resistance in pregnant women with GDM, which indicates that SBOS may play an important role in the control of GDM complications. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Brain insulin lowers circulating BCAA levels by inducing hepatic BCAA catabolism

    PubMed Central

    Shin, Andrew C.; Fasshauer, Martin; Filatova, Nika; Grundell, Linus A.; Zielinski, Elizabeth; Zhou, Jian-Ying; Scherer, Thomas; Lindtner, Claudia; White, Phillip J.; Lapworth, Amanda L.; Ilkayeva, Olga; Knippschild, Uwe; Wolf, Anna M.; Scheja, Ludger; Grove, Kevin L.; Smith, Richard D.; Qian, Wei-Jun; Lynch, Christopher J.; Newgard, Christopher B.; Buettner, Christoph

    2014-01-01

    Summary Circulating branched-chain amino acid (BCAA) levels are elevated in obesity/diabetes and are a sensitive predictor for type 2 diabetes. Here we show in rats that insulin dose-dependently lowers plasma BCAA levels through induction of hepatic protein expression and activity of branched-chain α keto-acid dehydrogenase (BCKDH), the rate-limiting enzyme in the BCAA degradation pathway. Selective induction of hypothalamic insulin signaling in rats and genetic modulation of brain insulin receptors in mice demonstrate that brain insulin signaling is a major regulator of BCAA metabolism by inducing hepatic BCKDH. Short-term overfeeding impairs the ability of brain insulin to lower BCAAs in rats. High-fat feeding in non-human primates and obesity and/or diabetes in humans is associated with reduced BCKDH protein in liver. These findings support the concept that decreased hepatic BCKDH is a major cause of increased plasma BCAAs, and that hypothalamic insulin resistance may account for impaired BCAA metabolism in obesity and diabetes. PMID:25307860

  7. Isolation and characterization of a novel metagenomic enzyme capable of degrading bacterial phytotoxin toxoflavin

    PubMed Central

    Lee, Boyoung; Park, Ji Hyun; Oh, Joon Young; Choi, Jung Sup; Kim, Jin-Cheol

    2018-01-01

    Toxoflavin, a 7-azapteridine phytotoxin produced by the bacterial pathogens such as Burkholderia glumae and Burkholderia gladioli, has been known as one of the key virulence factors in crop diseases. Because the toxoflavin had an antibacterial activity, a metagenomic E. coli clone capable of growing well in the presence of toxoflavin (30 μg/ml) was isolated and the first metagenome-derived toxoflavin-degrading enzyme, TxeA of 140 amino acid residues, was identified from the positive E. coli clone. The conserved amino acids for metal-binding and extradiol dioxygenase activity, Glu-12, His-8 and Glu-130, were revealed by the sequence analysis of TxeA. The optimum conditions for toxoflavin degradation were evaluated with the TxeA purified in E. coli. Toxoflavin was totally degraded at an initial toxoflavin concentration of 100 μg/ml and at pH 5.0 in the presence of Mn2+, dithiothreitol and oxygen. The final degradation products of toxoflavin and methyltoxoflavin were fully identified by MS and NMR as triazines. Therefore, we suggested that the new metagenomic enzyme, TxeA, provided the clue to applying the new metagenomic enzyme to resistance development of crop plants to toxoflavin-mediated disease as well as to biocatalysis for Baeyer-Villiger type oxidation. PMID:29293506

  8. Filter Paper Blood Spot Enzyme Linked Immunoassay for Insulin and Application in the Evaluation of Determinants of Child Insulin Resistance

    PubMed Central

    Martin, Richard M.; Patel, Rita; Zinovik, Alexander; Kramer, Michael S.; Oken, Emily; Vilchuck, Konstantin; Bogdanovich, Natalia; Sergeichick, Natalia; Gunnarsson, Robert; Grufman, Lisa; Foo, Ying; Gusina, Nina

    2012-01-01

    Background In large-scale epidemiology, bloodspot sampling by fingerstick onto filter paper has many advantages, including ease and low costs of collection, processing and transport. We describe the development of an enzyme-linked immunoassay (ELISA) for quantifying insulin from dried blood spots and demonstrate its application in a large trial. Methods We adapted an existing commercial kit (Mercodia Human Insulin ELISA, 10-1113-01) to quantify insulin from two 3-mm diameter discs (≈6 µL of blood) punched from whole blood standards and from trial samples. Paediatricians collected dried blood spots in a follow-up of 13,879 fasted children aged 11.5 years (interquartile range 11.3–11.8 years) from 31 trial sites across Belarus. We quantified bloodspot insulin levels and examined their distribution by demography and anthropometry. Results Mean intra-assay (n = 157) coefficients of variation were 15% and 6% for ‘low’ (6.7 mU/L) and ‘high’ (23.1 mU/L) values, respectively; the respective inter-assay values (n = 33) were 23% and 11%. The intraclass correlation coefficient between 50 paired whole bloodspot versus serum samples, collected simultaneously, was 0.90 (95% confidence interval 0.85 to 0.95). Bloodspot insulin was stable for at least 31 months at −80°C, for one week at +30°C and following four freeze-thaw cycles. Paediatricians collected a median of 8 blood spots from 13,487 (97%) children. The geometric mean insulin (log standard deviation) concentrations amongst 12,812 children were 3.0 mU/L (1.1) in boys and 4.0 mU/L (1.0) in girls and were positively associated with pubertal stage, measures of central and peripheral adiposity, height and fasting glucose. Conclusions Our simple and convenient bloodspot assay is suitable for the measurement of insulin in very small volumes of blood collected on filter paper cards and can be applied to large-scale epidemiology studies of the early-life determinants of circulating insulin. PMID:23056434

  9. Optimization of liquid-state fermentation conditions for the glyphosate degradation enzyme production of strain Aspergillus oryzae by ultraviolet mutagenesis.

    PubMed

    Fu, Gui-Ming; Li, Ru-Yi; Li, Kai-Min; Hu, Ming; Yuan, Xiao-Qiang; Li, Bin; Wang, Feng-Xue; Liu, Cheng-Mei; Wan, Yin

    2016-11-16

    This study aimed to obtain strains with high glyphosate-degrading ability and improve the ability of glyphosate degradation enzyme by the optimization of fermentation conditions. Spore from Aspergillus oryzae A-F02 was subjected to ultraviolet mutagenesis. Single-factor experiment and response surface methodology were used to optimize glyphosate degradation enzyme production from mutant strain by liquid-state fermentation. Four mutant strains were obtained and named as FUJX 001, FUJX 002, FUJX 003, and FUJX 004, in which FUJX 001 gave the highest total enzyme activity. Starch concentration at 0.56%, GP concentration at 1,370 mg/l, initial pH at 6.8, and temperature at 30°C were the optimum conditions for the improved glyphosate degradation endoenzyme production of A. oryzae FUJX 001. Under these conditions, the experimental endoenzyme activity was 784.15 U/100 ml fermentation liquor. The result (784.15 U/100 ml fermentation liquor) was approximately 14-fold higher than that of the original strain. The result highlights the potential of glyphosate degradation enzyme to degrade glyphosate.

  10. Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of baker's yeast.

    PubMed

    Greiner, R; Alminger, M L; Carlsson, N G

    2001-05-01

    During food processing such as baking, phytate is dephosphorylated to produce degradation products, such as myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphates. Certain myo-inositol phosphates have been proposed to have positive effects on human health. The position of the phosphate groups on the myo-inositol ring is thereby of great significance for their physiological functions. Using a combination of high-performance ion chromatography analysis and kinetic studies the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme from baker's yeast (Saccharomyces cerevisiae) was established. The data demonstrate that the phytate-degrading enzyme from baker's yeast dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), D-Ins(1,2)P(2), to finally Ins(2)P (notation 3/4/5/6/1). Knowledge of the absolute stereochemical specificity of the baker's yeast phytase allows use of the enzyme to produce defined myo-inositol phosphates for kinetic and physiological studies.

  11. Identification of Small Peptides in Human Cerebrospinal Fluid upon Amyloid-β Degradation.

    PubMed

    Mizuta, Naoki; Yanagida, Kanta; Kodama, Takashi; Tomonaga, Takeshi; Takami, Mako; Oyama, Hiroshi; Kudo, Takashi; Ikeda, Manabu; Takeda, Masatoshi; Tagami, Shinji; Okochi, Masayasu

    2017-01-01

    Amyloid-β (Aβ) degradation in brains of Alzheimer disease patients is a crucial focus for the clarification of disease pathogenesis. Nevertheless, the mechanisms underlying Aβ degradation in the human brain remain unclear. This study aimed to quantify the levels of small C-terminal Aβ fragments generated upon Aβ degradation in human cerebrospinal fluid (CSF). A fraction containing small peptides was isolated and purified from human CSF by high-pressure liquid chromatography. Degradation products of Aβ C termini were identified and measured by liquid chromatography-tandem mass spectrometry. The C-terminal fragments of Aβ in the conditioned medium of cultured cells transfected with the Swedish variant of βAPP (sw βAPP) were analyzed. These fragments in brains of PS1 I213T knock-in transgenic mice, overexpressing sw βAPP, were also analyzed. The peptide fragments GGVV and GVV, produced by the cleavage of Aβ40, were identified in human CSF as well as in the brains of the transgenic mice and in the conditioned medium of the cultured cells. Relative to Aβ40 levels, GGVV and GVV levels were 7.6 ± 0.81 and 1.5 ± 0.18%, respectively, in human CSF. Levels of the GGVV fragment did not increase by the introduction of genes encoding neprilysin and insulin-degrading enzyme to the cultured cells. Our results indicate that a substantial amount of Aβ40 in human brains is degraded via a neprilysin- or insulin-degrading enzyme-independent pathway. © 2017 S. Karger AG, Basel.

  12. Myostatin induces insulin resistance via Casitas B-lineage lymphoma b (Cblb)-mediated degradation of insulin receptor substrate 1 (IRS1) protein in response to high calorie diet intake.

    PubMed

    Bonala, Sabeera; Lokireddy, Sudarsanareddy; McFarlane, Craig; Patnam, Sreekanth; Sharma, Mridula; Kambadur, Ravi

    2014-03-14

    To date a plethora of evidence has clearly demonstrated that continued high calorie intake leads to insulin resistance and type-2 diabetes with or without obesity. However, the necessary signals that initiate insulin resistance during high calorie intake remain largely unknown. Our results here show that in response to a regimen of high fat or high glucose diets, Mstn levels were induced in muscle and liver of mice. High glucose- or fat-mediated induction of Mstn was controlled at the level of transcription, as highly conserved carbohydrate response and sterol-responsive (E-box) elements were present in the Mstn promoter and were revealed to be critical for ChREBP (carbohydrate-responsive element-binding protein) or SREBP1c (sterol regulatory element-binding protein 1c) regulation of Mstn expression. Further molecular analysis suggested that the increased Mstn levels (due to high glucose or fatty acid loading) resulted in increased expression of Cblb in a Smad3-dependent manner. Casitas B-lineage lymphoma b (Cblb) is an ubiquitin E3 ligase that has been shown to specifically degrade insulin receptor substrate 1 (IRS1) protein. Consistent with this, our results revealed that elevated Mstn levels specifically up-regulated Cblb, resulting in enhanced ubiquitin proteasome-mediated degradation of IRS1. In addition, over expression or knock down of Cblb had a major impact on IRS1 and pAkt levels in the presence or absence of insulin. Collectively, these observations strongly suggest that increased glucose levels and high fat diet, both, result in increased circulatory Mstn levels. The increased Mstn in turn is a potent inducer of insulin resistance by degrading IRS1 protein via the E3 ligase, Cblb, in a Smad3-dependent manner.

  13. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase

    PubMed Central

    Schumacher, Marc M; Elsabrouty, Rania; Seemann, Joachim; Jo, Youngah; DeBose-Boyd, Russell A

    2015-01-01

    Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD. DOI: http://dx.doi.org/10.7554/eLife.05560.001 PMID:25742604

  14. Effect of insulin analog initiation therapy on LDL/HDL subfraction profile and HDL associated enzymes in type 2 diabetic patients.

    PubMed

    Aslan, Ibrahim; Kucuksayan, Ertan; Aslan, Mutay

    2013-04-24

    Insulin treatment can lead to good glycemic control and result in improvement of lipid parameters in type 2 diabetic patients. This study was designed to evaluate the effect of insulin analog initiation therapy on low-density lipoprotein (LDL)/ high-density lipoprotein (HDL) sub-fractions and HDL associated enzymes in type 2 diabetic patients during early phase. Twenty four type 2 diabetic patients with glycosylated hemoglobin (HbA1c) levels above 10% despite ongoing combination therapy with sulphonylurea and metformin were selected. Former treatment regimen was continued for the first day followed by substitution of sulphonylurea therapy with different insulin analogs (0.4 U/kg/day) plus metformin. Glycemic profiles were determined over 72 hours by continuous glucose monitoring system (CGMS) and blood samples were obtained from all patients at 24 and 72 hours. Plasma levels of cholesteryl ester transfer protein (CETP), lecithin-cholesterol acyltransferase (LCAT), apolipoprotein B (apoB) and apolipoprotein A-1 (apoA-I) were determined by enzyme-linked immunosorbent assay (ELISA). Measurement of CETP and LCAT activity was performed via fluorometric analysis. Paraoxonase (PON1) enzyme activity was assessed from the rate of enzymatic hydrolysis of phenyl acetate to phenol formation. LDL and HDL subfraction analysis was done by continuous disc polyacrylamide gel electrophoresis. Mean blood glucose, total cholesterol (TC), triglyceride (TG) and very low-density lipoprotein cholesterol (VLDL-C) levels were significantly decreased while HDL-C levels were significantly increased after insulin treatment. Although LDL-C levels were not significantly different before and after insulin initiation therapy a significant increase in LDL-1 subgroup and a significant reduction in atherogenic LDL-3 and LDL-4 subgroups were observed. Insulin analog initiation therapy caused a significant increase in HDL-large, HDL- intermediate and a significant reduction in HDL-small subfractions

  15. Characterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175.

    PubMed

    Hanphakphoom, Srisuda; Maneewong, Narisara; Sukkhum, Sukhumaporn; Tokuyama, Shinji; Kitpreechavanich, Vichien

    2014-01-01

    Eleven strains of poly(L-lactide) (PLLA)-degrading thermophilic bacteria were isolated from forest soils and selected based on clear zone formation on an emulsified PLLA agar plate at 50°C. Among the isolates, strain LP175 showed the highest PLLA-degrading ability. It was closely related to Laceyella sacchari, with 99.9% similarity based on the 16S rRNA gene sequence. The PLLA-degrading enzyme produced by the strain was purified to homogeneity by 48.1% yield and specific activity of 328 U·mg-protein-1 with a 15.3-fold purity increase. The purified enzyme was strongly active against specific substrates such as casein and gelatin and weakly active against Suc-(Ala)₃-pNA. Optimum enzyme activity was exhibited at a temperature of 60°C with thermal stability up to 50°C and a pH of 9.0 with pH stability in a range of 8.5-10.5. Molecular weight of the enzyme was approximately 28.0 kDa, as determined by gel filtration and SDS-PAGE. The inhibitors phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetate (EDTA), and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) strongly inhibited enzyme activity, but the activity was not inhibited by 1 mM 1,10-phenanthroline (1,10-phen). The N-terminal amino acid sequences had 100% homology with thermostable serine protease (thermitase) from Thermoactinomyces vulgaris. The results obtained suggest that the PLLA-degrading enzyme produced by L. sacchari strain LP175 is serine protease.

  16. Influence of nutrient restriction and melatonin supplementation of pregnant ewes on maternal and fetal pancreatic digestive enzymes and insulin-containing clusters.

    PubMed

    Keomanivong, F E; Lemley, C O; Camacho, L E; Yunusova, R; Borowicz, P P; Caton, J S; Meyer, A M; Vonnahme, K A; Swanson, K C

    2016-03-01

    Primiparous ewes (n=32) were assigned to dietary treatments in a 2×2 factorial arrangement to determine effects of nutrient restriction and melatonin supplementation on maternal and fetal pancreatic weight, digestive enzyme activity, concentration of insulin-containing clusters and plasma insulin concentrations. Treatments consisted of nutrient intake with 60% (RES) or 100% (ADQ) of requirements and melatonin supplementation at 0 (CON) or 5 mg/day (MEL). Treatments began on day 50 of gestation and continued until day 130. On day 130, blood was collected under general anesthesia from the uterine artery, uterine vein, umbilical artery and umbilical vein for plasma insulin analysis. Ewes were then euthanized and the pancreas removed from the ewe and fetus, trimmed of mesentery and fat, weighed and snap-frozen until enzyme analysis. In addition, samples of pancreatic tissue were fixed in 10% formalin solution for histological examination including quantitative characterization of size and distribution of insulin-containing cell clusters. Nutrient restriction decreased (P⩽0.001) maternal pancreatic mass (g) and α-amylase activity (U/g, kU/pancreas, U/kg BW). Ewes supplemented with melatonin had increased pancreatic mass (P=0.03) and α-amylase content (kU/pancreas and U/kg BW). Melatonin supplementation decreased (P=0.002) maternal pancreatic insulin-positive tissue area (relative to section of tissue), and size of the largest insulin-containing cell cluster (P=0.04). Nutrient restriction decreased pancreatic insulin-positive tissue area (P=0.03) and percent of large (32 001 to 512 000 µm2) and giant (⩾512 001 µm2) insulin-containing cell clusters (P=0.04) in the fetus. Insulin concentrations in plasma from the uterine vein, umbilical artery and umbilical vein were greater (P⩽0.01) in animals receiving 100% requirements. When comparing ewes to fetuses, ewes had a greater percentage of medium insulin-containing cell clusters (2001 to 32 000 µm2) while fetuses

  17. Acetyl-CoA carboxylase in Reuber hepatoma cells: variation in enzyme activity, insulin regulation, and cellular lipid content.

    PubMed

    Bianchi, A; Evans, J L; Nordlund, A C; Watts, T D; Witters, L A

    1992-01-01

    Reuber hepatoma cells are useful cultured lines for the study of insulin action, lipid and lipoprotein metabolism, and the regulation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis. During investigations in different clonal lines of these cells, we have uncovered marked intercellular variability in the activity, enzyme content, and insulin regulation of ACC paralleled by differences in cellular neutral lipid (triglyceride) content. Two contrasting clonal lines, Fao and H356A-1, have been studied in detail. Several features distinguish these two lines, including differences in ACC activity and enzyme kinetics, the content of the two major hepatic ACC isozymes (Mr 280,000 and 265,000 Da) and their heteroisozymic complex, the extent of ACC phosphorylation, and the ability of ACC to be activated on stimulation by insulin and insulinomimetic agonists. As studied by Nile Red staining and fluorescence-activated cell sorting, these two lines also display marked differences in neutral lipid content, which correlates with both basal levels of ACC activity and inhibition of ACC by the fatty acid analog, 5-(tetradecyloxy)-2-furoic acid (TOFA). These results emphasize the importance of characterization of any particular clonal line of Reuber cells for studies of enzyme regulation, substrate metabolism, and hormone action. With respect to ACC, studies in contrasting clonal lines of Reuber cells could provide valuable clues to understanding both the complex mechanisms of intracellular ACC regulation in the absence and presence of hormones and its regulatory role(s) in overall hepatic lipid metabolism.

  18. Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides.

    PubMed

    Luis, Ana S; Briggs, Jonathon; Zhang, Xiaoyang; Farnell, Benjamin; Ndeh, Didier; Labourel, Aurore; Baslé, Arnaud; Cartmell, Alan; Terrapon, Nicolas; Stott, Katherine; Lowe, Elisabeth C; McLean, Richard; Shearer, Kaitlyn; Schückel, Julia; Venditto, Immacolata; Ralet, Marie-Christine; Henrissat, Bernard; Martens, Eric C; Mosimann, Steven C; Abbott, D Wade; Gilbert, Harry J

    2018-02-01

    The major nutrients available to human colonic Bacteroides species are glycans, exemplified by pectins, a network of covalently linked plant cell wall polysaccharides containing galacturonic acid (GalA). Metabolism of complex carbohydrates by the Bacteroides genus is orchestrated by polysaccharide utilization loci (PULs). In Bacteroides thetaiotaomicron, a human colonic bacterium, the PULs activated by different pectin domains have been identified; however, the mechanism by which these loci contribute to the degradation of these GalA-containing polysaccharides is poorly understood. Here we show that each PUL orchestrates the metabolism of specific pectin molecules, recruiting enzymes from two previously unknown glycoside hydrolase families. The apparatus that depolymerizes the backbone of rhamnogalacturonan-I is particularly complex. This system contains several glycoside hydrolases that trim the remnants of other pectin domains attached to rhamnogalacturonan-I, and nine enzymes that contribute to the degradation of the backbone that makes up a rhamnose-GalA repeating unit. The catalytic properties of the pectin-degrading enzymes are optimized to protect the glycan cues that activate the specific PULs ensuring a continuous supply of inducing molecules throughout growth. The contribution of Bacteroides spp. to metabolism of the pectic network is illustrated by cross-feeding between organisms.

  19. Ubiquitinated CD36 sustains insulin-stimulated Akt activation by stabilizing insulin receptor substrate 1 in myotubes.

    PubMed

    Sun, Shishuo; Tan, Pengcheng; Huang, Xiaoheng; Zhang, Wei; Kong, Chen; Ren, Fangfang; Su, Xiong

    2018-02-16

    Both the magnitude and duration of insulin signaling are important in executing its cellular functions. Insulin-induced degradation of insulin receptor substrate 1 (IRS1) represents a key negative feedback loop that restricts insulin signaling. Moreover, high concentrations of fatty acids (FAs) and glucose involved in the etiology of obesity-associated insulin resistance also contribute to the regulation of IRS1 degradation. The scavenger receptor CD36 binds many lipid ligands, and its contribution to insulin resistance has been extensively studied, but the exact regulation of insulin sensitivity by CD36 is highly controversial. Herein, we found that CD36 knockdown in C2C12 myotubes accelerated insulin-stimulated Akt activation, but the activated signaling was sustained for a much shorter period of time as compared with WT cells, leading to exacerbated insulin-induced insulin resistance. This was likely due to enhanced insulin-induced IRS1 degradation after CD36 knockdown. Overexpression of WT CD36, but not a ubiquitination-defective CD36 mutant, delayed IRS1 degradation. We also found that CD36 functioned through ubiquitination-dependent binding to IRS1 and inhibiting its interaction with cullin 7, a key component of the multisubunit cullin-RING E3 ubiquitin ligase complex. Moreover, dissociation of the Src family kinase Fyn from CD36 by free FAs or Fyn knockdown/inhibition accelerated insulin-induced IRS1 degradation, likely due to disrupted IRS1 interaction with CD36 and thus enhanced binding to cullin 7. In summary, we identified a CD36-dependent FA-sensing pathway that plays an important role in negative feedback regulation of insulin activation and may open up strategies for preventing or managing type 2 diabetes mellitus. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Effect of enzyme additions on methane production and lignin degradation of landfilled sample of municipal solid waste.

    PubMed

    Jayasinghe, P A; Hettiaratchi, J P A; Mehrotra, A K; Kumar, Sunil

    2011-04-01

    Operation of waste cells as landfill bioreactors with leachate recirculation is known to accelerate waste degradation and landfill gas generation. However, waste degradation rates in landfill bioreactors decrease with time, with the accumulation of difficult to degrade materials, such as lignin-rich waste. Although, potential exists to modify the leachate quality to promote further degradation of such waste, very little information is available in literature. The objective of this study was to determine the viability of augmenting leachate with enzymes to increase the rate of degradation of lignin-rich waste materials. Among the enzymes evaluated MnP enzyme showed the best performance in terms of methane yield and substrate (lignin) utilization. Methane production of 200 mL CH(4)/g VS was observed for the MnP amended reactor as compared to 5.7 mL CH(4)/g VS for the control reactor. The lignin reduction in the MnP amended reactor and control reactor was 68.4% and 6.2%, respectively. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Extracellular Enzymes Produced by the Cultivated Mushroom Lentinus edodes during Degradation of a Lignocellulosic Medium

    PubMed Central

    Leatham, Gary F.

    1985-01-01

    Although the commercially important mushroom Lentinus (= Lentinula) edodes (Berk.) Sing. can be rapidly cultivated on supplemented wood particles, fruiting is not reliable. This study addressed the problem by developing more information about growth and development on a practical oakwood-oatmeal medium. The study determined (i) the components degraded during a 150-day incubation at 22°C, (ii) the apparent vegetative growth pattern, (iii) the likely growth-limiting nutrient, and (iv) assays that can be used to study key extracellular enzymes. All major components of the medium were degraded, lignin selectively so. The vegetative growth rate was most rapid during the initial 90 days, during which weight loss correlated with glucosamine accumulation (assayed after acid hydrolysis). The rate then slowed; in apparent preparation for fruiting, the cultures rapidly accumulated glucosamine (or its oligomer or polymer). Nitrogen was growth limiting. Certain enzyme activities were associated with the pattern of medium degradation, with growth, or with development. They included cellulolytic system enzymes, hemicellulases, the ligninolytic system, (gluco-)amylase, pectinase, acid protease, cell wall lytic enzymes (laminarinase, 1,4-β-d-glucosidase, β-N-acetyl-d-glucosaminidase, α-d-galactosidase, β-d-mannosidase), acid phosphatase, and laccase. Enzyme activities over the 150-day incubation period with and without a fruiting stimulus are reported. These results provide a basis for future investigations into the physiology and biochemistry of growth and fruiting. PMID:16346918

  2. In-vitro evaluation of enteric coated insulin tablets containing absorption enhancer and enzyme inhibitor.

    PubMed

    Wong, Chun Y; Martinez, Jorge; Carnagarin, Revathy; Dass, Crispin R

    2017-03-01

    The aim of this study was to develop an enteric coated insulin tablet formulation using polymers, absorption enhancer and enzyme inhibitor, which protect the tablets in acidic pH and enhance systemic bioavailability. In this study, the influence of coating by cellulose acetate hydrogen phthalate solution and chosen excipients on Glut-4 transporter translocation in C2C12 skeletal muscle cells was examined. Following the determination of optimum number of coating layers, two dissolution buffers such as 0.01 m hydrochloric acid, pH 2, and 50 mm phosphate, pH 7.4, were employed to determine the in-vitro release of insulin. Insulin was protected by the coating during the dissolution process. Five (5-CL) coating layers and eight (8-CL) coating layers had minimal insulin release in hydrochloric acid, but not three (3-CL) coating layers. Glut-4 translocation in C2C12 cells was promoted by the chosen excipients. No detrimental metabolic effects were observed in these cells. To date, limited studies combine the overall effectiveness of multiple excipients. Our study showed that the coated tablets have an immediate release effect in phosphate buffer. In Glut-4 translocation assay, insulin was still functional after releasing from the tablet. Such tablet formulation can be potentially beneficial to type 1 diabetes patients. © 2017 Royal Pharmaceutical Society.

  3. Duodenal-jejunal bypass surgery up-regulates the expression of the hepatic insulin signaling proteins and the key regulatory enzymes of intestinal gluconeogenesis in diabetic Goto-Kakizaki rats.

    PubMed

    Sun, Dong; Wang, Kexin; Yan, Zhibo; Zhang, Guangyong; Liu, Shaozhuang; Liu, Fengjun; Hu, Chunxiao; Hu, Sanyuan

    2013-11-01

    Duodenal-jejunal bypass (DJB), which is not routinely applied in metabolic surgery, is an effective surgical procedure in terms of type 2 diabetes mellitus resolution. However, the underlying mechanisms are still undefined. Our aim was to investigate the diabetic improvement by DJB and to explore the changes in hepatic insulin signaling proteins and regulatory enzymes of gluconeogenesis after DJB in a non-obese diabetic rat model. Sixteen adult male Goto-Kakizaki rats were randomly divided into DJB and sham-operated groups. The body weight, food intake, hormone levels, and glucose metabolism were measured. The levels of protein expression and phosphorylation of insulin receptor-beta (IR-β) and insulin receptor substrate 2 (IRS-2) were evaluated in the liver. We also detected the expression of key regulatory enzymes of gluconeogenesis [phosphoenoylpyruvate carboxykinase-1 (PCK1), glucose-6-phosphatase-alpha (G6Pase-α)] in small intestine and liver. DJB induced significant diabetic improvement with higher postprandial glucagons-like peptide 1, peptide YY, and insulin levels, but without weight loss. The DJB group exhibited increased expression and phosphorylation of IR-β and IRS-2 in liver, up-regulated the expression of PCK1 and G6Pase-α in small intestine, and down-regulated the expression of these enzymes in liver. DJB is effective in up-regulating the expression of the key proteins in the hepatic insulin signaling pathway and the key regulatory enzymes of intestinal gluconeogenesis and down-regulating the expression of the key regulatory enzymes of hepatic gluconeogenesis without weight loss. Our study helps to reveal the potential role of hepatic insulin signaling pathway and intestinal gluconeogenesis in ameliorating insulin resistance after metabolic surgery.

  4. Potential of insulin nanoparticle formulations for oral delivery and diabetes treatment.

    PubMed

    Wong, Chun Y; Al-Salami, Hani; Dass, Crispin R

    2017-10-28

    Nanoparticles have demonstrated significant advancements in potential oral delivery of insulin. In this publication, we review the current status of polymeric, inorganic and solid-lipid nanoparticles designed for oral administration of insulin. Firstly, the structure and physiological function of insulin are examined. Then, the efficiency and shortcomings of insulin nanoparticle are discussed. These include the susceptibility to digestive enzyme degradation, instability in the acidic pH environment, poor mucus diffusion and inadequate permeation through the gastrointestinal epithelium. In order to optimise the nanocarriers, the following considerations, including polymer nature, surface charge, size, polydispersity index and morphology of nanoparticles, have to be taken into account. Some novel designs such as chitosan-based glucose-responsive nanoparticles, layer by layer technique-based nanoparticles and zwitterion nanoparticles are being adopted to overcome the physiological challenges. The review ends with some future directions and challenges to be addressed for the success of oral delivery of insulin-loaded nanoparticle formulation. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. [Application of repair enzymes to improve the quality of degraded DNA templates for PCR amplification].

    PubMed

    Dovgerd, A P; Zharkov, D O

    2014-01-01

    PCR amplification of severely degraded DNA, including ancient DNA, forensic samples, and preparations from deeply processed foodstuffs, is a serious problem. Living organisms have a set of enzymes to repair lesions in their DNA. In this work, we have developed and characterized model systems of degraded high-molecular-weight DNA with a predominance of different types of damage. It was shown that depurination and oxidation of the model plasmid DNA template led to a decrease in the PCR efficiency. A set of enzymes performing a full cycle of excision repair of some lesions was determined. The treatment of model-damaged substrates with this set of enzymes resulted in an increased PCR product yield as compared with that of the unrepaired samples.

  6. Degradation rate of lyophilized insulin, exhibiting an apparent Arrhenius behavior around glass transition temperature regardless of significant contribution of molecular mobility.

    PubMed

    Yoshioka, Sumie; Miyazaki, Tamaki; Aso, Yukio

    2006-12-01

    The relative influences of chemical activation energy and molecular mobility in determining chemical reactivity were evaluated for insulin lyophilized with alpha,beta-poly(N-hydroxyethyl)-L-aspartamide (PHEA), and compared with that for insulin lyophilized with trehalose, which had been found to have the ability to decrease the molecular mobility of insulin at low humidity. The ratio of the observed rate constant k(obs) to the chemical activation energy-controlled rate constant k(act) (k(obs)/k(act)) at glass transition temperature (T(g)) was estimated to be approximately 0.6 and 0.8 at 6% RH and 12% RH, respectively, indicating that the degradation rate is significantly affected by molecular mobility at lower humidity conditions. However, these k(obs)/k(act) values at T(g) were larger than those for the insulin-trehalose system, and changes in the temperature-dependent slope around T(g) were less obvious than those for the insulin-trehalose system. Thus, the contribution of molecular mobility to the degradation rate in the insulin-PHEA system appeared to be less intense than that in the insulin-trehalose system. The subtle change in the temperature-dependent slope around T(g) observed in the insulin-PHEA system brought about a significant bias in shelf-life estimation when the reaction rate was extrapolated from temperatures above T(g) according to the Arrhenius equation. (c) 2006 Wiley-Liss, Inc. and the American Pharmacists Association

  7. New hydroxamate inhibitors of neurotensin-degrading enzymes. Synthesis and enzyme active-site recognition.

    PubMed

    Bourdel, E; Doulut, S; Jarretou, G; Labbe-Jullie, C; Fehrentz, J A; Doumbia, O; Kitabgi, P; Martinez, J

    1996-08-01

    Selective and mixed inhibitors of the three zinc metallopeptidases that degrade neurotensin (NT), e.g. endopeptidase 24-16 (EC 3.4.24.16), endopeptidase 24-11 (EC 3.4.24.11 or neutral endopeptidase, NEP) and endopeptidase 24-15 (EC 3.4.24.15), and leucine-aminopeptidase (type IV-S), that degrades the NT-related peptides, Neuromedin N (NN), are of great interest. On the structural basis of compound JMV 390-1 (N-[3-[(hydroxyamino)carbonyl]-1-oxo-2(R)-benzylpropyl]-L- isoleucyl-L-leucine), which was a full inhibitor of the major NT degrading enzymes, several hydroxamate inhibitors corresponding to the general formula HONHCO-CH2-CH(CH2-C6H5)CO-X-Y-OH (with X-Y = dipeptide) have been synthesized. Compound 7a (X-Y = Ile-Ala) was nearly 40-times more potent in inhibiting EC 24-16 than NEP and more than 800-times more potent than EC 24-15, with an IC50 (12 nM) almost equivalent to that of compound JMV 390-1. Therefore, this compound is an interesting selective inhibitor of EC 24-16, and should be an interesting probe to explore the physiological involvement of EC 24-16 in the metabolism of neurotensin.

  8. Effects of realimentation after nutrient restriction during mid- to late gestation on pancreatic digestive enzymes, serum insulin and glucose levels, and insulin-containing cell cluster morphology.

    PubMed

    Keomanivong, F E; Camacho, L E; Lemley, C O; Kuemper, E A; Yunusova, R D; Borowicz, P P; Kirsch, J D; Vonnahme, K A; Caton, J S; Swanson, K C

    2017-06-01

    This study examined effects of stage of gestation and nutrient restriction with subsequent realimentation on maternal and foetal bovine pancreatic function. Dietary treatments were assigned on day 30 of pregnancy and included: control (CON; 100% requirements; n = 18) and restricted (R; 60% requirements; n = 30). On day 85, cows were slaughtered (CON, n = 6; R, n = 6), remained on control (CC; n = 12) and restricted (RR; n = 12), or realimented to control (RC; n = 11). On day 140, cows were slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5) or realimented to control (RRC, n = 6). On day 254, the remaining cows were slaughtered and serum samples were collected from the maternal jugular vein and umbilical cord to determine insulin and glucose concentrations. Pancreases from cows and foetuses were removed, weighed, and subsampled for enzyme and histological analysis. As gestation progressed, maternal pancreatic α-amylase activity decreased and serum insulin concentrations increased (p ≤ 0.03). Foetal pancreatic trypsin activity increased (p < 0.001) with advancing gestation. Foetal pancreases subjected to realimentation (CCC vs. RCC and RRC) had increased protein and α-amylase activity at day 254 (p ≤ 0.02), while trypsin (U/g protein; p = 0.02) demonstrated the opposite effect. No treatment effects were observed for maternal or foetal pancreatic insulin-containing cell clusters. Foetal serum insulin and glucose levels were reduced with advancing gestation (p ≤ 0.03). The largest maternal insulin-containing cell cluster was not influenced by advancing gestation, while foetal clusters grew throughout (p = 0.01). These effects indicate that maternal digestive enzymes are influenced by nutrient restriction and there is a potential for programming of increased foetal digestive enzyme production resulting from previous maternal nutrient restriction. Journal of Animal Physiology and Animal

  9. Daily chocolate consumption is inversely associated with insulin resistance and liver enzymes in the Observation of Cardiovascular Risk Factors in Luxembourg study.

    PubMed

    Alkerwi, Ala'a; Sauvageot, Nicolas; Crichton, Georgina E; Elias, Merrill F; Stranges, Saverio

    2016-05-01

    This study examined the association of chocolate consumption with insulin resistance and serum liver enzymes in a national sample of adults in Luxembourg. A random sample of 1153 individuals, aged 18-69 years, was recruited to participate in the cross-sectional Observation of Cardiovascular Risk Factors in Luxembourg study. Chocolate consumption (g/d) was obtained from a semi-quantitative FFQ. Blood glucose and insulin levels were used for the homoeostasis model assessment of insulin resistance (HOMA-IR). Hepatic biomarkers such as serum γ-glutamyl-transpeptidase (γ-GT), serum aspartate transaminase and serum alanine transaminase (ALT) (mg/l) were assessed using standard laboratory assays. Chocolate consumers (81·8 %) were more likely to be younger, physically active, affluent people with higher education levels and fewer chronic co-morbidities. After excluding subjects taking antidiabetic medications, higher chocolate consumption was associated with lower HOMA-IR (β=-0·16, P=0·004), serum insulin levels (β=-0·16, P=0·003) and γ-GT (β=-0·12, P=0·009) and ALT (β=-0·09, P=0·004), after adjustment for age, sex, education, lifestyle and dietary confounding factors, including intakes of fruits and vegetables, alcohol, polyphenol-rich coffee and tea. This study reports an independent inverse relationship between daily chocolate consumption and levels of insulin, HOMA-IR and liver enzymes in adults, suggesting that chocolate consumption may improve liver enzymes and protect against insulin resistance, a well-established risk factor for cardiometabolic disorders. Further observational prospective research and well-designed randomised-controlled studies are needed to confirm this cross-sectional relationship and to comprehend the role and mechanisms that different types of chocolate may play in insulin resistance and cardiometabolic disorders.

  10. The 46 kDa dimeric protein from Variovorax paradoxus shows faster methotrexate degrading activity in its nanoform compare to the native enzyme.

    PubMed

    Bayineni, Venkata Krishna; Venkatesh, Krishna; Sahu, Chandan Kumar; Kadeppagari, Ravi-Kumar

    2016-04-01

    Methotrexate degrading enzymes are required to overcome the toxicity of the methotrexate while treating the cancer. The enzyme from Variovorax paradoxus converts the methotrexate in to non toxic products. Methotrexate degrading enzyme from V. paradoxus is a dimeric protein with a molecular mass of 46 kDa and it acts on casein and gelatin. This enzyme is optimally active at pH 7.5 and 40°C and nanoparticles of this enzyme were prepared by desolvation-crosslinking method. Enzyme nanoparticles could degrade methotrexate faster than the native enzyme and they show lower Km compare to the native enzyme. Enzyme nanoparticles show better thermostability and they were stable for much longer time in the serum compare to the native enzyme. Enzyme nanoparticles show better functionality than the native enzyme while clearing the methotrexate added to the serum suggesting their advantage over the native enzyme for the therapeutic and biotechnological applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. [Effect of Jinlida on changes in expression of skeletal muscle lipid transport enzymes in fat-induced insulin resistance ApoE -/- mice].

    PubMed

    Jin, Xin; Zhang, Hui-xin; Zhang, Yan-fen; Cui, Wen-wen; Bi, Yao; He, Qi-long; Zhou, Sheng-shan

    2015-03-01

    To study the effect of Jinlida on changes in expression of skeletal muscle lipid transport enzymes in fat-induced insulin resistance ApoE -/- mice. Eight male C57BL/6J mice were selected in the normal group (NF), 40 male ApoE -/- mice were fed for 16 weeks, divided into the model group (HF), the rosiglitazone group ( LGLT), the Jinlida low-dose group (JLDL), the Jinlida medium-dose group (JLDM), the Jinlida high-dose group (JLDH) and then orally given drugs for 8 weeks. The organization free fatty acids, BCA protein concentration determination methods were used to determine the skeletal muscle FFA content. The Real-time fluorescent quantitative reverse transcription PCR ( RT-PCR) and Western blot method were adopted to determine mRNA and protein expressions of mice fatty acids transposition enzyme (FAT/CD36), carnitine palm acyltransferase 1 (CPT1), peroxide proliferators-activated receptor α( PPAR α). Jinlida could decrease fasting blood glucose (FBG), cholesterol (TC), triglyceride (TG), free fatty acid (FFA) and fasting insulin (FIns) and raise insulin sensitive index (ISI) in mice to varying degrees. It could also up-regulate mRNA and protein expressions of CPT1 and PPARα, and down-regulate mRNA and protein levels of FAT/CD36. Jinlida can improve fat-induced insulin resistance ApoE -/- in mice by adjusting the changes in expression of skeletal muscle lipid transport enzymes.

  12. Screening of SDS-degrading bacteria from car wash wastewater and study of the alkylsulfatase enzyme activity

    PubMed Central

    Shahbazi, Razieh; Kasra-Kermanshahi, Roha; Gharavi, Sara; Moosavi-Nejad, Zahra; Borzooee, Faezeh

    2013-01-01

    Background and Objectives Sodium dodecyl sulfate (SDS) is one of the main surfactant components in detergents and cosmetics, used in high amounts as a detergent in products such as shampoos, car wash soap and toothpaste. Therefore, its bioremediation by suitable microorganisms is important. Alkylsulfatase is an enzyme that hydrolyses sulfate -ester bonds to give inorganic sulfate and alcohol. The purpose of this study was to isolate SDS–degrading bacteria from Tehran city car wash wastewater, study bacterial alkylsulfatase enzyme activity and identify the alkylsulfatase enzyme coding gene. Materials and Methods Screening of SDS-degrading bacteria was carried out on basal salt medium containing SDS as the sole source of carbon. Amount of SDS degraded was assayed by methylene blue active substance (MBAS). Results and Conclusion Identification of the sdsA gene was carried by PCR and subsequent sequencing of the 16S rDNA gene and biochemical tests identified Pseudomonas aeruginosa. This bacterium is able to degrade 84% of SDS after four days incubation. Bacteria isolated from car wash wastewater were shown to carry the sdsA gene (670bp) and the alkylsulfatase enzyme specific activity expressed from this gene was determined to be 24.3 unit/mg. The results presented in this research indicate that Pseudomonas aeruginosa is a suitable candidate for SDS biodegradation. PMID:23825734

  13. Co-targeting the HER and IGF/insulin receptor axis in breast cancer, with triple targeting with endocrine therapy for hormone-sensitive disease.

    PubMed

    Chakraborty, Ashok; Hatzis, Christos; DiGiovanna, Michael P

    2017-05-01

    Interactions between HER2, estrogen receptor (ER), and insulin-like growth factor I receptor (IGF1R) are implicated in resistance to monotherapies targeting these receptors. We have previously shown in pre-clinical studies synergistic anti-tumor effects for co-targeting each pairwise combination of HER2, IGF1R, and ER. Strikingly, synergy for HER2/IGF1R targeting occurred not only in a HER2+ model, but also in a HER2-normal model. The purpose of the current study was therefore to determine the generalizability of synergistic anti-tumor effects of co-targeting HER2/IGF1R, the anti-tumor activity of triple-targeting HER2/IGF1R/ER in hormone-dependent cell lines, and the effect of using the multi-targeting drugs neratinib (pan-HER) and BMS-754807 (dual IGF1R/insulin receptor). Proliferation and apoptosis assays were performed in a large panel of cell lines representing varying receptor expression levels. Mechanistic effects were studied using phospho-protein immunoblotting. Analyses of drug interaction effects were performed using linear mixed-effects regression models. Enhanced anti-proliferative effects of HER/IGF-insulin co-targeting were seen in most, though not all, cell lines, including HER2-normal lines. For ER+ lines, triple targeting with inclusion of anti-estrogen generally resulted in the greatest anti-tumor effects. Double or triple targeting generally resulted in marked increases in apoptosis in the sensitive lines. Mechanistic studies demonstrated that the synergy between drugs was correlated with maximal inhibition of Akt and ERK pathway signaling. Dual HER/IGF-insulin targeting, and triple targeting with inclusion of anti-estrogen drugs, shows striking anti-tumor activity across breast cancer types, and drugs with broader receptor specificity may be more effective than single receptor selective drugs, particularly for ER- cells.

  14. Degradation Signals Recognized by the Ubc6p-Ubc7p Ubiquitin-Conjugating Enzyme Pair

    PubMed Central

    Gilon, Tamar; Chomsky, Orna; Kulka, Richard G.

    2000-01-01

    Proteolysis by the ubiquitin-proteasome system is highly selective. Specificity is achieved by the cooperation of diverse ubiquitin-conjugating enzymes (Ubcs or E2s) with a variety of ubiquitin ligases (E3s) and other ancillary factors. These recognize degradation signals characteristic of their target proteins. In a previous investigation, we identified signals directing the degradation of β-galactosidase and Ura3p fusion proteins via a subsidiary pathway of the ubiquitin-proteasome system involving Ubc6p and Ubc7p. This pathway has recently been shown to be essential for the degradation of misfolded and regulated proteins in the endoplasmic reticulum (ER) lumen and membrane, which are transported to the cytoplasm via the Sec61p translocon. Mutant backgrounds which prevent retrograde transport of ER proteins (hrd1/der3Δ and sec61-2) did not inhibit the degradation of the β-galactosidase and Ura3p fusions carrying Ubc6p/Ubc7p pathway signals. We therefore conclude that the ubiquitination of these fusion proteins takes place on the cytosolic face of the ER without prior transfer to the ER lumen. The contributions of different sequence elements to a 16-amino-acid-residue Ubc6p-Ubc7p-specific signal were analyzed by mutation. A patch of bulky hydrophobic residues was an essential element. In addition, positively charged residues were found to be essential. Unexpectedly, certain substitutions of bulky hydrophobic or positively charged residues with alanine created novel degradation signals, channeling the degradation of fusion proteins to an unidentified proteasomal pathway not involving Ubc6p and Ubc7p. PMID:10982838

  15. Brain insulin lowers circulating BCAA levels by inducing hepatic BCAA catabolism.

    PubMed

    Shin, Andrew C; Fasshauer, Martin; Filatova, Nika; Grundell, Linus A; Zielinski, Elizabeth; Zhou, Jian-Ying; Scherer, Thomas; Lindtner, Claudia; White, Phillip J; Lapworth, Amanda L; Ilkayeva, Olga; Knippschild, Uwe; Wolf, Anna M; Scheja, Ludger; Grove, Kevin L; Smith, Richard D; Qian, Wei-Jun; Lynch, Christopher J; Newgard, Christopher B; Buettner, Christoph

    2014-11-04

    Circulating branched-chain amino acid (BCAA) levels are elevated in obesity/diabetes and are a sensitive predictor for type 2 diabetes. Here we show in rats that insulin dose-dependently lowers plasma BCAA levels through induction of hepatic protein expression and activity of branched-chain α-keto acid dehydrogenase (BCKDH), the rate-limiting enzyme in the BCAA degradation pathway. Selective induction of hypothalamic insulin signaling in rats and genetic modulation of brain insulin receptors in mice demonstrate that brain insulin signaling is a major regulator of BCAA metabolism by inducing hepatic BCKDH. Short-term overfeeding impairs the ability of brain insulin to lower BCAAs in rats. High-fat feeding in nonhuman primates and obesity and/or diabetes in humans is associated with reduced BCKDH protein in liver. These findings support the concept that decreased hepatic BCKDH is a major cause of increased plasma BCAAs and that hypothalamic insulin resistance may account for impaired BCAA metabolism in obesity and diabetes. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Metabolism and insulin signaling in common metabolic disorders and inherited insulin resistance.

    PubMed

    Højlund, Kurt

    2014-07-01

    muscle in vivo by activation of the insulin signaling cascade to glucose transport through the enzymes IRS1, PI3K, Akt2, AS160/TBC1D4 and RAC1, and to glycogen synthesis through Akt2, inhibition of GSK3 and activation of glycogen synthase (GS) via dephosphorylation of serine residues in both the NH2-terminal (site 2+2a) and the COOH-terminal end (site 3a+3b). In type 2 diabetes, obesity and PCOS, there is, although with some variation from study to study, defects in insulin signaling through IRS1, PI3K, Akt2 and AS160/TBC1D4, which can explain reduced insulin action on glucose transport. In type 2 diabetes an altered intracellular distribution of SNAP23 and impaired activation of RAC1 also seem to play a role for reduced insulin action on glucose transport. In all common metabolic disorders, we observed an impaired insulin activation of GS, which seems to be caused by attenuated dephosphorylation of GS at site 2+2a, whereas as the inhibition of GSK3 and the dephosphorylation of GS at its target sites, site 3a+3a, appeared to be completely normal. In individuals with inherited insulin resistance, we observed largely the same defects in insulin action on IRS1, PI3K, Akt2 and GS, as well as a normal inhibition of GSK3 and dephosphorylation of GS at site 3a+3b. In these individuals, however, a markedly reduced insulin clearance seems to partially rescue insulin signaling to glucose transport and GS. Adiponectin is thought to improve insulin sensitivity primarily by increasing lipid oxidation through activation of the enzyme AMPK, and possibly via cross-talking of adiponectin with insulin signaling, and hence glucose transport and glycogen synthesis. We demonstrated a strong correlation between plasma adiponectin and insulin action on glucose disposal and glycogen synthesis in obesity, type 2 diabetes and PCOS. In individuals with inherited insulin resistance, plasma adiponectin was normal, but the correlation of adiponectin with insulin-stimulated glucose uptake and glycogen

  17. Probiotic activity of lignocellulosic enzyme as bioactivator for rice husk degradation

    NASA Astrophysics Data System (ADS)

    Lamid, Mirni; Al-Arif, Anam; Warsito, Sunaryo Hadi

    2017-02-01

    The utilization of lignocellulosic enzyme will increase nutritional value of rice husk. Cellulase consists of C1 (β-1, 4-glucan cellobiohydrolase or exo-β-1,4glucanase), Cc (endo-β-1,4-glucanase) and component and cellobiose (β-glucocidase). Hemicellulase enzyme consists of endo-β-1,4-xilanase, β-xilosidase, α-L arabinofuranosidase, α-D-glukuronidaseand asetil xilan esterase. This research aimed to study the activity of lignocellulosic enzyme, produced by cows in their rumen, which can be used as a bioactivator in rice husk degradation. This research resulted G6 and G7 bacteria, producing xylanase and cellulase with the activity of 0.004 U mL-1 and 0.021 U mL-1; 0.003 ( U mL-1) and 0.026 (U mL-1) respectively.

  18. The Role of Mammalian Target of Rapamycin (mTOR) in Insulin Signaling.

    PubMed

    Yoon, Mee-Sup

    2017-10-27

    The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that controls a wide spectrum of cellular processes, including cell growth, differentiation, and metabolism. mTOR forms two distinct multiprotein complexes known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which are characterized by the presence of raptor and rictor, respectively. mTOR controls insulin signaling by regulating several downstream components such as growth factor receptor-bound protein 10 (Grb10), insulin receptor substrate (IRS-1), F-box/WD repeat-containing protein 8 (Fbw8), and insulin like growth factor 1 receptor/insulin receptor (IGF-IR/IR). In addition, mTORC1 and mTORC2 regulate each other through a feedback loop to control cell growth. This review outlines the current understanding of mTOR regulation in insulin signaling in the context of whole body metabolism.

  19. Development of Liver-Targeting Insulin

    DTIC Science & Technology

    2017-08-01

    decision unless so designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188 Public reporting burden for this...have been done in the design of better insulin, problems still exist with the current therapies. For example, frequent subcutaneous injections are always...ligands will be designed and synthesized. The ligands molecular weight is a fraction of insulin, and therefore should result in an insulin analog with

  20. Degradative Enzymes from the Pharmacy or Health Food Store: Interesting Examples for Introductory Biology Laboratories

    ERIC Educational Resources Information Center

    Deutch, Charles E.

    2007-01-01

    Degradative enzymes in over-the-counter products from pharmacies and health food stores provide good examples of biological catalysis. These include [beta]-galactosidase in Lactaid[TM], [alpha]-galactosidase in Beano[R], [alpha]-amylase and proteases in digestive aids, and proteases in contact lens cleaners. These enzymes can be studied…

  1. Selective Targeting of Proteins within Secretory Pathway for Endoplasmic Reticulum-associated Degradation

    PubMed Central

    Vecchi, Lara; Petris, Gianluca; Bestagno, Marco; Burrone, Oscar R.

    2012-01-01

    The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity. PMID:22523070

  2. Cysteine degradation gene yhaM, encoding cysteine desulfidase, serves as a genetic engineering target to improve cysteine production in Escherichia coli.

    PubMed

    Nonaka, Gen; Takumi, Kazuhiro

    2017-12-01

    Cysteine is an important amino acid for various industries; however, there is no efficient microbial fermentation-based production method available. Owing to its cytotoxicity, bacterial intracellular levels of cysteine are stringently controlled via several modes of regulation, including cysteine degradation by cysteine desulfhydrases and cysteine desulfidases. In Escherichia coli, several metabolic enzymes are known to exhibit cysteine degradative activities, however, their specificity and physiological significance for cysteine detoxification via degradation are unclear. Relaxing the strict regulation of cysteine is crucial for its overproduction; therefore, identifying and modulating the major degradative activity could facilitate the genetic engineering of a cysteine-producing strain. In the present study, we used genetic screening to identify genes that confer cysteine resistance in E. coli and we identified yhaM, which encodes cysteine desulfidase and decomposes cysteine into hydrogen sulfide, pyruvate, and ammonium. Phenotypic characterization of a yhaM mutant via growth under toxic concentrations of cysteine followed by transcriptional analysis of its response to cysteine showed that yhaM is cysteine-inducible, and its physiological role is associated with resisting the deleterious effects of cysteine in E. coli. In addition, we confirmed the effects of this gene on the fermentative production of cysteine using E. coli-based cysteine-producing strains. We propose that yhaM encodes the major cysteine-degrading enzyme and it has the most significant role in cysteine detoxification among the numerous enzymes reported in E. coli, thereby providing a core target for genetic engineering to improve cysteine production in this bacterium.

  3. Major amyloid-β-degrading enzymes, endothelin-converting enzyme-2 and neprilysin, are expressed by distinct populations of GABAergic interneurons in hippocampus and neocortex.

    PubMed

    Pacheco-Quinto, Javier; Eckman, Christopher B; Eckman, Elizabeth A

    2016-12-01

    Impaired clearance of amyloid-β peptide (Aβ) has been postulated to significantly contribute to the amyloid accumulation typical of Alzheimer's disease. Among the enzymes known to degrade Aβ in vivo are endothelin-converting enzyme (ECE)-1, ECE-2, and neprilysin (NEP), and evidence suggests that they regulate independent pools of Aβ that may be functionally significant. To better understand the differential regulation of Aβ concentration by its physiological degrading enzymes, we characterized the cell and region-specific expression pattern of ECE-1, ECE-2, and NEP by in situ hybridization and immunohistochemistry in brain areas relevant to Alzheimer's disease. In contrast to the broader distribution of ECE-1, ECE-2 and NEP were found enriched in GABAergic neurons. ECE-2 was majorly expressed by somatostatin-expressing interneurons and was active in isolated synaptosomes. NEP messenger RNA was found mainly in parvalbumin-expressing interneurons, with NEP protein localized to perisomatic parvalbuminergic synapses. The identification of somatostatinergic and parvalbuminergic synapses as hubs for Aβ degradation is consistent with the possibility that Aβ may have a physiological function related to the regulation of inhibitory signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Measuring phospholipase D activity in insulin-secreting pancreatic beta-cells and insulin-responsive muscle cells and adipocytes.

    PubMed

    Cazzolli, Rosanna; Huang, Ping; Teng, Shuzhi; Hughes, William E

    2009-01-01

    Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels.

  5. The insulin receptor.

    PubMed

    Kaplan, S A

    1984-03-01

    Cells are endowed with specific cognitive molecules that function as receptors for hormones, neurotransmitters, and other intercellular messengers. The receptor molecules may be present in the plasma membrane, cytoplasm, or nucleus. When occupied by the messenger, the receptor is coupled to the cellular machinery that responds to the message-bearing molecules. For some hormones the events following attachment of the messenger to the receptor are well known. An example is the generation of cAMP after combination of glucagon with its receptor and the series of steps culminating in activation of phosphorylase. In the case of many other messengers, including insulin, the nature of these coupling steps is not known. Receptors are subject to the regulatory processes of synthesis, degradation, and conformational change; alterations in receptor properties may have significant effects on the qualitative and quantitative responses of the cell to the extracellular messenger. The insulin receptor is located in the plasma membrane, is composed of two pairs of subunits, and has a molecular weight of about 350,000. It is located in cells such as adipocytes, hepatocytes, and skeletal muscle cells as well as in cells not considered to be typical target organ cells. Insulin receptors in nonfetal cells are downregulated by exposure of the cells to high concentrations of insulin. Other factors that regulate insulin binding include muscular exercise, diet, thyroid hormones, glucocorticoids, androgens, estrogens, and cyclic nucleotides. The fetus has high concentrations of insulin receptors in several tissues. These begin to appear early in fetal life and may outnumber those found in adult tissues. Fetal insulin receptors are unusual in that they may not undergo downregulation but may experience the opposite when exposed to insulin in high concentrations. Thus the offspring of a mother with poorly controlled diabetes may be placed in double jeopardy by fetal hyperinsulinemia and

  6. DDB1-Mediated CRY1 Degradation Promotes FOXO1-Driven Gluconeogenesis in Liver.

    PubMed

    Tong, Xin; Zhang, Deqiang; Charney, Nicholas; Jin, Ethan; VanDommelen, Kyle; Stamper, Kenneth; Gupta, Neil; Saldate, Johnny; Yin, Lei

    2017-10-01

    Targeted protein degradation through ubiquitination is an important step in the regulation of glucose metabolism. Here, we present evidence that the DDB1-CUL4A ubiquitin E3 ligase functions as a novel metabolic regulator that promotes FOXO1-driven hepatic gluconeogenesis. In vivo, hepatocyte-specific Ddb1 deletion leads to impaired hepatic gluconeogenesis in the mouse liver but protects mice from high-fat diet-induced hyperglycemia. Lack of Ddb1 downregulates FOXO1 protein expression and impairs FOXO1-driven gluconeogenic response. Mechanistically, we discovered that DDB1 enhances FOXO1 protein stability via degrading the circadian protein cryptochrome 1 (CRY1), a known target of DDB1 E3 ligase. In the Cry1 depletion condition, insulin fails to reduce the nuclear FOXO1 abundance and suppress gluconeogenic gene expression. Chronic depletion of Cry1 in the mouse liver not only increases FOXO1 protein but also enhances hepatic gluconeogenesis. Thus, we have identified the DDB1-mediated CRY1 degradation as an important target of insulin action on glucose homeostasis. © 2017 by the American Diabetes Association.

  7. Brain insulin lowers circulating BCAA levels by inducing hepatic BCAA catabolism

    DOE PAGES

    Shin, Andrew C.; Fasshauer, Martin; Filatova, Nika; ...

    2014-10-09

    Circulating branched-chain amino acid (BCAA) levels are elevated in obesity and diabetes and are a sensitive predictor for type 2 diabetes. Here we show in rats that insulin dose-dependently lowers plasma BCAA levels through induction of protein expression and activity of branched-chain α-keto acid dehydrogenase (BCKDH), the rate-limiting enzyme in the BCAA degradation pathway in the liver. Selective induction of hypothalamic insulin signaling in rats as well as inducible and lifelong genetic modulation of brain insulin receptor expression in mice both demonstrate that brain insulin signaling is a major regulator of BCAA metabolism by inducing hepatic BCKDH. Further, short-term overfeedingmore » impairs the ability of brain insulin to lower circulating BCAA levels in rats. Chronic high-fat feeding in primates and obesity and/or type 2 diabetes in humans is associated with reduced BCKDH protein expression in liver, further supporting the concept that decreased hepatic BCKDH is a primary cause of increased plasma BCAA levels in insulin-resistant states. These findings demonstrate that neuroendocrine pathways control BCAA homeostasis and that hypothalamic insulin resistance can be a cause of impaired BCAA metabolism in obesity and diabetes.« less

  8. Brain insulin lowers circulating BCAA levels by inducing hepatic BCAA catabolism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shin, Andrew C.; Fasshauer, Martin; Filatova, Nika

    Circulating branched-chain amino acid (BCAA) levels are elevated in obesity and diabetes and are a sensitive predictor for type 2 diabetes. Here we show in rats that insulin dose-dependently lowers plasma BCAA levels through induction of protein expression and activity of branched-chain α-keto acid dehydrogenase (BCKDH), the rate-limiting enzyme in the BCAA degradation pathway in the liver. Selective induction of hypothalamic insulin signaling in rats as well as inducible and lifelong genetic modulation of brain insulin receptor expression in mice both demonstrate that brain insulin signaling is a major regulator of BCAA metabolism by inducing hepatic BCKDH. Further, short-term overfeedingmore » impairs the ability of brain insulin to lower circulating BCAA levels in rats. Chronic high-fat feeding in primates and obesity and/or type 2 diabetes in humans is associated with reduced BCKDH protein expression in liver, further supporting the concept that decreased hepatic BCKDH is a primary cause of increased plasma BCAA levels in insulin-resistant states. These findings demonstrate that neuroendocrine pathways control BCAA homeostasis and that hypothalamic insulin resistance can be a cause of impaired BCAA metabolism in obesity and diabetes.« less

  9. Individualized correction of insulin measurement in hemolyzed serum samples.

    PubMed

    Wu, Zhi-Qi; Lu, Ju; Chen, Huanhuan; Chen, Wensen; Xu, Hua-Guo

    2017-06-01

    Insulin measurement plays a key role in the investigation of patients with hypoglycemia, subtype classification of diabetes mellitus, insulin resistance, and impaired beta cell function. However, even slight hemolysis can negatively affect insulin measurement due to RBC insulin-degrading enzyme (IDE). Here, we derived and validated an individualized correction equation in an attempt to eliminate the effects of hemolysis on insulin measurement. The effects of hemolysis on insulin measurement were studied by adding lysed self-RBCs to serum. A correction equation was derived, accounting for both percentage and exposure time of hemolysis. The performance of this individualized correction was evaluated in intentionally hemolyzed samples. Insulin concentration decreased with increasing percentage and exposure time of hemolysis. Based on the effects of hemolysis on insulin measurement of 17 donors (baseline insulin concentrations ranged from 156 to 2119 pmol/L), the individualized hemolysis correction equation was derived: INS corr  = INS meas /(0.705lgHb plasma /Hb serum  - 0.001Time - 0.612). This equation can revert insulin concentrations of the intentionally hemolyzed samples to values that were statistically not different from the corresponding insulin baseline concentrations (p = 0.1564). Hemolysis could lead to a negative interference on insulin measurement; by individualized hemolysis correction equation for insulin measurement, we can correct and report reliable serum insulin results for a wide range of degrees of sample hemolysis. This correction would increase diagnostic accuracy, reduce inappropriate therapeutic decisions, and improve patient satisfaction with care.

  10. FBW7 targets KLF10 for ubiquitin-dependent degradation.

    PubMed

    Yu, Su; Wang, Feng; Tan, Xiao; Gao, Guo-Li; Pan, Wei-Juan; Luan, Yi; Ge, Xin

    2018-01-08

    FBW7, a key component of SCF FBW7 E3 ubiquitin ligase, targets various proteins for degradation via the conserved Cdc4 phosphodegron (CPD) in substrates. In this study, we report that KLF10 is degraded by FBW7 via a conserved CPD. Through systematic analysis of the degradation of KLF transcription factors by FBW7, we identified KLF10 as a novel degradation target of FBW7. Ectopic expression of FBW7 markedly promoted the degradation of KLF10 while knockdown of endogenous FBW7 increased the protein levels of KLF10. In addition, simultaneous mutations of both threonine 82 (T82) and serine 86 (S86) significantly reduced the FBW7-mediated KLF10 degradation. Moreover, KLF10 containing a conserved putative CPD (TPPXSP) from amino acids 82 to 87, directly interacted with WD40 domain of FBW7 in a phosphorylation-dependent manner. Importantly, FBW7 could reverse the KLF10-mediated inhibition of Smad7 activity. Thus, our study uncovers a novel regulatory mechanism underlying which KLF10 stability and its biological function are mediated by FBW7. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Glutathione Degradation.

    PubMed

    Bachhawat, Anand Kumar; Kaur, Amandeep

    2017-11-20

    Glutathione degradation has for long been thought to occur only on noncytosolic pools. This is because there has been only one enzyme known to degrade glutathione (γ-glutamyl transpeptidase) and this localizes to either the plasma membrane (mammals, bacteria) or the vacuolar membrane (yeast, plants) and acts on extracellular or vacuolar pools. The last few years have seen the discovery of several new enzymes of glutathione degradation that function in the cytosol, throwing new light on glutathione degradation. Recent Advances: The new enzymes that have been identified in the last few years that can initiate glutathione degradation include the Dug enzyme found in yeast and fungi, the ChaC1 enzyme found among higher eukaryotes, the ChaC2 enzyme found from bacteria to man, and the RipAY enzyme found in some bacteria. These enzymes play roles ranging from housekeeping functions to stress responses and are involved in processes such as embryonic neural development and pathogenesis. In addition to delineating the pathways of glutathione degradation in detail, a critical issue is to find how these new enzymes impact cellular physiology and homeostasis. Glutathione degradation plays a far greater role in cellular physiology than previously envisaged. The differential regulation and differential specificities of various enzymes, each acting on distinct pools, can lead to different consequences to the cell. It is likely that the coming years will see these downstream effects being unraveled in greater detail and will lead to a better understanding and appreciation of glutathione degradation. Antioxid. Redox Signal. 27, 1200-1216.

  12. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides.

    PubMed

    Cravatt, B F; Giang, D K; Mayfield, S P; Boger, D L; Lerner, R A; Gilula, N B

    1996-11-07

    Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.

  13. Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation.

    PubMed

    Zheng, Jing; Tan, Chunyan; Xue, Pengcheng; Cao, Jiakun; Liu, Feng; Tan, Ying; Jiang, Yuyang

    2016-02-19

    Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Different Forms of Vanadate on Sugar Transport in Insulin Target and Nontarget Cells

    PubMed Central

    2002-01-01

    The effects of several vanadates (ie, orthovanadate, pervanadate, and two stable peroxovanadium compounds) on basal and insulin-stimulated 2-DG transport in insulin target and nontarget cell lines are reported, herein. In nontarget cells, exposure to vanadates (5 × 10−6 to 10−4 mol/L) resulted in 2-DG transport stimulatory responses similar to those observed in 2-DG transport post exposure to 667 nmol/L insulin alone, or insulin in combination with vanadates. In 3T3-L1 adipocytes and L6 myotubes, exposure to a vanadate compound or 67 nmol/L insulin, stimulated 2-DG transport dramatically. Again, this effect on stimulated transport was similar to 2-DG transport post-treatment with the effective vanadates in combination with insulin. While pervanadate or stable peroxovanadates stimulated 2-DG transport at 10−5 to 10−6 mol/L, orthovanadate up to 10−4 mol/L was not effective in stimulating 2-DG transport in any of the cell lines tested. The data indicate that the various peroxovanadates are clearly superior insulin mimetics while a more limited insulin mimesis is observed with orthovanadate over a wide variety of cell types. PMID:12488596

  15. Different Forms of Vanadate on Sugar Transport in Insulin Target and Nontarget Cells.

    PubMed

    Germinario, Ralph J.; Colby-Germinario, Susan P.; Posner, Barry I.; Nahm, K.

    2002-01-01

    The effects of several vanadates (ie, orthovanadate, pervanadate, and two stable peroxovanadium compounds) on basal and insulin-stimulated 2-DG transport in insulin target and nontarget cell lines are reported, herein. In nontarget cells, exposure to vanadates (5 x 10(-6) to 10(-4) mol/L) resulted in 2-DG transport stimulatory responses similar to those observed in 2-DG transport post exposure to 667 nmol/L insulin alone, or insulin in combination with vanadates. In 3T3-L1 adipocytes and L6 myotubes, exposure to a vanadate compound or 67 nmol/L insulin, stimulated 2-DG transport dramatically. Again, this effect on stimulated transport was similar to 2-DG transport post-treatment with the effective vanadates in combination with insulin. While pervanadate or stable peroxovanadates stimulated 2-DG transport at 10(-5) to 10(-6) mol/L, orthovanadate up to 10(-4) mol/L was not effective in stimulating 2-DG transport in any of the cell lines tested. The data indicate that the various peroxovanadates are clearly superior insulin mimetics while a more limited insulin mimesis is observed with orthovanadate over a wide variety of cell types.

  16. DNA Looping Facilitates Targeting of a Chromatin Remodeling Enzyme

    PubMed Central

    Yadon, Adam N; Singh, Badri Nath; Hampsey, Michael; Tsukiyama, Toshio

    2013-01-01

    Summary ATP-dependent chromatin remodeling enzymes are highly abundant and play pivotal roles regulating DNA-dependent processes. The mechanisms by which they are targeted to specific loci have not been well understood on a genome-wide scale. Here we present evidence that a major targeting mechanism for the Isw2 chromatin remodeling enzyme to specific genomic loci is through sequence-specific transcription factor (TF)-dependent recruitment. Unexpectedly, Isw2 is recruited in a TF-dependent fashion to a large number of loci without TF binding sites. Using the 3C assay, we show that Isw2 can be targeted by Ume6- and TFIIB-dependent DNA looping. These results identify DNA looping as a previously unknown mechanism for the recruitment of a chromatin remodeling enzyme and defines a novel function for DNA looping. We also present evidence suggesting that Ume6-dependent DNA looping is involved in chromatin remodeling and transcriptional repression, revealing a mechanism by which the three-dimensional folding of chromatin affects DNA-dependent processes. PMID:23478442

  17. Transcriptional response of lignin-degrading enzymes to 17α-ethinyloestradiol in two white rots

    PubMed Central

    Přenosilová, L; Křesinová, Z; Amemori, A Slavíková; Cajthaml, T; Svobodová, K

    2013-01-01

    Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase (MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17α-ethinyloestradiol (EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2 ± 6.7 U g−1 of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene (lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T. versicolor, Irpex lacteus was also able to remove 10 mg l−1 EE2 within 3 days of cultivation. While an increase to I. lacteus MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation. PMID:23170978

  18. Insulin degludec/insulin aspart versus biphasic insulin aspart 30 twice daily in insulin-experienced Japanese subjects with uncontrolled type 2 diabetes: Subgroup analysis of a Pan-Asian, treat-to-target Phase 3 Trial.

    PubMed

    Taneda, Shinji; Hyllested-Winge, Jacob; Gall, Mari-Anne; Kaneko, Shizuka; Hirao, Koichi

    2017-03-01

    The present study was a subgroup analysis of a Pan-Asian Phase 3 open-label randomized treat-to-target trial evaluating insulin degludec/insulin aspart (IDegAsp) and biphasic insulin aspart 30 (BIAsp 30) in Japanese subjects with type 2 diabetes inadequately controlled on insulin. Eligible subjects (n = 178) were randomized (2: 1) to twice-daily (b.i.d.) IDegAsp or BIAsp 30 with or without metformin for 26 weeks, titrated to a blood glucose target of between 3.9 and <5.0 mmol/L. Changes in HbA 1c , the proportion of responders reaching the HbA 1c target, and changes in fasting plasma glucose, nine-point self-monitored plasma glucose profiles, and body weight were assessed. At 26 weeks, the decrease in HbA 1c was similar in both groups. Fasting plasma glucose was lower with IDegAsp than BIAsp 30 (estimated treatment difference -1.50 mmol/L; 95 % confidence interval [CI] -1.98, -1.01). Overall confirmed hypoglycemia rates were similar; the nocturnal confirmed hypoglycemia rate was lower with IDegAsp than BIAsp 30 (estimated rate ratio 0.44; 95 % CI 0.20, 0.99). No severe hypoglycemic episodes were reported. The results indicate that IDegAsp b.i.d. improves glycemic control and, compared with BIAsp 30, lowers the rate of nocturnal confirmed hypoglycemia. © 2016 The Authors. Journal of Diabetes published John Wiley & Sons Australia, Ltd and Ruijin Hospital, Shanghai Jiaotong University School of Medicine.

  19. Newly isolated Penicillium oxalicum A592-4B secretes enzymes that degrade milled rice straw with high efficiency.

    PubMed

    Aoyama, Akihisa; Kurane, Ryuichiro; Matsuura, Akira; Nagai, Kazuo

    2015-01-01

    An enzyme producing micro-organism, which can directly saccharify rice straw that has only been crushed without undergoing the current acid or alkaline pretreatment, was found. From the homology with the ITS, 28S rDNA sequence, the strain named A592-4B was identified as Penicillium oxalicum. Activities of the A592-4B enzymes and commercial enzyme preparations were compared by Novozymes Cellic CTec2 and Genencore GC220. In the present experimental condition, activity of A592-4B enzymes was 2.6 times higher than that of CTec2 for degrading milled rice straw. Furthermore, even when a quarter amount of A592-4B enzyme was applied to the rice straw, the conversion rate was still higher than that by CTec2. By utilizing A592-4B enzymes, improved lignocellulose degradation yields can be achieved without pre-treatment of the substrates; thus, contributing to cost reduction as well as reducing environmental burden.

  20. Reduced Insulin Receptor Expression Enhances Proximal Tubule Gluconeogenesis.

    PubMed

    Pandey, Gaurav; Shankar, Kripa; Makhija, Ekta; Gaikwad, Anil; Ecelbarger, Carolyn; Mandhani, Anil; Srivastava, Aneesh; Tiwari, Swasti

    2017-02-01

    Reduced insulin receptor protein levels have been reported in the kidney cortex from diabetic humans and animals. We recently reported that, targeted deletion of insulin receptor (IR) from proximal tubules (PT) resulted in hyperglycemia in non-obese mice. To elucidate the mechanism, we examined human proximal tubule cells (hPTC) and C57BL/6 mice fed with high-fat diet (HFD, 60% fat for 20 weeks). Immunoblotting revealed a significantly lower protein level of IR in HFD compare to normal chow diet (NCD). Furthermore, a blunted rise in p-AKT 308 levels in the kidney cortex of HFD mice was observed in response to acute insulin (0.75 IU/kg body weight, i.p) relative to NCD n = 8/group, P < 0.05). Moreover, we found significantly higher transcript levels of phosphoenolpyruvate carboxykinase (PEPCK, a key gluconeogenic enzyme) in the kidney cortex from HFD, relative to mice on NCD. The higher level of PEPCK in HFD was confirmed by immunoblotting. However, no significant differences were observed in cortical glucose-6-phosphatase (G6Pase) or fructose-1,6, bisphosphosphatase (FBPase) enzyme transcript levels. Furthermore, we demonstrated insulin inhibited glucose production in hPTC treated with cyclic AMP and dexamethasone (cAMP/DEXA) to stimulate gluconeogenesis. Transcript levels of the gluconeogenic enzyme PEPCK were significantly increased in cAMP/DEXA-stimulated hPTC cells (n = 3, P < 0.05), and insulin attenuated this upregulation Furthermore, the effect of insulin on cAMP/DEXA-induced gluconeogenesis and PEPCK induction was significantly attenuated in IR (siRNA) silenced hPTC (n = 3, P < 0.05). Overall the above data indicate a direct role for IR expression as a determinant of PT-gluconeogenesis. Thus reduced insulin signaling of the proximal tubule may contribute to hyperglycemia in the metabolic syndrome via elevated gluconeogenesis. J. Cell. Biochem. 118: 276-285, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Production and characterization of multi-polysaccharide degrading enzymes from Aspergillus aculeatus BCC199 for saccharification of agricultural residues.

    PubMed

    Suwannarangsee, Surisa; Arnthong, Jantima; Eurwilaichitr, Lily; Champreda, Verawat

    2014-10-01

    Enzymatic hydrolysis of lignocellulosic biomass into fermentable sugars is a key step in the conversion of agricultural by-products to biofuels and value-added chemicals. Utilization of a robust microorganism for on-site production of biomass-degrading enzymes has gained increasing interest as an economical approach for supplying enzymes to biorefinery processes. In this study, production of multi-polysaccharide-degrading enzymes from Aspergillus aculeatus BCC199 by solid-state fermentation was improved through the statistical design approach. Among the operational parameters, yeast extract and soybean meal as well as the nonionic surfactant Tween 20 and initial pH were found as key parameters for maximizing production of cellulolytic and hemicellulolytic enzymes. Under the optimized condition, the production of FPase, endoglucanase, β-glucosidase, xylanase, and β-xylosidase was achieved at 23, 663, 88, 1,633, and 90 units/g of dry substrate, respectively. The multi-enzyme extract was highly efficient in the saccharification of alkaline-pretreated rice straw, corn cob, and corn stover. In comparison with commercial cellulase preparations, the BCC199 enzyme mixture was able to produce remarkable yields of glucose and xylose, as it contained higher relative activities of β-glucosidase and core hemicellulases (xylanase and β-xylosidase). These results suggested that the crude enzyme extract from A. aculeatus BCC199 possesses balanced cellulolytic and xylanolytic activities required for the efficient saccharification of lignocellulosic biomass feedstocks, and supplementation of external β-glucosidase or xylanase was dispensable. The work thus demonstrates the high potential of A. aculeatus BCC199 as a promising producer of lignocellulose-degrading enzymes for the biomass conversion industry.

  2. Transcriptional targets of DAF-16 insulin signaling pathway protect C. elegans from extreme hypertonic stress.

    PubMed

    Lamitina, S Todd; Strange, Kevin

    2005-02-01

    All cells adapt to hypertonic stress by regulating their volume after shrinkage, by accumulating organic osmolytes, and by activating mechanisms that protect against and repair hypertonicity-induced damage. In mammals and nematodes, inhibition of signaling from the DAF-2/IGF-1 insulin receptor activates the DAF-16/FOXO transcription factor, resulting in increased life span and resistance to some types of stress. We tested the hypothesis that inhibition of insulin signaling in Caenorhabditis elegans also increases hypertonic stress resistance. Genetic inhibition of DAF-2 or its downstream target, the AGE-1 phosphatidylinositol 3-kinase, confers striking resistance to a normally lethal hypertonic shock in a DAF-16-dependent manner. However, insulin signaling is not inhibited by or required for adaptation to hypertonic conditions. Microarray studies have identified 263 genes that are transcriptionally upregulated by DAF-16 activation. We identified 14 DAF-16-upregulated genes by RNA interference screening that are required for age-1 hypertonic stress resistance. These genes encode heat shock proteins, proteins of unknown function, and trehalose synthesis enzymes. Trehalose levels were elevated approximately twofold in age-1 mutants, but this increase was insufficient to prevent rapid hypertonic shrinkage. However, age-1 animals unable to synthesize trehalose survive poorly under hypertonic conditions. We conclude that increased expression of proteins that protect eukaryotic cells against environmental stress and/or repair stress-induced molecular damage confers hypertonic stress resistance in C. elegans daf-2/age-1 mutants. Elevated levels of solutes such as trehalose may also function in a cytoprotective manner. Our studies provide novel insights into stress resistance in animal cells and a foundation for new studies aimed at defining molecular mechanisms underlying these essential processes.

  3. Endothelin-converting enzyme-1 degrades internalized somatostatin-14.

    PubMed

    Roosterman, Dirk; Kempkes, Cordula; Cottrell, Graeme S; Padilla, Benjamin E; Bunnett, Nigel W; Turck, Christoph W; Steinhoff, Martin

    2008-05-01

    Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with beta-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized (125)I-Tyr(11)-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized (125)I-Tyr(1)-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A(1). ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

  4. Endothelin-Converting Enzyme-1 Degrades Internalized Somatostatin-14

    PubMed Central

    Roosterman, Dirk; Kempkes, Cordula; Cottrell, Graeme S.; Padilla, Benjamin E.; Bunnett, Nigel W.; Turck, Christoph W.; Steinhoff, Martin

    2008-01-01

    Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with β-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized 125I-Tyr11-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized 125I-Tyr1-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A1. ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis. PMID:18276747

  5. Insulin/IGF-driven cancer cell-stroma crosstalk as a novel therapeutic target in pancreatic cancer.

    PubMed

    Mutgan, Ayse Ceren; Besikcioglu, H Erdinc; Wang, Shenghan; Friess, Helmut; Ceyhan, Güralp O; Demir, Ihsan Ekin

    2018-02-23

    Pancreatic ductal adenocarcinoma (PDAC) is unrivalled the deadliest gastrointestinal cancer in the western world. There is substantial evidence implying that insulin and insulin-like growth factor (IGF) signaling axis prompt PDAC into an advanced stage by enhancing tumor growth, metastasis and by driving therapy resistance. Numerous efforts have been made to block Insulin/IGF signaling pathway in cancer therapy. However, therapies that target the IGF1 receptor (IGF-1R) and IGF subtypes (IGF-1 and IGF-2) have been repeatedly unsuccessful. This failure may not only be due to the complexity and homology that is shared by Insulin and IGF receptors, but also due to the complex stroma-cancer interactions in the pancreas. Shedding light on the interactions between the endocrine/exocrine pancreas and the stroma in PDAC is likely to steer us toward the development of novel treatments. In this review, we highlight the stroma-derived IGF signaling and IGF-binding proteins as potential novel therapeutic targets in PDAC.

  6. Identification of Pectin Degrading Enzymes Secreted by Xanthomonas oryzae pv. oryzae and Determination of Their Role in Virulence on Rice

    PubMed Central

    Tayi, Lavanya; Maku, Roshan V.; Patel, Hitendra Kumar; Sonti, Ramesh V.

    2016-01-01

    Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo. PMID:27907079

  7. Identification of Pectin Degrading Enzymes Secreted by Xanthomonas oryzae pv. oryzae and Determination of Their Role in Virulence on Rice.

    PubMed

    Tayi, Lavanya; Maku, Roshan V; Patel, Hitendra Kumar; Sonti, Ramesh V

    2016-01-01

    Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo.

  8. Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier–dependent pathway

    PubMed Central

    Ahner, Annette; Gong, Xiaoyan; Schmidt, Bela Z.; Peters, Kathryn W.; Rabeh, Wael M.; Thibodeau, Patrick H.; Lukacs, Gergely L.; Frizzell, Raymond A.

    2013-01-01

    Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27’s ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4’s impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin–proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein. PMID:23155000

  9. Biochemical characterisation of an allantoate-degrading enzyme from French bean (Phaseolus vulgaris): the requirement of phenylhydrazine.

    PubMed

    Raso, María José; Muñoz, Alfonso; Pineda, Manuel; Piedras, Pedro

    2007-10-01

    In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules is used for synthesis of the ureides allantoin and allantoic acid, the major long distance transport forms of organic nitrogen in these species. The purpose of this investigation was to characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined "in vivo" using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37 degrees C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.

  10. Subconjunctivally Implantable Hydrogels with Degradable and Thermoresponsive Properties for Sustained Release of Insulin to the Retina

    PubMed Central

    Misra, Gauri P.; Singh, Ravi S.J.; Aleman, Tomas S.; Jacobson, Samuel G.; Gardner, Thomas W.; Lowe, Tao L.

    2009-01-01

    The objective of this work is to develop subconjunctivally implantable, biodegradable hydrogels for sustained release of intact insulin to the retina to prevent and treat retinal neurovascular degeneration such as diabetic retinopathy. The hydrogels are synthesized by UV photopolymerization of N-isopropylacrylamide (NIPAAm) monomer and a dextran macromer containing multiple hydrolytically degradable oligolactate-(2-hydroxyetheyl methacrylate) units (Dex-lactateHEMA) in 25:75 (v:v) ethanol:water mixture solvent. Insulin is loaded into the hydrogels during the synthesis process with loading efficiency up to 98%. The hydrogels can release biologically active insulin in vitro for at least one week and the release kinetics can be modulated by varying the ratio between NIPAAm and Dex-lactateHEMA and altering the physical size of the hydrogels. The hydrogels are not toxic to R28 retinal neuron cells in culture medium with 100% cell viability. The hydrogels can be implanted under the conjunctiva without causing adverse effects to the retina based on hematoxylin and eosin stain, immunostaining for microglial cell activation, and electroretinography. These subconjunctivally implantable hydrogels have potential for long-term periocular delivery of insulin or other drugs to treat diabetic retinopathy and other retinal diseases. PMID:19709741

  11. Carbon-Degrading Enzyme Activities Stimulated by Increased Nutrient Availability in Arctic Tundra Soils

    PubMed Central

    Koyama, Akihiro; Wallenstein, Matthew D.; Simpson, Rodney T.; Moore, John C.

    2013-01-01

    Climate-induced warming of the Arctic tundra is expected to increase nutrient availability to soil microbes, which in turn may accelerate soil organic matter (SOM) decomposition. We increased nutrient availability via fertilization to investigate the microbial response via soil enzyme activities. Specifically, we measured potential activities of seven enzymes at four temperatures in three soil profiles (organic, organic/mineral interface, and mineral) from untreated native soils and from soils which had been fertilized with nitrogen (N) and phosphorus (P) since 1989 (23 years) and 2006 (six years). Fertilized plots within the 1989 site received annual additions of 10 g N⋅m-2⋅year-1 and 5 g P⋅m-2⋅year-1. Within the 2006 site, two fertilizer regimes were established – one in which plots received 5 g N⋅m-2⋅year-1 and 2.5 g P⋅m-2⋅year-1 and one in which plots received 10 g N⋅m-2⋅year-1 and 5 g P⋅m-2⋅year-1. The fertilization treatments increased activities of enzymes hydrolyzing carbon (C)-rich compounds but decreased phosphatase activities, especially in the organic soils. Activities of two enzymes that degrade N-rich compounds were not affected by the fertilization treatments. The fertilization treatments increased ratios of enzyme activities degrading C-rich compounds to those for N-rich compounds or phosphate, which could lead to changes in SOM chemistry over the long term and to losses of soil C. Accelerated SOM decomposition caused by increased nutrient availability could significantly offset predicted increased C fixation via stimulated net primary productivity in Arctic tundra ecosystems. PMID:24204773

  12. Insulin signalling mechanisms for triacylglycerol storage.

    PubMed

    Czech, M P; Tencerova, M; Pedersen, D J; Aouadi, M

    2013-05-01

    Insulin signalling is uniquely required for storing energy as fat in humans. While de novo synthesis of fatty acids and triacylglycerol occurs mostly in liver, adipose tissue is the primary site for triacylglycerol storage. Insulin signalling mechanisms in adipose tissue that stimulate hydrolysis of circulating triacylglycerol, uptake of the released fatty acids and their conversion to triacylglycerol are poorly understood. New findings include (1) activation of DNA-dependent protein kinase to stimulate upstream stimulatory factor (USF)1/USF2 heterodimers, enhancing the lipogenic transcription factor sterol regulatory element binding protein 1c (SREBP1c); (2) stimulation of fatty acid synthase through AMP kinase modulation; (3) mobilisation of lipid droplet proteins to promote retention of triacylglycerol; and (4) upregulation of a novel carbohydrate response element binding protein β isoform that potently stimulates transcription of lipogenic enzymes. Additionally, insulin signalling through mammalian target of rapamycin to activate transcription and processing of SREBP1c described in liver may apply to adipose tissue. Paradoxically, insulin resistance in obesity and type 2 diabetes is associated with increased triacylglycerol synthesis in liver, while it is decreased in adipose tissue. This and other mysteries about insulin signalling and insulin resistance in adipose tissue make this topic especially fertile for future research.

  13. Comparative metagenomic analysis of microcosm structures and lignocellulolytic enzyme systems of symbiotic biomass-degrading consortia.

    PubMed

    Wongwilaiwalin, Sarunyou; Laothanachareon, Thanaporn; Mhuantong, Wuttichai; Tangphatsornruang, Sithichoke; Eurwilaichitr, Lily; Igarashi, Yasuo; Champreda, Verawat

    2013-10-01

    Decomposition of lignocelluloses by cooperative microbial actions is an essential process of carbon cycling in nature and provides a basis for biomass conversion to fuels and chemicals in biorefineries. In this study, structurally stable symbiotic aero-tolerant lignocellulose-degrading microbial consortia were obtained from biodiversified microflora present in industrial sugarcane bagasse pile (BGC-1), cow rumen fluid (CRC-1), and pulp mill activated sludge (ASC-1) by successive subcultivation on rice straw under facultative anoxic conditions. Tagged 16S rRNA gene pyrosequencing revealed that all isolated consortia originated from highly diverse environmental microflora shared similar composite phylum profiles comprising mainly Firmicutes, reflecting convergent adaptation of microcosm structures, however, with substantial differences at refined genus level. BGC-1 comprising cellulolytic Clostridium and Acetanaerobacterium in stable coexistence with ligninolytic Ureibacillus showed the highest capability on degradation of agricultural residues and industrial pulp waste with CMCase, xylanase, and β-glucanase activities in the supernatant. Shotgun pyrosequencing of the BGC-1 metagenome indicated a markedly high relative abundance of genes encoding for glycosyl hydrolases, particularly for lignocellulytic enzymes in 26 families. The enzyme system comprised a unique composition of main-chain degrading and side-chain processing hydrolases, dominated by GH2, 3, 5, 9, 10, and 43, reflecting adaptation of enzyme profiles to the specific substrate. Gene mapping showed metabolic potential of BGC-1 for conversion of biomass sugars to various fermentation products of industrial importance. The symbiotic consortium is a promising simplified model for study of multispecies mechanisms on consolidated bioprocessing and a platform for discovering efficient synergistic enzyme systems for biotechnological application.

  14. Transcriptional response of lignin-degrading enzymes to 17α-ethinyloestradiol in two white rots.

    PubMed

    Přenosilová, L; Křesinová, Z; Amemori, A Slavíková; Cajthaml, T; Svobodová, K

    2013-05-01

    Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase (MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17α-ethinyloestradiol (EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2 ± 6.7 U g⁻¹ of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene (lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T. versicolor, Irpex lacteus was also able to remove 10 mg l⁻¹ EE2 within 3 days of cultivation. While an increase to I. lacteus MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  15. Targeting Anti-Insulin B Cell Receptors Improves Receptor Editing in Type 1 Diabetes-Prone Mice1, 2, 3

    PubMed Central

    Bonami, Rachel H.; Thomas, James W.

    2015-01-01

    Autoreactive B lymphocytes that commonly arise in the developing repertoire can be salvaged by receptor editing, a central tolerance mechanism that alters BCR specificity through continued L chain rearrangement. It is unknown whether autoantigens with weak cross-linking potential, such as insulin, elicit receptor editing, or if this process is dysregulated in related autoimmunity. To resolve these issues, an editing-competent model was developed in which anti-insulin Vκ125 was targeted to the Igκ locus and paired with anti-insulin VH125Tg. Physiologic, circulating insulin increased RAG-2 expression and was associated with BCR replacement that eliminated autoantigen recognition in a proportion of developing anti-insulin B lymphocytes. The proportion of anti-insulin B cells that underwent receptor editing was reduced in the type 1 diabetes-prone NOD strain relative to a non-autoimmune strain. Resistance to editing was associated with increased surface IgM expression on immature (but not transitional or mature) anti-insulin B cells in the NOD strain. The actions of mAb123 on central tolerance were also investigated, as selective targeting of insulin-occupied BCR by mAb123 eliminates anti-insulin B lymphocytes and prevents type 1 diabetes. Autoantigen-targeting by mAb123 increased RAG-2 expression and dramatically enhanced BCR replacement in newly developed B lymphocytes. Administering F(ab’)2123 induced IgM downregulation and reduced the frequency of anti-insulin B lymphocytes within the polyclonal repertoire of VH125Tg/NOD mice, suggesting enhanced central tolerance by direct BCR interaction. These findings indicate that weak or faulty checkpoints for central tolerance can be overcome by autoantigen-specific immunomodulatory therapy. PMID:26432895

  16. Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

    PubMed Central

    Payne, Christina M.; Resch, Michael G.; Chen, Liqun; Crowley, Michael F.; Himmel, Michael E.; Taylor, Larry E.; Sandgren, Mats; Ståhlberg, Jerry; Stals, Ingeborg; Tan, Zhongping; Beckham, Gregg T.

    2013-01-01

    Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls. PMID:23959893

  17. Fungal cellulose degradation by oxidative enzymes: from dysfunctional GH61 family to powerful lytic polysaccharide monooxygenase family.

    PubMed

    Morgenstern, Ingo; Powlowski, Justin; Tsang, Adrian

    2014-11-01

    Our understanding of fungal cellulose degradation has shifted dramatically in the past few years with the characterization of a new class of secreted enzymes, the lytic polysaccharide monooxygenases (LPMO). After a period of intense research covering structural, biochemical, theoretical and evolutionary aspects, we have a picture of them as wedge-like copper-dependent metalloenzymes that on reduction generate a radical copper-oxyl species, which cleaves mainly crystalline cellulose. The main biological function lies in the synergism of fungal LPMOs with canonical hydrolytic cellulases in achieving efficient cellulose degradation. Their important role in cellulose degradation is highlighted by the wide distribution and often numerous occurrences in the genomes of almost all plant cell-wall degrading fungi. In this review, we provide an overview of the latest achievements in LPMO research and consider the open questions and challenges that undoubtedly will continue to stimulate interest in this new and exciting group of enzymes. © The Author 2014. Published by Oxford University Press.

  18. Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants.

    PubMed

    Aylward, Frank O; Khadempour, Lily; Tremmel, Daniel M; McDonald, Bradon R; Nicora, Carrie D; Wu, Si; Moore, Ronald J; Orton, Daniel J; Monroe, Matthew E; Piehowski, Paul D; Purvine, Samuel O; Smith, Richard D; Lipton, Mary S; Burnum-Johnson, Kristin E; Currie, Cameron R

    2015-01-01

    Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.

  19. Enrichment and broad representation of plant biomass-degrading enzymes in the specialized hyphal swellings of Leucoagaricus gongylophorus, the fungal symbiont of leaf-cutter ants

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aylward, Frank O.; Khadempour, Lily; Tremmel, Daniel M.

    Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plantmore » biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.« less

  20. Enrichment and broad representation of plant biomass-degrading enzymes in the specialized hyphal swellings of Leucoagaricus gongylophorus, the fungal symbiont of leaf-cutter ants

    DOE PAGES

    Aylward, Frank O.; Khadempour, Lily; Tremmel, Daniel M.; ...

    2015-08-28

    Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plantmore » biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.« less

  1. Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants

    PubMed Central

    Aylward, Frank O.; Khadempour, Lily; Tremmel, Daniel M.; McDonald, Bradon R.; Nicora, Carrie D.; Wu, Si; Moore, Ronald J.; Orton, Daniel J.; Monroe, Matthew E.; Piehowski, Paul D.; Purvine, Samuel O.; Smith, Richard D.; Lipton, Mary S.; Burnum-Johnson, Kristin E.; Currie, Cameron R.

    2015-01-01

    Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass. PMID:26317212

  2. An angiotensin-(1–7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme

    PubMed Central

    Wilson, Bryan A.; Cruz-Diaz, Nildris; Marshall, Allyson C.; Pirro, Nancy T.; Su, Yixin; Gwathmey, TanYa M.; Rose, James C.

    2015-01-01

    Angiotensin 1–7 [ANG-(1–7)] is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic, and pro-oxidant effects of ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1–7) to ANG-(1–4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313–323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1–7) to ANG-(1–4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol·min−1·mg−1) compared with the tubules (96 ± 12 fmol·min−1·mg−1) and cortex (107 ± 9 fmol·min−1·mg−1). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1–7) and its endogenous analog [Ala1]-ANG-(1–7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp1]-ANG II, ANG I, and ANG-(1–12). Although the ANG-(1–7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1–7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1–7) tone. PMID:25568136

  3. Enhancement in multiple lignolytic enzymes production for optimized lignin degradation and selectivity in fungal pretreatment of sweet sorghum bagasse.

    PubMed

    Mishra, Vartika; Jana, Asim K; Jana, Mithu Maiti; Gupta, Antriksh

    2017-07-01

    The objective of this work was to study the increase in multiple lignolytic enzyme productions through the use of supplements in combination in pretreatment of sweet sorghum bagasse (SSB) by Coriolus versicolor such that enzymes act synergistically to maximize the lignin degradation and selectivity. Enzyme activities were enhanced by metallic salts and phenolic compound supplements in SSF. Supplement of syringic acid increased the activities of LiP, AAO and laccase; gallic acid increased MnP; CuSO 4 increased laccase and PPO to improve the lignin degradations and selectivity individually, higher than control. Combination of supplements optimized by RSM increased the production of laccase, LiP, MnP, PPO and AAO by 17.2, 45.5, 3.5, 2.4 and 3.6 folds respectively for synergistic action leading to highest lignin degradation (2.3 folds) and selectivity (7.1 folds). Enzymatic hydrolysis of pretreated SSB yielded ∼2.43 times fermentable sugar. This technique could be widely applied for pretreatment and enzyme productions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Identification of intracellular degradation intermediates of aldolase B by antiserum to the denatured enzyme.

    PubMed Central

    Reznick, A Z; Rosenfelder, L; Shpund, S; Gershon, D

    1985-01-01

    A method has been developed that enables us to identify intracellular degradation intermediates of fructose-bisphosphate aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). This method is based on the use of antibody against thoroughly denatured purified aldolase. This antibody has been shown to recognize only denatured molecules, and it did not interact with "native" enzyme. supernatants (24,000 X g for 30 min) of liver and kidney homogenates were incubated with antiserum to denatured enzyme. The antigen-antibody precipitates thus formed were subjected to NaDodSO4/PAGE, followed by electrotransfer to nitrocellulose paper and immunodecoration with antiserum to denatured enzyme and 125I-labeled protein A. Seven peptides with molecular weights ranging from 38,000 (that of the intact subunit) to 18,000, which cross-reacted antigenically with denatured fructose-bisphosphate aldolase, could be identified in liver. The longest three peptides were also present in kidney. The possibility that these peptides were artifacts of homogenization was ruled out as follows: 125I-labeled tagged purified native aldolase was added to the buffer prior to liver homogenization. The homogenates were than subjected to NaDodSO4/PAGE followed by autoradiography, and the labeled enzyme was shown to remain intact. This method is suggested for general use in the search for degradation products of other cellular proteins. Images PMID:3898080

  5. Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

    PubMed Central

    Geiser, Elena; Reindl, Michèle; Blank, Lars M.; Feldbrügge, Michael

    2016-01-01

    ABSTRACT The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCE This study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a

  6. Insulin resistance and improvements in signal transduction.

    PubMed

    Musi, Nicolas; Goodyear, Laurie J

    2006-02-01

    Type 2 diabetes and obesity are common metabolic disorders characterized by resistance to the actions of insulin to stimulate skeletal muscle glucose disposal. Insulin-resistant muscle has defects at several steps of the insulin-signaling pathway, including decreases in insulin-stimulated insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation, and phosphatidylinositol 3-kinase (PI 3-kinase) activation. One approach to increase muscle glucose disposal is to reverse/improve these insulin-signaling defects. Weight loss and thiazolidinediones (TZDs) improve glucose disposal, in part, by increasing insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation and PI 3-kinase activity. In contrast, physical training and metformin improve whole-body glucose disposal but have minimal effects on proximal insulin-signaling steps. A novel approach to reverse insulin resistance involves inhibition of the stress-activated protein kinase Jun N-terminal kinase (JNK) and the protein tyrosine phosphatases (PTPs). A different strategy to increase muscle glucose disposal is by stimulating insulin-independent glucose transport. AMP-activated protein kinase (AMPK) is an enzyme that works as a fuel gauge and becomes activated in situations of energy consumption, such as muscle contraction. Several studies have shown that pharmacologic activation of AMPK increases glucose transport in muscle, independent of the actions of insulin. AMPK activation is also involved in the mechanism of action of metformin and adiponectin. Moreover, in the hypothalamus, AMPK regulates appetite and body weight. The effect of AMPK to stimulate muscle glucose disposal and to control appetite makes it an important pharmacologic target for the treatment of type 2 diabetes and obesity.

  7. Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

    PubMed

    Okuhira, Keiichiro; Shoda, Takuji; Omura, Risa; Ohoka, Nobumichi; Hattori, Takayuki; Shibata, Norihito; Demizu, Yosuke; Sugihara, Ryo; Ichino, Asato; Kawahara, Haruka; Itoh, Yukihiro; Ishikawa, Minoru; Hashimoto, Yuichi; Kurihara, Masaaki; Itoh, Susumu; Saito, Hiroyuki; Naito, Mikihiko

    2017-03-01

    Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein. Copyright © 2017 by

  8. Role of Insulin in the Regulation of Proprotein Convertase Subtilisin/Kexin Type 9.

    PubMed

    Miao, Ji; Manthena, Praveen V; Haas, Mary E; Ling, Alisha V; Shin, Dong-Ju; Graham, Mark J; Crooke, Rosanne M; Liu, Jingwen; Biddinger, Sudha B

    2015-07-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds the low-density lipoprotein receptor and targets it for degradation, has emerged as an important regulator of serum cholesterol levels and cardiovascular disease risk. Although much work is currently focused on developing therapies for inhibiting PCSK9, the endogenous regulation of PCSK9, particularly by insulin, remains unclear. The objective of these studies was to determine the effects of insulin on PCSK9 in vitro and in vivo. Using rat hepatoma cells and primary rat hepatocytes, we found that insulin increased PCSK9 expression and increased low-density lipoprotein receptor degradation in a PCSK9-dependent manner. In parallel, hepatic Pcsk9 mRNA and plasma PCSK9 protein levels were reduced by 55% to 75% in mice with liver-specific knockout of the insulin receptor; 75% to 88% in mice made insulin-deficient with streptozotocin; and 65% in ob/ob mice treated with antisense oligonucleotides against the insulin receptor. However, antisense oligonucleotide-mediated knockdown of insulin receptor in lean, wild-type mice had little effect. In addition, we found that fasting was able to reduce PCSK9 expression by 80% even in mice that lack hepatic insulin signaling. Taken together, these data indicate that although insulin induces PCSK9 expression, it is not the sole or even dominant regulator of PCSK9 under all conditions. © 2015 American Heart Association, Inc.

  9. Polyphenols as enzyme inhibitors in different degraded peat soils: Implication for microbial metabolism in rewetted peatlands

    NASA Astrophysics Data System (ADS)

    Zak, Dominik; Roth, Cyril; Gelbrecht, Jörg; Fenner, Nathalie; Reuter, Hendrik

    2015-04-01

    Recently, more than 30,000 ha of drained minerotrophic peatlands (= fens) in NE Germany were rewetted to restore their ecological functions. Due to an extended drainage history, a re-establishment of their original state is not expected in the short-term. Elevated concentrations of dissolved organic carbon, ammonium and phosphate have been measured in the soil porewater of the upper degraded peat layers of rewetted fens at levels of one to three orders higher than the values in pristine systems; an indicator of increased microbial activity in the upper degraded soil layers. On the other hand there is evidence that the substrate availability within the degraded peat layer is lowered since the organic matter has formerly been subject to intense decomposition over the decades of drainage and intense agricultural use of the areas. Previously however, it was suggested that inhibition of hydrolytic enzymes by polyphenolic substances is suspended during aeration of peat soils mainly due to the decomposition of the inhibiting polyphenols by oxidising enzymes such as phenol oxidase. Accordingly we hypothesised a lack of enzyme inhibiting polyphenols in degraded peat soils of rewetted fens compared to less decomposed peat of more natural fens. We collected both peat samples at the soil surface (0-20 cm) and fresh roots of dominating vascular plants and mosses (as peat parent material) from five formerly drained rewetted sites and five more natural sites of NE Germany and NW Poland. Less decomposed peat and living roots were used to obtain an internal standard for polyphenol analysis and to run enzyme inhibition tests. For all samples we determined the total phenolic contents and in addition we distinguished between the contents of hydrolysable and condensed tannic substances. From a methodical perspective the advantage of internal standards compared to the commercially available standards cyanidin chloride and tannic acid became apparent. Quantification with cyanidin or

  10. Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

    USDA-ARS?s Scientific Manuscript database

    Discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly c...

  11. Identification of food-grade subtilisins as gluten-degrading enzymes to treat celiac disease

    PubMed Central

    Wei, Guoxian; Tian, Na; Siezen, Roland; Schuppan, Detlef

    2016-01-01

    Gluten are proline- and glutamine-rich proteins present in wheat, barley, and rye and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISAs. The gliadin-degrading Rothia enzymes were identified by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC). A search with the Basic Local Alignment Search Tool revealed that these are subtilisin-like serine proteases belonging to the peptidase S8 family. Alignment of the major Rothia subtilisins indicated that all contain the catalytic triad with Asp (D), His (H), and Ser (S) in the D-H-S order. They cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. The Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in CD. PMID:27469368

  12. Tight glycemic control with insulin does not affect skeletal muscle degradation during the early post-operative period following pediatric cardiac surgery

    PubMed Central

    Fisher, Jeremy G.; Sparks, Eric A.; Khan, Faraz A.; Alexander, Jamin L.; Asaro, Lisa A.; Wypij, David; Gaies, Michael; Modi, Biren P.; Duggan, Christopher; Agus, Michael S.D.; Yu, Yong-Ming; Jaksic, Tom

    2015-01-01

    Objective Critical illness is associated with significant catabolism and persistent protein loss correlates with increased morbidity and mortality. Insulin is a potent anti-catabolic hormone; high-dose insulin decreases skeletal muscle protein breakdown in critically ill pediatric surgical patients. However, insulin's effect on protein catabolism when given at clinically utilized doses has not been studied. The objective was to evaluate the effect of post-operative tight glycemic control and clinically-dosed insulin on skeletal muscle degradation in children after cardiac surgery with cardiopulmonary bypass. Design Secondary analysis of a two-center, prospective randomized trial comparing tight glycemic control with standard care. Randomization was stratified by study center. Patients Children 0-36 months who were admitted to the ICU after cardiac surgery requiring cardiopulmonary bypass. Interventions In the tight glycemic control (TGC) arm, insulin was titrated to maintain blood glucose between 80-110 mg/dL. Patients in the control arm received standard care. Skeletal muscle breakdown was quantified by a ratio of urinary 3-methylhistidine to urinary creatinine (3MH:Cr). Main Results A total of 561 patients were included: 281 in the TGC arm and 280 receiving standard care. There was no difference in 3MH:Cr between groups (TGC 249 ± 127 vs. standard care 253 ± 112, mean ± standard deviation in μmol/g, P=0.72). In analyses restricted to the TGC patients, higher 3MH:Cr correlated with younger age as well as lower weight, weight-for-age z-score, length, and body surface area (P<0.005 for each), and lower post-operative day 3 serum creatinine (r=-0.17, P=0.02). Sex, prealbumin, and albumin were not associated with 3MH:Cr. During urine collection, 245 patients (87%) received insulin. However, any insulin exposure did not impact 3MH:Cr (t-test, P=0.45), and there was no dose-dependent effect of insulin on 3MH:Cr (r=-0.03, P=0.60). Conclusion Though high-dose insulin

  13. Reaching the Melting Point: Degradative Enzymes and Protease Inhibitors Involved in Baculovirus Infection and Dissemination

    PubMed Central

    Ishimwe, Egide; Hodgson, Jeffrey J.; Clem, Rollie J.; Passarelli, A. Lorena

    2015-01-01

    Baculovirus infection of a host insect involves several steps, beginning with initiation of virus infection in the midgut, followed by dissemination of infection from the midgut to other tissues in the insect, and finally culminating in “melting” or liquefaction of the host, which allows for horizontal spread of infection to other insects. While all of the viral gene products are involved in ultimately reaching this dramatic infection endpoint, this review focuses on two particular types of baculovirus-encoded proteins: degradative enzymes and protease inhibitors. Neither of these types of proteins is commonly found in other virus families, but they both play important roles in baculovirus infection. The types of degradative enzymes and protease inhibitors encoded by baculoviruses are discussed, as are the roles of these proteins in the infection process. PMID:25724418

  14. Leptin inhibits amyloid β-protein degradation through decrease of neprilysin expression in primary cultured astrocytes.

    PubMed

    Yamamoto, Naoki; Tanida, Mamoru; Ono, Yoko; Kasahara, Rika; Fujii, Yuko; Ohora, Kentaro; Suzuki, Kenji; Sobue, Kazuya

    2014-02-28

    Pathogenesis of Alzheimer's disease (AD) is characterized by accumulation of extracellular deposits of amyloid β-protein (Aβ) in the brain. The steady state level of Aβ in the brain is determined by the balance between its production and removal; the latter occurring through egress across blood and CSF barriers as well as Aβ degradation. The major Aβ-degrading enzymes in the brain are neprilysin (NEP) and insulin-degrading enzyme (IDE), which may promote Aβ deposition in patients with sporadic late-onset AD. Epidemiological studies have suggested an inverse relationship between the adipocytokine leptin levels and the onset of AD. However, the mechanisms underlying the relationship remain uncertain. We investigated whether leptin is associated with Aβ degradation by inducing NEP and IDE expression within primary cultured astrocytes. Leptin significantly decreased the expression of NEP but not IDE in a concentration- and time-dependent manner through the activation of extracellular signal-regulated kinase (ERK) in cultured rat astrocytes. Furthermore, leptin inhibited the degradation of exogenous Aβ in primary cultured astrocytes. These results suggest that leptin suppresses Aβ degradation by NEP through activation of ERK. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Atrazine degradation by fungal co-culture enzyme extracts under different soil conditions.

    PubMed

    Chan-Cupul, Wilberth; Heredia-Abarca, Gabriela; Rodríguez-Vázquez, Refugio

    2016-01-01

    This investigation was undertaken to determine the atrazine degradation by fungal enzyme extracts (FEEs) in a clay-loam soil microcosm contaminated at field application rate (5 μg g(-1)) and to study the influence of different soil microcosm conditions, including the effect of soil sterilization, water holding capacity, soil pH and type of FEEs used in atrazine degradation through a 2(4) factorial experimental design. The Trametes maxima-Paecilomyces carneus co-culture extract contained more laccase activity and hydrogen peroxide (H2O2) content (laccase = 18956.0 U mg protein(-1), H2O2 = 6.2 mg L(-1)) than the T. maxima monoculture extract (laccase = 12866.7 U mg protein(-1), H2O2 = 4.0 mg L(-1)). Both extracts were able to degrade atrazine at 100%; however, the T. maxima monoculture extract (0.32 h) achieved a lower half-degradation time than its co-culture with P. carneus (1.2 h). The FEE type (p = 0.03) and soil pH (p = 0.01) significantly affected atrazine degradation. The best degradation rate was achieved by the T. maxima monoculture extract in an acid soil (pH = 4.86). This study demonstrated that both the monoculture extracts of the native strain T. maxima and its co-culture with P. carneus can efficiently and quickly degrade atrazine in clay-loam soils.

  16. Highly stable and degradable multifunctional microgel for self-regulated insulin delivery under physiological conditions

    NASA Astrophysics Data System (ADS)

    Zhang, Xinjie; Lü, Shaoyu; Gao, Chunmei; Chen, Chen; Zhang, Xuan; Liu, Mingzhu

    2013-06-01

    The response to glucose, pH and temperature, high drug loading capacity, self-regulated drug delivery and degradation in vivo are simultaneously probable by applying a multifunctional microgel under a rational design in a colloid chemistry method. Such multifunctional microgels are fabricated with N-isopropylacrylamide (NIPAAm), (2-dimethylamino)ethyl methacrylate (DMAEMA) and 3-acrylamidephenylboronic acid (AAPBA) through a precipitation emulsion method and cross-linked by reductive degradable N,N'-bis(arcyloyl)cystamine (BAC). This novel kind of microgel with a narrow size distribution (~250 nm) is suitable for diabetes because it can adapt to the surrounding medium of different glucose concentrations over a clinically relevant range (0-20 mM), control the release of preloaded insulin and is highly stable under physiological conditions (pH 7.4, 0.15 M NaCl, 37 °C). When synthesized multifunctional microgels regulate drug delivery, they gradually degrade as time passes and, as a result, show enhanced biocompatibility. This exhibits a new proof-of-concept for diabetes treatment that takes advantage of the properties of each building block from a multifunctional micro-object. These highly stable and versatile multifunctional microgels have the potential to be used for self-regulated therapy and monitoring of the response to treatment, or even simultaneous diagnosis as nanobiosensors.The response to glucose, pH and temperature, high drug loading capacity, self-regulated drug delivery and degradation in vivo are simultaneously probable by applying a multifunctional microgel under a rational design in a colloid chemistry method. Such multifunctional microgels are fabricated with N-isopropylacrylamide (NIPAAm), (2-dimethylamino)ethyl methacrylate (DMAEMA) and 3-acrylamidephenylboronic acid (AAPBA) through a precipitation emulsion method and cross-linked by reductive degradable N,N'-bis(arcyloyl)cystamine (BAC). This novel kind of microgel with a narrow size

  17. Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation.

    PubMed

    Balamurugan, Appakalai N; Green, Michael L; Breite, Andrew G; Loganathan, Gopalakrishnan; Wilhelm, Joshua J; Tweed, Benjamin; Vargova, Lenka; Lockridge, Amber; Kuriti, Manikya; Hughes, Michael G; Williams, Stuart K; Hering, Bernhard J; Dwulet, Francis E; McCarthy, Robert C

    2016-01-01

    Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

  18. Brain Insulin Resistance and Deficiency as Therapeutic Targets in Alzheimer's Disease

    PubMed Central

    de la Monte, Suzanne M

    2012-01-01

    Alzheimer's disease [AD] is the most common cause of dementia in North America. Despite 30+ years of intense investigation, the field lacks consensus regarding the etiology and pathogenesis of sporadic AD, and therefore we still do not know the best strategies for treating and preventing this debilitating and costly disease. However, growing evidence supports the concept that AD is fundamentally a metabolic disease with substantial and progressive derangements in brain glucose utilization and responsiveness to insulin and insulin-like growth factor [IGF] stimulation. Moreover, AD is now recognized to be heterogeneous in nature, and not solely the end-product of aberrantly processed, misfolded, and aggregated oligomeric amyloid-beta peptides and hyperphosphorylated tau. Other factors, including impairments in energy metabolism, increased oxidative stress, inflammation, insulin and IGF resistance, and insulin/IGF deficiency in the brain should be incorporated into all equations used to develop diagnostic and therapeutic approaches to AD. Herein, the contributions of impaired insulin and IGF signaling to AD-associated neuronal loss, synaptic disconnection, tau hyperphosphorylation, amyloid-beta accumulation, and impaired energy metabolism are reviewed. In addition, we discuss current therapeutic strategies and suggest additional approaches based on the hypothesis that AD is principally a metabolic disease similar to diabetes mellitus. Ultimately, our ability to effectively detect, monitor, treat, and prevent AD will require more efficient, accurate and integrative diagnostic tools that utilize clinical, neuroimaging, biochemical, and molecular biomarker data. Finally, it is imperative that future therapeutic strategies for AD abandon the concept of uni-modal therapy in favor of multi-modal treatments that target distinct impairments at different levels within the brain insulin/IGF signaling cascades. PMID:22329651

  19. Characterization of Hyaluronan-Degrading Enzymes from Yeasts.

    PubMed

    Smirnou, Dzianis; Krčmář, Martin; Kulhánek, Jaromír; Hermannová, Martina; Bobková, Lenka; Franke, Lukáš; Pepeliaev, Stanislav; Velebný, Vladimír

    2015-10-01

    Hyaluronidases (HAases) from yeasts were characterized for the first time. The study elucidated that hyaluronate 4-glycanohydrolase and hyaluronan (HA) lyase can be produced by yeasts. Six yeasts producing HAases were found through express screening of activities. The extracellular HAases from two of the yeast isolates, Pseudozyma aphidis and Cryptococcus laurentii, were characterized among them. P. aphidis HAase hydrolyzed β-1,4 glycosidic bonds of HA, yielding even-numbered oligosaccharides with N-acetyl-D-glucosamine at the reducing end. C. laurentii produced hyaluronan lyase, which cleaved β-1,4 glycosidic bonds of HA in β-elimination reaction, and the products of HA degradation were different-sized even-numbered oligosaccharides. The shortest detected HA oligomer was dimer. The enzymes' pH and temperature optima were pH 3.0 and 37-45 °C (P. aphidis) and pH 6.0 and 37 °C (C. laurentii), respectively. Both HAases showed good thermostability.

  20. Enhanced Gene Detection Assays for Fumarate-Adding Enzymes Allow Uncovering of Anaerobic Hydrocarbon Degraders in Terrestrial and Marine Systems

    PubMed Central

    von Netzer, Frederick; Pilloni, Giovanni; Kleindienst, Sara; Krüger, Martin; Knittel, Katrin; Gründger, Friederike

    2013-01-01

    The detection of anaerobic hydrocarbon degrader populations via catabolic gene markers is important for the understanding of processes at contaminated sites. Fumarate-adding enzymes (FAEs; i.e., benzylsuccinate and alkylsuccinate synthases) have already been established as specific functional marker genes for anaerobic hydrocarbon degraders. Several recent studies based on pure cultures and laboratory enrichments have shown the existence of new and deeply branching FAE gene lineages, such as clostridial benzylsuccinate synthases and homologues, as well as naphthylmethylsuccinate synthases. However, established FAE gene detection assays were not designed to target these novel lineages, and consequently, their detectability in different environments remains obscure. Here, we present a new suite of parallel primer sets for detecting the comprehensive range of FAE markers known to date, including clostridial benzylsuccinate, naphthylmethylsuccinate, and alkylsuccinate synthases. It was not possible to develop one single assay spanning the complete diversity of FAE genes alone. The enhanced assays were tested with a range of hydrocarbon-degrading pure cultures, enrichments, and environmental samples of marine and terrestrial origin. They revealed the presence of several, partially unexpected FAE gene lineages not detected in these environments before: distinct deltaproteobacterial and also clostridial bssA homologues as well as environmental nmsA homologues. These findings were backed up by dual-digest terminal restriction fragment length polymorphism diagnostics to identify FAE gene populations independently of sequencing. This allows rapid insights into intrinsic degrader populations and degradation potentials established in aromatic and aliphatic hydrocarbon-impacted environmental systems. PMID:23124238

  1. High Potential Source for Biomass Degradation Enzyme Discovery and Environmental Aspects Revealed through Metagenomics of Indian Buffalo Rumen

    PubMed Central

    Singh, K. M.; Reddy, Bhaskar; Patel, Dishita; Patel, A. K.; Patel, J. B.; Joshi, C. G.

    2014-01-01

    The complex microbiomes of the rumen functions as an effective system for plant cell wall degradation, and biomass utilization provide genetic resource for degrading microbial enzymes that could be used in the production of biofuel. Therefore the buffalo rumen microbiota was surveyed using shot gun sequencing. This metagenomic sequencing generated 3.9 GB of sequences and data were assembled into 137270 contiguous sequences (contigs). We identified potential 2614 contigs encoding biomass degrading enzymes including glycoside hydrolases (GH: 1943 contigs), carbohydrate binding module (CBM: 23 contigs), glycosyl transferase (GT: 373 contigs), carbohydrate esterases (CE: 259 contigs), and polysaccharide lyases (PE: 16 contigs). The hierarchical clustering of buffalo metagenomes demonstrated the similarities and dissimilarity in microbial community structures and functional capacity. This demonstrates that buffalo rumen microbiome was considerably enriched in functional genes involved in polysaccharide degradation with great prospects to obtain new molecules that may be applied in the biofuel industry. PMID:25136572

  2. Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis.

    PubMed

    Muro, Silvia; Schuchman, Edward H; Muzykantov, Vladimir R

    2006-01-01

    Enzyme replacement therapy, a state-of-the-art treatment for many lysosomal storage disorders, relies on carbohydrate-mediated binding of recombinant enzymes to receptors that mediate lysosomal delivery via clathrin-dependent endocytosis. Suboptimal glycosylation of recombinant enzymes and deficiency of clathrin-mediated endocytosis in some lysosomal enzyme-deficient cells limit delivery and efficacy of enzyme replacement therapy for lysosomal disorders. We explored a novel delivery strategy utilizing nanocarriers targeted to a glycosylation- and clathrin-independent receptor, intercellular adhesion molecule (ICAM)-1, a glycoprotein expressed on diverse cell types, up-regulated and functionally involved in inflammation, a hallmark of many lysosomal disorders. We targeted recombinant human acid sphingomyelinase (ASM), deficient in types A and B Niemann-Pick disease, to ICAM-1 by loading this enzyme to nanocarriers coated with anti-ICAM. Anti-ICAM/ASM nanocarriers, but not control ASM or ASM nanocarriers, bound to ICAM-1-positive cells (activated endothelial cells and Niemann-Pick disease patient fibroblasts) via ICAM-1, in a glycosylation-independent manner. Anti-ICAM/ASM nanocarriers entered cells via CAM-mediated endocytosis, bypassing the clathrin-dependent pathway, and trafficked to lysosomes, where delivered ASM displayed stable activity and alleviated lysosomal lipid accumulation. Therefore, lysosomal enzyme targeting using nanocarriers targeted to ICAM-1 bypasses defunct pathways and may improve the efficacy of enzyme replacement therapy for lysosomal disorders, such as Niemann-Pick disease.

  3. Metagenomic mining pectinolytic microbes and enzymes from an apple pomace-adapted compost microbial community.

    PubMed

    Zhou, Man; Guo, Peng; Wang, Tao; Gao, Lina; Yin, Huijun; Cai, Cheng; Gu, Jie; Lü, Xin

    2017-01-01

    Degradation of pectin in lignocellulosic materials is one of the key steps for biofuel production. Biological hydrolysis of pectin, i.e., degradation by pectinolytic microbes and enzymes, is an attractive paradigm because of its obvious advantages, such as environmentally friendly procedures, low in energy demand for lignin removal, and the possibility to be integrated in consolidated process. In this study, a metagenomics sequence-guided strategy coupled with enrichment culture technique was used to facilitate targeted discovery of pectinolytic microbes and enzymes. An apple pomace-adapted compost (APAC) habitat was constructed to boost the enrichment of pectinolytic microorganisms. Analyses of 16S rDNA high-throughput sequencing revealed that microbial communities changed dramatically during composting with some bacterial populations being greatly enriched. Metagenomics data showed that apple pomace-adapted compost microbial community (APACMC) was dominated by Proteobacteria and Bacteroidetes . Functional analysis and carbohydrate-active enzyme profiles confirmed that APACMC had been successfully enriched for the targeted functions. Among the 1756 putative genes encoding pectinolytic enzymes, 129 were predicted as novel (with an identity <30% to any CAZy database entry) and only 1.92% were more than 75% identical with proteins in NCBI environmental database, demonstrating that they have not been observed in previous metagenome projects. Phylogenetic analysis showed that APACMC harbored a broad range of pectinolytic bacteria and many of them were previously unrecognized. The immensely diverse pectinolytic microbes and enzymes found in our study will expand the arsenal of proficient degraders and enzymes for lignocellulosic biofuel production. Our study provides a powerful approach for targeted mining microbes and enzymes in numerous industries.

  4. Genome wide comprehensive analysis and web resource development on cell wall degrading enzymes from phyto-parasitic nematodes.

    PubMed

    Rai, Krishan Mohan; Balasubramanian, Vimal Kumar; Welker, Cassie Marie; Pang, Mingxiong; Hii, Mei Mei; Mendu, Venugopal

    2015-08-01

    The plant cell wall serves as a primary barrier against pathogen invasion. The success of a plant pathogen largely depends on its ability to overcome this barrier. During the infection process, plant parasitic nematodes secrete cell wall degrading enzymes (CWDEs) apart from piercing with their stylet, a sharp and hard mouthpart used for successful infection. CWDEs typically consist of cellulases, hemicellulases, and pectinases, which help the nematode to infect and establish the feeding structure or form a cyst. The study of nematode cell wall degrading enzymes not only enhance our understanding of the interaction between nematodes and their host, but also provides information on a novel source of enzymes for their potential use in biomass based biofuel/bioproduct industries. Although there is comprehensive information available on genome wide analysis of CWDEs for bacteria, fungi, termites and plants, but no comprehensive information available for plant pathogenic nematodes. Herein we have performed a genome wide analysis of CWDEs from the genome sequenced phyto pathogenic nematode species and developed a comprehensive publicly available database. In the present study, we have performed a genome wide analysis for the presence of CWDEs from five plant parasitic nematode species with fully sequenced genomes covering three genera viz. Bursaphelenchus, Glorodera and Meloidogyne. Using the Hidden Markov Models (HMM) conserved domain profiles of the respective gene families, we have identified 530 genes encoding CWDEs that are distributed among 24 gene families of glycoside hydrolases (412) and polysaccharide lyases (118). Furthermore, expression profiles of these genes were analyzed across the life cycle of a potato cyst nematode. Most genes were found to have moderate to high expression from early to late infectious stages, while some clusters were invasion stage specific, indicating the role of these enzymes in the nematode's infection and establishment process

  5. Prostate Cancer Relevant Antigens and Enzymes for Targeted Drug Delivery

    PubMed Central

    Barve, Ashutosh; Jin, Wei; Cheng, Kun

    2014-01-01

    Chemotherapy is one of the most widely used approaches in combating advanced prostate cancer, but its therapeutic efficacy is usually insufficient due to lack of specificity and associated toxicity. Lack of targeted delivery to prostate cancer cells is also the primary obstacles in achieving feasible therapeutic effect of other promising agents including peptide, protein, and nucleic acid. Consequently, there remains a critical need for strategies to increase the selectivity of anti-prostate cancer agents. This review will focus on various prostate cancer-specific antigens and enzymes that could be exploited for prostate cancer targeted drug delivery. Among various targeting strategies, active targeting is the most advanced approach to specifically deliver drugs to their designated cancer cells. In this approach, drug carriers are modified with targeting ligands that can specifically bind to prostate cancer-specific antigens. Moreover, there are several specific enzymes in the tumor microenvironment of prostate cancer that can be exploited for stimulus-responsive drug delivery systems. These systems can specifically release the active drug in the tumor microenvironment of prostate cancer, leading to enhanced tumor penetration efficiency. PMID:24878184

  6. Germline TRAV5D-4 T-Cell Receptor Sequence Targets a Primary Insulin Peptide of NOD Mice

    PubMed Central

    Nakayama, Maki; Castoe, Todd; Sosinowski, Tomasz; He, XiangLing; Johnson, Kelly; Haskins, Kathryn; Vignali, Dario A.A.; Gapin, Laurent; Pollock, David; Eisenbarth, George S.

    2012-01-01

    There is accumulating evidence that autoimmunity to insulin B chain peptide, amino acids 9–23 (insulin B:9–23), is central to development of autoimmune diabetes of the NOD mouse model. We hypothesized that enhanced susceptibility to autoimmune diabetes is the result of targeting of insulin by a T-cell receptor (TCR) sequence commonly encoded in the germline. In this study, we aimed to demonstrate that a particular Vα gene TRAV5D-4 with multiple junction sequences is sufficient to induce anti-islet autoimmunity by studying retrogenic mouse lines expressing α-chains with different Vα TRAV genes. Retrogenic NOD strains expressing Vα TRAV5D-4 α-chains with many different complementarity determining region (CDR) 3 sequences, even those derived from TCRs recognizing islet-irrelevant molecules, developed anti-insulin autoimmunity. Induction of insulin autoantibodies by TRAV5D-4 α-chains was abrogated by the mutation of insulin peptide B:9–23 or that of two amino acid residues in CDR1 and 2 of the TRAV5D-4. TRAV13–1, the human ortholog of murine TRAV5D-4, was also capable of inducing in vivo anti-insulin autoimmunity when combined with different murine CDR3 sequences. Targeting primary autoantigenic peptides by simple germline-encoded TCR motifs may underlie enhanced susceptibility to the development of autoimmune diabetes. PMID:22315318

  7. MicroRNA-99a inhibits insulin-induced proliferation, migration, dedifferentiation, and rapamycin resistance of vascular smooth muscle cells by inhibiting insulin-like growth factor-1 receptor and mammalian target of rapamycin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Zi-wei; Department of Cardiology, Kunming General Hospital of Chengdu Military Area; Guo, Rui-wei

    Patients with type 2 diabetes mellitus (T2DM) are characterized by insulin resistance and are subsequently at high risk for atherosclerosis. Hyperinsulinemia has been associated with proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) during the pathogenesis of atherosclerosis. Moreover, insulin-like growth factor-1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) have been demonstrated to be the underlying signaling pathways. Recently, microRNA-99a (miR-99a) has been suggested to regulate the phenotypic changes of VSMCs in cancer cells. However, whether it is involved in insulin-induced changes of VSCMs has not been determined. In this study, we found that insulin induced proliferation,more » migration, and dedifferentiation of mouse VSMCs in a dose-dependent manner. Furthermore, the stimulating effects of high-dose insulin on proliferation, migration, and dedifferentiation of mouse VSMCs were found to be associated with the attenuation of the inhibitory effects of miR-99a on IGF-1R and mTOR signaling activities. Finally, we found that the inducing effect of high-dose insulin on proliferation, migration, and dedifferentiation of VSMCs was partially inhibited by an active mimic of miR-99a. Taken together, these results suggest that miR-99a plays a key regulatory role in the pathogenesis of insulin-induced proliferation, migration, and phenotype conversion of VSMCs at least partly via inhibition of IGF-1R and mTOR signaling. Our results provide evidence that miR-99a may be a novel target for the treatment of hyperinsulinemia-induced atherosclerosis. - Highlights: • Suggesting a new mechanism of insulin-triggered VSMC functions. • Providing a new therapeutic strategies that target atherosclerosis in T2DM patients. • Providing a new strategies that target in-stent restenosis in T2DM patients.« less

  8. Quorum-Quenching and Matrix-Degrading Enzymes in Multilayer Coatings Synergistically Prevent Bacterial Biofilm Formation on Urinary Catheters.

    PubMed

    Ivanova, Kristina; Fernandes, Margarida M; Francesko, Antonio; Mendoza, Ernest; Guezguez, Jamil; Burnet, Michael; Tzanov, Tzanko

    2015-12-16

    Bacteria often colonize in-dwelling medical devices and grow as complex biofilm communities of cells embedded in a self-produced extracellular polymeric matrix, which increases their resistance to antibiotics and the host immune system. During biofilm growth, bacterial cells cooperate through specific quorum-sensing (QS) signals. Taking advantage of this mechanism of biofilm formation, we hypothesized that interrupting the communication among bacteria and simultaneously degrading the extracellular matrix would inhibit biofilm growth. To this end, coatings composed of the enzymes acylase and α-amylase, able to degrade bacterial QS molecules and polysaccharides, respectively, were built on silicone urinary catheters using a layer-by-layer deposition technique. Multilayer coatings of either acylase or amylase alone suppressed the biofilm formation of corresponding Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus. Further assembly of both enzymes in hybrid nanocoatings resulted in stronger biofilm inhibition as a function of acylase or amylase position in the layers. Hybrid coatings, with the QS-signal-degrading acylase as outermost layer, demonstrated 30% higher antibiofilm efficiency against medically relevant Gram-negative bacteria compared to that of the other assemblies. These nanocoatings significantly reduced the occurrence of single-species (P. aeruginosa) and mixed-species (P. aeruginosa and Escherichia coli) biofilms on silicone catheters under both static and dynamic conditions. Moreover, in an in vivo animal model, the quorum quenching and matrix degrading enzyme assemblies delayed the biofilm growth up to 7 days.

  9. Insulin‐degrading enzyme is genetically associated with Alzheimer's disease in the Finnish population

    PubMed Central

    Vepsäläinen, Saila; Parkinson, Michele; Helisalmi, Seppo; Mannermaa, Arto; Soininen, Hilkka; Tanzi, Rudolph E; Bertram, Lars; Hiltunen, Mikko

    2007-01-01

    The gene for insulin‐degrading enzyme (IDE), which is located at chromosome 10q24, has been previously proposed as a candidate gene for late‐onset Alzheimer's disease (AD) based on its ability to degrade amyloid β‐protein. Genotyping of single nucleotide polymorphisms (SNPs) in the IDE gene in Finnish patients with AD and controls revealed SNPs rs4646953 and rs4646955 to be associated with AD, conferring an approximately two‐fold increased risk. Single locus findings were corroborated by the results obtained from haplotype analyses. This suggests that genetic alterations in or near the IDE gene may increase the risk for developing AD. PMID:17496198

  10. MicroRNA-320 involves in the cardioprotective effect of insulin against myocardial ischemia by targeting survivin.

    PubMed

    Yang, Ni; Wu, Liuzhong; Zhao, Ying; Zou, Ning; Liu, Chunfeng

    2018-04-01

    It is generally accepted that insulin exerts an antiapoptotic effect against ischemia/reperfusion through the activation of PI3K/Akt/mTOR pathway. MicroRNAs involve in multiple cardiac pathophysiological processes, including ischemia/reperfusion-induced cardiac injury. However, the regulation of microRNAs in the cardioprotective effect of insulin is rarely discussed. In this study, using a cell model of ischemia through culturing H9C2 cardiac myocytes in serum-free medium with hypoxia, we demonstrated that pretreatment with insulin significantly inhibited cell apoptosis and downregulated microRNA-320 (miR-320) expression. Interestingly, miR-320 mimic impaired the cardioprotective effect of insulin against myocardial ischemia injury by targeting survivin, which is a member of the family of inhibitor of apoptosis proteins. Suppression miR-320 expression by miR-320 inhibitor in H9C2 cells with myocardial ischemia mimics the cardioprotective effect of insulin by maintaining survivin expression. Taken together, miR-320-mediated survivin expression involves in cardioprotective effect of insulin against myocardial ischemia injury. Myocardial ischemia/reperfusion (I/R) injury remains an important clinical problem with extremely deficient clinical therapies. Insulin exerts an antiapoptotic effect against I/R through the activation of PI3K/Akt/mTOR pathway. Here, we provided evidences to show that microRNA-320 involves in the cardioprotective effect of insulin by targeting survivin, which is an inhibitor of apoptosis protein and functions as a key regulator in cell apoptosis and involves in the tumour genesis and progression. Our findings may provide a new potential therapeutic strategy for I/R injury and ischemic heart disease. Copyright © 2018 John Wiley & Sons, Ltd.

  11. Efficacy and safety of once-daily insulin degludec/insulin aspart compared with once-daily insulin glargine in participants with Type 2 diabetes: a randomized, treat-to-target study.

    PubMed

    Kumar, S; Jang, H C; Demirağ, N G; Skjøth, T V; Endahl, L; Bode, B

    2017-02-01

    To investigate, in a 26-week, open-label, randomized, treat-to-target trial, the efficacy and safety of insulin degludec/insulin aspart (IDegAsp) once daily vs insulin glargine (IGlar) once daily in adults with Type 2 diabetes, inadequately controlled on basal insulin. Participants were randomized (1:1) to IDegAsp once daily or IGlar once daily in combination with existing oral antidiabetic drugs. IDegAsp once daily was administered with the main evening meal or the largest meal of the day (agreed at baseline); dosing time was maintained throughout the trial. Participants titrated their insulin dose weekly to a mean pre-breakfast self-measured plasma glucose target [3.9-4.9 mmol/l (70-89 mg/dl)]. IDegAsp once daily was non-inferior to IGlar once daily in reducing HbA 1c after 26 weeks [mean estimated treatment difference IDegAsp once daily - IGlar once daily: -0.03% (95% CI -0.20, 0.14)]. The evening meal glucose increment was significantly lower with IDegAsp once daily vs IGlar once daily [estimated treatment difference IDegAsp once daily - IGlar once daily: -1.32 mmol/l (95% CI -1.93, -0.72); P < 0.05]. The overall confirmed hypoglycaemia rate was higher with IDegAsp once daily (estimated rate ratio 1.43; 95% CI 1.07, 1.92; P < 0.05). The rate of nocturnal hypoglycaemia did not significantly differ between the IDegAsp and IGlar groups [estimated rate ratio 0.80 (95% CI 0.49, 1.30); not significant]. In participants with Type 2 diabetes inadequately controlled on basal insulin, IDegAsp once daily improved glycaemic control and was non-inferior to IGlar once daily. IDegAsp led to higher rates of overall hypoglycaemia than IGlar, with no significant difference in rates of nocturnal hypoglycaemia. © 2016 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.

  12. Optimisation of synergistic biomass-degrading enzyme systems for efficient rice straw hydrolysis using an experimental mixture design.

    PubMed

    Suwannarangsee, Surisa; Bunterngsook, Benjarat; Arnthong, Jantima; Paemanee, Atchara; Thamchaipenet, Arinthip; Eurwilaichitr, Lily; Laosiripojana, Navadol; Champreda, Verawat

    2012-09-01

    Synergistic enzyme system for the hydrolysis of alkali-pretreated rice straw was optimised based on the synergy of crude fungal enzyme extracts with a commercial cellulase (Celluclast™). Among 13 enzyme extracts, the enzyme preparation from Aspergillus aculeatus BCC 199 exhibited the highest level of synergy with Celluclast™. This synergy was based on the complementary cellulolytic and hemicellulolytic activities of the BCC 199 enzyme extract. A mixture design was used to optimise the ternary enzyme complex based on the synergistic enzyme mixture with Bacillus subtilis expansin. Using the full cubic model, the optimal formulation of the enzyme mixture was predicted to the percentage of Celluclast™: BCC 199: expansin=41.4:37.0:21.6, which produced 769 mg reducing sugar/g biomass using 2.82 FPU/g enzymes. This work demonstrated the use of a systematic approach for the design and optimisation of a synergistic enzyme mixture of fungal enzymes and expansin for lignocellulosic degradation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Thrombus Degradation by Fibrinolytic Enzyme of Stenotrophomonas sp. Originated from Indonesian Soybean-Based Fermented Food on Wistar Rats

    PubMed Central

    Tjandrawinata, Raymond R.

    2016-01-01

    Objective. To evaluate thrombus degrading effect of a fibrinolytic enzyme from food origin Stenotrophomonas sp. of Indonesia. Methods. Prior to animal study, the enzyme safety was tested using cell culture. The effect on expression of tissue plasminogen activator was also analysed in the cell culture. For in vivo studies, 25 Wistar rats were used: normal control, negative control, treatment groups with crude and semipurified enzyme given orally at 25 mg/kg, and positive control group which received Lumbrokinase at 25 mg/kg. Blood clot in the tail was induced by kappa carrageenan injection at 1 mg/kg BW. Results. Experiment with cell culture confirmed the enzyme safety at the concentration used and increased expression of tPA. Decreasing of thrombus was observed in the positive group down to 70.35 ± 23.11% of the negative control animals (100%). The thrombus observed in the crude enzyme treatment was down to 56.99 ± 15.95% and 71.5 ± 15.7% for semipurified enzyme. Scanning electron microscopy showed clearly that bood clots were found in the animals injected with kappa carrageenan; however, in the treatment and positive groups, the clot was much reduced. Conclusions. Oral treatment of enzyme from Stenotrophomonas sp. of Indonesian fermented food was capable of degrading thrombus induced in Wistar rats. PMID:27635131

  14. Alternative translation initiation of Caveolin-2 desensitizes insulin signaling through dephosphorylation of insulin receptor by PTP1B and causes insulin resistance.

    PubMed

    Kwon, Hayeong; Jang, Donghwan; Choi, Moonjeong; Lee, Jaewoong; Jeong, Kyuho; Pak, Yunbae

    2018-06-01

    Insulin resistance, defined as attenuated sensitivity responding to insulin, impairs insulin action. Direct causes and molecular mechanisms of insulin resistance have thus far remained elusive. Here we show that alternative translation initiation (ATI) of Caveolin-2 (Cav-2) regulates insulin sensitivity. Cav-2β isoform yielded by ATI desensitizes insulin receptor (IR) via dephosphorylation by protein-tyrosine phosphatase 1B (PTP1B), and subsequent endocytosis and lysosomal degradation of IR, causing insulin resistance. Blockage of Cav-2 ATI protects against insulin resistance by preventing Cav-2β-PTP1B-directed IR desensitization, thereby normalizing insulin sensitivity and glucose uptake. Our findings show that Cav-2β is a negative regulator of IR signaling, and identify a mechanism causing insulin resistance through control of insulin sensitivity via Cav-2 ATI. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Autodigestion: Proteolytic Degradation and Multiple Organ Failure in Shock

    PubMed Central

    Altshuler, Angelina E.; Kistler, Erik B.; Schmid-Schönbein, Geert W.

    2015-01-01

    There is currently no effective treatment for multiorgan failure following shock other than alleviation supportive care. A better understanding of the pathogenesis of these sequelae to shock is required. The intestine plays a central role in multiorgan failure. It was previously suggested that bacteria and their toxins are responsible for the organ failure seen in circulatory shock, but clinical trials in septic patients have not confirmed this hypothesis. Instead, we review here evidence that the digestive enzymes, synthesized in the pancreas and discharged into the small intestine as requirement for normal digestion, may play a role in multi-organ failure. These powerful enzymes are non-specific, highly concentrated and fully activated in the lumen of the intestine. During normal digestion they are compartmentalized in the lumen of the intestine by the mucosal epithelial barrier. However, if this barrier becomes permeable, e.g. in an ischemic state, the digestive enzymes escape into the wall of the intestine. They digest tissues in the mucosa and generate small molecular weight cytotoxic fragments such as unbound free fatty acids. Digestive enzymes may also escape into the systemic circulation and activate other degrading proteases. These proteases have the ability to clip the ectodomain of surface receptors and compromise their function; for example cleaving the insulin receptor causing insulin resistance. The combination of digestive enzymes and cytotoxic fragments leaking into the central circulation causes cell and organ dysfunction, and ultimately may lead to complete organ failure and death. We summarize current evidence suggesting that enteral blockade of digestive enzymes inside the lumen of the intestine may serve to reduce acute cell and organ damage and improve survival in experimental shock. PMID:26717111

  16. The transcription factor Prep1 controls hepatic insulin sensitivity and gluconeogenesis by targeting nuclear localization of FOXO1.

    PubMed

    Kulebyakin, Konstantin; Penkov, Dmitry; Blasi, Francesco; Akopyan, Zhanna; Tkachuk, Vsevolod

    2016-12-02

    Liver plays a key role in controlling body carbohydrate homeostasis by switching between accumulation and production of glucose and this way maintaining constant level of glucose in blood. Increased blood glucose level triggers release of insulin from pancreatic β-cells. Insulin represses hepatic glucose production and increases glucose accumulation. Insulin resistance is the main cause of type 2 diabetes and hyperglycemia. Currently thiazolidinediones (TZDs) targeting transcriptional factor PPARγ are used as insulin sensitizers for treating patients with type 2 diabetes. However, TZDs are reported to be associated with cardiovascular and liver problems and stimulate obesity. Thus, it is necessary to search new approaches to improve insulin sensitivity. A promising candidate is transcriptional factor Prep1, as it was shown earlier it could affect insulin sensitivity in variety of insulin-sensitive tissues. The aim of the present study was to evaluate a possible involvement of transcriptional factor Prep1 in control of hepatic glucose accumulation and production. We created mice with liver-specific Prep1 knockout and discovered that hepatocytes derived from these mice are much more sensitive to insulin, comparing to their WT littermates. Incubation of these cells with 100 nM insulin results in almost complete inhibition of gluconeogenesis, while in WT cells this repression is only partial. However, Prep1 doesn't affect gluconeogenesis in the absence of insulin. Also, we observed that nuclear content of gluconeogenic transcription factor FOXO1 was greatly reduced in Prep1 knockout hepatocytes. These findings suggest that Prep1 may control hepatic insulin sensitivity by targeting FOXO1 nuclear stability. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Degradation of Linuron and Some Other Herbicides and Fungicides by a Linuron-Inducible Enzyme Obtained from Bacillus sphaericus

    PubMed Central

    Engelhardt, G.; Wallnöfer, P. R.; Plapp, R.

    1971-01-01

    Linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea] induces the formation of an enzyme (acylamidase) responsible for the degradation of a large variety of different herbicides and fungicides of the acylanilide and phenylurea type. The former type is degraded at a rate at least 10 times higher than the latter. PMID:5119200

  18. Titration of basal insulin or immediate addition of rapid acting insulin in patients not at target using basal insulin supported oral antidiabetic treatment - A prospective observational study in 2202 patients.

    PubMed

    Siegmund, Thorsten; Pfohl, Martin; Forst, Thomas; Pscherer, Stefan; Bramlage, Peter; Foersch, Johannes; Borck, Anja; Seufert, Jochen

    Optimal treatment intensification strategies in patients with type-2 diabetes mellitus (T2DM) receiving basal insulin supported oral antidiabetic therapy (BOT) remain controversial. The objective of the present study was to compare outcomes of BOT-intensification by either the uptitration of long-acting insulin glargine or by the immediate addition of a rapid acting insulin analogue (RAIA). This was a prospective, observational, 24-week study in T2DM patients with BOT using insulin glargine and baseline glycated hemoglobin (HbA1c) between 7.0 and 8.5%. Patients were stratified by their physicians to one of the following treatment intensification strategies: Basal insulin titration to target with discretionary subsequent addition of RAIA at weeks 12 or 24 (GLAR), or immediate addition of RAIA at baseline (GLARplus). A total of 3266 patients were prescreened of whom 2202 fulfilled the selection criteria. Of these, 1684 patients were documented in the GLAR group and 518 in the GLARplus group. In the GLAR group, in 91 (5.5%) and 21 patients (1.3%) RAIA was added at weeks 12 and 24, respectively. The groups displayed similar baseline characteristics; except, mean diabetes duration was slightly shorter in the GLAR group (8.7 vs. 9.4 years). During the study, insulin glargine dose was increased from 18.7 to 26.4U (plus 7.7U) in GLAR and from 24.9 to 27.3U (plus 2.4U) in GLARplus patients. Mean RAIA dose was 9.6±4.7U at the final visit. After 24 weeks, HbA1c was reduced by 0.8 and 0.9% in the GLAR and GLARplus groups, respectively (both p<0.001). An HbA1c of ≤7.0% was achieved in 49.2% of GLAR and 48.5% of GLARplus patients. In both groups, we observed improvements in cardiovascular risk factors such as lipids and blood pressure. The rates of symptomatic (1.6 vs. 1.7%) and severe (0.18 vs. 0.19%) hypoglycemic episodes were low and comparable in both groups. These findings provide evidence that treatment intensification in patients with type 2 diabetes not at glycemic

  19. Liver Proteome in Diabetes Type 1 Rat Model: Insulin-Dependent and -Independent Changes.

    PubMed

    Braga, Camila Pereira; Boone, Cory H T; Grove, Ryan A; Adamcova, Dana; Fernandes, Ana Angélica Henrique; Adamec, Jiri; de Magalhães Padilha, Pedro

    2016-12-01

    Diabetes mellitus type 1 (DM1) is a major public health problem that continues to burden the healthcare systems worldwide, costing exponentially more as the epidemic grows. Innovative strategies and omics system diagnostics for earlier diagnosis or prognostication of DM1 are essential to prevent secondary complications and alleviate the associated economic burden. In a preclinical study design that involved streptozotocin (STZ)-induced DM1, insulin-treated STZ-induced DM1, and control rats, we characterized the insulin-dependent and -independent changes in protein profiles in liver samples. Digested proteins were subjected to LC-MS E for proteomic data. Progenesis QI data processing and analysis of variance were utilized for statistical analyses. We found 305 proteins with significantly altered abundance among the control, DM1, and insulin-treated DM1 groups (p < 0.05). These differentially regulated proteins were related to enzymes that function in key metabolic pathways and stress responses. For example, gluconeogenesis appeared to return to control levels in the DM1 group after insulin treatment, with the restoration of gluconeogenesis regulatory enzyme, FBP1. Insulin administration to DM1 rats also restored the blood glucose levels and enzymes of general stress and antioxidant response systems. These observations are crucial for insights on DM1 pathophysiology and new molecular targets for future clinical biomarkers, drug discovery, and development. Additionally, we underscore that proteomics offers much potential in preclinical biomarker discovery for diabetes as well as common complex diseases such as cancer, dementia, and infectious disorders.

  20. Comparative analysis of sugarcane bagasse metagenome reveals unique and conserved biomass-degrading enzymes among lignocellulolytic microbial communities.

    PubMed

    Mhuantong, Wuttichai; Charoensawan, Varodom; Kanokratana, Pattanop; Tangphatsornruang, Sithichoke; Champreda, Verawat

    2015-01-01

    As one of the most abundant agricultural wastes, sugarcane bagasse is largely under-exploited, but it possesses a great potential for the biofuel, fermentation, and cellulosic biorefinery industries. It also provides a unique ecological niche, as the microbes in this lignocellulose-rich environment thrive in relatively high temperatures (50°C) with varying microenvironments of aerobic surface to anoxic interior. The microbial community in bagasse thus presents a good resource for the discovery and characterization of new biomass-degrading enzymes; however, it remains largely unexplored. We have constructed a fosmid library of sugarcane bagasse and obtained the largest bagasse metagenome to date. A taxonomic classification of the bagasse metagenome reviews the predominance of Proteobacteria, which are also found in high abundance in other aerobic environments. Based on the functional characterization of biomass-degrading enzymes, we have demonstrated that the bagasse microbial community benefits from a large repertoire of lignocellulolytic enzymes, which allows them to digest different components of lignocelluoses into single molecule sugars. Comparative genomic analyses with other lignocellulolytic and non-lignocellulolytic metagenomes show that microbial communities are taxonomically separable by their aerobic "open" or anoxic "closed" environments. Importantly, a functional analysis of lignocellulose-active genes (based on the CAZy classifications) reveals core enzymes highly conserved within the lignocellulolytic group, regardless of their taxonomic compositions. Cellulases, in particular, are markedly more pronounced compared to the non-lignocellulolytic group. In addition to the core enzymes, the bagasse fosmid library also contains some uniquely enriched glycoside hydrolases, as well as a large repertoire of the newly defined auxiliary activity proteins. Our study demonstrates a conservation and diversification of carbohydrate-active genes among diverse

  1. Multiple E2 ubiquitin-conjugating enzymes regulate human cytomegalovirus US2-mediated immunoreceptor downregulation.

    PubMed

    van de Weijer, Michael L; Schuren, Anouk B C; van den Boomen, Dick J H; Mulder, Arend; Claas, Frans H J; Lehner, Paul J; Lebbink, Robert Jan; Wiertz, Emmanuel J H J

    2017-09-01

    Misfolded endoplasmic reticulum (ER) proteins are dislocated towards the cytosol and degraded by the ubiquitin-proteasome system in a process called ER-associated protein degradation (ERAD). During infection with human cytomegalovirus (HCMV), the viral US2 protein targets HLA class I molecules (HLA-I) for degradation via ERAD to avoid elimination by the immune system. US2-mediated degradation of HLA-I serves as a paradigm of ERAD and has facilitated the identification of TRC8 (also known as RNF139) as an E3 ubiquitin ligase. No specific E2 enzymes had previously been described for cooperation with TRC8. In this study, we used a lentiviral CRISPR/Cas9 library targeting all known human E2 enzymes to assess their involvement in US2-mediated HLA-I downregulation. We identified multiple E2 enzymes involved in this process, of which UBE2G2 was crucial for the degradation of various immunoreceptors. UBE2J2, on the other hand, counteracted US2-induced ERAD by downregulating TRC8 expression. These findings indicate the complexity of cellular quality control mechanisms, which are elegantly exploited by HCMV to elude the immune system. © 2017. Published by The Company of Biologists Ltd.

  2. Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo.

    PubMed

    Parisi, Federica; Riccardo, Sara; Daniel, Margaret; Saqcena, Mahesh; Kundu, Nandini; Pession, Annalisa; Grifoni, Daniela; Stocker, Hugo; Tabak, Esteban; Bellosta, Paola

    2011-09-27

    Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Our biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β ) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways

  3. Factors associated with reaching or not reaching target HbA1c after initiation of basal or premixed insulin in patients with type 2 diabetes.

    PubMed

    Scheen, A J; Schmitt, H; Jiang, H H; Ivanyi, T

    2017-02-01

    To evaluate factors associated with reaching or not reaching target glycated haemoglobin (HbA 1c ) levels by analysing the respective contributions of fasting hyperglycaemia (FHG), also referred to as basal hyperglycaemia, vs postprandial hyperglycaemia (PHG) before and after initiation of a basal or premixed insulin regimen in patients with type 2 diabetes. This post-hoc analysis of insulin-naïve patients in the DURABLE study randomised to receive either insulin glargine or insulin lispro mix 25 evaluated the percentages of patients achieving a target HbA 1c of <7.0% (<53mmol/mol) per baseline HbA 1c quartiles, and the effect of each insulin regimen on the relative contributions of PHG and FHG to overall hyperglycaemia. Patients had comparable demographic characteristics and similar HbA 1c and FHG values at baseline in each HbA 1c quartile regardless of whether they reached the target HbA 1c . The higher the HbA 1c quartile, the greater was the decrease in HbA 1c , but also the smaller the percentage of patients achieving the target HbA 1c . HbA 1c and FHG decreased more in patients reaching the target, resulting in significantly lower values at endpoint in all baseline HbA 1c quartiles with either insulin treatment. Patients not achieving the target HbA 1c had slightly higher insulin doses, but lower total hypoglycaemia rates. Smaller decreases in FHG were associated with not reaching the target HbA 1c , suggesting a need to increase basal or premixed insulin doses to achieve targeted fasting plasma glucose and improve patient response before introducing more intensive prandial insulin regimens. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  4. Induction Specificity and Catabolite Repression of the Early Enzymes in Camphor Degradation by Pseudomonas putida

    PubMed Central

    Hartline, Richard A.; Gunsalus, I. C.

    1971-01-01

    The ability of bornane and substituted bornanes to induce the early enzymes for d(+)-camphor degradation and control of these enzymes by catabolite repression were studied in a strain of a Pseudomonas putida. Bornane and 20 substituted bornane compounds showed induction. Of these 21 compounds, bornane and 8 of the substituted bornanes provided induction without supporting growth. Oxygen, but not nitrogen, enhanced the inductive potency of the unsubstituted bornane ring. All bornanedione isomers caused induction, and those with substituents on each of the three consecutive carbon atoms, including the methyl group at the bridgehead carbon, showed induction without supporting growth. Although it was not possible to obtain experimental data for a case of absolute gratuitous induction by compounds not supporting growth, indirect evidence in support of gratuitous induction is presented. It is proposed that the ability of P. putida to tolerate the unusually high degree of possible gratuitous induction observed for camphor catabolism may be related to the infrequent occurrence of bicyclic ring structures in nature. Survival of an organism with a broad specificity for gratuitous induction is discussed. Glucose and succinate, but not glutamate, produced catabolite repression of the early camphor-degrading enzymes. Pathway enzymes differ in their degree of sensitivity to succinate-provoked catabolite repression. The ability of a compound to produce catabolite repression is not, however, directly related to the duration of the lag period (diauxic lag) between growth on camphor and growth on the repressing compound. PMID:5573731

  5. The Inhibitory Effect of Natural Products on Protein Fibrillation May Be Caused by Degradation Products--A Study Using Aloin and Insulin.

    PubMed

    Lobbens, Eva S; Foderà, Vito; Nyberg, Nils T; Andersen, Kirsten; Jäger, Anna K; Jorgensen, Lene; van de Weert, Marco

    2016-01-01

    Protein fibrillation is the pathological hallmark of several neurodegenerative diseases and also complicates the manufacturing and use of protein drugs. As a case study, the inhibitory activity of the natural compound aloin against insulin fibrillation was investigated. Based on Thioflavin T assays, high-performance liquid chromatography and transmission electron microscopy it was found that a degradation product of aloin, formed over weeks of storage, was able to significantly inhibit insulin fibrillation. The activity of the stored aloin was significantly reduced in the presence of small amounts of sodium azide or ascorbic acid, suggesting the active compound to be an oxidation product. A high-performance liquid chromatography method and a liquid chromatography-mass spectrometry method were developed to investigate the degradation products in the aged aloin solution. We found that the major compounds in the solution were aloin A and aloin B. In addition, 10-hydroxy aloin and elgonica dimers were detected in smaller amounts. The identified compounds were isolated and tested for activity by means of Thioflavin T assays, but no activity was observed. Thus, the actual fibrillation inhibitor is an as yet unidentified and potentially metastable degradation product of aloin. These results suggest that degradation products, and in particular oxidation products, are to be considered thoroughly when natural products are investigated for activity against protein fibrillation.

  6. Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

    PubMed Central

    Duckworth, Benjamin P.; Nelson, Kathryn M.; Aldrich, Courtney C.

    2013-01-01

    Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases. PMID:22283817

  7. A New Strategy for Smoking Cessation: Characterization of a Bacterial Enzyme for the Degradation of Nicotine.

    PubMed

    Xue, Song; Schlosburg, Joel E; Janda, Kim D

    2015-08-19

    Smoking is the leading cause of preventable diseases; thus, effective smoking cessation aids are crucial for reducing the prevalence of cigarette smoking and smoking-related illnesses. In our current campaign we offer a nicotine-degrading enzyme from Pseudomonas putida, NicA2, a flavin-containing protein. To explore its potential, a kinetic evaluation of the enzyme was conducted, which included determination of K(m), k(cat), buffer/serum half-life, and thermostability. Additionally, the catabolism profile of NicA2 was elucidated to assess the potential toxicity of the nicotine-derived products. In characterizing the enzyme, a favorable biochemical profile of the enzyme was discovered, making NicA2 a prospective therapeutic candidate. This approach provides a new avenue for the field of nicotine addiction therapy.

  8. [Effect of new peptide bioregulators livagen and epitalon on enkephalin-degrading enzymes in human serum].

    PubMed

    Kost, N V; Sokolov, O Iu; Gabaeva, M V; Zolotarev, Iu A; Malinin, V V; Khavinson, V Kh

    2003-01-01

    The effect of new peptide bioregulators--Livagen (Lys-Glu-Asp-Ala) and Epitalon (Ala-Glu-Asp-Gly)--on endogenous opioid system was studied, particularly, their ability to change the activity of enkephalin-degrading enzymes from serum and interact with opioid receptors of the brain membrane fraction. Enkephalinase activity was assayed in vitro by the rate of 3H-Leu-enkephalin hydrolysis in the presence of the tested peptides. Livagen and Epitalon inhibited enkephalin-degrading enzymes from human serum. Livagen proved to be more efficient also as compared to well-known peptidase inhibitors such as puromycin, leupeptin, and D-PAM. The dose-inhibitory effect curves for Livagen and Epitalon were plotted; their IC50 equaled 20 and 500 microM, respectively. The interaction between the peptides and opioid receptors was estimated using a radioreceptor method with [3H][D-Ala2, D-Leu5]-enkephalin. No interaction was observed between the tested peptides and mu- or delta-opioid receptors of the membrane fraction from the rat brain.

  9. PDK1-dependent activation of atypical PKC leads to degradation of the p21 tumour modifier protein

    PubMed Central

    Scott, Mary T.; Ingram, Angela; Ball, Kathryn L.

    2002-01-01

    p21WAF1/CIP1 contributes to positive and negative growth control on multiple levels. We previously mapped phosphorylation sites within the C-terminal domain of p21 that regulate proliferating cell nucear antigen binding. In the current study, a kinase has been fractionated from mammalian cells that stoichiometrically phosphorylates p21 at the Ser146 site, and the enzyme has been identified as an insulin-responsive atypical protein kinase C (aPKC). Expression of PKCζ or activation of the endogenous kinase by 3-phosphoinositide dependent protein kinase-1 (PDK1) decreased the half-life of p21. Conversely, dnPKCζ or dnPDK1 increased p21 protein half-life, and a PDK1-dependent increase in the rate of p21 degradation was mediated by aPKC. Insulin stimulation gave a biphasic response with a rapid transient decrease in p21 protein levels during the initial signalling phase that was dependent on phosphatidylinositol 3- kinase, PKC and proteasome activity. Thus, aPKC provides a physiological signal for the degradation of p21. The rapid degradation of p21 protein during the signalling phase of insulin stimulation identifies a novel link between energy metabolism and a key modulator of cell cycle progression. PMID:12485998

  10. [Effect of soy isoflavone on gene expression of leptin and insulin sensibility in insulin-resistant rats].

    PubMed

    Chen, Shi-wei; Zhang, Li-shi; Zhang, Hong-min; Feng, Xiao-fan; Peng, Xiao-li

    2006-04-18

    To explore the effects of soy isoflavone (SIF) on gene expression of leptin and insulin sensibility in insulin-resistant (IR) rats induced by high-fat, and to reveal the mechanisms of SIF in ameliorating insulin sensibility. IR rats were randomly divided into four groups based on their insulin-resistant indexes (IRI): one model control group and three SIF groups that were gavaged with water solutions with SIF at doses of 0 mg/kg, 50 mg/kg, 150 mg/kg, and 450 mg/kg, respectively. After one month, fasting glucose, fasting insulin, leptin in serum, and leptin mRNA in the perirenal adipocyte were detected by enzymic method, radioimmunoassay, enzyme linked immunosorbent assay, and real time quantitative RT-PCR, respectively. The model control group was used to compare against the other groups: (1) Insulin and IRI were lower in the 150 mg/kg and 450 mg/kg groups; (2) In the 450 mg/kg group, body weight and leptin mRNA expression were lower, serum leptin content was higher. These results indicate that soy isoflavone might decrease body weight of rats and leptin mRNA, increase serum leptin level, and ameliorate leptin and insulin sensitivities.

  11. The transcription factor Prep1 controls hepatic insulin sensitivity and gluconeogenesis by targeting nuclear localization of FOXO1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kulebyakin, Konstantin; Penkov, Dmitry; IFOM – the FIRC Institute of Molecular Oncology, Via Adamello 16, Milan, 20139

    Liver plays a key role in controlling body carbohydrate homeostasis by switching between accumulation and production of glucose and this way maintaining constant level of glucose in blood. Increased blood glucose level triggers release of insulin from pancreatic β-cells. Insulin represses hepatic glucose production and increases glucose accumulation. Insulin resistance is the main cause of type 2 diabetes and hyperglycemia. Currently thiazolidinediones (TZDs) targeting transcriptional factor PPARγ are used as insulin sensitizers for treating patients with type 2 diabetes. However, TZDs are reported to be associated with cardiovascular and liver problems and stimulate obesity. Thus, it is necessary to searchmore » new approaches to improve insulin sensitivity. A promising candidate is transcriptional factor Prep1, as it was shown earlier it could affect insulin sensitivity in variety of insulin-sensitive tissues. The aim of the present study was to evaluate a possible involvement of transcriptional factor Prep1 in control of hepatic glucose accumulation and production. We created mice with liver-specific Prep1 knockout and discovered that hepatocytes derived from these mice are much more sensitive to insulin, comparing to their WT littermates. Incubation of these cells with 100 nM insulin results in almost complete inhibition of gluconeogenesis, while in WT cells this repression is only partial. However, Prep1 doesn't affect gluconeogenesis in the absence of insulin. Also, we observed that nuclear content of gluconeogenic transcription factor FOXO1 was greatly reduced in Prep1 knockout hepatocytes. These findings suggest that Prep1 may control hepatic insulin sensitivity by targeting FOXO1 nuclear stability. - Highlights: • A novel model of liver-specific Prep1 knockout is established. • Ablation of Prep1 in hepatocytes increases insulin sensitivity. • Prep1 controls hepatic insulin sensitivity by regulating localization of FOXO1. • Prep1 regulates

  12. Identification of BACE2 as an avid ß-amyloid-degrading protease

    PubMed Central

    2012-01-01

    Background Proteases that degrade the amyloid ß-protein (Aß) have emerged as key players in the etiology and potential treatment of Alzheimer’s disease (AD), but it is unlikely that all such proteases have been identified. To discover new Aß-degrading proteases (AßDPs), we conducted an unbiased, genome-scale, functional cDNA screen designed to identify proteases capable of lowering net Aß levels produced by cells, which were subsequently characterized for Aß-degrading activity using an array of downstream assays. Results The top hit emerging from the screen was ß-site amyloid precursor protein-cleaving enzyme 2 (BACE2), a rather unexpected finding given the well-established role of its close homolog, BACE1, in the production of Aß. BACE2 is known to be capable of lowering Aß levels via non-amyloidogenic processing of APP. However, in vitro, BACE2 was also found to be a particularly avid AßDP, with a catalytic efficiency exceeding all known AßDPs except insulin-degrading enzyme (IDE). BACE1 was also found to degrade Aß, albeit ~150-fold less efficiently than BACE2. Aß is cleaved by BACE2 at three peptide bonds—Phe19-Phe20, Phe20-Ala21, and Leu34-Met35—with the latter cleavage site being the initial and principal one. BACE2 overexpression in cultured cells was found to lower net Aß levels to a greater extent than multiple, well-established AßDPs, including neprilysin (NEP) and endothelin-converting enzyme-1 (ECE1), while showing comparable effectiveness to IDE. Conclusions This study identifies a new functional role for BACE2 as a potent AßDP. Based on its high catalytic efficiency, its ability to degrade Aß intracellularly, and other characteristics, BACE2 represents a particulary strong therapeutic candidate for the treatment or prevention of AD. PMID:22986058

  13. Potential of semiarid soil from Caatinga biome as a novel source for mining lignocellulose-degrading enzymes.

    PubMed

    Lacerda Júnior, Gileno V; Noronha, Melline F; de Sousa, Sanderson Tarciso P; Cabral, Lucélia; Domingos, Daniela F; Sáber, Mírian L; de Melo, Itamar S; Oliveira, Valéria M

    2017-02-01

    The litterfall is the major organic material deposited in soil of Brazilian Caatinga biome, thus providing the ideal conditions for plant biomass-degrading microorganisms to thrive. Herein, the phylogenetic composition and lignocellulose-degrading capacity have been explored for the first time from a fosmid library dataset of Caatinga soil by sequence-based screening. A complex bacterial community dominated by Proteobacteria and Actinobacteria was unraveled. SEED subsystems-based annotations revealed a broad range of genes assigned to carbohydrate and aromatic compounds metabolism, indicating microbial ability to utilize plant-derived material. CAZy-based annotation identified 7275 genes encoding 37 glycoside hydrolases (GHs) families related to hydrolysis of cellulose, hemicellulose, oligosaccharides and other lignin-modifying enzymes. Taxonomic affiliation of genes showed high genetic potential of the phylum Acidobacteria for hemicellulose degradation, whereas Actinobacteria members appear to play an important role in celullose hydrolysis. Additionally, comparative analyses revealed greater GHs profile similarity among soils as compared to the digestive tract of animals capable of digesting plant biomass, particularly in the hemicellulases content. Combined results suggest a complex synergistic interaction of community members required for biomass degradation into fermentable sugars. This large repertoire of lignocellulolytic enzymes opens perspectives for mining potential candidates of biochemical catalysts for biofuels production from renewable resources and other environmental applications. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Down-regulation of the miR-543 alleviates insulin resistance through targeting the SIRT1.

    PubMed

    Hu, Xiaojing; Chi, Liyi; Zhang, Wentao; Bai, Tiao; Zhao, Wei; Feng, Zhanbin; Tian, Hongyan

    2015-12-25

    Insulin resistance plays an important role in the development of hypertension, which is seriously detrimental to human health. Recently, Sirtuin-1 (SIRT1) has been found to participate in regulation of insulin resistance. Therefore, further studies focused on the SIRT1 regulators might provide a potential approach for combating insulin resistance and hypertension. Interestingly, in this study, we found that SIRT1 was the target gene of the miR-543 by the Dual-Luciferase Reporter Assay. Moreover, the miR-543 expression notably increased in the insulin-resistant HepG2 cells induced by TNF-α. Further analysis showed that the overexpression of the miR-543 lowered the SIRT1 mRNA and protein levels, resulting in the insulin resistance in the HepG2 cells; the inhibition of miR-543, however, enhanced the mRNA and protein expression of the SIRT1, and alleviated the insulin resistance. Furthermore, the SIRT1 overexpression abrogated the effect of miR-543 on insulin resistance. In addition, the overexpression of the miR-543 by the lentivirus-mediated gene transfer markedly impaired the insulin signaling assessed by the Western blot analysis of the glycogen synthesis and the phosphorylation of Akt and GSK3β. In summary, our study suggested that the downregulation of the miR-543 could alleviate the insulin resistance via the modulation of the SIRT1 expression, which might be a potential new strategy for treating insulin resistance and a promising therapeutic method for hypertension. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Development of a genetically programed vanillin-sensing bacterium for high-throughput screening of lignin-degrading enzyme libraries.

    PubMed

    Sana, Barindra; Chia, Kuan Hui Burton; Raghavan, Sarada S; Ramalingam, Balamurugan; Nagarajan, Niranjan; Seayad, Jayasree; Ghadessy, Farid J

    2017-01-01

    Lignin is a potential biorefinery feedstock for the production of value-added chemicals including vanillin. A huge amount of lignin is produced as a by-product of the paper industry, while cellulosic components of plant biomass are utilized for the production of paper pulp. In spite of vast potential, lignin remains the least exploited component of plant biomass due to its extremely complex and heterogenous structure. Several enzymes have been reported to have lignin-degrading properties and could be potentially used in lignin biorefining if their catalytic properties could be improved by enzyme engineering. The much needed improvement of lignin-degrading enzymes by high-throughput selection techniques such as directed evolution is currently limited, as robust methods for detecting the conversion of lignin to desired small molecules are not available. We identified a vanillin-inducible promoter by RNAseq analysis of Escherichia coli cells treated with a sublethal dose of vanillin and developed a genetically programmed vanillin-sensing cell by placing the 'very green fluorescent protein' gene under the control of this promoter. Fluorescence of the biosensing cell is enhanced significantly when grown in the presence of vanillin and is readily visualized by fluorescence microscopy. The use of fluorescence-activated cell sorting analysis further enhances the sensitivity, enabling dose-dependent detection of as low as 200 µM vanillin. The biosensor is highly specific to vanillin and no major response is elicited by the presence of lignin, lignin model compound, DMSO, vanillin analogues or non-specific toxic chemicals. We developed an engineered E. coli cell that can detect vanillin at a concentration as low as 200 µM. The vanillin-sensing cell did not show cross-reactivity towards lignin or major lignin degradation products including vanillin analogues. This engineered E. coli cell could potentially be used as a host cell for screening lignin-degrading enzymes that

  16. XRN2 is required for the degradation of target RNAs by RNase H1-dependent antisense oligonucleotides

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hori, Shin-Ichiro; Yamamoto, Tsuyoshi; Obika, Satoshi, E-mail: obika@phs.osaka-u.ac.jp

    Antisense oligonucleotides (ASOs) can suppress the expression of a target gene by cleaving pre-mRNA and/or mature mRNA via RNase H1. Following the initial endonucleolytic cleavage by RNase H1, the target RNAs are degraded by a mechanism that is poorly understood. To better understand this degradation pathway, we depleted the expression of two major 5′ to 3′ exoribonucleases (XRNs), named XRN1 and XRN2, and analyzed the levels of 3′ fragments of the target RNAs in vitro. We found that the 3′ fragments of target pre-mRNA generated by ASO were almost completely degraded from their 5′ ends by nuclear XRN2 after RNase H1-mediatedmore » cleavage, whereas the 3′ fragments of mature mRNA were partially degraded by XRN2. In contrast to ASO, small interference RNA (siRNA) could reduce the expression level of only mature mRNA, and the 3′ fragment was degraded by cytoplasmic XRN1. Our findings indicate that the RNAs targeted by RNase H1-dependent ASO are rapidly degraded in the nucleus, contrary to the cytoplasmic degradation pathway mediated by siRNA. - Highlights: • We compared the degradation mechanism of the transcript targeted by ASO and siRNA. • We focused on two 5′ to 3′ exoribonucleases, cytoplasmic XRN1, and nuclear XRN2. • The 3′ fragment of target pre-mRNA generated by ASO was degraded by XRN2. • The 3′ fragment of target mRNA generated by ASO was partially degraded by XRN2. • XRN1 depletion promoted accumulation of the 3′ fragment of mRNA generated by siRNA.« less

  17. Effect of Pterocarpus santalinus bark, on blood glucose, serum lipids, plasma insulin and hepatic carbohydrate metabolic enzymes in streptozotocin-induced diabetic rats.

    PubMed

    Kondeti, Vinay Kumar; Badri, Kameswara Rao; Maddirala, Dilip Rajasekhar; Thur, Sampath Kumar Mekala; Fatima, Shaik Sameena; Kasetti, Ramesh Babu; Rao, Chippada Appa

    2010-05-01

    The present study was designed to investigate the effect of bark of Pterocarpus santalinus, an ethnomedicinal plant, on blood glucose, plasma insulin, serum lipids and the activities of hepatic glucose metabolizing enzymes in streptozotocin-induced diabetic rats. Streptozotocin-induced diabetic rats were treated (acute/short-term and long-term) with ethyl acetate:methanol fractions of ethanolic extract of the bark of P. santalinus. Fasting blood glucose, HbA(1C), plasma insulin and protein were estimated before and after the treatment, along with hepatic glycogen, and activities of hexokinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase and glucose-6-phosphate dehydrogenase. Further anti-hyperlipidemic activity was studied by measuring the levels of serum lipids and lipoproteins. Phytochemical analysis of active fraction showed the presence of flavonoids, glycosides and phenols. Biological testing of the active fraction demonstrated a significant antidiabetic activity by reducing the elevated blood glucose levels and glycosylated hemoglobin, improving hyperlipidemia and restoring the insulin levels in treated experimental induced diabetic rats. Further elucidation of mechanism of action showed improvement in the hepatic carbohydrate metabolizing enzymes after the treatment. Our present investigation suggests that active fraction of ethanolic extract of bark of P. santalinus decreases streptozotocin induced hyperglycemia by increasing glycolysis and decreasing gluconeogenesis. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  18. Genomewide analysis of polysaccharides degrading enzymes in 11 white- and brown-rot Polyporales provides insight into mechanisms of wood decay

    Treesearch

    Chiaki Hori; Jill Gaskell; Kiyohiko Igarashi; Masahiro Samejima; David Hibbett; Bernard Henrissat; Dan Cullen

    2013-01-01

    To degrade the polysaccharides, wood-decay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrate-active enzymes (CAZys) and their appended carbohydrate-binding modules (CBM). Oxidative enzymes, such as cellobiose dehydrogenase (CDH) and lytic polysaccharide monooxygenase (...

  19. Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance.

    PubMed

    Koves, Timothy R; Ussher, John R; Noland, Robert C; Slentz, Dorothy; Mosedale, Merrie; Ilkayeva, Olga; Bain, James; Stevens, Robert; Dyck, Jason R B; Newgard, Christopher B; Lopaschuk, Gary D; Muoio, Deborah M

    2008-01-01

    Previous studies have suggested that insulin resistance develops secondary to diminished fat oxidation and resultant accumulation of cytosolic lipid molecules that impair insulin signaling. Contrary to this model, the present study used targeted metabolomics to find that obesity-related insulin resistance in skeletal muscle is characterized by excessive beta-oxidation, impaired switching to carbohydrate substrate during the fasted-to-fed transition, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle. In cultured myotubes, lipid-induced insulin resistance was prevented by manipulations that restrict fatty acid uptake into mitochondria. These results were recapitulated in mice lacking malonyl-CoA decarboxylase (MCD), an enzyme that promotes mitochondrial beta-oxidation by relieving malonyl-CoA-mediated inhibition of carnitine palmitoyltransferase 1. Thus, mcd(-/-) mice exhibit reduced rates of fat catabolism and resist diet-induced glucose intolerance despite high intramuscular levels of long-chain acyl-CoAs. These findings reveal a strong connection between skeletal muscle insulin resistance and lipid-induced mitochondrial stress.

  20. Insulin-like growth factor 1, liver enzymes, and insulin resistance in patients with PCOS and hirsutism.

    PubMed

    Çakir, Evrim; Topaloğlu, Oya; Çolak Bozkurt, Nujen; Karbek Bayraktar, Başak; Güngüneş, Aşkın; Sayki Arslan, Müyesser; Öztürk Ünsal, İlknur; Tutal, Esra; Uçan, Bekir; Delıbaşi, Tuncay

    2014-01-01

    Hyperinsulinemia and insulin resistance are commonly seen in patients with hirsutism and polycystic ovary syndrome (PCOS), and are associated with cardiovascular disease risk. However, it is not yet known whether insulin-like growth factor I (IGF-I) and alanine transaminase (ALT) produced by the liver play roles in hyperinsulinemia and subclinical atherosclerotic process in patients with PCOS and idiopathic hirsutism (IH). This was a prospective case-controlled study. The study population consisted of 25 reproductive-age PCOS women, 33 women with IH, and 25 control subjects. Mean IGF-I levels and median ALT levels were higher in patients with IH and PCOS than controls, but these differences were not statistically significant. The participants who had a homeostasis model assessment insulin resistance index (HOMA-IR) greater than 2.7 had significantly higher IGF-1 and ALT levels. ALT levels were positively correlated with body mass index, FG, insulin and HOMA-IR. The study illustrated that IGF-1 and ALT levels were significantly higher in patients with increased insulin resistance. Due to short disease duration in younger participants, we did not observe any correlation between IGF-1 and hyperinsulinemia. These findings suggest that increased hepatic production of IGF-I and ALT might be an early indicator of insulin resistance in hirsutism.

  1. Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells

    PubMed Central

    Azizi, Paymon M.; Zyla, Roman E.; Guan, Sha; Wang, Changsen; Liu, Jun; Bolz, Steffen-Sebastian; Heit, Bryan; Klip, Amira; Lee, Warren L.

    2015-01-01

    Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel–derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to study insulin transcytosis across individual, primary human adipose microvascular endothelial cells (HAMECs), involving insulin uptake followed by vesicle-mediated exocytosis visualized by total internal reflection fluorescence microscopy. In this setting, fluorophore-conjugated insulin exocytosis depended on its initial binding and uptake, which was saturable and much greater than in muscle cells. Unlike its degradation within muscle cells, insulin was stable within HAMECs and escaped lysosomal colocalization. Insulin transcytosis required dynamin but was unaffected by caveolin-1 knockdown or cholesterol depletion. Instead, insulin transcytosis was significantly inhibited by the clathrin-mediated endocytosis inhibitor Pitstop 2 or siRNA-mediated clathrin depletion. Accordingly, insulin internalized for 1 min in HAMECs colocalized with clathrin far more than with caveolin-1. This study constitutes the first evidence of vesicle-mediated insulin transcytosis and highlights that its initial uptake is clathrin dependent and caveolae independent. PMID:25540431

  2. The selenazal drug ebselen potently inhibits indoleamine 2,3-dioxygenase by targeting enzyme cysteine residues.

    PubMed

    Terentis, Andrew C; Freewan, Mohammed; Sempértegui Plaza, Tito S; Raftery, Mark J; Stocker, Roland; Thomas, Shane R

    2010-01-26

    The heme enzyme indoleamine 2,3-dioxygenase (IDO) plays an important immune regulatory role by catalyzing the oxidative degradation of l-tryptophan. Here we show that the selenezal drug ebselen is a potent IDO inhibitor. Exposure of human macrophages to ebselen inhibited IDO activity in a manner independent of changes in protein expression. Ebselen inhibited the activity of recombinant human IDO (rIDO) with an apparent inhibition constant of 94 +/- 17 nM. Optical and resonance Raman spectroscopy showed that ebselen altered the active site heme of rIDO by inducing a transition of the ferric heme iron from the predominantly high- to low-spin form and by lowering the vibrational frequency of the Fe-CO stretch of the CO complex, indicating an opening of the distal heme pocket. Substrate binding studies showed that ebselen enhanced nonproductive l-tryptophan binding, while circular dichroism indicated that the drug reduced the helical content and protein stability of rIDO. Thiol labeling and mass spectrometry revealed that ebselen reacted with multiple cysteine residues of IDO. Removal of cysteine-bound ebselen with dithiothreitol reversed the effects of the drug on the heme environment and significantly restored enzyme activity. These findings indicate that ebselen inhibits IDO activity by reacting with the enzyme's cysteine residues that result in changes to protein conformation and active site heme, leading to an increase in the level of nonproductive substrate binding. This study highlights that modification of cysteine residues is a novel and effective means of inhibiting IDO activity. It also suggests that IDO is under redox control and that the enzyme represents a previously unrecognized in vivo target of ebselen.

  3. Arterial gastroduodenal infusion of cholecystokinin-33 stimulates the exocrine pancreatic enzyme release via an enteropancreatic reflex, without affecting the endocrine insulin secretion in pigs.

    PubMed

    Rengman, Sofia; Weström, Björn; Ahrén, Bo; Pierzynowski, Stefan G

    2009-03-01

    Cholecystokinin (CCK)-dependent exocrine pancreatic regulation seems to involve different pathways in different species. The aims were to explore the enteropancreatic reflex in the CCK-mediated regulation of the exocrine pancreas and to evaluate a possible involvement of this reflex in the endocrine insulin release. In anesthetized pigs, CCK-33 in increasing doses (4-130 pmol kg 10 min) was infused locally to the gastroduodenal artery, or systemically via the jugular vein. Also, a low CCK-33 dose (13 pmol kg) was injected to the duodenum/antrum area before and after a bilateral truncal vagotomy. Cholecystokinin-33 in the physiological dose range 4 to 32 pmol kg 10 min increased protein and trypsin outputs after local infusion to the antral-duodenal area, whereas it had no effect after systemic infusion. Cholecystokinin-33 in the pharmacological dose range 64 to 130 pmol kg 10 min further increased the secretion after both local and systemic infusions. Only CCK-33 infusions in the pharmacological dose range were able to elevate the plasma insulin levels. Vagotomy had no effect on CCK-33-mediated stimulation of the enzyme release, whereas it had a significant effect on the plasma insulin level. Cholecystokinin-33 in the physiological dose range 4 to 32 pmol kg 10 min stimulates the enzyme secretion but had no effect on the insulin release via a short enteropancreatic pathway in pigs.

  4. Targeted Overexpression of Inducible 6-Phosphofructo-2-kinase in Adipose Tissue Increases Fat Deposition but Protects against Diet-induced Insulin Resistance and Inflammatory Responses*

    PubMed Central

    Huo, Yuqing; Guo, Xin; Li, Honggui; Xu, Hang; Halim, Vera; Zhang, Weiyu; Wang, Huan; Fan, Yang-Yi; Ong, Kuok Teong; Woo, Shih-Lung; Chapkin, Robert S.; Mashek, Douglas G.; Chen, Yanming; Dong, Hui; Lu, Fuer; Wei, Lai; Wu, Chaodong

    2012-01-01

    Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues. PMID:22556414

  5. A viral deubiquitylating enzyme targets viral RNA-dependent RNA polymerase and affects viral infectivity

    PubMed Central

    Chenon, Mélanie; Camborde, Laurent; Cheminant, Soizic; Jupin, Isabelle

    2012-01-01

    Selective protein degradation via the ubiquitin-proteasome system (UPS) plays an essential role in many major cellular processes, including host–pathogen interactions. We previously reported that the tightly regulated viral RNA-dependent RNA polymerase (RdRp) of the positive-strand RNA virus Turnip yellow mosaic virus (TYMV) is degraded by the UPS in infected cells, a process that affects viral infectivity. Here, we show that the TYMV 98K replication protein can counteract this degradation process thanks to its proteinase domain. In-vitro assays revealed that the recombinant proteinase domain is a functional ovarian tumour (OTU)-like deubiquitylating enzyme (DUB), as is the 98K produced during viral infection. We also demonstrate that 98K mediates in-vivo deubiquitylation of TYMV RdRp protein—its binding partner within replication complexes—leading to its stabilization. Finally, we show that this DUB activity contributes to viral infectivity in plant cells. The identification of viral RdRp as a specific substrate of the viral DUB enzyme thus reveals the intricate interplay between ubiquitylation, deubiquitylation and the interaction between viral proteins in controlling levels of RdRp and viral infectivity. PMID:22117220

  6. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    NASA Astrophysics Data System (ADS)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  7. Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes.

    PubMed

    Hagberg, Carolina E; Mehlem, Annika; Falkevall, Annelie; Muhl, Lars; Fam, Barbara C; Ortsäter, Henrik; Scotney, Pierre; Nyqvist, Daniel; Samén, Erik; Lu, Li; Stone-Elander, Sharon; Proietto, Joseph; Andrikopoulos, Sofianos; Sjöholm, Ake; Nash, Andrew; Eriksson, Ulf

    2012-10-18

    The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved β-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.

  8. Uronic polysaccharide degrading enzymes.

    PubMed

    Garron, Marie-Line; Cygler, Miroslaw

    2014-10-01

    In the past several years progress has been made in the field of structure and function of polysaccharide lyases (PLs). The number of classified polysaccharide lyase families has increased to 23 and more detailed analysis has allowed the identification of more closely related subfamilies, leading to stronger correlation between each subfamily and a unique substrate. The number of as yet unclassified polysaccharide lyases has also increased and we expect that sequencing projects will allow many of these unclassified sequences to emerge as new families. The progress in structural analysis of PLs has led to having at least one representative structure for each of the families and for two unclassified enzymes. The newly determined structures have folds observed previously in other PL families and their catalytic mechanisms follow either metal-assisted or Tyr/His mechanisms characteristic for other PL enzymes. Comparison of PLs with glycoside hydrolases (GHs) shows several folds common to both classes but only for the β-helix fold is there strong indication of divergent evolution from a common ancestor. Analysis of bacterial genomes identified gene clusters containing multiple polysaccharide cleaving enzymes, the Polysaccharides Utilization Loci (PULs), and their gene complement suggests that they are organized to process completely a specific polysaccharide. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Degradation of toluene by ortho cleavage enzymes in Burkholderia fungorum FLU100

    PubMed Central

    Dobslaw, Daniel; Engesser, Karl-Heinrich

    2015-01-01

    Burkholderia fungorum FLU100 simultaneously oxidized any mixture of toluene, benzene and mono-halogen benzenes to (3-substituted) catechols with a selectivity of nearly 100%. Further metabolism occurred via enzymes of ortho cleavage pathways with complete mineralization. During the transformation of 3-methylcatechol, 4-carboxymethyl-2-methylbut-2-en-4-olide (2-methyl-2-enelactone, 2-ML) accumulated transiently, being further mineralized only after a lag phase of 2 h in case of cells pre-grown on benzene or mono-halogen benzenes. No lag phase, however, occurred after growth on toluene. Cultures inhibited by chloramphenicol after growth on benzene or mono-halogen benzenes were unable to metabolize 2-ML supplied externally, even after prolonged incubation. A control culture grown with toluene did not show any lag phase and used 2-ML as a substrate. This means that 2-ML is an intermediate of toluene degradation and converted by specific enzymes. The conversion of 4-methylcatechol as a very minor by-product of toluene degradation in strain FLU100 resulted in the accumulation of 4-carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone, 4-ML) as a dead-end product, excluding its nature as a possible intermediate. Thus, 3-methylcyclohexa-3,5-diene-1,2-diol, 3-methylcatechol, 2-methyl muconate and 2-ML were identified as central intermediates of productive ortho cleavage pathways for toluene metabolism in B. fungorum FLU100. PMID:25130674

  10. Insulin degludec and insulin aspart: novel insulins for the management of diabetes mellitus

    PubMed Central

    Atkin, Stephen; Javed, Zeeshan; Fulcher, Gregory

    2015-01-01

    Patients with type 2 diabetes mellitus require insulin as disease progresses to attain or maintain glycaemic targets. Basal insulin is commonly prescribed initially, alone or with one or more rapid-acting prandial insulin doses, to limit mealtime glucose excursions (a basal–bolus regimen). Both patients and physicians must balance the advantages of improved glycaemic control with the risk of hypoglycaemia and increasing regimen complexity. The rapid-acting insulin analogues (insulin aspart, insulin lispro and insulin glulisine) all have similar pharmacokinetic and pharmacodynamic characteristics and clinical efficacy/safety profiles. However, there are important differences in the pharmacokinetic and pharmacodynamic profiles of basal insulins (insulin glargine, insulin detemir and insulin degludec). Insulin degludec is an ultra-long-acting insulin analogue with a flat and stable glucose-lowering profile, a duration of action exceeding 30 h and less inter-patient variation in glucose-lowering effect than insulin glargine. In particular, the chemical properties of insulin degludec have allowed the development of a soluble co-formulation with prandial insulin aspart (insulin degludec/insulin aspart) that provides basal insulin coverage for at least 24 h with additional mealtime insulin for one or two meals depending on dose frequency. Pharmacokinetic and pharmacodynamic studies have shown that the distinct, long basal glucose-lowering action of insulin degludec and the prandial glucose-lowering effect of insulin aspart are maintained in the co-formulation. Evidence from pivotal phase III clinical trials indicates that insulin degludec/insulin aspart translate into sustained glycaemic control with less hypoglycaemia and the potential for a simpler insulin regimen with fewer daily injections. PMID:26568812

  11. Sulfur isotopic fractionation of carbonyl sulfide during degradation by soil bacteria and enzyme

    NASA Astrophysics Data System (ADS)

    Kamezaki, Kazuki; Hattori, Shohei; Ogawa, Takahiro; Toyoda, Sakae; Kato, Hiromi; Katayama, Yoko; Yoshida, Naohiro

    2017-04-01

    Carbonyl sulfide (COS) is an atmospheric trace gas that possess great potential for tracer of carbon cycle (Campbell et al., 2008). COS is taken up by vegetation during photosynthesis like absorption of carbon dioxide but COS can not emit by respiration of vegetation, suggesting possible tracer for gross primary production. However, some studies show the COS-derived GPP is larger than the estimates by using carbon dioxide flux because COS flux by photolysis and soil flux are not distinguished (e.g. Asaf et al., 2013). Isotope analysis is a useful tool to trace sources and transformations of trace gases. Recently our group developed a promising new analytical method for measuring the stable sulfur isotopic compositions of COS using nanomole level samples: the direct isotopic analytical technique of on-line gas chromatography-isotope ratio mass spectrometry (GC-IRMS) using fragmentation ions S+ enabling us to easily analyze sulfur isotopes in COS (Hattori et al., 2015). Soil is thought to be important as both a source and a sink of COS in the troposphere. In particular, soil has been reported as a large environmental sink for atmospheric COS. Bacteria isolated from various soils actively degrade COS, with various enzymes such as carbonic anhydrase and COSase (Ogawa et al., 2013) involved in COS degradation. However, the mechanism and the magnitude of bacterial contribution in terms of a sink for atmospheric COS is still uncertain. Therefore, it is important to quantitatively evaluate this contribution using COS sulfur isotope analysis. We present isotopic fractionation constants for COS by laboratory incubation experiments during degradation by soil bacteria and COSase. Incubation experiments were conducted using strains belonging to the genera Mycobacterium, Williamsia, Cupriavidus, and Thiobacillus, isolated from natural soil or activated sludge and enzyme purified from a bacteria. As a result, the isotopic compositions of OCS were increased during degradation of

  12. Lignocellulose-Degrading Microbial Communities in Landfill Sites Represent a Repository of Unexplored Biomass-Degrading Diversity.

    PubMed

    Ransom-Jones, Emma; McCarthy, Alan J; Haldenby, Sam; Doonan, James; McDonald, James E

    2017-01-01

    The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, to address the paucity of information on biomass-degrading microbial diversity beyond the gastrointestinal tract, cellulose (cotton) "baits" were incubated in landfill leachate microcosms to enrich the landfill cellulolytic microbial community for taxonomic and functional characterization. Metagenome and 16S rRNA gene amplicon sequencing demonstrated the dominance of Firmicutes , Bacteroidetes , Spirochaetes , and Fibrobacteres in the landfill cellulolytic community. Functional metagenome analysis revealed 8,371 carbohydrate active enzymes (CAZymes) belonging to 244 CAZyme families. In addition to observing biomass-degrading enzymes of anaerobic bacterial "cellulosome" systems of members of the Firmicutes , we report the first detection of the Fibrobacter cellulase system and the Bacteroidetes polysaccharide utilization locus (PUL) in landfill sites. These data provide evidence for the presence of multiple mechanisms of biomass degradation in the landfill microbiome and highlight the extraordinary functional diversity of landfill microorganisms as a rich source of biomass-degrading enzymes of potential biotechnological significance. IMPORTANCE The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused

  13. Lignocellulose-Degrading Microbial Communities in Landfill Sites Represent a Repository of Unexplored Biomass-Degrading Diversity

    PubMed Central

    Ransom-Jones, Emma; McCarthy, Alan J.; Haldenby, Sam; Doonan, James

    2017-01-01

    ABSTRACT The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, to address the paucity of information on biomass-degrading microbial diversity beyond the gastrointestinal tract, cellulose (cotton) “baits” were incubated in landfill leachate microcosms to enrich the landfill cellulolytic microbial community for taxonomic and functional characterization. Metagenome and 16S rRNA gene amplicon sequencing demonstrated the dominance of Firmicutes, Bacteroidetes, Spirochaetes, and Fibrobacteres in the landfill cellulolytic community. Functional metagenome analysis revealed 8,371 carbohydrate active enzymes (CAZymes) belonging to 244 CAZyme families. In addition to observing biomass-degrading enzymes of anaerobic bacterial “cellulosome” systems of members of the Firmicutes, we report the first detection of the Fibrobacter cellulase system and the Bacteroidetes polysaccharide utilization locus (PUL) in landfill sites. These data provide evidence for the presence of multiple mechanisms of biomass degradation in the landfill microbiome and highlight the extraordinary functional diversity of landfill microorganisms as a rich source of biomass-degrading enzymes of potential biotechnological significance. IMPORTANCE The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have

  14. Pac-Man for biotechnology: co-opting degrons for targeted protein degradation to control and alter cell function.

    PubMed

    Yu, Geng; Rosenberg, Julian N; Betenbaugh, Michael J; Oyler, George A

    2015-12-01

    Protein degradation in normal living cells is precisely regulated to match the cells' physiological requirements. The selectivity of protein degradation is determined by an elaborate degron-tagging system. Degron refers to an amino acid sequence that encodes a protein degradation signal, which is oftentimes a poly-ubiquitin chain that can be transferred to other proteins. Current understanding of ubiquitination dependent and independent protein degradation processes has expanded the application of degrons for targeted protein degradation and novel cell engineering strategies. Recent findings suggest that small molecules inducing protein association can be exploited to create degrons that target proteins for degradation. Here, recent applications of degron-based targeted protein degradation in eukaryotic organisms are reviewed. The degron mediated protein degradation represents a rapidly tunable methodology to control protein abundance, which has broad application in therapeutics and cellular function control and monitoring. Copyright © 2015. Published by Elsevier Ltd.

  15. Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis.

    PubMed

    McKleroy, William; Lee, Ting-Hein; Atabai, Kamran

    2013-06-01

    Pulmonary fibrosis is a vexing clinical problem with no proven therapeutic options. In the normal lung there is continuous collagen synthesis and collagen degradation, and these two processes are precisely balanced to maintain normal tissue architecture. With lung injury there is an increase in the rate of both collagen production and collagen degradation. The increase in collagen degradation is critical in preventing the formation of permanent scar tissue each time the lung is exposed to injury. In pulmonary fibrosis, collagen degradation does not keep pace with collagen production, resulting in extracellular accumulation of fibrillar collagen. Collagen degradation occurs through both extracellular and intracellular pathways. The extracellular pathway involves cleavage of collagen fibrils by proteolytic enzyme including the metalloproteinases. The less-well-described intracellular pathway involves binding and uptake of collagen fragments by fibroblasts and macrophages for lysosomal degradation. The relationship between these two pathways and their relevance to the development of fibrosis is complex. Fibrosis in the lung, liver, and skin has been associated with an impaired degradative environment. Much of the current scientific effort in fibrosis is focused on understanding the pathways that regulate increased collagen production. However, recent reports suggest an important role for collagen turnover and degradation in regulating the severity of tissue fibrosis. The objective of this review is to evaluate the roles of the extracellular and intracellular collagen degradation pathways in the development of fibrosis and to examine whether pulmonary fibrosis can be viewed as a disease of impaired matrix degradation rather than a disease of increased matrix production.

  16. Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis

    PubMed Central

    McKleroy, William; Lee, Ting-Hein

    2013-01-01

    Pulmonary fibrosis is a vexing clinical problem with no proven therapeutic options. In the normal lung there is continuous collagen synthesis and collagen degradation, and these two processes are precisely balanced to maintain normal tissue architecture. With lung injury there is an increase in the rate of both collagen production and collagen degradation. The increase in collagen degradation is critical in preventing the formation of permanent scar tissue each time the lung is exposed to injury. In pulmonary fibrosis, collagen degradation does not keep pace with collagen production, resulting in extracellular accumulation of fibrillar collagen. Collagen degradation occurs through both extracellular and intracellular pathways. The extracellular pathway involves cleavage of collagen fibrils by proteolytic enzyme including the metalloproteinases. The less-well-described intracellular pathway involves binding and uptake of collagen fragments by fibroblasts and macrophages for lysosomal degradation. The relationship between these two pathways and their relevance to the development of fibrosis is complex. Fibrosis in the lung, liver, and skin has been associated with an impaired degradative environment. Much of the current scientific effort in fibrosis is focused on understanding the pathways that regulate increased collagen production. However, recent reports suggest an important role for collagen turnover and degradation in regulating the severity of tissue fibrosis. The objective of this review is to evaluate the roles of the extracellular and intracellular collagen degradation pathways in the development of fibrosis and to examine whether pulmonary fibrosis can be viewed as a disease of impaired matrix degradation rather than a disease of increased matrix production. PMID:23564511

  17. Cometabolic Degradation of Dibenzofuran and Dibenzothiophene by a Naphthalene-Degrading Comamonas sp. JB.

    PubMed

    Ji, Xiangyu; Xu, Jing; Ning, Shuxiang; Li, Nan; Tan, Liang; Shi, Shengnan

    2017-12-01

    Comamonas sp. JB was used to investigate the cometabolic degradation of dibenzofuran (DBF) and dibenzothiophene (DBT) with naphthalene as the primary substrate. Dehydrogenase and ATPase activity of the growing system with the presence of DBF and DBT were decreased when compared to only naphthalene in the growing system, indicating that the presence of DBF and DBT inhibited the metabolic activity of strain JB. The pathways and enzymes involved in the cometabolic degradation were tested. Examination of metabolites elucidated that strain JB cometabolically degraded DBF to 1,2-dihydroxydibenzofuran, subsequently to 2-hydroxy-4-(3'-oxo-3'H-benzofuran-2'-yliden)but-2-enoic acid, and finally to catechol. Meanwhile, strain JB cometabolically degraded DBT to 1,2-dihydroxydibenzothiophene and subsequently to the ring cleavage product. A series of naphthalene-degrading enzymes including naphthalene dioxygenase, 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase, salicylate hydroxylase, and catechol 2,3-oxygenase have been detected, confirming that naphthalene was the real inducer of expression the degradation enzymes and metabolic pathways were controlled by naphthalene-degrading enzymes.

  18. Chemical approaches to targeted protein degradation through modulation of the ubiquitin-proteasome pathway.

    PubMed

    Collins, Ian; Wang, Hannah; Caldwell, John J; Chopra, Raj

    2017-03-15

    Manipulation of the ubiquitin-proteasome system to achieve targeted degradation of proteins within cells using chemical tools and drugs has the potential to transform pharmacological and therapeutic approaches in cancer and other diseases. An increased understanding of the molecular mechanism of thalidomide and its analogues following their clinical use has unlocked small-molecule modulation of the substrate specificity of the E3 ligase cereblon (CRBN), which in turn has resulted in the advancement of new immunomodulatory drugs (IMiDs) into the clinic. The degradation of multiple context-specific proteins by these pleiotropic small molecules provides a means to uncover new cell biology and to generate future drug molecules against currently undruggable targets. In parallel, the development of larger bifunctional molecules that bring together highly specific protein targets in complexes with CRBN, von Hippel-Lindau, or other E3 ligases to promote ubiquitin-dependent degradation has progressed to generate selective chemical compounds with potent effects in cells and in vivo models, providing valuable tools for biological target validation and with future potential for therapeutic use. In this review, we survey recent breakthroughs achieved in these two complementary methods and the discovery of new modes of direct and indirect engagement of target proteins with the proteasome. We discuss the experimental characterisation that validates the use of molecules that promote protein degradation as chemical tools, the preclinical and clinical examples disclosed to date, and the future prospects for this exciting area of chemical biology. © 2017 The Author(s).

  19. Hepatic Insulin Resistance is Sufficient to Produce Dyslipidemia and Susceptibility to Atherosclerosis

    PubMed Central

    Biddinger, Sudha B.; Hernandez-Ono, Antonio; Rask-Madsen, Christian; Haas, Joel T.; Alemán, José O.; Suzuki, Ryo; Scapa, Erez F.; Agarwal, Chhavi; Carey, Martin C.; Stephanopoulos, Gregory; Cohen, David E.; King, George L.; Ginsberg, Henry; Kahn, C. Ronald

    2014-01-01

    Insulin resistance plays a central role in the development of the metabolic syndrome, but how it relates to cardiovascular disease remains controversial. Liver insulin receptor knockout (LIRKO) mice have pure hepatic insulin resistance. On a chow diet, LIRKO mice have a proatherogenic lipoprotein profile with reduced HDL cholesterol and VLDL particles that are markedly enriched in cholesterol. This is due to increased secretion and decreased clearance of apoB-containing lipoproteins, coupled with decreased triglyceride secretion secondary to increased expression of PGC-1β, which promotes VLDL secretion, but decreased expression of SREBP-1c, SREBP-2 and their targets, the lipogenic enzymes and the LDL receptor. Within twelve weeks on an atherogenic diet, LIRKO mice show marked hypercholesterolemia, and 100% of LIRKO mice, but 0% of controls, develop severe atherosclerosis. Thus, insulin resistance at the level of the liver is sufficient to produce the dyslipidemia and increased risk of atherosclerosis associated with the metabolic syndrome. PMID:18249172

  20. Histidine augments the suppression of hepatic glucose production by central insulin action.

    PubMed

    Kimura, Kumi; Nakamura, Yusuke; Inaba, Yuka; Matsumoto, Michihiro; Kido, Yoshiaki; Asahara, Shun-Ichiro; Matsuda, Tomokazu; Watanabe, Hiroshi; Maeda, Akifumi; Inagaki, Fuyuhiko; Mukai, Chisato; Takeda, Kiyoshi; Akira, Shizuo; Ota, Tsuguhito; Nakabayashi, Hajime; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

    2013-07-01

    Glucose intolerance in type 2 diabetes is related to enhanced hepatic glucose production (HGP) due to the increased expression of hepatic gluconeogenic enzymes. Previously, we revealed that hepatic STAT3 decreases the expression of hepatic gluconeogenic enzymes and suppresses HGP. Here, we show that increased plasma histidine results in hepatic STAT3 activation. Intravenous and intracerebroventricular (ICV) administration of histidine-activated hepatic STAT3 reduced G6Pase protein and mRNA levels and augmented HGP suppression by insulin. This suppression of hepatic gluconeogenesis by histidine was abolished by hepatic STAT3 deficiency or hepatic Kupffer cell depletion. Inhibition of HGP by histidine was also blocked by ICV administration of a histamine H1 receptor antagonist. Therefore, histidine activates hepatic STAT3 and suppresses HGP via central histamine action. Hepatic STAT3 phosphorylation after histidine ICV administration was attenuated in histamine H1 receptor knockout (Hrh1KO) mice but not in neuron-specific insulin receptor knockout (NIRKO) mice. Conversely, hepatic STAT3 phosphorylation after insulin ICV administration was attenuated in NIRKO but not in Hrh1KO mice. These findings suggest that central histidine action is independent of central insulin action, while both have additive effects on HGP suppression. Our results indicate that central histidine/histamine-mediated suppression of HGP is a potential target for the treatment of type 2 diabetes.

  1. Histidine Augments the Suppression of Hepatic Glucose Production by Central Insulin Action

    PubMed Central

    Kimura, Kumi; Nakamura, Yusuke; Inaba, Yuka; Matsumoto, Michihiro; Kido, Yoshiaki; Asahara, Shun-ichiro; Matsuda, Tomokazu; Watanabe, Hiroshi; Maeda, Akifumi; Inagaki, Fuyuhiko; Mukai, Chisato; Takeda, Kiyoshi; Akira, Shizuo; Ota, Tsuguhito; Nakabayashi, Hajime; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

    2013-01-01

    Glucose intolerance in type 2 diabetes is related to enhanced hepatic glucose production (HGP) due to the increased expression of hepatic gluconeogenic enzymes. Previously, we revealed that hepatic STAT3 decreases the expression of hepatic gluconeogenic enzymes and suppresses HGP. Here, we show that increased plasma histidine results in hepatic STAT3 activation. Intravenous and intracerebroventricular (ICV) administration of histidine-activated hepatic STAT3 reduced G6Pase protein and mRNA levels and augmented HGP suppression by insulin. This suppression of hepatic gluconeogenesis by histidine was abolished by hepatic STAT3 deficiency or hepatic Kupffer cell depletion. Inhibition of HGP by histidine was also blocked by ICV administration of a histamine H1 receptor antagonist. Therefore, histidine activates hepatic STAT3 and suppresses HGP via central histamine action. Hepatic STAT3 phosphorylation after histidine ICV administration was attenuated in histamine H1 receptor knockout (Hrh1KO) mice but not in neuron-specific insulin receptor knockout (NIRKO) mice. Conversely, hepatic STAT3 phosphorylation after insulin ICV administration was attenuated in NIRKO but not in Hrh1KO mice. These findings suggest that central histidine action is independent of central insulin action, while both have additive effects on HGP suppression. Our results indicate that central histidine/histamine-mediated suppression of HGP is a potential target for the treatment of type 2 diabetes. PMID:23474485

  2. Immobilization of oxalate-degrading enzymes into p(HEMA) for inhibiting encrustation on ureteral stents

    NASA Astrophysics Data System (ADS)

    Mellman, James Kenneth

    Ureteral stents develop calcium-bearing deposits, called encrustation, that diminish their biocompatibility due to complications, such as chronic abrasion to the lumen of the ureter wall and subsequent infection. A reduction of encrustation, namely calcium oxalate, will improve the lifetime, health care costs, and infection resistance of such devices. The purpose of this research project is to study oxalate-degrading enzymes entrapped into a coating material that will control the interface to the urinary environment for ureteral stents. The coating material was a lightly crosslinked poly(2-hydroxyethyl methacrylate) (p(HEMA)) matrix in which the active enzymes were entrapped within the bulk material's free volume. The swelling of p(HEMA) films was comparable in ddH2O and urine. This hydrophilic matrix allows oxalate anions to diffuse into the bulk so that enzyme activity against oxalate can lower its local concentration, and thereby reduce the supersaturation of calcium oxalate. Oxalate oxidase (OxO) and oxalate decarboxylase (OxDc) were the oxalate-degrading enzymes examined herein. Michaelis Menten kinetic models were applied to free and immobilized enzyme activity. A substrate inhibition model was applied to OxO. The free form of OxO had a Vmax of 1.8 +/- 0.1 muM/min-mug, a km of 1.8 +/- 0.1 mM, and a ks of 35.4 +/- 3.7 mM while the immobilized form had a Vmax of 1.2 +/- 0.2 muM/min-mug, a km of 4.1 +/- 0.6 mM, and a ks of 660 +/- 140 mM. The free form of OxDc had a Vmax of 23.5 +/- 1.4 muM/min-mug and a km of 0.5 +/- 0.1 mM while the immobilized form had a Vmax of 5.0 +/- 1.9 muM/min-mug and km of 23.2 +/- 9.1 mM. The enzyme activity was measured to indicate viable application conditions for the coating, such as storing the films in urine over time. The maximum activity was shown at pH 4.2 to 4.5 and activity drops to be negligible by pH 7.0. Storing the enzyme at pH 6.1 exhibited a larger retained activity than storing at pH 4.2, yet storing in urine showed

  3. Effects of insulin and exercise training on FGF21, its receptors and target genes in obesity and type 2 diabetes.

    PubMed

    Kruse, Rikke; Vienberg, Sara G; Vind, Birgitte F; Andersen, Birgitte; Højlund, Kurt

    2017-10-01

    Pharmacological doses of FGF21 improve glucose tolerance, lipid metabolism and energy expenditure in rodents. Induced expression and secretion of FGF21 from muscle may increase browning of white adipose tissue (WAT) in a myokine-like manner. Recent studies have reported that insulin and exercise increase FGF21 in plasma. Obesity and type 2 diabetes are potentially FGF21-resistant states, but to what extent FGF21 responses to insulin and exercise training are preserved, and whether FGF21, its receptors and target genes are altered, remains to be established. The effects of insulin during euglycaemic-hyperinsulinaemic clamps and 10 week endurance training on serum FGF21 were examined in individuals with type 2 diabetes and in glucose tolerant overweight/obese and lean individuals. Gene expression of FGF21, its receptors and target genes in muscle and WAT biopsies was evaluated by quantitative real-time PCR (qPCR). Insulin increased serum and muscle FGF21 independent of overweight/obesity or type 2 diabetes, and there were no effects associated with exercise training. The insulin-induced increases in serum FGF21 and muscle FGF21 expression correlated tightly (p < 0.001). In WAT, overweight/obesity with and without type 2 diabetes led to reduced expression of KLB, but increased FGFR1c expression. However, the expression of most FGF21 target genes was unaltered except for reduced CIDEA expression in individuals with type 2 diabetes. Insulin-induced expression of muscle FGF21 correlates strongly with a rise in serum FGF21, and this response appears intact in overweight/obesity and type 2 diabetes. FGF21 resistance may involve reduced KLB expression in WAT. However, increased FGFR1c expression or other mechanisms seem to ensure adequate expression of most FGF21 target genes in WAT.

  4. A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation

    PubMed Central

    Shen, Yang; Zeng, Lin; Novosyadlyy, Ruslan; Forest, Amelie; Zhu, Aiping; Korytko, Andrew; Zhang, Haifan; Eastman, Scott W; Topper, Michael; Hindi, Sagit; Covino, Nicole; Persaud, Kris; Kang, Yun; Burtrum, Douglas; Surguladze, David; Prewett, Marie; Chintharlapalli, Sudhakar; Wroblewski, Victor J; Shen, Juqun; Balderes, Paul; Zhu, Zhenping; Snavely, Marshall; Ludwig, Dale L

    2015-01-01

    Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor – type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique “capture-for-degradation” mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions. PMID:26073904

  5. Reversal of diet-induced obesity and insulin resistance by inducible genetic ablation of GRK2

    PubMed Central

    Vila-Bedmar, Rocio; Cruces-Sande, Marta; Lucas, Elisa; Willemen, Hanneke L.D.M.; Heijnen, Cobi J.; Kavelaars, Annemieke; Mayor, Federico; Murga, Cristina

    2015-01-01

    Insulin resistance is a common feature of obesity and predisposes individuals to various prevalent pathological conditions. G protein-coupled receptor kinase 2 (GRK2) integrates several signal transduction pathways and is emerging as a physiologically relevant inhibitor of insulin signaling. GRK2 abundanceis increased in humans with metabolic syndrome and in different murine models of insulin resistance. To support GRK2 as a potential drug target in type 2 diabetes and obesity, we investigated whether lowering GRK2 abundance reversed an ongoing systemic insulin-resistant phenotype, using a mouse model of tamoxifen-induced GRK2 ablation after high fat diet-dependent obesity and insulin resistance. Tamoxifen-triggered GRK2 deletion impeded further body weight gain, normalized fa sting glycemia, improved glucose tolerance and was associated with preserved insulin sensitivity in skeletal muscle and liver, thereby maintaining whole body glucose homeostasis. Moreover, when continued to be fed a high fat diet, these animals displayed reduced fat mass and smaller adipocytes, were resistant to the development of liver steatosis, and showed reduced expression of pro-inflammatory markers in the liver. Our results indicate that GRK2 acts as a hub to control metabolic functions in different tissues, which is key to controlling insulin resistance development in vivo. These data suggest that inhibiting GRK2 could reverse an established insulin-resistant and obese phenotype, thereby putting forward this enzyme as a potential therapeutic target linking glucose homeostasis and regulation of adiposity. PMID:26198359

  6. Effect of an aqueous extract of Scoparia dulcis on blood glucose, plasma insulin and some polyol pathway enzymes in experimental rat diabetes.

    PubMed

    Latha, M; Pari, L

    2004-04-01

    The effects of an aqueous extract of the plant Scoparia dulcis (200 mg/kg) on the polyol pathway and lipid peroxidation were examined in the liver of streptozotocin adult diabetic male albino Wistar rats. The diabetic control rats (N = 6) presented a significant increase in blood glucose, sorbitol dehydrogenase, glycosylated hemoglobin and lipid peroxidation markers such as thiobarbituric acid reactive substances (TBARS) and hydroperoxides, and a significant decrease in plasma insulin and antioxidant enzymes such as glutathione peroxidase (GPx), glutathione-S-transferase (GST) and reduced glutathione (GSH) compared to normal rats (N = 6). Scoparia dulcis plant extract (SPEt, 200 mg kg-1 day-1) and glibenclamide (600 microg kg-1 day-1), a reference drug, were administered by gavage for 6 weeks to diabetic rats (N = 6 for each group) and significantly reduced blood glucose, sorbitol dehydrogenase, glycosylated hemoglobin, TBARS, and hydroperoxides, and significantly increased plasma insulin, GPx, GST and GSH activities in liver. The effect of the SPEt was compared with that of glibenclamide. The effect of the extract may have been due to the decreased influx of glucose into the polyol pathway leading to increased activities of antioxidant enzymes and plasma insulin and decreased activity of sorbitol dehydrogenase. These results indicate that the SPEt was effective in attenuating hyperglycemia in rats and their susceptibility to oxygen free radicals.

  7. [The optimal blood glucose target in critically ill patient: comparison of two intensive insulin therapy protocols].

    PubMed

    Raurell Torredà, Marta; del Llano Serrano, César; Almirall Solsona, Dolors; Catalan Ibars, Rosa María; Nicolás Arfelis, José María

    2014-03-04

    Recent studies in critically ill patients receiving insulin intravenous therapy (IIT) have shown an increased incidence of severe hypoglycemia, while intermittent subcutaneous insulin «sliding scales» (conventional insulin therapy [CIT]) is associated with hyperglycemia. The objective of this study is to assess whether glycemic control range IIT can affect glucose levels and their variability and to compare it with CIT. Prospective comparative cohort study in intensive care unit, with 2 study periods: Period 1, IIT with glycemic target range 110-140 mg/dL, and Period 2, IIT of 140-180 mg/dL. In both periods CIT glycemic target was 110-180 mg/dL. We assessed severe hypoglycemia (< 50 mg/dL), moderate hypoglycemia (51-79 mg/dL), hyperglycemia (> 216 mg/L) and the variability of blood glucose. We studied 221 patients with 12.825 blood glucose determinations. Twenty-six and 17% of patients required IIT for glycemic control in Period 1 and 2, respectively. Hypoglycemia was associated with a discontinuous nutritional intake, glycemic target 110-140 mg/dL and low body mass index (BMI) (P = .002). Hyperglycemia was exclusively associated with a history of diabetes mellitus (OR 2.6 [95% CI 1.6 to 4.5]). Glycemic variability was associated with a discontinuous nutritional intake, low BMI, CIT insulinization, diabetes mellitus, elderly and high APACHE II (P < .001). The use of IIT is useful to reduce the variability of blood glucose. Although the 140-180 mg/dL range would be more secure as to presenting greater variability and hyperglycemia, the 110-140 mg/dL range is most suitable. Copyright © 2012 Elsevier España, S.L. All rights reserved.

  8. Molecular Mechanisms of Insulin Resistance in Chronic Kidney Disease

    PubMed Central

    Thomas, Sandhya S.; Zhang, Liping; Mitch, William E.

    2015-01-01

    Insulin resistance refers to reduced sensitivity of organs to insulin-initiated biologic processes that result in metabolic defects. Insulin resistance is common in patients with end-stage renal disease but also occurs in patients with chronic kidney disease (CKD), even when the serum creatinine is minimally increased. Following insulin binding to its receptor, auto-phosphorylation of the insulin receptor is followed by kinase reactions that phosphorylate insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3K) and Akt. In fact, low levels of Akt phosphorylation (p-Akt) identifies the presence of the insulin resistance that leads to metabolic defects in insulin-initiated metabolism of glucose, lipids and muscle proteins. Besides CKD, other complex conditions (e.g., inflammation, oxidative stress, metabolic acidosis, aging and excess angiotensin II) reduce p-Akt resulting in insulin resistance. Insulin resistance in each of these conditions is due to activation of different, E3 ubiquitin ligases which specifically conjugate ubiquitin to IRS-1 marking it for degradation in the ubiquitin-proteasome system (UPS). Consequently, IRS-1 degradation suppresses insulin-induced intracellular signaling, causing insulin resistance. Understanding mechanisms of insulin resistance could lead to therapeutic strategies that improve the metabolism of patients with CKD. PMID:26444029

  9. The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation.

    PubMed

    Baumberger, Nicolas; Tsai, Ching-Hsui; Lie, Miranda; Havecker, Ericka; Baulcombe, David C

    2007-09-18

    Plant and animal viruses encode suppressor proteins of an adaptive immunity mechanism in which viral double-stranded RNA is processed into 21-25 nt short interfering (si)RNAs. The siRNAs guide ARGONAUTE (AGO) proteins so that they target viral RNA. Most viral suppressors bind long dsRNA or siRNAs and thereby prevent production of siRNA or binding of siRNA to AGO. The one exception is the 2b suppressor of Cucumoviruses that binds to and inhibits AGO1. Here we describe a novel suppressor mechanism in which a Polerovirus-encoded F box protein (P0) targets the PAZ motif and its adjacent upstream sequence in AGO1 and mediates its degradation. F box proteins are components of E3 ubiquitin ligase complexes that add polyubiquitin tracts on selected lysine residues and thereby mark a protein for proteasome-mediated degradation. With P0, however, the targeted degradation of AGO is insensitive to inhibition of the proteasome, indicating that the proteasome is not involved. We also show that P0 does not block a mobile signal of silencing, indicating that the signal molecule does not have AGO protein components. The ability of P0 to block silencing without affecting signal movement may contribute to the phloem restriction of viruses in the Polerovirus group.

  10. Cbl-independent degradation of Met: ways to avoid agonism of bivalent Met-targeting antibody.

    PubMed

    Lee, J M; Kim, B; Lee, S B; Jeong, Y; Oh, Y M; Song, Y-J; Jung, S; Choi, J; Lee, S; Cheong, K H; Kim, D U; Park, H W; Han, Y K; Kim, G W; Choi, H; Song, P H; Kim, K A

    2014-01-02

    The Met receptor tyrosine kinase, found to be constitutively activated in many tumors, has become a leading target for cancer therapy. Disruptions in Met downregulation have been associated with aggressive tumor progression with several therapeutic strategies addressing this aspect of Met biology. Castias B-lineage lymphoma (Cbl) E3 ligase-mediated degradation, which attenuates Met signaling via ligand-dependent Met internalization, is a major negative regulator of Met expression. It is believed that one of the mechanisms by which the therapeutic anti-Met antibodies induce cancer cell death in Met overexpressing tumors is via internalization and subsequent degradation of Met from the cell surface. However, a previously reported Met-targeting antibody demonstrated intrinsic agonistic activity while being capable of inducing Cbl-mediated degradation of Met, suggesting that Cbl-mediated degradation requires receptor activation and impedes therapeutic application. We have developed a potent and selective bivalent Met-targeting antibody (SAIT301) that invokes Met degradation using an alternative regulator LRIG1. In this report, we demonstrate that LRIG1 mediates degradation of Met by SAIT301 and this degradation does not require Met activation. Furthermore, SAIT301 was able to downregulate Met and dramatically inhibit growth of tumors with low or no Cbl expression, as well as tumors with Met exon 14 deletion that prevents Met binding to Cbl. In summary, we demonstrate the enhanced therapeutic potential of a novel tumor-inhibiting anti-Met antibody, SAIT301, which utilizes a Cbl-independent, LRIG1-mediated Met degradation pathway and thereby avoids the agonism that limits the effectiveness of previously reported anti-Met antibodies.

  11. Mitochondrial-Targeted Catalase Protects Against High-Fat Diet-Induced Muscle Insulin Resistance by Decreasing Intramuscular Lipid Accumulation.

    PubMed

    Lee, Hui-Young; Lee, Jae Sung; Alves, Tiago; Ladiges, Warren; Rabinovitch, Peter S; Jurczak, Michael J; Choi, Cheol Soo; Shulman, Gerald I; Samuel, Varman T

    2017-08-01

    We explored the role of reactive oxygen species (ROS) in the pathogenesis of muscle insulin resistance. We assessed insulin action in vivo with a hyperinsulinemic-euglycemic clamp in mice expressing a mitochondrial-targeted catalase (MCAT) that were fed regular chow (RC) or a high-fat diet (HFD) or underwent an acute infusion of a lipid emulsion. RC-fed MCAT mice were similar to littermate wild-type (WT) mice. However, HFD-fed MCAT mice were protected from diet-induced insulin resistance. In contrast, an acute lipid infusion caused muscle insulin resistance in both MCAT and WT mice. ROS production was decreased in both HFD-fed and lipid-infused MCAT mice and cannot explain the divergent response in insulin action. MCAT mice had subtly increased energy expenditure and muscle fat oxidation with decreased intramuscular diacylglycerol (DAG) accumulation, protein kinase C-θ (PKCθ) activation, and impaired insulin signaling with HFD. In contrast, the insulin resistance with the acute lipid infusion was associated with increased muscle DAG content in both WT and MCAT mice. These studies suggest that altering muscle mitochondrial ROS production does not directly alter the development of lipid-induced insulin resistance. However, the altered energy balance in HFD-fed MCAT mice protected them from DAG accumulation, PKCθ activation, and impaired muscle insulin signaling. © 2017 by the American Diabetes Association.

  12. Protection against the synaptic targeting and toxicity of Alzheimer's-associated Aβ oligomers by insulin mimetic chiro-inositols

    PubMed Central

    Pitt, Jason; Thorner, Michael; Brautigan, David; Larner, Joseph; Klein, William L.

    2013-01-01

    Alzheimer's disease (AD) is a progressive dementia that correlates highly with synapse loss. This loss appears due to the synaptic accumulation of toxic Aβ oligomers (ADDLs), which damages synapse structure and function. Although it has been reported that oligomer binding and toxicity can be prevented by stimulation of neuronal insulin signaling with PPARγ agonists, these agonists have problematic side effects. We therefore investigated the therapeutic potential of chiro-inositols, insulin-sensitizing compounds safe for human consumption. Chiro-inositols have been studied extensively for treatment of diseases associated with peripheral insulin resistance, but their insulin mimetic function in memory-relevant central nervous system (CNS) cells is unknown. Here we demonstrate that mature cultures of hippocampal neurons respond to d-chiro-inositol (DCI), pinitol (3-O-methyl DCI), and the inositol glycan INS-2 (pinitol β-1-4 galactosamine) with increased phosphorylation in key upstream components in the insulin-signaling pathway (insulin receptor, insulin receptor substrate-1, and Akt). Consistent with insulin stimulation, DCI treatment promotes rapid withdrawal of dendritic insulin receptors. With respect to neuroprotection, DCI greatly enhances the ability of insulin to prevent ADDL-induced synapse damage (EC50 of 90 nM). The mechanism comprises inhibition of oligomer binding at synapses and requires insulin/IGF signaling. DCI showed no effects on Aβ oligomerization. We propose that inositol glycans and DCI, a compound already established as safe for human consumption, have potential as AD therapeutics by protecting CNS synapses against Aβ oligomers through their insulin mimetic activity.—Pitt, J., Thorner, M., Brautigan, D., Larner, J., Klein, W. L. Protection against the synaptic targeting and toxicity of Alzheimer's-associated Aβ oligomers by insulin mimetic chiro-inositols. PMID:23073831

  13. siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

    PubMed

    Vickers, Timothy A; Crooke, Stanley T

    2012-07-01

    While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

  14. Insulin-induced translocation of IR to the nucleus in insulin responsive cells requires a nuclear translocation sequence.

    PubMed

    Kesten, Dov; Horovitz-Fried, Miriam; Brutman-Barazani, Tamar; Sampson, Sanford R

    2018-04-01

    Insulin binding to its cell surface receptor (IR) activates a cascade of events leading to its biological effects. The Insulin-IR complex is rapidly internalized and then is either recycled back to the plasma membrane or sent to lysosomes for degradation. Although most of the receptor is recycled or degraded, a small amount may escape this pathway and migrate to the nucleus of the cell where it might be important in promulgation of receptor signals. In this study we explored the mechanism by which insulin induces IR translocation to the cell nucleus. Experiments were performed cultured L6 myoblasts, AML liver cells and 3T3-L1 adipocytes. Insulin treatment induced a rapid increase in nuclear IR protein levels within 2 to 5 min. Treatment with WGA, an inhibitor of nuclear import, reduced insulin-induced increases nuclear IR protein; IR was, however, translocated to a perinuclear location. Bioinformatics tools predicted a potential nuclear localization sequence (NLS) on IR. Immunofluorescence staining showed that a point mutation on the predicted NLS blocked insulin-induced IR nuclear translocation. In addition, blockade of nuclear IR activation in isolated nuclei by an IR blocking antibody abrogated insulin-induced increases in IR tyrosine phosphorylation and nuclear PKCδ levels. Furthermore, over expression of mutated IR reduced insulin-induced glucose uptake and PKB phosphorylation. When added to isolated nuclei, insulin induced IR phosphorylation but had no effect on nuclear IR protein levels. These results raise questions regarding the possible role of nuclear IR in IR signaling and insulin resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. In vitro growth and cell wall degrading enzyme production by Argentinean isolates of Macrophomina phaseolina, the causative agent of charcoal rot in corn.

    PubMed

    Ramos, Araceli M; Gally, Marcela; Szapiro, Gala; Itzcovich, Tatiana; Carabajal, Maira; Levin, Laura

    Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on many commercially important species. Damages caused by this pathogen in soybean and maize crops in Argentina during drought and hot weather have increased due its ability to survive as sclerotia in soil and crop debris under non-till practices. In this work, we explored the in vitro production of plant cell wall-degrading enzymes [pectinases (polygalacturonase and polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the ligninolytic enzyme laccase] by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina-maize interaction. The isolates were grown in liquid synthetic medium supplemented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source. Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3U/ml and polymethylgalacturonase between 0.15 and 1.3U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration followed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36U/l and 63U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  16. Production of cell wall-degrading enzymes by Aspergillus nidulans: a model system for fungal pathogenesis of plants.

    PubMed Central

    Dean, R A; Timberlake, W E

    1989-01-01

    The cell wall-degrading enzymes polygalacturonase and pectate lyase have been suggested to be crucial for penetration and colonization of plant tissues by some fungal pathogens. We have found that Aspergillus nidulans (= Emericella nidulans), a saprophytic Ascomycete, produces levels of these enzymes equal to those produced by soft-rotting Erwinia species. Induction of polygacturonase and pectate lyase in A. nidulans requires substrate and is completely repressed by glucose. Surprisingly, inoculation of excised plant tissues with A. nidulans conidia leads to formation of necrotic, water-soaked lesions within which the organism sporulates. Thus, A. nidulans has phytopathogenic potential. The release of glucose and other sugars from wounded tissues may repress pectolytic enzyme production and limit disease development. Therefore, we tested creA204, a mutation that relieves glucose repression of some A. nidulans carbon utilization enzymes, for its effect on production of pectolytic enzymes. creA204 failed to relieve catabolite repression of polygalacturonase or pectate lyase and had no effect on disease severity. PMID:2535501

  17. Ring finger protein 145 (RNF145) is a ubiquitin ligase for sterol-induced degradation of HMG-CoA reductase.

    PubMed

    Jiang, Lu-Yi; Jiang, Wei; Tian, Na; Xiong, Yan-Ni; Liu, Jie; Wei, Jian; Wu, Kai-Yue; Luo, Jie; Shi, Xiong-Jie; Song, Bao-Liang

    2018-03-16

    Cholesterol biosynthesis is tightly regulated in the cell. For example, high sterol concentrations can stimulate degradation of the rate-limiting cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, HMGCR). HMGCR is broken down by the endoplasmic reticulum membrane-associated protein complexes consisting of insulin-induced genes (Insigs) and the E3 ubiquitin ligase gp78. Here we found that HMGCR degradation is partially blunted in Chinese hamster ovary (CHO) cells lacking gp78 ( gp78 -KO). To identify other ubiquitin ligase(s) that may function together with gp78 in triggering HMGCR degradation, we performed a small-scale short hairpin RNA-based screening targeting endoplasmic reticulum-localized E3s. We found that knockdown of both ring finger protein 145 ( Rnf145 ) and gp78 genes abrogates sterol-induced degradation of HMGCR in CHO cells. We also observed that RNF145 interacts with Insig-1 and -2 proteins and ubiquitinates HMGCR. Moreover, the tetrapeptide sequence YLYF in the sterol-sensing domain and the Cys-537 residue in the RING finger domain were essential for RNF145 binding to Insigs and RNF145 E3 activity, respectively. Of note, amino acid substitutions in the YLYF or of Cys-537 completely abolished RNF145-mediated HMGCR degradation. In summary, our study reveals that RNF145, along with gp78, promotes HMGCR degradation in response to elevated sterol levels and identifies residues essential for RNF145 function. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. A comparative genomic analysis of the oxidative enzymes potentially involved in lignin degradation by Agaricus bisporus.

    PubMed

    Doddapaneni, Harshavardhan; Subramanian, Venkataramanan; Fu, Bolei; Cullen, Dan

    2013-06-01

    The oxidative enzymatic machinery for degradation of organic substrates in Agaricus bisporus (Ab) is at the core of the carbon recycling mechanisms in this fungus. To date, 156 genes have been tentatively identified as part of this oxidative enzymatic machinery, which includes 26 peroxidase encoding genes, nine copper radical oxidase [including three putative glyoxal oxidase-encoding genes (GLXs)], 12 laccases sensu stricto and 109 cytochrome P450 monooxygenases. Comparative analyses of these enzymes in Ab with those of the white-rot fungus, Phanerochaete chrysosporium, the brown-rot fungus, Postia placenta, the coprophilic litter fungus, Coprinopsis cinerea and the ectomychorizal fungus, Laccaria bicolor, revealed enzyme diversity consistent with adaptation to substrates rich in humic substances and partially degraded plant material. For instance, relative to wood decay fungi, Ab cytochrome P450 genes were less numerous (109 gene models), distributed among distinctive families, and lacked extensive duplication and clustering. Viewed together with P450 transcript accumulation patterns in three tested growth conditions, these observations were consistent with the unique Ab lifestyle. Based on tandem gene arrangements, a certain degree of gene duplication seems to have occurred in this fungus in the copper radical oxidase (CRO) and the laccase gene families. In Ab, high transcript levels and regulation of the heme-thiolate peroxidases, two manganese peroxidases and the three GLX-like genes are likely in response to complex natural substrates, including lignocellulose and its derivatives, thereby suggesting an important role in lignin degradation. On the other hand, the expression patterns of the related CROs suggest a developmental role in this fungus. Based on these observations, a brief comparative genomic overview of the Ab oxidative enzyme machinery is presented. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. New strategy for renal fibrosis: Targeting Smad3 proteins for ubiquitination and degradation.

    PubMed

    Wang, Xin; Feng, Shaozhen; Fan, Jinjin; Li, Xiaoyan; Wen, Qiong; Luo, Ning

    2016-09-15

    Smad3 is a critical signaling protein in renal fibrosis. Proteolysis targeting chimeric molecules (PROTACs) are small molecules designed to degrade target proteins via ubiquitination. They have three components: (1) a recognition motif for E3 ligase; (2) a linker; and (3) a ligand for the target protein. We aimed to design a new PROTAC to prevent renal fibrosis by targeting Smad3 proteins and using hydroxylated pentapeptide of hypoxia-inducible factor-1α as the recognition motif for von Hippel-Lindau (VHL) ubiquitin ligase (E3). Computer-aided drug design was used to find a specific ligand targeting Smad3. Surface plasmon resonance (SPR) was used to verify and optimize screening results. Synthesized PROTAC was validated by two-stage mass spectrometry. The PROTAC's specificity for VHL (E3 ligase) was proved with two human renal carcinoma cell lines, 786-0 (VHL(-)) and ACHN (VHL(+)), and its anti-fibrosis effect was tested in renal fibrosis cell models. Thirteen small molecular compounds (SMCs) were obtained from the Enamine library using GLIDE molecular docking program. SPR results showed that #8 SMC (EN300-72284) combined best with Smad3 (KD=4.547×10(-5)M). Mass spectrometry showed that synthesized PROTAC had the correct peptide molecular weights. Western blot showed Smad3 was degraded by PROTAC with whole-cell lysate of ACHN but not 786-0. Degradation, but not ubiquitination, of Smad3 was inhibited by proteasome inhibitor MG132. The upregulation of fibronectin and Collagen I induced by TGF-β1 in both renal fibroblast and mesangial cells were inhibited by PROTAC. The new PROTAC might prevent renal fibrosis by targeting Smad3 for ubiquitination and degradation. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Evaluation of Mn-superoxide dismutase and catalase gene expression in childhood obesity: its association with insulin resistance.

    PubMed

    Mohseni, Roohollah; Arab Sadeghabadi, Zahra; Goodarzi, Mohammad Taghi; Teimouri, Maryam; Nourbakhsh, Mitra; Razzaghy Azar, Maryam

    2018-06-28

    Obesity is associated with oxidative stress. Superoxide dismutase (SOD) is the first line of defense against reactive oxygen species (ROS), eliminating the strong superoxide radical and producing H2O2, which can then be degraded by catalase (CAT). The main objective of this study was to evaluate the gene expression antioxidant enzymes (Mn-SOD and CAT) in peripheral blood mononuclear cells (PBMCs) of obese and normal-weight children, and its association with anthropometric and biochemical parameters. Thirty obese and 30 control subjects between the ages of 8 and 16 years were enrolled in this study. Serum insulin levels were measured using enzyme-linked immunosorbent assay (ELISA), and insulin resistance was calculated using the homeostasis model assessment of insulin resistance (HOMA-IR). Biochemical parameters were also measured. PBMCs of the subjects were separated and Mn-SOD and CAT gene expression was measured using real-time polymerase chain reaction (PCR). Mn-SOD and CAT gene expression was significantly lower in the obese group compared with the control group (p<0.01). Also, a positive correlation was observed between the gene expression of Mn-SOD and CAT and body mass index (BMI), fasting blood sugar, insulin resistance, low density lipoprotein-cholesterol (LDL-C) cholesterol, triglycerides (TG) and systolic blood pressure (SBP). Induction of antioxidants, especially Mn-SOD and CAT, can lead to reduction of oxidative stress and prevent the complications of obesity in children.

  1. Melanoma cell therapy: Endothelial progenitor cells as shuttle of the MMP12 uPAR-degrading enzyme

    PubMed Central

    Laurenzana, Anna; Biagioni, Alessio; D'Alessio, Silvia; Bianchini, Francesca; Chillà, Anastasia; Margheri, Francesca; Luciani, Cristina; Mazzanti, Benedetta; Pimpinelli, Nicola; Torre, Eugenio; Danese, Silvio; Calorini, Lido; Rosso, Mario Del; Fibbi, Gabriella

    2014-01-01

    The receptor for the urokinase-type plasminogen activator (uPAR) accounts for many features of cancer progression, and is therefore considered a target for anti-tumoral therapy. Only full length uPAR mediates tumor progression. Matrix-metallo-proteinase-12 (MMP12)-dependent uPAR cleavage results into the loss of invasion properties and angiogenesis. MMP12 can be employed in the field of “targeted therapies” as a biological drug to be delivered directly in patient's tumor mass. Endothelial Progenitor Cells (EPCs) are selectively recruited within the tumor and could be used as cellular vehicles for delivering anti-cancer molecules. The aim of our study is to inhibit cancer progression by engeneering ECFCs, a subset of EPC, with a lentivirus encoding the anti-tumor uPAR-degrading enzyme MMP12. Ex vivo manipulated ECFCs lost the capacity to perform capillary morphogenesis and acquired the anti-tumor and anti-angiogenetic activity. In vivo MMP12-engineered ECFCs cleaved uPAR within the tumor mass and strongly inhibited tumor growth, tumor angiogenesis and development of lung metastasis. The possibility to exploit tumor homing and activity of autologous MMP12-engineered ECFCs represents a novel way to combat melanoma by a “personalized therapy”, without rejection risk. The i.v. injection of radiolabelled MMP12-ECFCs can thus provide a new theranostic approach to control melanoma progression and metastasis. PMID:25003596

  2. Occurrence and function of enzymes for lignocellulose degradation in commercial Agaricus bisporus cultivation.

    PubMed

    Kabel, Mirjam A; Jurak, Edita; Mäkelä, Miia R; de Vries, Ronald P

    2017-06-01

    The white button mushroom Agaricus bisporus is economically the most important commercially produced edible fungus. It is grown on carbon- and nitrogen-rich substrates, such as composted cereal straw and animal manure. The commercial mushroom production process is usually performed in buildings or tunnels under highly controlled environmental conditions. In nature, the basidiomycete A. bisporus has a significant impact on the carbon cycle in terrestrial ecosystems as a saprotrophic decayer of leaf litter. In this mini-review, the fate of the compost plant cell wall structures, xylan, cellulose and lignin, is discussed. A comparison is made from the structural changes observed to the occurrence and function of enzymes for lignocellulose degradation present, with a special focus on the extracellular enzymes produced by A. bisporus. In addition, recent advancements in whole genome level molecular studies in various growth stages of A. bisporus in compost are reviewed.

  3. Lindane degradation by Candida VITJzN04, a newly isolated yeast strain from contaminated soil: kinetic study, enzyme analysis and biodegradation pathway.

    PubMed

    Salam, Jaseetha Abdul; Das, Nilanjana

    2014-04-01

    A new yeast strain was isolated from sugarcane cultivation field which was able to utilize lindane as sole carbon source for growth in mineral medium. The yeast was identified and named as Candida sp. VITJzN04 based on a polyphasic approach using morphological, biochemical and 18S rDNA, D1/D2 and ITS sequence analysis. The isolated yeast strain efficiently degraded 600 mg L⁻¹ of lindane within 6 days in mineral medium under the optimal conditions (pH 7; temperature 30 °C and inoculum dosage 0.06 g L⁻¹) with the least half-life of 1.17 days and degradation constant of 0.588 per day. Lindane degradation was tested with various kinetic models and results revealed that the reaction could be described best by first-order and pseudo first-order models. In addition, involvement of the enzymes viz. dechlorinase, dehalogenase, dichlorohydroquinone reductive dechlorinase, lignin peroxidase and manganese peroxidase was noted during lindane degradation. Addition of H2O2 in the mineral medium showed 32 % enhancement of lindane degradation within 3 days. Based on the metabolites identified by GC-MS and FTIR analysis, sequential process of lindane degradation by Candida VITJzN04 was proposed. To the best of our knowledge, this is the first report of isolation and characterization of lindane-degrading Candida sp. and elucidation of enzyme systems during the degradation process.

  4. Mir-338-3p Mediates Tnf-A-Induced Hepatic Insulin Resistance by Targeting PP4r1 to Regulate PP4 Expression.

    PubMed

    Dou, Lin; Wang, Shuyue; Sun, Libo; Huang, Xiuqing; Zhang, Yang; Shen, Tao; Guo, Jun; Man, Yong; Tang, Weiqing; Li, Jian

    2017-01-01

    Insulin resistance is a critical factor contributing to the pathogenesis of type 2 diabetes and other metabolic diseases. Recent studies have indicated that miR-338-3p plays an important role in cancer. Here, we investigated whether miR-338-3p mediates tumour necrosis factor-α (TNF-α)-induced hepatic insulin resistance. The activation of the insulin signalling pathway and the level of glycogenesis were examined in the livers of the db/db and high fat diet (HFD)-fed mice and in HEP1-6 cells transfected with miR-338-3p mimic or inhibitor. Computational prediction of microRNA target, luciferase assay and Western blot were used to assess the miR-338-3p target. Chromatin immunoprecipitation (ChIP) assay was used to determine the transcriptional regulator of miR-338-3p. miR-338-3p was down-regulated in the livers of the db/db, HFD-fed and TNF-α-treated C57BL/6J mice, as well as in mouse HEP1-6 hepatocytes treated with TNF-α. Importantly the down-regulation of miR-338-3p induced insulin resistance, as indicated by impaired glucose tolerance and insulin tolerance. Further research showed that the down-regulated miR-338-3p resulted in the impaired AKT/ glycogen synthase kinase 3 beta (GSl·Gβ) signalling pathway and glycogen synthesis. In contrast, hepatic over-expression of miR-338-3p rescued the TNF-α-induced insulin resistance. Moreover, protein phosphatase 4 regulator subunit 1 (PP4R1) was identified as a direct target of miR-338-3p that mediated hepatic insulin signalling by regulating protein phosphatase 4 (PP4). Finally we identified hepatic nuclear factor 4 alpha (HNF-4α) as the transcriptional regulator of miRNA-338-3p. Our studies provide novel insight into the critical role and molecular mechanism by which miR-338-3p is involved in TNF-α-induced hepatic insulin resistance. miR-338-3p might mediate TNF-α-induced hepatic insulin resistance by targeting PP4R1 to regulate PP4 expression. © 2017 The Author(s). Published by S. Karger AG, Basel.

  5. Controlled enzyme-catalyzed degradation of polymeric capsules templated on CaCO₃: influence of the number of LbL layers, conditions of degradation, and disassembly of multicompartments.

    PubMed

    Marchenko, Irina; Yashchenok, Alexey; Borodina, Tatiana; Bukreeva, Tatiana; Konrad, Manfred; Möhwald, Helmuth; Skirtach, Andre

    2012-09-28

    Enzyme-catalyzed degradation of CaCO₃-templated capsules is presented. We investigate a) biodegradable, b) mixed biodegradable/synthetic, and c) multicompartment polyelectrolyte multilayer capsules with different numbers of polymer layers. Using confocal laser scanning microscopy we observed the kinetics of the non-specific protease Pronase-induced degradation of capsules is slowed down on the order of hours by either increasing the number of layers in the wall of biodegradable capsules, or by inserting synthetic polyelectrolyte multilayers into the shell comprised of biodegradable polymers. The degradation rate increases with the concentration of Pronase. Controlled detachment of subcompartments of multicompartment capsules, with potential for intracellular delivery or in-vivo applications, is also shown. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Recent advances in targeting protein arginine methyltransferase enzymes in cancer therapy.

    PubMed

    Smith, Emily; Zhou, Wei; Shindiapina, Polina; Sif, Said; Li, Chenglong; Baiocchi, Robert A

    2018-05-21

    Exploration in the field of epigenetics has revealed the diverse roles of the protein arginine methyltransferase (PRMT) family of proteins in multiple disease states. These findings have led to the development of specific inhibitors and discovery of several new classes of drugs with potential to treat both benign and malignant conditions. Areas covered: We provide an overview on the role of PRMT enzymes in healthy and malignant cells, highlighting the role of arginine methylation in specific pathways relevant to cancer pathogenesis. Additionally, we describe structure and catalytic activity of PRMT and discuss the mechanisms of action of novel small molecule inhibitors of specific members of the arginine methyltransferase family. Expert opinion: As the field of PRMT biology advances, it's becoming clear that this class of enzymes is highly relevant to maintaining normal physiologic processes as well and disease pathogenesis. We discuss the potential impact of PRMT inhibitors as a broad class of drugs, including the pleiotropic effects, off target effects the need for more detailed PRMT-centric interactomes, and finally, the potential for targeting this class of enzymes in clinical development of experimental therapeutics for cancer.

  7. Novel strategy for a bispecific antibody: induction of dual target internalization and degradation.

    PubMed

    Lee, J M; Lee, S H; Hwang, J-W; Oh, S J; Kim, B; Jung, S; Shim, S-H; Lin, P W; Lee, S B; Cho, M-Y; Koh, Y J; Kim, S Y; Ahn, S; Lee, J; Kim, K-M; Cheong, K H; Choi, J; Kim, K-A

    2016-08-25

    Activation of the extensive cross-talk among the receptor tyrosine kinases (RTKs), particularly ErbB family-Met cross-talk, has emerged as a likely source of drug resistance. Notwithstanding brilliant successes were attained while using small-molecule inhibitors or antibody therapeutics against specific RTKs in multiple cancers over recent decades, a high recurrence rate remains unsolved in patients treated with these targeted inhibitors. It is well aligned with multifaceted properties of cancer and cross-talk and convergence of signaling pathways of RTKs. Thereby many therapeutic interventions have been actively developed to overcome inherent or acquired resistance. To date, no bispecific antibody (BsAb) showed complete depletion of dual RTKs from the plasma membrane and efficient dual degradation. In this manuscript, we report the first findings of a target-specific dual internalization and degradation of membrane RTKs induced by designed BsAbs based on the internalizing monoclonal antibodies and the therapeutic values of these BsAbs. Leveraging the anti-Met mAb able to internalize and degrade by a unique mechanism, we generated the BsAbs for Met/epidermal growth factor receptor (EGFR) and Met/HER2 to induce an efficient EGFR or HER2 internalization and degradation in the presence of Met that is frequently overexpressed in the invasive tumors and involved in the resistance against EGFR- or HER2-targeted therapies. We found that Met/EGFR BsAb ME22S induces dissociation of the Met-EGFR complex from Hsp90, followed by significant degradation of Met and EGFR. By employing patient-derived tumor models we demonstrate therapeutic potential of the BsAb-mediated dual degradation in various cancers.

  8. [Impact of exogenous paraquat on enzyme exudation and biochemical changes of lignin degradation fungi].

    PubMed

    Zhao, Yunchen; Li, Jianlong; Chen, Yuru; Huang, Haixia; Yu, Zui

    2009-08-01

    To study the effect of exogenous oxygen, we added water solution of paraquat to 7 d cultures of Coriolus versicolor for the next 148 h. Enzyme exudation and biochemical process were investigated on the addition of paraquat. We found that compared with the control (without paraquat), the addition of 30 micromol/L paraquat stimulated the activity of manganese dependent peroxidase (MnP), lignin peroxidase (LiP), and laccases (Lac) 7, 2.5 and 1.3 times, respectively. Also, addition of paraquat enhanced activity of superoxide dismutase (SOD) and catalase (CAT) in the first 48 h. Impact of paraquat on ligninolytic enzymes was significant than that on antioxidant enzyme. Addition of paraquat enhanced phenolic compounds and formaldehyde of cultures too. And concentration of malondialdehyde was increased in the first 24 h. The results showed that addition of paraquat promoted oxidative stress, but the antioxidant systems of the fungal strain are sufficient to prevent mycelia from oxidative stress. As exogenous oxygen, paraquat might be a useful substrate in degradation of lignocellulose.

  9. Heterogeneous Expression and Functional Characterization of Cellulose-Degrading Enzymes from Aspergillus niger for Enzymatic Hydrolysis of Alkali Pretreated Bamboo Biomass.

    PubMed

    Ali, Nasir; Ting, Zhang; Li, Hailong; Xue, Yong; Gan, Lihui; Liu, Jian; Long, Minnan

    2015-09-01

    Enzymatic hydrolysis of cellulosic biomass has caught much attention because of modest reaction conditions and environment friendly conditions. To reduce the cost and to achieve good quantity of cellulases, a heterologous expression system is highly favored. In this study, cellulose-degrading enzymes, GH3 family β-glucosidase (BGL), GH7 family-related cellobiohydrolases (CBHs), and endoglucanase (EG) from a newly isolated Aspergillus niger BE-2 are highly expressed in Pichia pastoris GS115. The strain produced EG, CBHs, and BGL enzymatic concentration of 0.56, 0.11, and 22 IU/mL, respectively. Mode of actions of the recombinant enzymes for substrate specificity and end product analysis are verified and found specific for cellulose degradation. Bamboo biomass saccharification with A. niger cellulase released a high level of fermentable sugars. Hydrolysis parameters are optimized to obtain reducing sugars level of 3.18 g/L. To obtain reducing sugars from a cellulosic biomass, A. niger could be a good candidate for enzymes resource of cellulase to produce reducing sugars from a cellulosic biomass. This study also facilitates the development of highly efficient enzyme cocktails for the bioconversion of lignocellulosic biomass into monosaccharides and oligosaccharides.

  10. Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find a new cancer treatment approach.

    PubMed

    Glorieux, Christophe; Calderon, Pedro Buc

    2017-09-26

    This review is centered on the antioxidant enzyme catalase and will present different aspects of this particular protein. Among them: historical discovery, biological functions, types of catalases and recent data with regard to molecular mechanisms regulating its expression. The main goal is to understand the biological consequences of chronic exposure of cells to hydrogen peroxide leading to cellular adaptation. Such issues are of the utmost importance with potential therapeutic extrapolation for various pathologies. Catalase is a key enzyme in the metabolism of H2O2 and reactive nitrogen species, and its expression and localization is markedly altered in tumors. The molecular mechanisms regulating the expression of catalase, the oldest known and first discovered antioxidant enzyme, are not completely elucidated. As cancer cells are characterized by an increased production of reactive oxygen species (ROS) and a rather altered expression of antioxidant enzymes, these characteristics represent an advantage in terms of cell proliferation. Meanwhile, they render cancer cells particularly sensitive to an oxidant insult. In this context, targeting the redox status of cancer cells by modulating catalase expression is emerging as a novel approach to potentiate chemotherapy.

  11. [RAAS and insulin resistance].

    PubMed

    Motoshima, Hiroyuki; Araki, Eiichi

    2012-09-01

    The role of the renin-angiotensin-aldosterone system (RAAS) on the development of insulin resistance and type 2 diabetes (T2DM) is an area of growing interest. Most of the deleterious actions of the RAAS on insulin signals appear to be mediated through activation of the serine/threonine kinase, oxidative stress and tissue-inflammation in insulin-sensitive organs. Both experimental and clinical studies demonstrated that angiotensin II (Ang II) and aldosterone could play a role in the development of insulin resistance, diabetes and cardiovascular diseases. Large randomized clinical trials revealed that blockade of the RAAS with either angiotensin I converting enzyme inhibitors or AT1 receptor blockers results in decreased T2DM incidence, with a minor attenuation of markers for insulin resistance. This review focuses on the role of RAAS in the pathogenesis of insulin resistance, as well as on clinical relevance of RAAS blockade in the prevention and treatment of the metabolic syndrome and pre-diabetes.

  12. The Draft Genome of the Invasive Walking Stick, Medauroidea extradendata, Reveals Extensive Lineage-Specific Gene Family Expansions of Cell Wall Degrading Enzymes in Phasmatodea

    PubMed Central

    Brand, Philipp; Lin, Wei; Johnson, Brian R.

    2018-01-01

    Plant cell wall components are the most abundant macromolecules on Earth. The study of the breakdown of these molecules is thus a central question in biology. Surprisingly, plant cell wall breakdown by herbivores is relatively poorly understood, as nearly all early work focused on the mechanisms used by symbiotic microbes to breakdown plant cell walls in insects such as termites. Recently, however, it has been shown that many organisms make endogenous cellulases. Insects, and other arthropods, in particular have been shown to express a variety of plant cell wall degrading enzymes in many gene families with the ability to break down all the major components of the plant cell wall. Here we report the genome of a walking stick, Medauroidea extradentata, an obligate herbivore that makes uses of endogenously produced plant cell wall degrading enzymes. We present a draft of the 3.3Gbp genome along with an official gene set that contains a diversity of plant cell wall degrading enzymes. We show that at least one of the major families of plant cell wall degrading enzymes, the pectinases, have undergone a striking lineage-specific gene family expansion in the Phasmatodea. This genome will be a useful resource for comparative evolutionary studies with herbivores in many other clades and will help elucidate the mechanisms by which metazoans breakdown plant cell wall components. PMID:29588379

  13. [Characteristics of soil microbes and enzyme activities in different degraded alpine meadows].

    PubMed

    Yin, Ya Li; Wang, Yu Qin; Bao, Gen Sheng; Wang, Hong Sheng; Li, Shi Xiong; Song, Mei Ling; Shao, Bao Lian; Wen, Yu Cun

    2017-12-01

    Soil microbial biomass C and N, microbial diversities and enzyme activity in 0-10 cm and 10-20 cm soil layers of different degraded grasslands (non-degradation, ND; light degradation, LD; moderate degradation, MD; sever degradation, SD; and black soil beach, ED) were measured by Biolog and other methods. The results showed that: 1) There were significant diffe-rences between 0-10 cm and 10-20 cm soil layers in soil microbial biomass, diversities and inver-tase activities in all grasslands. 2) The ratio of soil microbial biomass C to N decreased significantly with the grassland degradation. In the 0-10 cm soil layer, microbial biomass C and N in ND and LD were significantly higher than that in MD, SD and ED. Among the latter three kinds of grasslands, there was no difference for microbial biomass C, but microbial biomass N was lower in MD than in the other grasslands. The average color change rate (AWCD) and McIntosh Index (U) also decreased with grassland degradation, but only the reduction from ND to MD was significant. There were no differences among all grasslands for Shannon index (H) and Simpson Index (D). The urease activity was highest in MD and SD, and the activity of phosphatase and invertase was lowest in ED. In the 10-20 cm soil layer, microbial biomass C in ND and LD were significantly higher than that in the other grasslands. Microbial biomass N in LD and ED were significantly higher than that in the other grasslands. Carbon metabolism index in MD was significantly lower than that in LD and SD. AWCD and U index in ND and LD were significantly higher than that in ED. H index and D index showed no difference among different grasslands. The urease activity in ND and MD was significantly higher than that in the other grasslands. The phosphatase activity was highest in MD, and the invertase activity was lowest in MD. 3) The belowground biomass was significantly positively correlated with microbial biomass, carbon metabolic index and phosphatase activity

  14. Fibroblast activation protein (FAP) as a novel metabolic target.

    PubMed

    Sánchez-Garrido, Miguel Angel; Habegger, Kirk M; Clemmensen, Christoffer; Holleman, Cassie; Müller, Timo D; Perez-Tilve, Diego; Li, Pengyun; Agrawal, Archita S; Finan, Brian; Drucker, Daniel J; Tschöp, Matthias H; DiMarchi, Richard D; Kharitonenkov, Alexei

    2016-10-01

    Fibroblast activation protein (FAP) is a serine protease belonging to a S9B prolyl oligopeptidase subfamily. This enzyme has been implicated in cancer development and recently reported to regulate degradation of FGF21, a potent metabolic hormone. Using a known FAP inhibitor, talabostat (TB), we explored the impact of FAP inhibition on metabolic regulation in mice. To address this question we evaluated the pharmacology of TB in various mouse models including those deficient in FGF21, GLP1 and GIP signaling. We also studied the ability of FAP to process FGF21 in vitro and TB to block FAP enzymatic activity. TB administration to diet-induced obese (DIO) animals led to profound decreases in body weight, reduced food consumption and adiposity, increased energy expenditure, improved glucose tolerance and insulin sensitivity, and lowered cholesterol levels. Total and intact plasma FGF21 were observed to be elevated in TB-treated DIO mice but not lean animals where the metabolic impact of TB was significantly attenuated. Furthermore, and in stark contrast to naïve DIO mice, the administration of TB to obese FGF21 knockout animals demonstrated no appreciable effect on body weight or any other measures of metabolism. In support of these results we observed no enzymatic degradation of human FGF21 at either end of the protein when FAP was inhibited in vitro by TB. We conclude that pharmacological inhibition of FAP enhances levels of FGF21 in obese mice to provide robust metabolic benefits not observed in lean animals, thus validating this enzyme as a novel drug target for the treatment of obesity and diabetes.

  15. Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

    PubMed Central

    Berg Miller, Margret E.; Antonopoulos, Dionysios A.; Rincon, Marco T.; Band, Mark; Bari, Albert; Akraiko, Tatsiana; Hernandez, Alvaro; Thimmapuram, Jyothi; Henrissat, Bernard; Coutinho, Pedro M.; Borovok, Ilya; Jindou, Sadanari; Lamed, Raphael; Flint, Harry J.; Bayer, Edward A.; White, Bryan A.

    2009-01-01

    Background Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. Methodology/Principal Findings The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. Conclusions/Significance The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has

  16. Diversity and strain specificity of plant cell wall degrading enzymes revealed by the draft genome of Ruminococcus flavefaciens FD-1.

    PubMed

    Berg Miller, Margret E; Antonopoulos, Dionysios A; Rincon, Marco T; Band, Mark; Bari, Albert; Akraiko, Tatsiana; Hernandez, Alvaro; Thimmapuram, Jyothi; Henrissat, Bernard; Coutinho, Pedro M; Borovok, Ilya; Jindou, Sadanari; Lamed, Raphael; Flint, Harry J; Bayer, Edward A; White, Bryan A

    2009-08-14

    Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes

  17. Preparation of chitosan-based multifunctional nanocarriers overcoming multiple barriers for oral delivery of insulin.

    PubMed

    Li, Lei; Jiang, Guohua; Yu, Weijiang; Liu, Depeng; Chen, Hua; Liu, Yongkun; Tong, Zaizai; Kong, Xiangdong; Yao, Juming

    2017-01-01

    To overcome multiple barriers for oral delivery of insulin, the chitosan-based multifunctional nanocarriers modified by L-valine (LV, used as a target ligand to facilitate the absorption of the small intestine) and phenylboronic acid (PBA, used as a glucose-responsive unit) have been designed and evaluated in this study. The resultant nanocarriers exhibited low cytotoxicity against HT-29 cells and excellent stability against protein solution. The insulin release behaviors were evaluated triggered by pH and glucose in vitro. The chemical stability of loaded insulin against digestive enzyme were established in presence of simulated gastric fluid (SGF) containing pepsin and simulated intestinal fluid (SIF) containing pancreatin, respectively. The uptake behavior of HT-29 cells was evaluated by confocal laser scanning microscope. After oral administration to the diabetic rats, an effective hypoglycemic effect was obtained compared with subcutaneous injection of insulin. This work suggests that L-valine modified chitosan-based multifunctional nanocarriers may be a promising drug delivery carrier for oral administration of insulin. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Appropriate insulin initiation dosage for insulin-naive type 2 diabetes outpatients receiving insulin monotherapy or in combination with metformin and/or pioglitazone.

    PubMed

    Liao, Lin; Yang, Ming; Qiu, Lu-Lu; Mou, Ya-Ru; Zhao, Jia-Jun; Dong, Jian-Jun

    2010-12-01

    Few studies have given suggestions on appropriate initiation insulin dosage when combined with oral antidiabetic drugs (OADs). This research was to investigate appropriate initiation insulin doses for insulin-naive type 2 diabetes patients with different combinations and the relationship between insulin dosage and relevant factors. This was a randomized, open-label, treat to target study. The target was 20% decrease of both fasting plasma glucose (FPG) and 2 hours post-breakfast blood glucose (P2hBG). One hundred and forty-seven insulin-naive Chinese patients recruited were randomly assigned to 3 groups: group A, patients received insulin monotherapy; group B, received insulin plus metformin (0.5 g, tid) and group C, received insulin plus metformin (0.5 g, tid) and pioglitazone (15 mg, qd). Insulin doses were initiated with a dose of 0.3 U×kg(-1)×d(-1) and titrated according to FPG and P2hBG till reached the targets. Both the time of getting 20% reduction of FPG and P2hBG showed significant differences among the three groups. The time was shortest in Group C. The insulin doses needed to achieve glucose reduction of 20% in three treatment groups were (0.40 ± 0.04) U×kg(-1)×d(-1) for Group A, (0.37 ± 0.04) U×kg(-1)×d(-1) for Group B, and (0.35 ± 0.03) U×kg(-1)×d(-1) for Group C, respectively. Multiple linear stepwise regression analysis showed that insulin doses correlated with body weight, FPG, diabetes duration, age and history of sulfonylurea treatment. The standardized regression coefficients were 0.871, 0.322, 0.089, 0.067 and 0.063 (with all P < 0.05). To achieve blood glucose's reduction of 20% within safety context, initial insulin doses were recommended as the following: 0.40 U×kg(-1)×d(-1) for insulin mono-therapy, 0.37 U×kg(-1)×d(-1) for insulin plus metformin treatment, and 0.35 U×kg(-1)×d(-1) for insulin plus metformin and pioglitazone treatment in Chinese type 2 diabetes outpatients. Body weight is found the most closely related factor

  19. Isolation and characterization of an ether-type polyurethane-degrading micro-organism and analysis of degradation mechanism by Alternaria sp.

    PubMed

    Matsumiya, Y; Murata, N; Tanabe, E; Kubota, K; Kubo, M

    2010-06-01

    To degrade ether-type polyurethane (ether-PUR), ether-PUR-degrading micro-organism was isolated. Moreover, ether-PUR-degrading mechanisms were analysed using model compounds of ether-PUR. A fungus designated as strain PURDK2, capable of changing the configuration of ether-PUR, has been isolated. This isolated fungus was identified as Alternaria sp. Using a scanning electron microscope, the grid structure of ether-PUR was shown to be melted and disrupted by the fungus. The degradation of ether-PUR by the fungus was analysed, and the ether-PUR was degraded by the fungus by about 27.5%. To analyse the urethane-bond degradation by the fungus, a degraded product of ethylphenylcarbamate was analysed using GC/MS. Aniline and ethanol were detected by degradation with the supernatant, indicating that the fungus secreted urethane-bond-degrading enzyme(s). PURDK2 also degraded urea bonds when diphenylmethane-4,4'-dibutylurea was used as a substrate. The enzyme(s) from PURDK2 degraded urethane and urea bonds to convert the high molecular weight structure of ether-PUR to small molecules; and then the fungus seems to use the small molecules as an energy source. Ether-PUR-degrading fungus, strain PURDK2, was isolated, and the urethane- and urea-bonds-degrading enzymes from strain PURDK2 could contribute to the material recycling of ether-PUR.

  20. The role of the renin-angiotensin system in the development of insulin resistance in skeletal muscle.

    PubMed

    Henriksen, Erik J; Prasannarong, Mujalin

    2013-09-25

    The canonical renin-angiotensin system (RAS) involves the initial action of renin to cleave angiotensinogen to angiotensin I (ANG I), which is then converted to ANG II by the angiotensin converting enzyme (ACE). ANG II plays a critical role in numerous physiological functions, and RAS overactivity underlies many conditions of cardiovascular dysregulation. In addition, ANG II, by acting on both endothelial and myocellular AT1 receptors, can induce insulin resistance by increasing cellular oxidative stress, leading to impaired insulin signaling and insulin-stimulated glucose transport activity. This insulin resistance associated with RAS overactivity, when coupled with progressive ß-cell dysfunction, eventually leads to the development of type 2 diabetes. Interventions that target RAS overactivity, including ACE inhibitors, ANG II receptor blockers, and, most recently, renin inhibitors, are effective both in reducing hypertension and in improving whole-body and skeletal muscle insulin action, due at least in part to enhanced Akt-dependent insulin signaling and insulin-dependent glucose transport activity. ANG-(1-7), which is produced from ANG II by the action of ACE2 and acts via Mas receptors, can counterbalance the deleterious actions of the ACE/ANG II/AT1 receptor axis on the insulin-dependent glucose transport system in skeletal muscle. This beneficial effect of the ACE2/ANG-(1-7)/Mas receptor axis appears to depend on the activation of Akt. Collectively, these findings underscore the importance of RAS overactivity in the multifactorial etiology of insulin resistance in skeletal muscle, and provide support for interventions that target the RAS to ameliorate both cardiovascular dysfunctions and insulin resistance in skeletal muscle tissue. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  1. Calcium phosphate-PEG-insulin-casein (CAPIC) particles as oral delivery systems for insulin.

    PubMed

    Morçöl, T; Nagappan, P; Nerenbaum, L; Mitchell, A; Bell, S J D

    2004-06-11

    An oral delivery system for insulin was developed and functional activity was tested in a non-obese diabetic (NOD) mice model. Calcium phosphate particles containing insulin was synthesized in the presence of PEG-3350 and modified by aggregating the particles with caseins to obtain the calcium phosphate-PEG-insulin-casein (CAPIC) oral insulin delivery system. Single doses of CAPIC formulation were tested in NOD mice under fasting or fed conditions to evaluate the glycemic activity. The blood glucose levels were monitored every 1-2h for 12h following the treatments using an ACCU CHECK blood glucose monitoring system. Orally administered and subcutaneously injected free insulin solution served as controls in the study. Based on the results obtained we propose that: (1). the biological activity of insulin is preserved in CAPIC formulation; (2). insulin in CAPIC formulations, but not the free insulin, displays a prolonged hypoglycemic effect after oral administration to diabetic mice; (3). CAPIC formulation protects insulin from degradation while passing through the acidic environment of the GI track until it is released in the less acidic environment of the intestines where it can be absorbed in its biologically active form; (4). CAPIC formulation represents a new and unique oral delivery system for insulin and other macromolecules.

  2. Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung

    Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the ‘fed’ condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1more » plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology. - Highlights: • Insulin treatment increases CARM1 expression in hepatocytes. • CARM1 overexpression does not increase the expression of lipogenic proteins. • CARM1 knockdown does not influence insulin sensitivity. • Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation.« less

  3. Enzyme technology for precision functional food ingredient processes.

    PubMed

    Meyer, Anne S

    2010-03-01

    A number of naturally occurring dietary substances may exert physiological benefits. The production of enhanced levels or particularly tailored versions of such candidate functional compounds can be targeted by enzymatic catalysis. The recent literature contains examples of enhancing bioavailability of iron via enzyme-catalyzed degradation of phytate in wheat bran, increasing diacyl-glycerol and conjugated linoleic acid levels by lipase action, enhancing the absorption of the citrus flavonoid hesperetin via rhamnosidase treatment, and obtaining solubilized dietary fiber via enzymatic modification of potato starch processing residues. Such targeted enzyme-catalyzed reactions provide new invention opportunities for designing functional foods with significant health benefits. The provision of well-defined naturally structured compounds can, moreover, assist in obtaining the much-needed improved understanding of the physiological benefits of complex natural substances.

  4. Therapeutics of diabetes mellitus: focus on insulin analogues and insulin pumps.

    PubMed

    Valla, Vasiliki

    2010-01-01

    Inadequately controlled diabetes accounts for chronic complications and increases mortality. Its therapeutic management aims in normal HbA1C, prandial and postprandial glucose levels. This review discusses diabetes management focusing on the latest insulin analogues, alternative insulin delivery systems and the artificial pancreas. Intensive insulin therapy with multiple daily injections (MDI) allows better imitation of the physiological rhythm of insulin secretion. Longer-acting, basal insulin analogues provide concomitant improvements in safety, efficacy and variability of glycaemic control, followed by low risks of hypoglycaemia. Continuous subcutaneous insulin infusion (CSII) provides long-term glycaemic control especially in type 1 diabetic patients, while reducing hypoglycaemic episodes and glycaemic variability. Continuous subcutaneous glucose monitoring (CGM) systems provide information on postprandial glucose excursions and nocturnal hypo- and/or hyperglycemias. This information enhances treatment options, provides a useful tool for self-monitoring and allows safer achievement of treatment targets. In the absence of a cure-like pancreas or islets transplants, artificial "closed-loop" systems mimicking the pancreatic activity have been also developed. Individualized treatment plans for insulin initiation and administration mode are critical in achieving target glycaemic levels. Progress in these fields is expected to facilitate and improve the quality of life of diabetic patients.

  5. Targeting endogenous proteins for degradation through the affinity-directed protein missile system.

    PubMed

    Fulcher, Luke J; Hutchinson, Luke D; Macartney, Thomas J; Turnbull, Craig; Sapkota, Gopal P

    2017-05-01

    Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel-Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. © 2017 The Authors.

  6. Targeting endogenous proteins for degradation through the affinity-directed protein missile system

    PubMed Central

    Fulcher, Luke J.; Hutchinson, Luke D.; Macartney, Thomas J.; Turnbull, Craig

    2017-01-01

    Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel–Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. PMID:28490657

  7. A Proteomic Study of Pectin Degrading Enzymes Secreted by Botrytis cinerea Grown in Liquid Culture

    PubMed Central

    Shah, Punit; Gutierrez-Sanchez, Gerardo; Orlando, Ron; Bergmann, Carl

    2009-01-01

    Botrytis cinerea is a pathogenic filamentous fungus which infects more than 200 plant species. The enzymes secreted by B. cinerea play an important role in the successful colonization of a host plant. Some of the secreted enzymes are involved in the degradation of pectin, a major component of the plant cell wall. A total of 126 proteins secreted by B. cinerea were identified by growing the fungus on highly or partially esterified pectin, or on sucrose in liquid culture. Sixty-seven common proteins were identified in each of the growth conditions, of which 50 proteins exhibited a Signal P motif. Thirteen B. cinerea proteins with functions related to pectin degradation were identified in both pectin growth conditions, while only four were identified in sucrose. Our results indicate it is unlikely that the activation of B. cinerea from the dormant state to active infection is solely dependent on changes in the degree of esterification of the pectin component of the plant cell wall. Further, these results suggest that future studies of the B. cinerea secretome in infections of ripe and unripe fruits will provide important information that will describe the mechanisms that the fungus employs to access nutrients and decompose tissues. PMID:19526562

  8. Phage lytic enzymes: a history.

    PubMed

    Trudil, David

    2015-02-01

    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  9. Resveratrol Influences Pancreatic Islets by Opposing Effects on Electrical Activity and Insulin Release.

    PubMed

    Brouwer, Simone; Hoffmeister, Theresa; Gresch, Anne; Schönhoff, Lisa; Düfer, Martina

    2018-03-01

    Resveratrol is suggested to improve glycemic control by activation of sirtuin 1 (SIRT1) and has already been tested clinically. Our investigation characterizes the targets of resveratrol in pancreatic beta cells and their contribution to short- and long-term effects on insulin secretion. Islets or beta cells are isolated from C57BL/6N mice. Electrophysiology is performed with microelectrode arrays and patch-clamp technique, insulin secretion and content are determined by radioimmunoassay, cAMP is measured by enzyme-linked immunosorbent assay, and cytosolic Ca 2+ concentration by fluorescence methods. Resveratrol (25 μmol L -1 ) elevates [Ca 2+ ] c and potentiates glucose-stimulated insulin secretion. These effects are associated with increased intracellular cAMP and are sensitive to the SIRT1 blocker Ex-527. Inhibition of EPAC1 by CE3F4 also abolishes the stimulatory effect of resveratrol. The underlying mechanism does not involve membrane depolarization as resveratrol even reduces electrical activity despite blocking K ATP channels. Importantly, after prolonged exposure to resveratrol (14 days), the beneficial influence of the polyphenol on insulin release is lost. Resveratrol addresses multiple targets in pancreatic islets. Potentiation of insulin secretion is mediated by SIRT1-dependent activation of cAMP/EPAC1. Considering resveratrol as therapeutic supplement for patients with type 2 diabetes mellitus, the inhibitory influence on electrical excitability attenuates positive effects. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Natural products inhibiting the ubiquitin-proteasome proteolytic pathway, a target for drug development.

    PubMed

    Tsukamoto, Sachiko; Yokosawa, Hideyoshi

    2006-01-01

    The ubiquitin-proteasome proteolytic pathway plays a major role in selective protein degradation and regulates various cellular events including cell cycle progression, transcription, DNA repair, signal transduction, and immune response. Ubiquitin, a highly conserved small protein in eukaryotes, attaches to a target protein prior to degradation. The polyubiquitin chain tagged to the target protein is recognized by the 26S proteasome, a high-molecular-mass protease subunit complex, and the protein portion is degraded by the 26S proteasome. The potential of specific proteasome inhibitors, which act as anti-cancer agents, is now under intensive investigation, and bortezomib (PS-341), a proteasome inhibitor, has been recently approved by FDA for multiple myeloma treatment. Since ubiquitination of proteins requires the sequential action of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3), and polyubiquitination is a prerequisite for proteasome-mediated protein degradation, inhibitors of E1, E2, and E3 are reasonably thought to be drug candidates for treatment of diseases related to ubiquitination. Recently, various compounds inhibiting the ubiquitin-proteasome pathway have been isolated from natural resources. We also succeeded in isolating inhibitors against the proteasome and E1 enzyme from marine natural resources. In this review, we summarize the structures and biological activities of natural products that inhibit the ubiquitin-proteasome proteolytic pathway.

  11. Mercaptosuccinate Dioxygenase, a Cysteine Dioxygenase Homologue, from Variovorax paradoxus Strain B4 Is the Key Enzyme of Mercaptosuccinate Degradation

    PubMed Central

    Brandt, Ulrike; Schürmann, Marc; Steinbüchel, Alexander

    2014-01-01

    The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent Km of 0.4 mm, and a specific activity (Vmax) of 20.0 μmol min−1 mg−1 corresponding to a kcat of 7.7 s−1. Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase. PMID:25228698

  12. Atrazine degradation and enzyme activities in an agricultural soil under two tillage systems.

    PubMed

    Mahía, Jorge; Martín, Angela; Carballas, Tarsy; Díaz-Raviña, Montserrat

    2007-05-25

    The content of atrazine and its metabolites (hydroxyatrazine, deethylatrazine and deisopropylatrazine) as well as the activities of two soil enzymes (urease and beta-glucosidase) were evaluated in an acid agricultural soil, located in a temperate humid zone (Galicia, NW Spain), with an annual ryegrass-maize rotation under conventional tillage (CT) and no tillage (NT). Samples were collected during two consecutive years from the arable layer at two depths (0-5 cm and 5-20 cm) and different times after atrazine application. Hydroxyatrazine and deisopropylatrazine were the main metabolites resulting from atrazine degradation in the acid soil studied, the highest levels being detected in the surface layer of the NT treatment. A residual effect of atrazine was observed since hydroxyatrazine was detected in the arable layer (0-5 cm, 5-20 cm) even one year after the herbicide application. Soil enzyme activities in the upper 5 cm layer under NT were consistently higher than those in the same layer under CT. Urease and beta-glucosidase activities decreased with depth in the profile under NT but they did not show any differences between the two depths for the plots under CT. For both tillage systems enzyme activities also reflected temporal changes during the maize cultivation; however, no consistent effect of the herbicide application was observed.

  13. Use of salsalate to target inflammation in the treatment of insulin resistance and type 2 diabetes.

    PubMed

    Goldfine, Allison B; Silver, Robert; Aldhahi, Waleed; Cai, Dongsheng; Tatro, Elizabeth; Lee, Jongsoon; Shoelson, Steven E

    2008-05-01

    Chronic subacute inflammation is implicated in the pathogenesis of insulin resistance and type 2 diabetes. Salicylates were shown years ago to lower glucose and more recently to inhibit NF-kappaB activity. Salsalate, a prodrug form of salicylate, has seen extensive clinical use and has a favorable safety profile. We studied the efficacy of salsalate in reducing glycemia and insulin resistance and potential mechanisms of action to validate NF-kappaB as a potential pharmacologic target in diabetes. In open label studies, both high (4.5 g/d) and standard (3.0 g/d) doses of salsalate reduced fasting and postchallenge glucose levels after 2 weeks of treatment. Salsalate increased glucose utilization during euglycemic hyperinsulinemic clamps, by approximately 50% and 15% at the high and standard doses, respectively, and insulin clearance was decreased. Dose-limiting tinnitus occurred only at the higher dose. In a third, double-masked, placebo-controlled trial, 1 month of salsalate at maximum tolerable dose (no tinnitus) improved fasting and postchallenge glucose levels. Circulating free fatty acids were reduced and adiponectin increased in all treated subjects. These data demonstrate that salsalate improves in vivo glucose and lipid homeostasis, and support targeting of inflammation and NF-kappaB as a therapeutic approach in type 2 diabetes.

  14. Insulin and insulin-like growth factor-1 induce pronounced hypertrophy of skeletal myofibers in tissue culture

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.; Karlisch, Patricia; Shansky, Janet

    1990-01-01

    Skeletal myofibers differentiated from primary avian myoblasts in tissue culture can be maintained in positive nitrogen balance in a serum-free medium for at least 6 to 7 days when embedded in a three dimensional collagen gel matrix. The myofibers are metabolically sensitive to physiological concentrations of insulin but these concentrations do not stimulate cell growth. Higher insulin concentrations stimulate both cell hyperplasia and myofiber hypertrophy. Cell growth results from a long term 42 percent increase in total protein synthesis and a 38 percent increase in protein degradation. Myofiber diameters increase by 71 to 98 percent after 6 to 7 days in insulin-containing medium. Insulin-like growth factor-1 but not insulin-like growth factor-2, at 250 ng/ml, is as effective as insulin in stimulating cell hyperplasia and myofiber hypertrophy. This model system provides a new method for studying the long-term anabolic effects of insulin and insulin-like growth factors on myofiber hypertrophy under defined tissue culture conditions.

  15. Microbial surface displayed enzymes based biofuel cell utilizing degradation products of lignocellulosic biomass for direct electrical energy.

    PubMed

    Fan, Shuqin; Hou, Chuantao; Liang, Bo; Feng, Ruirui; Liu, Aihua

    2015-09-01

    In this work, a bacterial surface displaying enzyme based two-compartment biofuel cell for the direct electrical energy conversion from degradation products of lignocellulosic biomass is reported. Considering that the main degradation products of the lignocellulose are glucose and xylose, xylose dehydrogenase (XDH) displayed bacteria (XDH-bacteria) and glucose dehydrogenase (GDH) displayed bacteria (GDH-bacteria) were used as anode catalysts in anode chamber with methylene blue as electron transfer mediator. While the cathode chamber was constructed with laccase/multi-walled-carbon nanotube/glassy-carbon-electrode. XDH-bacteria exhibited 1.75 times higher catalytic efficiency than GDH-bacteria. This assembled enzymatic fuel cell exhibited a high open-circuit potential of 0.80 V, acceptable stability and energy conversion efficiency. Moreover, the maximum power density of the cell could reach 53 μW cm(-2) when fueled with degradation products of corn stalk. Thus, this finding holds great potential to directly convert degradation products of biomass into electrical energy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Lignocellulose degradation patterns, structural changes, and enzyme secretion by Inonotus obliquus on straw biomass under submerged fermentation.

    PubMed

    Xu, Xiangqun; Xu, Zhiqi; Shi, Song; Lin, Mengmeng

    2017-10-01

    This study examined the white rot fungus I. obliquus on the degradation of three types of straw biomass and the production of extracellular lignocellulolytic enzymes under submerged fermentation. The fungus process resulted in a highest lignin loss of 72%, 39%, and 47% in wheat straw, rice straw, and corn stover within 12days, respectively. In merely two days, the fungus selectively degraded wheat straw lignin by 37%, with only limited cellulose degradation (13%). Fourier transform infrared spectroscopy revealed that the fungus most effectively degraded the wheat straw lignin and rice straw crystalline cellulose. Scanning electronic microscopy showed the most pronounced structural changes in wheat straw. High activities of manganese peroxidase (159.0U/mL) and lignin peroxidase (123.4U/mL) were observed in wheat straw culture on Day 2 and 4, respectively. Rice straw was the best substrate to induce the production of cellulase and xylanase. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Decomposition of insoluble and hard-to-degrade animal proteins by enzyme E77 and its potential applications.

    PubMed

    Zhao, Hui; Mitsuiki, Shinji; Takasugi, Mikako; Sakai, Masashi; Goto, Masatoshi; Kanouchi, Hiroaki; Oka, Tatsuzo

    2012-04-01

    Insoluble and hard-to-degrade animal proteins are group of troublesome proteins, such as collagen, elastin, keratin, and prion proteins that are largely generated by the meat industry and ultimately converted to industrial wastes. We analyzed the ability of the abnormal prion protein-degrading enzyme E77 to degrade insoluble and hard-to-degrade animal proteins including keratin, collagen, and elastin. The results indicate that E77 has a much higher keratinolytic activity than proteinase K and subtilisin. Maximal E77 keratinolytic activity was observed at pH 12.0 and 65 °C. E77 was also adsorbed by keratin in a pH-independent manner. E77 showed lower collagenolytic and elastinolytic specificities than proteinase K and subtilisin. Moreover, E77 treatment did not damage collagens in ovine small intestines but did almost completely remove the muscles. We consider that E77 has the potential ability for application in the processing of animal feedstuffs and sausages.

  18. Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana.

    PubMed

    Pedrini, Nicolás; Zhang, Shizhu; Juárez, M Patricia; Keyhani, Nemat O

    2010-08-01

    The insect epicuticle or waxy layer comprises a heterogeneous mixture of lipids that include abundant levels of long-chain alkanes, alkenes, wax esters and fatty acids. This structure represents the first barrier against microbial attack and for broad-host-range insect pathogens, such as Beauveria bassiana, it is the initial interface mediating the host-pathogen interaction, since these organisms do not require any specialized mode of entry and infect target hosts via the cuticle. B. bassiana is able to grow on straight chain alkanes up to n-C(33) as a sole source of carbon and energy. The cDNA and genomic sequences, including putative regulatory elements, for eight cytochrome P450 enzymes, postulated to be involved in alkane and insect epicuticle degradation, were isolated and characterized. Expression studies using a range of alkanes as well as an insect-derived epicuticular extract from the blood-sucking bug Triatomas infestans revealed a differential expression pattern for the P450 genes examined, and suggest that B. bassiana contains a series of hydrocarbon-assimilating enzymes with overlapping specificity in order to target the surface lipids of insect hosts. Phylogenetic analysis of the translated ORFs of the sequences revealed that the enzyme which displayed the highest levels of induction on both alkanes and the insect epicuticular extract represents the founding member of a new cytochrome P450 family, with three of the other sequences assigned as the first members of new P450 subfamilies. The remaining four proteins clustered with known P450 families whose members include alkane monooxygenases.

  19. Insulin fibrillation and protein design: topological resistance of single-chain analogs to thermal degradation with application to a pump reservoir.

    PubMed

    Phillips, Nelson B; Whittaker, Jonathan; Ismail-Beigi, Faramarz; Weiss, Michael A

    2012-03-01

    Insulin is susceptible to thermal fibrillation, a misfolding process that leads to nonnative cross-β assembly analogous to pathological amyloid deposition. Pharmaceutical formulations are ordinarily protected from such degradation by sequestration of the susceptible monomer within native protein assemblies. With respect to the safety and efficacy of insulin pumps, however, this strategy imposes an intrinsic trade-off between pharmacokinetic goals (rapid absorption and clearance) and the requisite physical properties of a formulation (prolonged shelf life and stability within the reservoir). Available rapid-acting formulations are suboptimal in both respects; susceptibility to fibrillation is exacerbated even as absorption is delayed relative to the ideal specifications of a closed-loop system. To circumvent this molecular trade-off, we exploited structural models of insulin fibrils and amyloidogenic intermediates to define an alternative protective mechanism. Single-chain insulin (SCI) analogs were shown to be refractory to thermal fibrillation with maintenance of biological activity for more than 3 months under conditions that promote the rapid fibrillation and inactivation of insulin. The essential idea exploits an intrinsic incompatibility between SCI topology and the geometry of cross-β assembly. A peptide tether was thus interposed between the A- and B-chains whose length was (a) sufficiently long to provide the "play" needed for induced fit of the hormone on receptor binding and yet (b) sufficiently short to impose a topological barrier to fibrillation. Our findings suggest that ultrastable monomeric SCI analogs may be formulated without protective self-assembly and so permit simultaneous optimization of pharmacokinetics and reservoir life. © 2012 Diabetes Technology Society.

  20. Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification.

    PubMed

    Longoni, Paolo; Leelavathi, Sadhu; Doria, Enrico; Reddy, Vanga Siva; Cella, Rino

    2015-01-01

    Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry.

  1. Tumor necrosis factor alpha converting enzyme: an encouraging target for various inflammatory disorders.

    PubMed

    Bahia, Malkeet S; Silakari, Om

    2010-05-01

    Tumor necrosis factor alpha is one of the most common pro-inflammatory cytokines responsible for various inflammatory disorders. It plays an important role in the origin and progression of rheumatoid arthritis and also in other autoimmune disease conditions. Some anti-tumor necrosis factor alpha antibodies like Enbrel, Humira and Remicade have been successfully used in these disease conditions as antagonists of tumor necrosis factor alpha. Inhibition of generation of active form of tumor necrosis factor alpha is a promising therapy for various inflammatory disorders. Therefore, the inhibition of an enzyme (tumor necrosis factor alpha converting enzyme), which is responsible for processing inactive form of tumor necrosis factor alpha into its active soluble form, is an encouraging target. Many tumor necrosis factor alpha converting enzyme inhibitors have been the candidates of clinical trials but none of them have reached in to the market because of their broad spectrum inhibitory activity for other matrix metalloproteases. Selectivity of tumor necrosis factor alpha converting enzyme inhibition over matrix metalloproteases is of utmost importance. If selectivity is achieved successfully, side-effects can be over-ruled and this approach may become a novel therapy for treatment of rheumatoid arthritis and other inflammatory disorders. This cytokine not only plays a pivotal role in inflammatory conditions but also in some cancerous conditions. Thus, successful targeting of tumor necrosis factor alpha converting enzyme may result in multifunctional therapy.

  2. Determinants of Swe1p Degradation in Saccharomyces cerevisiae

    PubMed Central

    McMillan, John N.; Theesfeld, Chandra L.; Harrison, Jacob C.; Bardes, Elaine S. G.; Lew, Daniel J.

    2002-01-01

    Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCFMet30 acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCFMet30) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist. PMID:12388757

  3. Degradation of Diuron by Phanerochaete chrysosporium: Role of Ligninolytic Enzymes and Cytochrome P450

    PubMed Central

    Coelho-Moreira, Jaqueline da Silva; de Souza, Aline Cristine da Silva; Oliveira, Roselene Ferreira; de Sá-Nakanishi, Anacharis Babeto; de Souza, Cristina Giatti Marques; Peralta, Rosane Marina

    2013-01-01

    The white-rot fungus Phanerochaete chrysosporium was investigated for its capacity to degrade the herbicide diuron in liquid stationary cultures. The presence of diuron increased the production of lignin peroxidase in relation to control cultures but only barely affected the production of manganese peroxidase. The herbicide at the concentration of 7 μg/mL did not cause any reduction in the biomass production and it was almost completely removed after 10 days. Concomitantly with the removal of diuron, two metabolites, DCPMU [1-(3,4-dichlorophenyl)-3-methylurea] and DCPU [(3,4-dichlorophenyl)urea], were detected in the culture medium at the concentrations of 0.74 μg/mL and 0.06 μg/mL, respectively. Crude extracellular ligninolytic enzymes were not efficient in the in vitro degradation of diuron. In addition, 1-aminobenzotriazole (ABT), a cytochrome P450 inhibitor, significantly inhibited both diuron degradation and metabolites production. Significant reduction in the toxicity evaluated by the Lactuca sativa L. bioassay was observed in the cultures after 10 days of cultivation. In conclusion, P. chrysosporium can efficiently metabolize diuron without the accumulation of toxic products. PMID:24490150

  4. Conformational Dynamics of Insulin

    PubMed Central

    Hua, Qing-Xin; Jia, Wenhua; Weiss, Michael A.

    2011-01-01

    We have exploited a prandial insulin analog to elucidate the underlying structure and dynamics of insulin as a monomer in solution. A model was provided by insulin lispro (the active component of Humalog®; Eli Lilly and Co.). Whereas NMR-based modeling recapitulated structural relationships of insulin crystals (T-state protomers), dynamic anomalies were revealed by amide-proton exchange kinetics in D2O. Surprisingly, the majority of hydrogen bonds observed in crystal structures are only transiently maintained in solution, including key T-state-specific inter-chain contacts. Long-lived hydrogen bonds (as defined by global exchange kinetics) exist only at a subset of four α-helical sites (two per chain) flanking an internal disulfide bridge (cystine A20–B19); these sites map within the proposed folding nucleus of proinsulin. The anomalous flexibility of insulin otherwise spans its active surface and may facilitate receptor binding. Because conformational fluctuations promote the degradation of pharmaceutical formulations, we envisage that “dynamic re-engineering” of insulin may enable design of ultra-stable formulations for humanitarian use in the developing world. PMID:22649374

  5. Insulin/IGF-1 signaling mutants reprogram ER stress response regulators to promote longevity.

    PubMed

    Henis-Korenblit, Sivan; Zhang, Peichuan; Hansen, Malene; McCormick, Mark; Lee, Seung-Jae; Cary, Michael; Kenyon, Cynthia

    2010-05-25

    When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response is activated. This ER stress response restores ER homeostasis by coordinating processes that decrease translation, degrade misfolded proteins, and increase the levels of ER-resident chaperones. Ribonuclease inositol-requiring protein-1 (IRE-1), an endoribonuclease that mediates unconventional splicing, and its target, the XBP-1 transcription factor, are key mediators of the unfolded protein response. In this study, we show that in Caenorhabditis elegans insulin/IGF-1 pathway mutants, IRE-1 and XBP-1 promote lifespan extension and enhance resistance to ER stress. We show that these effects are not achieved simply by increasing the level of spliced xbp-1 mRNA and expression of XBP-1's normal target genes. Instead, in insulin/IGF-1 pathway mutants, XBP-1 collaborates with DAF-16, a FOXO-transcription factor that is activated in these mutants, to enhance ER stress resistance and to activate new genes that promote longevity.

  6. Enzyme sensitive smart inulin-dehydropeptide conjugate self-assembles into nanostructures useful for targeted delivery of ornidazole.

    PubMed

    Shivhare, Kriti; Garg, Charu; Priyam, Ayushi; Gupta, Alka; Sharma, Ashwani Kumar; Kumar, Pradeep

    2018-01-01

    Molecular self-assembly of biodegradable amphiphilic polymers allows rational design of biocompatible nanomaterials for drug delivery. Use of substituted polysaccharides for such applications offers the ease of design and synthesis, and provides higher biofunctionality and biocompatibility to nanomaterials. The present work focuses on the synthesis, characterization and potential biomedical applications of self-assembled polysaccharide-based materials. We demonstrated that the synthesized amphiphilic inulin self-assembled in aqueous medium into nanostructures with average size in the range of 146-486nm and encapsulated hydrophobic therapeutic molecule, ornidazole. Hydrophophic dehydropeptide was conjugated with inulin via a biocompatible ester linkage. Dehydrophenylalanine, an unusual amino acid, was incorporated in the peptide to make it stable at a broader range of pH as well as against proteases. The resulting core-shell type of nanostructures could encapsulate ornidazole in the hydrophobic core and released it in a controlled fashion. By taking the advantage of inulin, which gets degraded in the colon by colonic bacteria, the effect of enzyme, inulinase, present in the microflora of the large intestine, on inulin-peptide degradation followed by drug release has been studied. Altogether, small peptide conjugated to inulin offers novel scaffold for the future design of nanostructures with potential applications in the field of targeted drug delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Insulin, cognition, and dementia

    PubMed Central

    Cholerton, Brenna; Baker, Laura D.; Craft, Suzanne

    2015-01-01

    Cognitive disorders of aging represent a serious threat to the social and economic welfare of current society. It is now widely recognized that pathology related to such conditions, particularly Alzheimer’s disease, likely begins years or decades prior to the onset of clinical dementia symptoms. This revelation has led researchers to consider candidate mechanisms precipitating the cascade of neuropathological events that eventually lead to clinical Alzheimer’s disease. Insulin, a hormone with potent effects in the brain, has recently received a great deal of attention for its potential beneficial and protective role in cognitive function. Insulin resistance, which refers to the reduced sensitivity of target tissues to the favorable effects of insulin, is related to multiple chronic conditions known to impact cognition and increase dementia risk. With insulin resistance-associated conditions reaching epidemic proportions, the prevalence of Alzheimer’s disease and other cognitive disorders will continue to rise exponentially. Fortunately, these chronic insulin-related conditions are amenable to pharmacological intervention. As a result, novel therapeutic strategies that focus on increasing insulin sensitivity in the brain may be an important target for protecting or treating cognitive decline. The following review will highlight our current understanding of the role of insulin in brain, potential mechanisms underlying the link between insulin resistance and dementia, and current experimental therapeutic strategies aimed at improving cognitive function via modifying the brain’s insulin sensitivity. PMID:24070815

  8. Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)

    PubMed Central

    2017-01-01

    Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets. PMID:29240418

  9. Targeting the Autophagy/Lysosomal Degradation Pathway in Parkinson's Disease.

    PubMed

    Rivero-Ríos, Pilar; Madero-Pérez, Jesús; Fernández, Belén; Hilfiker, Sabine

    2016-01-01

    Autophagy is a cellular quality control mechanism crucial for neuronal homeostasis. Defects in autophagy are critically associated with mechanisms underlying Parkinson's disease (PD), a common and debilitating neurodegenerative disorder. Autophagic dysfunction in PD can occur at several stages of the autophagy/lysosomal degradative machinery, contributing to the formation of intracellular protein aggregates and eventual neuronal cell death. Therefore, autophagy inducers may comprise a promising new therapeutic approach to combat neurodegeneration in PD. Several currently available FDA-approved drugs have been shown to enhance autophagy, which may allow for their repurposing for use in novel clinical conditions including PD. This review summarizes our current knowledge of deficits in the autophagy/lysosomal degradation pathways associated with PD, and highlight current approaches which target this pathway as possible means towards novel therapeutic strategies.

  10. Mcl-1 Degradation Is Required for Targeted Therapeutics to Eradicate Colon Cancer Cells.

    PubMed

    Tong, Jingshan; Wang, Peng; Tan, Shuai; Chen, Dongshi; Nikolovska-Coleska, Zaneta; Zou, Fangdong; Yu, Jian; Zhang, Lin

    2017-05-01

    The Bcl-2 family protein Mcl-1 is often degraded in cancer cells subjected to effective therapeutic treatment, and defective Mcl-1 degradation has been associated with intrinsic and acquired drug resistance. However, a causal relationship between Mcl-1 degradation and anticancer drug responses has not been directly established, especially in solid tumor cells where Mcl-1 inhibition alone is insufficient to trigger cell death. In this study, we present evidence that Mcl-1 participates directly in determining effective therapeutic responses in colon cancer cells. In this setting, Mcl-1 degradation was induced by a variety of multikinase inhibitor drugs, where it relied upon GSK3β phosphorylation and FBW7-dependent ubiquitination. Specific blockade by genetic knock-in (KI) abolished apoptotic responses and conferred resistance to kinase inhibitors. Mcl-1 -KI also suppressed the antiangiogenic and anti-hypoxic effects of kinase inhibitors in the tumor microenvironment. Interestingly, these same inhibitors also induced the BH3-only Bcl-2 family protein PUMA, which is required for apoptosis. Degradation-resistant Mcl-1 bound and sequestered PUMA from other prosurvival proteins to maintain cell survival, which was abolished by small-molecule Mcl-1 inhibitors. Our findings establish a pivotal role for Mcl-1 degradation in the response of colon cancer cells to targeted therapeutics, and they provide a useful rational platform to develop Mcl-1-targeting agents that can overcome drug resistance. Cancer Res; 77(9); 2512-21. ©2017 AACR . ©2017 American Association for Cancer Research.

  11. Enzymes for improved biomass conversion

    DOEpatents

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  12. Degradation and polymerization of monolignols by Abortiporus biennis, and induction of its degradation with a reducing agent.

    PubMed

    Hong, Chang-Young; Park, Se-Yeong; Kim, Seon-Hong; Lee, Su-Yeon; Choi, Won-Sil; Choi, In-Gyu

    2016-10-01

    This study was carried out to better understand the characteristic modification mechanisms of monolignols by enzyme system of Abortiporus biennis and to induce the degradation of monolignols. Degradation and polymerization of monolignols were simultaneously induced by A. biennis. Whole cells of A. biennis degraded coniferyl alcohol to vanillin and coniferyl aldehyde, and degraded sinapyl alcohol to 2,6-dimethoxybenzene- 1,4-diol, with the production of dimers. The molecular weight of monolignols treated with A. biennis increased drastically. The activities of lignin degrading enzymes were monitored for 24 h to determine whether there was any correlation between monolignol biomodification and ligninolytic enzymes. We concluded that complex enzyme systems were involved in the degradation and polymerization of monolignols. To degrade monolignols, ascorbic acid was added to the culture medium as a reducing agent. In the presence of ascorbic acid, the molecular weight was less increased in the case of coniferyl alcohol, while that of sinapyl alcohol was similar to that of the control. Furthermore, the addition of ascorbic acid led to the production of various degraded compounds: syringaldehyde and acid compounds. Accordingly, these results demonstrated that ascorbic acid prevented the rapid polymerization of monolignols, thus stabilizing radicals generated by enzymes of A. biennis. Thereafter, A. biennis catalyzed the oxidation of stable monolignols. As a result, ascorbic acid facilitated predominantly monolignols degradation by A. biennis through the stabilization of radicals. These findings showed outstanding ability of A. biennis to modify the lignin compounds rapidly and usefully.

  13. Production of plant cell wall degrading enzymes by monoculture and co-culture of Aspergillus niger and Aspergillus terreus under SSF of banana peels.

    PubMed

    Rehman, Shazia; Aslam, Hina; Ahmad, Aqeel; Khan, Shakeel Ahmed; Sohail, Muhammad

    2014-01-01

    Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE), in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase) using a novel substrate, Banana Peels (BP) for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes.

  14. Production of plant cell wall degrading enzymes by monoculture and co-culture of Aspergillus niger and Aspergillus terreus under SSF of banana peels

    PubMed Central

    Rehman, Shazia; Aslam, Hina; Ahmad, Aqeel; Khan, Shakeel Ahmed; Sohail, Muhammad

    2014-01-01

    Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE), in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase) using a novel substrate, Banana Peels (BP) for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes. PMID:25763058

  15. The role of the ubiquitin proteasome pathway in keratin intermediate filament protein degradation.

    PubMed

    Rogel, Micah R; Jaitovich, Ariel; Ridge, Karen M

    2010-02-01

    Lung injury, whether caused by hypoxic or mechanical stresses, elicits a variety of responses at the cellular level. Alveolar epithelial cells respond and adapt to such injurious stimuli by reorganizing the cellular cytoskeleton, mainly accomplished through modification of the intermediate filament (IF) network. The structural and mechanical integrity in epithelial cells is maintained through this adaptive reorganization response. Keratin, the predominant IF expressed in epithelial cells, displays highly dynamic properties in response to injury, sometimes in the form of degradation of the keratin IF network. Post-translational modification, such as phosphorylation, targets keratin proteins for degradation in these circumstances. As with other structural and regulatory proteins, turnover of keratin is regulated by the ubiquitin (Ub)-proteasome pathway. The degradation process begins with activation of Ub by the Ub-activating enzyme (E1), followed by the exchange of Ub to the Ub-conjugating enzyme (E2). E2 shuttles the Ub molecule to the substrate-specific Ub ligase (E3), which then delivers the Ub to the substrate protein, thereby targeting it for degradation. In some cases of injury and IF-related disease, aggresomes form in epithelial cells. The mechanisms that regulate aggresome formation are currently unknown, although proteasome overload may play a role. Therefore, a more complete understanding of keratin degradation--causes, mechanisms, and consequences--will allow for a greater understanding of epithelial cell biology and lung pathology alike.

  16. Effects of hydroalcoholic extract of Rhus coriaria seed on glucose and insulin related biomarkers, lipid profile, and hepatic enzymes in nicotinamide-streptozotocin-induced type II diabetic male mice.

    PubMed

    Ahangarpour, Akram; Heidari, Hamid; Junghani, Majid Salehizade; Absari, Reza; Khoogar, Mehdi; Ghaedi, Ehsan

    2017-10-01

    Type 2 diabetes often leads to dislipidemia and abnormal activity of hepatic enzymes. The purpose of this study was to evaluate the antidiabetic and hypolipidemic properties of Rhus coriaria ( R. coriaria ) seed extrac on nicotinamide-streptozotocin induced type 2 diabetic mice. In this experimental study, 56 male Naval Medical Research Institute mice (30-35 g) were randomly separated into seven groups: control, diabetic group, diabetic mice treated with glibenclamide (0.25 mg/kg, as standard antidiabetic drug) or R. coriaria seed extract in doses of 200 and 300 mg/kg, and control groups received these two doses of extract orally for 28 days. Induction of diabetes was done by intraperitoneal injection of nicotinamide and streptozotocin. Ultimately, body weight of mice, blood levels of glucose, insulin, hepatic enzymes, leptin, and lipid profile were assayed. After induction of type 2 diabetes, level of glucose, cholesterol, low density lipoprotein, serum glutamic oxaloacetic transaminase, and serum glutamic pyruvic transaminase increased and level of insulin and high density lipoprotein decreased remarkably. Administration of both doses of extract decreased level of glucose and cholesterol significantly in diabetic mice. LDL level decreased in treated group with dose of 300 mg/kg of the extract. Although usage of the extract improved level of other lipid profiles, insulin and hepatic enzymes, changes weren't significant. This study showed R. coriaria seeds administration has a favorable effect in controlling some blood parameters in type 2 diabetes. Therefore it may be beneficial in the treatment of diabetes.

  17. Whole genome analysis of six organophosphate-degrading rhizobacteria reveals putative agrochemical degradation enzymes with broad substrate specificity.

    PubMed

    Iyer, Rupa; Iken, Brian; Damania, Ashish; Krieger, Jerry

    2018-05-01

    Six organophosphate-degrading bacterial strains collected from farm and ranch soil rhizospheres across the Houston-metropolitan area were identified as strains of Pseudomonas putida (CBF10-2), Pseudomonas stutzeri (ODKF13), Ochrobactrum anthropi (FRAF13), Stenotrophomonas maltophilia (CBF10-1), Achromobacter xylosoxidans (ADAF13), and Rhizobium radiobacter (GHKF11). Whole genome sequencing data was assessed for relevant genes, proteins, and pathways involved in the breakdown of agrochemicals. For comparative purposes, this analysis was expanded to also include data from deposited strains in the National Center for Biotechnology Information's (NCBI) database. This study revealed Zn-dependent metallo-β-lactamase (MBL)-fold proteins similar to OPHC2 first identified in P. pseudoalcaligenes as the likely agents of organophosphate (OP) hydrolysis in A. xylosoxidans ADAF13, S. maltophilia CBF10-1, O. anthropi FRAF13, and R. radiobacter GHKF11. A search of similar proteins within NCBI identified over 200 hits for bacterial genera and species with a similar OPHC2 domain. Taken together, we conclude from this data that intrinsic low-level OP hydrolytic activity is likely prevalent across the rhizosphere stemming from widespread OPHC2-like metalloenzymes. In addition, P. stutzeri ODKF13, P. putida CBF10-2, O. anthropi FRAF13, and R. radiobacter GHKF11 were found to harbor glycine oxidase (GO) enzymes that putatively possess low-level activity against the herbicide glyphosate. These bacterial GOs are reported to catalyze the degradation of glyphosate to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and suggest a possible link to AMPA that can be found in glyphosate-contaminated agricultural soil. The presence of aromatic degradation proteins were also detected in five of six study strains, but are attributed primarily to components of the widely distributed β-ketoadipate pathway found in many soil bacteria.

  18. Targeting the Allosteric Site of Oncoprotein BCR-ABL as an Alternative Strategy for Effective Target Protein Degradation.

    PubMed

    Shimokawa, Kenichiro; Shibata, Norihito; Sameshima, Tomoya; Miyamoto, Naoki; Ujikawa, Osamu; Nara, Hiroshi; Ohoka, Nobumichi; Hattori, Takayuki; Cho, Nobuo; Naito, Mikihiko

    2017-10-12

    Protein degradation technology based on hybrid small molecules is an emerging drug modality that has significant potential in drug discovery and as a unique method of post-translational protein knockdown in the field of chemical biology. Here, we report the first example of a novel and potent protein degradation inducer that binds to an allosteric site of the oncogenic BCR-ABL protein. BCR-ABL allosteric ligands were incorporated into the SNIPER (Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) platform, and a series of in vitro biological assays of binding affinity, target protein modulation, signal transduction, and growth inhibition were carried out. One of the designed compounds, 6 (SNIPER(ABL)-062), showed desirable binding affinities against ABL1, cIAP1/2, and XIAP and consequently caused potent BCR-ABL degradation.

  19. Targeted proteomics reveals strain-specific changes in the mouse insulin and central metabolic pathways after a sustained high-fat diet.

    PubMed

    Sabidó, Eduard; Wu, Yibo; Bautista, Lucia; Porstmann, Thomas; Chang, Ching-Yun; Vitek, Olga; Stoffel, Markus; Aebersold, Ruedi

    2013-07-16

    The metabolic syndrome is a collection of risk factors including obesity, insulin resistance and hepatic steatosis, which occur together and increase the risk of diseases such as diabetes, cardiovascular disease and cancer. In spite of intense research, the complex etiology of insulin resistance and its association with the accumulation of triacylglycerides in the liver and with hepatic steatosis remains not completely understood. Here, we performed quantitative measurements of 144 proteins involved in the insulin-signaling pathway and central metabolism in liver homogenates of two genetically well-defined mouse strains C57BL/6J and 129Sv that were subjected to a sustained high-fat diet. We used targeted mass spectrometry by selected reaction monitoring (SRM) to generate accurate and reproducible quantitation of the targeted proteins across 36 different samples (12 conditions and 3 biological replicates), generating one of the largest quantitative targeted proteomics data sets in mammalian tissues. Our results revealed rapid response to high-fat diet that diverged early in the feeding regimen, and evidenced a response to high-fat diet dominated by the activation of peroxisomal β-oxidation in C57BL/6J and by lipogenesis in 129Sv mice.

  20. Chitosan Nanofibers for Transbuccal Insulin Delivery

    PubMed Central

    Lancina, Michael G.; Shankar, Roopa Kanakatti; Yang, Hu

    2017-01-01

    Purpose In this work, we aimed at producing chitosan based nanofiber mats capable of delivering insulin via the buccal mucosa. Methods Chitosan was electrospun into nanofibers using poly (ethylene oxide) (PEO) as a carrier molecule in various feed ratios. The mechanical properties and degradation kinetics of the fibers were measured. Insulin release rates were determined in vitro using an ELISA assay. The bioactivity of released insulin was measured in terms of Akt activation in pre-adipocytes. Insulin permeation across the buccal mucosa was measured in an ex-vivo porcine transbuccal model. Results Fiber morphology, mechanical properties, and in vitro stability were dependent on PEO feed ratio. Lower PEO content blends produced smaller diameter fibers with significantly faster insulin release kinetics. Insulin showed no reduction in bioactivity due to electrospinning. Buccal permeation of insulin facilitated by high chitosan content blends was significantly higher than that of free insulin. Conclusions Taken together, our work demonstrates chitosan based nanofibers have the potential to serve as a transbuccal insulin delivery vehicle. PMID:28000386

  1. Degradation of Three Aromatic Dyes by White Rot Fungi and the Production of Ligninolytic Enzymes

    PubMed Central

    Jayasinghe, Chandana; Imtiaj, Ahmed; Lee, Geon Woo; Im, Kyung Hoan; Hur, Hyun; Lee, Min Woong; Yang, Hee-Sun

    2008-01-01

    This study was conducted to evaluate the degradation of aromatic dyes and the production of ligninolytic enzymes by 10 white rot fungi. The results of this study revealed that Pycnoporus cinnabarinus, Pleurotus pulmonarius, Ganoderma lucidum, Trametes suaveolens, Stereum ostrea and Fomes fomentarius have the ability to efficiently degrade congo red on solid media. However, malachite green inhibited the mycelial growth of these organisms. Therefore, they did not effectively decolorize malachite green on solid media. However, P. cinnabarinus and P. pulmonarius were able to effectively decolorize malachite green on solid media. T. suaveolens and F. rosea decolorized methylene blue more effectively than any of the other fungi evaluated in this study. In liquid culture, G. lucidum, P. cinnabarinus, Naematoloma fasciculare and Pycnoporus coccineus were found to have a greater ability to decolorize congo red. In addition, P. cinnabarinus, G. lucidum and T. suaveolens decolorized methylene blue in liquid media more effectively than any of the other organisms evaluated in this study. Only F. fomentarius was able to decolorize malachite green in liquid media, and its ability to do so was limited. To investigate the production of ligninolytic enzymes in media containing aromatic compounds, fungi were cultured in naphthalene supplemented liquid media. P. coccineus, Coriolus versicolor and P. cinnabarinus were found to produce a large amount of laccase when grown in medium that contained napthalene. PMID:23990745

  2. Impairment of Novel Object Recognition Memory and Brain Insulin Signaling in Fructose- but Not Glucose-Drinking Female Rats.

    PubMed

    Sangüesa, Gemma; Cascales, Mar; Griñán, Christian; Sánchez, Rosa María; Roglans, Núria; Pallàs, Mercè; Laguna, Juan Carlos; Alegret, Marta

    2018-01-26

    Excessive sugar intake has been related to cognitive alterations, but it remains unclear whether these effects are related exclusively to increased energy intake, and the molecular mechanisms involved are not fully understood. We supplemented Sprague-Dawley female rats with 10% w/v fructose in drinking water or with isocaloric glucose solution for 7 months. Cognitive function was assessed through the Morris water maze (MWM) and the novel object recognition (NOR) tests. Plasma parameters and protein/mRNA expression in the frontal cortex and hippocampus were determined. Results showed that only fructose-supplemented rats displayed postprandial and fasting hypertriglyceridemia (1.4 and 1.9-fold, p < 0.05) and a significant reduction in the discrimination index in the NOR test, whereas the results of the MWM test showed no differences between groups. Fructose-drinking rats displayed an abnormal glucose tolerance test and impaired insulin signaling in the frontal cortex, as revealed by significant reductions in insulin receptor substrate-2 protein levels (0.77-fold, p < 0.05) and Akt phosphorylation (0.72-fold, p < 0.05), and increased insulin-degrading enzyme levels (1.86-fold, p < 0.001). Fructose supplementation reduced the expression of antioxidant enzymes and altered the amount of proteins involved in mitochondrial fusion/fission in the frontal cortex. In conclusion, cognitive deficits induced by chronic liquid fructose consumption are not exclusively related to increased caloric intake and are correlated with hypertriglyceridemia, impaired insulin signaling, increased oxidative stress and altered mitochondrial dynamics, especially in the frontal cortex.

  3. FBXO32 suppresses breast cancer tumorigenesis through targeting KLF4 to proteasomal degradation.

    PubMed

    Zhou, H; Liu, Y; Zhu, R; Ding, F; Wan, Y; Li, Y; Liu, Z

    2017-06-08

    Krüppel-like factor 4 (KLF4, GKLF) is a zinc-finger transcription factor involved in a large variety of cellular processes, including apoptosis, cell cycle progression, as well as stem cell renewal. KLF4 is critical for cell fate decision and has an ambivalent role in tumorigenesis. Emerging data keep reminding us that KLF4 dysregulation either facilitates or impedes tumor progression, making it important to clarify the regulating network of KLF4. Like most transcription factors, KLF4 has a rather short half-life within the cell and its turnover must be carefully orchestrated by ubiquitination and ubiquitin-proteasome system. To better understand the mechanism of KLF4 ubiquitination, we performed a genome-wide screen of E3 ligase small interfering RNA library based on western blot and identified SCF-FBXO32 to be a new E3 ligase, which is responsible for KLF4 ubiquitination and degradation. The F-box domain is critical for FBXO32-dependent KLF4 ubiquitination and degradation. Furthermore, we demonstrated that FBXO32 physically interacts with the N-terminus (1-60 aa) of KLF4 via its C-terminus (228-355 aa) and directly targets KLF4 for ubiquitination and degradation. We also found out that p38 mitogen-activated protein kinase pathway may be implicated in FBXO32-mediated ubiquitination of KLF4, as p38 kinase inhibitor coincidently abrogates endogenous KLF4 ubiquitination and degradation, as well as FBXO32-dependent exogenous KLF4 ubiquitination and degradation. Finally, FBXO32 inhibits colony formation in vitro and primary tumor initiation and growth in vivo through targeting KLF4 into degradation. Our findings thus further elucidate the tumor-suppressive function of FBXO32 in breast cancer. These results expand our understanding of the posttranslational modification of KLF4 and of its role in breast cancer development and provide a potential target for diagnosis and therapeutic treatment of breast cancer.

  4. Insulin analogs for the treatment of diabetes mellitus: therapeutic applications of protein engineering.

    PubMed

    Berenson, Daniel F; Weiss, Allison R; Wan, Zhu-Li; Weiss, Michael A

    2011-12-01

    The engineering of insulin analogs represents a triumph of structure-based protein design. A framework has been provided by structures of insulin hexamers. Containing a zinc-coordinated trimer of dimers, such structures represent a storage form of the active insulin monomer. Initial studies focused on destabilization of subunit interfaces. Because disassembly facilitates capillary absorption, such targeted destabilization enabled development of rapid-acting insulin analogs. Converse efforts were undertaken to stabilize the insulin hexamer and promote higher-order self-assembly within the subcutaneous depot toward the goal of enhanced basal glycemic control with reduced risk of hypoglycemia. Current products either operate through isoelectric precipitation (insulin glargine, the active component of Lantus(®); Sanofi-Aventis) or employ an albumin-binding acyl tether (insulin detemir, the active component of Levemir(®); Novo-Nordisk). To further improve pharmacokinetic properties, modified approaches are presently under investigation. Novel strategies have recently been proposed based on subcutaneous supramolecular assembly coupled to (a) large-scale allosteric reorganization of the insulin hexamer (the TR transition), (b) pH-dependent binding of zinc ions to engineered His-X(3)-His sites at hexamer surfaces, or (c) the long-range vision of glucose-responsive polymers for regulated hormone release. Such designs share with wild-type insulin and current insulin products a susceptibility to degradation above room temperature, and so their delivery, storage, and use require the infrastructure of an affluent society. Given the global dimensions of the therapeutic supply chain, we envisage that concurrent engineering of ultra-stable protein analog formulations would benefit underprivileged patients in the developing world.

  5. Improved mortality of the Formosan subterranean termite by fungi, when amended with cuticle-degrading enzymes or eicosanoid biosynthesis inhibitors

    USDA-ARS?s Scientific Manuscript database

    Formosan subterranean termites (FST) were exposed to spores of the fungus Beauveria pseudobassiana (Bpb) strain 8046 to determine virulence of the fungus. Once Bpb was determined to cause mortality of FST it was combined with enzymes capable of degrading the insect cuticle to measure the potential ...

  6. Interaction of Carthamus tinctorius lignan arctigenin with the binding site of tryptophan-degrading enzyme indoleamine 2,3-dioxygenase☆

    PubMed Central

    Temml, Veronika; Kuehnl, Susanne; Schuster, Daniela; Schwaiger, Stefan; Stuppner, Hermann; Fuchs, Dietmar

    2013-01-01

    Mediterranean Carthamus tinctorius (Safflower) is used for treatment of inflammatory conditions and neuropsychiatric disorders. Recently C. tinctorius lignans arctigenin and trachelogenin but not matairesinol were described to interfere with the activity of tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) in peripheral blood mononuclear cells in vitro. We examined a potential direct influence of compounds on IDO enzyme activity applying computational calculations based on 3D geometry of the compounds. The interaction pattern analysis and force field-based minimization was performed within LigandScout 3.03, the docking simulation with MOE 2011.10 using the X-ray crystal structure of IDO. Results confirm the possibility of an intense interaction of arctigenin and trachelogenin with the binding site of the enzyme, while matairesinol had no such effect. PMID:24251110

  7. Reactive oxygen species enhance insulin sensitivity

    PubMed Central

    Loh, Kim; Deng, Haiyang; Fukushima, Atsushi; Cai, Xiaochu; Boivin, Benoit; Galic, Sandra; Bruce, Clinton; Shields, Benjamin J.; Skiba, Beata; Ooms, Lisa M.; Stepto, Nigel; Wu, Ben; Mitchell, Christina A.; Tonks, Nicholas K.; Watt, Matthew J.; Febbraio, Mark A.; Crack, Peter J.; Andrikopoulos, Sofianos; Tiganis, Tony

    2010-01-01

    SUMMARY Chronic reactive oxygen species (ROS) production by mitochondria may contribute to the development of insulin resistance, a primary feature of type 2 diabetes. In recent years it has become apparent that ROS generation in response to physiological stimuli such as insulin may also facilitate signaling by reversibly oxidizing and inhibiting protein tyrosine phosphatases (PTPs). Here we report that mice lacking one of the key enzymes involved in the elimination of physiological ROS, glutathione peroxidase 1 (Gpx1), were protected from high fat diet-induced insulin resistance. The increased insulin sensitivity in Gpx1−/− mice was attributed to insulin-induced phosphatidylinositol-3-kinase/Akt signaling and glucose uptake in muscle and could be reversed by the anti-oxidant N-acetylcysteine. Increased insulin signaling correlated with enhanced oxidation of the PTP family member PTEN, which terminates signals generated by phosphatidylinositol-3-kinase. These studies provide causal evidence for the enhancement of insulin signaling by ROS in vivo. PMID:19808019

  8. Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels

    PubMed Central

    Jung, Sang-Kyu; Parisutham, Vinuselvi; Jeong, Seong Hun; Lee, Sung Kuk

    2012-01-01

    A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such as Escherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinant E. coli or Z. mobilis and allow successful consolidated bioprocessing (CBP) in these microorganisms. In-planta expression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies. PMID:22911272

  9. Network Architecture Predisposes an Enzyme to Either Pharmacologic or Genetic Targeting.

    PubMed

    Jensen, Karin J; Moyer, Christian B; Janes, Kevin A

    2016-02-24

    Chemical inhibition and genetic knockdown of enzymes are not equivalent in cells, but network-level mechanisms that cause discrepancies between knockdown and inhibitor perturbations are not understood. Here we report that enzymes regulated by negative feedback are robust to knockdown but susceptible to inhibition. Using the Raf-MEK-ERK kinase cascade as a model system, we find that ERK activation is resistant to genetic knockdown of MEK but susceptible to a comparable degree of chemical MEK inhibition. We demonstrate that negative feedback from ERK to Raf causes this knockdown-versus-inhibitor discrepancy in vivo. Exhaustive mathematical modeling of three-tiered enzyme cascades suggests that this result is general: negative autoregulation or feedback favors inhibitor potency, whereas positive autoregulation or feedback favors knockdown potency. Our findings provide a rationale for selecting pharmacologic versus genetic perturbations in vivo and point out the dangers of using knockdown approaches in search of drug targets.

  10. Nanosized insulin-complexes based on biodegradable amine-modified graft polyesters poly[vinyl-3-(diethylamino)-propylcarbamate-co-(vinyl acetate)-co-(vinyl alcohol)]-graft-poly(L-lactic acid): protection against enzymatic degradation, interaction with Caco-2 cell monolayers, peptide transport and cytotoxicity.

    PubMed

    Simon, Michael; Behrens, Isabel; Dailey, Lea Anne; Wittmar, Matthias; Kissel, Thomas

    2007-05-01

    Non-parenteral insulin delivery by the oral route is limited by epithelial barriers and enzymatic degradation. Nanosized insulin-complexes based on amine modified comb-like polyesters were designed to overcome these barriers. Protection of insulin in nanocomplexes (NC) from enzymatic degradation was investigated. The interaction with enterocyte-like Caco-2 cells in terms of cytotoxicity, transport through and uptake in the cell layers was evaluated by measuring transepithelial electrical resistance (TEER), release of lactate dehydrogenase (LDH) and insulin transport. The protection capacity of NC and their interaction with Caco-2 cells varied strongly as a function of lactide-grafting (hydrophobicity). With increasing lactide-grafting (P(26) < or = P(26)-1(LL) < or = P(26)-2(LL)) NC protected up to 95% of the insulin against degradation by trypsin. Transport and uptake into cell monolayers increased with higher l-lactid grafting. About 25% of a 1.25mg/ml TRITC-insulin NC dispersion with P(26)-2(LL) was recovered in Caco-2 cells after 90 min. A concentration dependent cytotoxicity profile was observed showing elevated LDH release and decreased TEER values. The cytotoxicity correlates with the surfactant like character of the polymers, decreasing the surface tension to 46 mN/m for the amphiphilic P(26)-2(LL). The observed TEER decrease was reversible after 20 h, suggesting that the biodegradable comb-polyesters offer a promising approach to overcome mucosal barriers.

  11. [Enzymatic degradation of organophosphorus insecticide chlorpyrifos by fungus WZ-I].

    PubMed

    Xie, Hui; Zhu, Lu-sheng; Wang, Jun; Wang, Xiu-guo; Liu, Wei; Qian, Bo; Wang, Qian

    2005-11-01

    Degradation characteristics of chlorpyrifos insecticides was determined by the crude enzyme extracted from the isolated strain WZ-I ( Fusarium LK. ex Fx). The best separating condition and the degrading characteristic of chlorpyrifos were studied. Rate of degradation for chlorpyrifos by its intracellular enzyme, extracellular enzyme and cell fragment was 60.8%, 11.3% and 48%, respectively. The degrading enzyme was extracted after this fungus was incubated for 8 generations in the condition of noninducement, and its enzymic activity lost less, the results show that this enzyme is an intracellular and connatural enzyme. The solubility protein of the crude enzyme was determined with Albumin (bovine serum) as standard protein and the solubility protein of the crude enzyme was 3.36 mg x mL(-1). The pH optimum for crude enzyme was 6.8 for enzymatic degradation of chlorpyrifos, and it had comparatively high activity in the range of pH 6.0 - 9.0. The optimum temperature for enzymatic activity was at 40 degrees C, it still had comparatively high activity in the range of temperature 20-50 degrees C, the activity of enzyme rapidly reduced at 55 degrees C, its activity was 41% of the maximal activity. The crude enzyme showed Km value for chlorpyrifos of 1.049 26 mmol x L(-1), and the maximal enzymatic degradation rate was 0.253 5 micromol x (mg x min)(-1). Additional experimental evidence suggests that the enzyme had the stability of endure for temperature and pH, the crude enzyme of fungus WZ-I could effectively degrade chlorpyrifos.

  12. Insulin-induced Gene Protein (INSIG)-dependent Sterol Regulation of Hmg2 Endoplasmic Reticulum-associated Degradation (ERAD) in Yeast*

    PubMed Central

    Theesfeld, Chandra L.; Hampton, Randolph Y.

    2013-01-01

    Insulin-induced gene proteins (INSIGs) function in control of cellular cholesterol. Mammalian INSIGs exert control by directly interacting with proteins containing sterol-sensing domains (SSDs) when sterol levels are elevated. Mammalian 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR) undergoes sterol-dependent, endoplasmic-reticulum (ER)-associated degradation (ERAD) that is mediated by INSIG interaction with the HMGR SSD. The yeast HMGR isozyme Hmg2 also undergoes feedback-regulated ERAD in response to the early pathway-derived isoprene gernanylgeranyl pyrophosphate (GGPP). Hmg2 has an SSD, and its degradation is controlled by the INSIG homologue Nsg1. However, yeast Nsg1 promotes Hmg2 stabilization by inhibiting GGPP-stimulated ERAD. We have proposed that the seemingly disparate INSIG functions can be unified by viewing INSIGs as sterol-dependent chaperones of SSD clients. Accordingly, we tested the role of sterols in the Nsg1 regulation of Hmg2. We found that both Nsg1-mediated stabilization of Hmg2 and the Nsg1-Hmg2 interaction required the early sterol lanosterol. Lowering lanosterol in the cell allowed GGPP-stimulated Hmg2 ERAD. Thus, Hmg2-regulated degradation is controlled by a two-signal logic; GGPP promotes degradation, and lanosterol inhibits degradation. These data reveal that the sterol dependence of INSIG-client interaction has been preserved for over 1 billion years. We propose that the INSIGs are a class of sterol-dependent chaperones that bind to SSD clients, thus harnessing ER quality control in the homeostasis of sterols. PMID:23306196

  13. Use of a plastic insulin dosage guide to correct blood glucose levels out of the target range and for carbohydrate counting in subjects with type 1 diabetes.

    PubMed

    Kaufman, F R; Halvorson, M; Carpenter, S

    1999-08-01

    To improve glycemic control, a hand-held plastic Insulin Dosage Guide was developed to correct blood glucose levels outside of the target range. Protocol 1: Some 40 children (mean age 10.6+/-4.6 years) were randomly assigned for 3 months to use a written-on-paper algorithm or the Insulin Dosage Guide to correct abnormal blood glucose levels. Mean HbA1c and blood glucose levels and time to teach insulin dosage correction were compared. Protocol 2: The Insulin Dosage Guide was used by 83 subjects (mean age 11.4+/-4.3 years) for 1 year, and mean HbA1c levels, blood glucose levels, and number of consecutive high blood glucose values taken before and after the year were compared. Protocol 3: Some 20 patients (mean age 10.1+/-3.7 years) using rapid-acting insulin and 64 patients (mean age 15.9+/-3.6 years) using an insulin pump and rapid-acting insulin used the Insulin Dosage Guide and had mean blood glucose levels, HbA1c, and percentage of blood glucose levels outside of the target range determined. Protocol 1: There was a significant reduction in mean HbA1c (P = 0.04) and blood glucose levels (P = 0.05) and in the time needed to teach how to correct blood glucose values using the Insulin Dosage Guide compared with the paper algorithm. Protocol 2: There was a decrease in mean HbA1c levels (P = 0.0001) and a decrease in the mean number of consecutive blood glucose levels (P = 0.001) over the 1-year time period. Protocol 3: With rapid-acting insulin, there was a significant increase in the percentage of blood glucose levels within the target range (1 month, P = 0.04; at 3 months, P = 0.03). With the insulin pump, there was a high rate (90%) of blood glucose levels in the target range during pump initiation when the Insulin Dosage Guide was used. This inexpensive hand-held plastic card, which is portable and easy to use, may help patients improve glycemia and successfully manage diabetes.

  14. Post-Streptococcal Auto-Antibodies Inhibit Protein Disulfide Isomerase and Are Associated with Insulin Resistance

    PubMed Central

    Aran, Adi; Weiner, Karin; Lin, Ling; Finn, Laurel Ann; Greco, Mary Ann; Peppard, Paul; Young, Terry; Ofran, Yanay; Mignot, Emmanuel

    2010-01-01

    Post-streptococcal autoimmunity affects millions worldwide, targeting multiple organs including the heart, brain, and kidneys. To explore the post-streptococcal autoimmunity spectrum, we used western blot analyses, to screen 310 sera from healthy subjects with (33%) and without (67%) markers of recent streptococcal infections [anti-Streptolysin O (ASLO) or anti-DNAse B (ADB)]. A 58 KDa protein, reacting strongly with post-streptococcal sera, was identified as Protein Disulfide Isomerase (PDI), an abundant protein with pleiotropic metabolic, immunologic, and thrombotic effects. Anti-PDI autoantibodies, purified from human sera, targeted similar epitopes in Streptolysin O (SLO, P51-61) and PDI (P328-338). The correlation between post-streptococcal status and anti-human PDI auto-immunity was further confirmed in a total of 2987 samples (13.6% in 530 ASLO positive versus 5.6% in 2457 ASLO negative samples, p<0.0001). Finally, anti-PDI auto-antibodies inhibited PDI-mediated insulin degradation in vitro (n = 90, p<0.001), and correlated with higher serum insulin (14.1 iu/ml vs. 12.2 iu/ml, n = 1215, p = 0.039) and insulin resistance (Homeostatic Model Assessment (HOMA) 4.1 vs. 3.1, n = 1215, p = 0.004), in a population-based cohort. These results identify PDI as a major target of post-streptococcal autoimmunity, and establish a new link between infection, autoimmunity, and metabolic disturbances. PMID:20886095

  15. Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets--"Sand Out and Gold Stays".

    PubMed

    Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T; Wang, Hong; Yang, Xiao-feng

    2016-02-01

    To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: (1) Histone enzymes are differentially expressed in cardiovascular, immune, and other tissues; (2) our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, and histone methylation/demethylation are in the highest varieties; and (3) histone enzymes are more downregulated than upregulated in metabolic diseases and regulatory T cell (Treg) polarization/ differentiation, but not in tumors. These results have demonstrated a new working model of "Sand out and Gold stays," where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity.

  16. Degradable Magnetic Composites for Minimally Invasive Interventions: Device Fabrication, Targeted Drug Delivery, and Cytotoxicity Tests.

    PubMed

    Peters, Christian; Hoop, Marcus; Pané, Salvador; Nelson, Bradley J; Hierold, Christofer

    2016-01-20

    Superparamagnetic nanoparticles and a functional, degradable polymer matrix based on poly(ethylene glycol) are combined to enable fully degradable magnetic microdevices for minimally invasive biomedical applications. A bioinspired helical microrobot platform mimicking Escherichia coli bacteria is fabricated and actuated using weak rotating magnetic fields. Locomotion based on corkscrew propulsion, targeted drug delivery, and low-degradation-product cytotoxicity are demonstrated. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Substrate-Wrapped, Single-Walled Carbon Nanotube Probes for Hydrolytic Enzyme Characterization.

    PubMed

    Kallmyer, Nathaniel E; Musielewicz, Joseph; Sutter, Joel; Reuel, Nigel F

    2018-04-17

    Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze-thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans.

  18. Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aylward, Frank O.; Khadempour, Lily; Tremmel, Daniel

    Leaf-cutter ants are prolific and conspicuous Neotropical herbivores that derive energy from specialized fungus gardens they cultivate using foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain lignocellulases that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as it is foraged by the ants. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in thesemore » hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous lignocellulases likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three-quarters of all lignocellulases identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 23 lignocellulases enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.« less

  19. Induced oligomerization targets Golgi proteins for degradation in lysosomes.

    PubMed

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D

    2015-12-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. © 2015 Tewari et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  20. PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice.

    PubMed

    Zinker, Bradley A; Rondinone, Cristina M; Trevillyan, James M; Gum, Rebecca J; Clampit, Jill E; Waring, Jeffrey F; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E; Reilly, Regina M; Koterski, Sandra; Opgenorth, Terry J; Ulrich, Roger G; Crosby, Seth; Butler, Madeline; Murray, Susan F; McKay, Robert A; Bhanot, Sanjay; Monia, Brett P; Jirousek, Michael R

    2002-08-20

    The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA(1C). Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50alpha, were increased and PI3-kinase p85alpha expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

  1. [Purification, characterization and partial primary structure analysis of rutin-degrading enzyme in tartary buckwheat seeds].

    PubMed

    Zhang, Yuwei; Li, Jie; Yuan, Yong; Gu, Jijuan; Chen, Peng

    2017-05-25

    Rutin-degrading enzymes (RDE) can degrade rutin into poorly water soluble compound, quercetin, and cause the bitter taste in tartary buckwheat. In the present study RDE from Yu 6-21 tartary buckwheat seeds was purified by ammonium sulphate precipitation, followed by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, ion exchange chromatography on CM-Cellulose and gel filtration chromatography on Sephadex G-150. Purified RDE showed single band with molecular weight of 66 kDa on SDS-PAGE. The optimum pH and temperature of RDE were 5.0 and 50 ℃ respectively. The Km was 0.27 mmol/L, and the Vmax was 39.68 U/mg. The RDE activity could be inhibited by Cu²⁺, Zn²⁺, Mn²⁺ and EDTA, and showed tolerance to 50% methanol (V/V). The N terminal sequence (TVSRSSFPDGFLFGL) was obtained by Edman degradation method and 15 internal peptide sequences were determined by MALDI-TOF-MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). These results established the foundations for identification of the candidate gene of RDE via transcriptome data and further studying RDE biological function.

  2. Enantioselectivity in tebuconazole and myclobutanil non-target toxicity and degradation in soils.

    PubMed

    Li, Yuanbo; Dong, Fengshou; Liu, Xingang; Xu, Jun; Han, Yongtao; Zheng, Yongquan

    2015-03-01

    Tebuconazole and myclobutanil are two widely used triazole fungicides, both comprising two enantiomers with different fungicidal activity. However, their non-target toxicity and environmental behavior with respect to enantioselectivity have received limited attention. In the present study, tebuconazole and myclobutanil enantiomers were isolated and used to evaluate the occurrence of enantioselectivity in their acute toxicity to three non-target organisms (Scenedesmus obliquus, Daphnia magna, and Danio rerio). Significant differences were found: R-(-)-tebuconazole was about 1.4-5.9 times more toxic than S-(+)-tebuconazole; rac-myclobutanil was about 1.3-6.1 and 1.4-7.3 more toxic than (-)-myclobutanil and (+)-myclobutanil, respectively. Enantioselectivity was further investigated in terms of fungicide degradation in seven soil samples, which were selected to cover a broad range of soil properties. In aerobic or anaerobic soils, the S-(+)-tebuconazole degraded faster than R-(-)-tebuconazole, and the enantioselectivity showed a correlation with soil organic carbon content. (+)-Myclobutanil was preferentially degraded than (-)-myclobutanil in aerobic soils, whereas both enantiomers degraded at similar rates in anaerobic soils. Apparent correlations of enantioselectivity with soil pH and soil texture were observed for myclobutanil under aerobic conditions. In addition, both fungicides were configurationally stable in soils, i.e., no enantiomerization was found. Enantioselectivity may be a common phenomenon in both aquatic toxicity and biodegradation of chiral triazole fungicides, and this should be considered when assessing ecotoxicological risks of these compounds in the environment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Syrvatka, Vasyl J.; Slyvchuk, Yurij I.; Rozgoni, Ivan I.; Gevkan, Ivan I.; Overchuk, Marta O.

    2014-02-01

    Modern routine enzyme immunoassays for detection and quantification of biomolecules have several disadvantages such as high cost, insufficient sensitivity, complexity and long-term execution. The surface plasmon resonance of silver nanoparticles gives reasons of creating new in the basis of simple, highly sensitive and low cost colorimetric assays that can be applied to the detection of small molecules, DNA, proteins and pollutants. The main aim of the study was the improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles. For this purpose we developed method for synthesis of silver nanoparticles with hyaluronic acid and studied possibility of use these nanoparticles in direct determination of target molecules concentration (in particular proteins) and for improving of enzyme immunoassay. As model we used conventional enzyme immunoassays for determination of progesterone and estradiol concentration. We obtained the possibility to produce silver nanoparticles with hyaluronan homogeneous in size between 10 and 12 nm, soluble and stable in water during long term of storage using modified procedure of silver nanoparticles synthesis. New method allows to obtain silver nanoparticles with strong optical properties at the higher concentrations - 60-90 μg/ml with the peak of absorbance at the wavelength 400 nm. Therefore surface plasmon resonance of silver nanoparticles with hyaluronan and ultraviolet-visible spectroscopy provide an opportunity for rapid determination of target molecules concentration (especial protein). We used silver nanoparticles as enzyme carriers and signal enhancers. Our preliminary data show that silver nanoparticles increased absorbance of samples that allows improving upper limit of determination of estradiol and progesterone concentration.

  4. Cyclodextrin-insulin complex encapsulated polymethacrylic acid based nanoparticles for oral insulin delivery.

    PubMed

    Sajeesh, S; Sharma, Chandra P

    2006-11-15

    Present investigation was aimed at developing an oral insulin delivery system based on hydroxypropyl beta cyclodextrin-insulin (HPbetaCD-I) complex encapsulated polymethacrylic acid-chitosan-polyether (polyethylene glycol-polypropylene glycol copolymer) (PMCP) nanoparticles. Nanoparticles were prepared by the free radical polymerization of methacrylic acid in presence of chitosan and polyether in a solvent/surfactant free medium. Dynamic light scattering (DLS) experiment was conducted with particles dispersed in phosphate buffer (pH 7.4) and size distribution curve was observed in the range of 500-800 nm. HPbetaCD was used to prepare non-covalent inclusion complex with insulin and complex was analyzed by Fourier transform infrared (FTIR) and fluorescence spectroscopic studies. HPbetaCD complexed insulin was encapsulated into PMCP nanoparticles by diffusion filling method and their in vitro release profile was evaluated at acidic/alkaline pH. PMCP nanoparticles displayed good insulin encapsulation efficiency and release profile was largely dependent on the pH of the medium. Enzyme linked immunosorbent assay (ELISA) study demonstrated that insulin encapsulated inside the particles was biologically active. Trypsin inhibitory effect of PMCP nanoparticles was evaluated using N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and casein as substrates. Mucoadhesive studies of PMCP nanoparticles were conducted using freshly excised rat intestinal mucosa and the particles were found fairly adhesive. From the preliminary studies, cyclodextrin complexed insulin encapsulated mucoadhesive nanoparticles appear to be a good candidate for oral insulin delivery.

  5. Chitosan nanofibers for transbuccal insulin delivery.

    PubMed

    Lancina, Michael G; Shankar, Roopa Kanakatti; Yang, Hu

    2017-05-01

    In this work, they aimed at producing chitosan based nanofiber mats capable of delivering insulin via the buccal mucosa. Chitosan was electrospun into nanofibers using poly(ethylene oxide) (PEO) as a carrier molecule in various feed ratios. The mechanical properties and degradation kinetics of the fibers were measured. Insulin release rates were determined in vitro using an ELISA assay. The bioactivity of released insulin was measured in terms of Akt activation in pre-adipocytes. Insulin permeation across the buccal mucosa was measured in an ex-vivo porcine transbuccal model. Fiber morphology, mechanical properties, and in vitro stability were dependent on PEO feed ratio. Lower PEO content blends produced smaller diameter fibers with significantly faster insulin release kinetics. Insulin showed no reduction in bioactivity due to electrospinning. Buccal permeation of insulin facilitated by high chitosan content blends was significantly higher than that of free insulin. Taken together, the work demonstrates that chitosan-based nanofibers have the potential to serve as a transbuccal insulin delivery vehicle. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1252-1259, 2017. © 2017 Wiley Periodicals, Inc.

  6. Variability of Directly Measured First-Pass Hepatic Insulin Extraction and its Association With Insulin Sensitivity and Plasma Insulin.

    PubMed

    Asare-Bediako, Isaac; Paszkiewicz, Rebecca L; Kim, Stella P; Woolcott, Orison O; Kolka, Cathryn M; Burch, Miguel A; Kabir, Morvarid; Bergman, Richard N

    2018-05-11

    While the β-cells secrete insulin, it is the liver with its first-pass insulin extraction (FPE) that regulates the amount of insulin allowed into circulation for action on target tissues. The metabolic clearance rate of insulin, of which FPE is the dominant component, is reported to be a major determinant of insulin sensitivity (SI). We studied the intricate relationship between FPE, SI and fasting insulin. We used a direct method of measuring FPE, the paired portal/peripheral infusion protocol (PPII) where insulin is infused step-wise, either via the portal vein or a peripheral vein in healthy young dogs (n =12). FPE is calculated as the difference in clearance rates (slope of infusion rate vs. steady insulin plot) between the paired experiments. Significant correlations were found between FPE vs. clamp assessed SI (r s = 0.74); FPE vs. fasting insulin (r s = -0.64) and SI vs. fasting insulin (r s = - 0.67). Also, we found a wide variance in FPE (22.4 -77.2%; mean ± SD of 50.4 ± 19.1%) which is reflected in the variability of plasma insulin (48.1 ± 30.9pM) and SI (9.4 ± 5.8 x10 4 dL * kg -1 * min -1 * pM -1 ). FPE could be the nexus of regulation of both plasma insulin and SI. © 2018 by the American Diabetes Association.

  7. Lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier rather than by inducing nonproductive adsorption of enzymes.

    PubMed

    Djajadi, Demi T; Jensen, Mads M; Oliveira, Marlene; Jensen, Anders; Thygesen, Lisbeth G; Pinelo, Manuel; Glasius, Marianne; Jørgensen, Henning; Meyer, Anne S

    2018-01-01

    Lignin is known to hinder efficient enzymatic conversion of lignocellulose in biorefining processes. In particular, nonproductive adsorption of cellulases onto lignin is considered a key mechanism to explain how lignin retards enzymatic cellulose conversion in extended reactions. Lignin-rich residues (LRRs) were prepared via extensive enzymatic cellulose degradation of corn stover ( Zea mays subsp. mays L.), Miscanthus  ×  giganteus stalks (MS) and wheat straw ( Triticum aestivum L.) (WS) samples that each had been hydrothermally pretreated at three severity factors (log R 0 ) of 3.65, 3.83 and 3.97. The LRRs had different residual carbohydrate levels-the highest in MS; the lowest in WS. The residual carbohydrate was not traceable at the surface of the LRRs particles by ATR-FTIR analysis. The chemical properties of the lignin in the LRRs varied across the three types of biomass, but monolignols composition was not affected by the severity factor. When pure cellulose was added to a mixture of LRRs and a commercial cellulolytic enzyme preparation, the rate and extent of glucose release were unaffected by the presence of LRRs regardless of biomass type and severity factor, despite adsorption of the enzymes to the LRRs. Since the surface of the LRRs particles were covered by lignin, the data suggest that the retardation of enzymatic cellulose degradation during extended reaction on lignocellulosic substrates is due to physical blockage of the access of enzymes to the cellulose caused by the gradual accumulation of lignin at the surface of the biomass particles rather than by nonproductive enzyme adsorption. The study suggests that lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier blocking the access of enzymes to cellulose rather than by inducing retardation through nonproductive adsorption of enzymes.

  8. The renin-angiotensin system: a target of and contributor to dyslipidemias, altered glucose homeostasis, and hypertension of the metabolic syndrome

    PubMed Central

    Putnam, Kelly; Shoemaker, Robin; Yiannikouris, Frederique

    2012-01-01

    The renin-angiotensin system (RAS) is an important therapeutic target in the treatment of hypertension. Obesity has emerged as a primary contributor to essential hypertension in the United States and clusters with other metabolic disorders (hyperglycemia, hypertension, high triglycerides, low HDL cholesterol) defined within the metabolic syndrome. In addition to hypertension, RAS blockade may also serve as an effective treatment strategy to control impaired glucose and insulin tolerance and dyslipidemias in patients with the metabolic syndrome. Hyperglycemia, insulin resistance, and/or specific cholesterol metabolites have been demonstrated to activate components required for the synthesis [angiotensinogen, renin, angiotensin-converting enzyme (ACE)], degradation (ACE2), or responsiveness (angiotensin II type 1 receptors, Mas receptors) to angiotensin peptides in cell types (e.g., pancreatic islet cells, adipocytes, macrophages) that mediate specific disorders of the metabolic syndrome. An activated local RAS in these cell types may contribute to dysregulated function by promoting oxidative stress, apoptosis, and inflammation. This review will discuss data demonstrating the regulation of components of the RAS by cholesterol and its metabolites, glucose, and/or insulin in cell types implicated in disorders of the metabolic syndrome. In addition, we discuss data supporting a role for an activated local RAS in dyslipidemias and glucose intolerance/insulin resistance and the development of hypertension in the metabolic syndrome. Identification of an activated RAS as a common thread contributing to several disorders of the metabolic syndrome makes the use of angiotensin receptor blockers and ACE inhibitors an intriguing and novel option for multisymptom treatment. PMID:22227126

  9. Chemogenetic Characterization of Inositol Phosphate Metabolic Pathway Reveals Druggable Enzymes for Targeting Kinetoplastid Parasites.

    PubMed

    Cestari, Igor; Haas, Paige; Moretti, Nilmar Silvio; Schenkman, Sergio; Stuart, Ken

    2016-05-19

    Kinetoplastids cause Chagas disease, human African trypanosomiasis, and leishmaniases. Current treatments for these diseases are toxic and inefficient, and our limited knowledge of drug targets and inhibitors has dramatically hindered the development of new drugs. Here we used a chemogenetic approach to identify new kinetoplastid drug targets and inhibitors. We conditionally knocked down Trypanosoma brucei inositol phosphate (IP) pathway genes and showed that almost every pathway step is essential for parasite growth and infection. Using a genetic and chemical screen, we identified inhibitors that target IP pathway enzymes and are selective against T. brucei. Two series of these inhibitors acted on T. brucei inositol polyphosphate multikinase (IPMK) preventing Ins(1,4,5)P3 and Ins(1,3,4,5)P4 phosphorylation. We show that IPMK is functionally conserved among kinetoplastids and that its inhibition is also lethal for Trypanosoma cruzi. Hence, IP enzymes are viable drug targets in kinetoplastids, and IPMK inhibitors may aid the development of new drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Iontophoresis of monomeric insulin analogues in vitro: effects of insulin charge and skin pretreatment.

    PubMed

    Langkjaer, L; Brange, J; Grodsky, G M; Guy, R H

    1998-01-23

    The aim of this study was to investigate the influence of association state and net charge of human insulin analogues on the rate of iontophoretic transport across hairless mouse skin, and the effect of different skin pretreatments on said transport. No insulin flux was observed with anodal delivery probably because of degradation at the Ag/AgCl anode. The flux during cathodal iontophoresis through intact skin was insignificant for human hexameric insulin, and only low and variable fluxes were observed for monomeric insulins. Using stripped skin on the other hand, the fluxes of monomeric insulins with two extra negative charges were 50-100 times higher than that of hexameric human insulin. Introducing three additional charges led to a further 2-3-fold increase in flux. Wiping the skin gently with absolute alcohol prior to iontophoresis resulted in a 1000-fold increase in transdermal transport of insulin relative to that across untreated skin, i.e. to almost the same level as stripping the skin. The alcohol pretreatment reduced the electrical resistance of the skin, presumably by lipid extraction. In conclusion, monomeric insulin analogues with at least two extra negative charges can be iontophoretically delivered across hairless mouse skin, whereas insignificant flux is observed with human, hexameric insulin. Wiping the skin with absolute alcohol prior to iontophoresis gave substantially improved transdermal transport of monomeric insulins resulting in clinically relevant delivery rates for basal treatment.

  11. DGAT: novel therapeutic target for obesity and type 2 diabetes mellitus.

    PubMed

    Subauste, Angela; Burant, Charles F

    2003-12-01

    Obesity is currently an exceptionally common problem in humans. The last several years have produced a significant number of breakthroughs in obesity related areas of investigation. Triglycerides are considered the main form of storage of excess calories in fat. A key enzyme in the synthesis of triglycerides is acylCoA: diacylglycerol acyltransferase (DGAT). Recent studies have shown that mice deficient in this enzyme are resistant to diet induced obesity and have increased insulin and leptin sensitivity. These effects suggest that inhibition of DGAT in vivo may be a novel therapeutic target not only for obesity but also for diabetes.

  12. Insulin transport into the brain.

    PubMed

    Gray, Sarah M; Barrett, Eugene J

    2018-05-30

    While there is a growing consensus that insulin has diverse and important regulatory actions on the brain, seemingly important aspects of brain insulin physiology are poorly understood. Examples include: what is the insulin concentration within brain interstitial fluid under normal physiologic conditions; whether insulin is made in the brain and acts locally; does insulin from the circulation cross the blood-brain barrier or the blood-CSF barrier in a fashion that facilitates its signaling in brain; is insulin degraded within the brain; do privileged areas with a "leaky" blood-brain barrier serve as signaling nodes for transmitting peripheral insulin signaling; does insulin action in the brain include regulation of amyloid peptides; whether insulin resistance is a cause or consequence of processes involved in cognitive decline. Heretofore, nearly all studies examining brain insulin physiology have employed techniques and methodologies that do not appreciate the complex fluid compartmentation and flow throughout the brain. This review attempts to provide a status report on historical and recent work that begins to address some of these issues. It is undertaken in an effort to suggest a framework for studies going forward. Such studies are inevitably influenced by recent physiologic and genetic studies of insulin accessing and acting in brain, discoveries relating to brain fluid dynamics and the interplay of cerebrospinal fluid, brain interstitial fluid, and brain lymphatics, and advances in clinical neuroimaging that underscore the dynamic role of neurovascular coupling.

  13. [Risk for hyperkalemia during long-term treatment with angiotensin-converting enzyme inhibitors in insulin-dependent type 2 diabetics in relation to the glomerular filtration rate].

    PubMed

    Raml, A; Schmekal, B; Grafinger, P; Biesenbach, G

    2001-11-23

    The risk for hyperkalaemia during therapy with angiotensin-converting enzyme inhibitors is especially increased in the elderly diabetic because of a decrease in glomerular filtration rate (GFR), as well as the occurrence of hyporeninaemic hypoaldosteronism. We evaluated the risk for hyperkalaemia under long-term angiotensin-converting enyzme inhibition in 86 insulin-dependent type 2 diabetic patients in relation to their GFR. We compared the influence of a 3 to 6 months long treatment with angiotensin-converting enzyme inhibitors on the serum potassium levels, the creatinine clearance and the urinary albumin excretion in insulin-dependent type 2 diabetic patients with an initial creatinine clearance < 50 ml/min/1.73m(2) (n = 15, age 66 +/- 6 years) and >/= 50 ml/min/1.73m(2) respectively (n = 71, age 61 +/- 10 years). In addition, we also investigated the influence on the metabolic control and the blood pressure values in both groups of patients. In the patients with creatinine clearance >/= 50 ml/min/1,73m(2) the mean potassium level increased from 4.3 +/- 0.2 to 4.6 +/- 0.4 mmol/l (P < 0,01), while the incidence of a potassium level > 5 mmol/l was 17 %. In the group with a creatinine clearance < 50 ml/min/1.73m(2) the potassium level rose from 4.5 +/- 0.2 to 5.0 +/- 0.4 mmol/l (P < 0.01). The incidence of potassium levels > 5 mmol/l was 66 % (P < 0,01). In both patient groups the creatinine clearances did not change significantly during angiotensin-converting enzyme inhibition, and the urinary albumin excretion as well as the HbA(1c) values and blood pressure showed only a tendency towards a decrease. Long-term treatment with angiotensin-converting enzyme inhibitors in insulin-dependent type 2 diabetic patients leads to a significant increase in serum potassium. The incidence of hyperkalaemia with potassium levels > 5 mmol/l is significantly higher in the patients with initial creatinine clearance < 50 ml/min/1.73m(2). Severe hyperkalaemia with potassium levels

  14. Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme.

    PubMed

    Vallecillo-Viejo, Isabel C; Liscovitch-Brauer, Noa; Montiel-Gonzalez, Maria Fernanda; Eisenberg, Eli; Rosenthal, Joshua J C

    2018-01-02

    Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.

  15. Combining insulins for optimal blood glucose control in type 1 and 2 diabetes: Focus on insulin glulisine

    PubMed Central

    Ulrich, Heather; Snyder, Benjamin; K Garg, Satish

    2007-01-01

    Normalization of blood glucose is essential for the prevention of diabetes mellitus (DM)-related microvascular and macrovascular complications. Despite substantial literature to support the benefits of glucose lowering and clear treatment targets, glycemic control remains suboptimal for most people with DM in the United States. Pharmacokinetic limitations of conventional insulins have been a barrier to achieving treatment targets secondary to adverse effects such as hypoglycemia and weight gain. Recombinant DNA technology has allowed modification of the insulin molecule to produce insulin analogues that overcome these pharmacokinetic limitations. With time action profiles that more closely mimic physiologic insulin secretion, rapid acting insulin analogues (RAAs) reduce post-prandial glucose excursions and hypoglycemia when compared to regular human insulin (RHI). Insulin glulisine (Apidra®) is a rapid-acting insulin analogue created by substituting lysine for asparagine at position B3 and glutamic acid for lysine at position B29 on the B chain of human insulin. The quick absorption of insulin glulisine more closely reproduces physiologic first-phase insulin secretion and its rapid acting profile is maintained across patient subtypes. Clinical trials have demonstrated comparable or greater efficacy of insulin glulisine versus insulin lispro or RHI, respectively. Efficacy is maintained even when insulin glulisine is administered post-meal. In addition, glulisine appears to have a more rapid time action profile compared with insulin lispro across various body mass indexes (BMIs). The safety and tolerability profile of insulin glulisine is also comparable to that of insulin lispro or RHI in type 1 or 2 DM and it has been shown to be as safe and effective when used in a continuous subcutaneous insulin infusion (CSII). In summary, insulin glulisine is a safe, effective, and well tolerated rapid-acting insulin analogue across all BMIs and a worthy option for prandial

  16. Degradation of Perfluorooctanoic Acid and Perfluoroctane Sulfonate by Enzyme Catalyzed Oxidative Humification Reactions

    NASA Astrophysics Data System (ADS)

    Huang, Q.

    2016-12-01

    Poly- and perfluoroalkyl substances (PFASs) are alkyl based chemicals having multiple or all hydrogens replaced by fluorine atoms, and thus exhibit high thermal and chemical stability and other unusual characteristics. PFASs have been widely used in a wide variety of industrial and consumer products, and tend to be environmentally persistent. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are two representative PFASs that have drawn particular attention because of their ubiquitous presence in the environment, resistance to degradation and toxicity to animals. This study examined the decomposition of PFOA and PFOS in enzyme catalyzed oxidative humification reactions (ECOHR), a class of reactions that are ubiquitous in the environment involved in natural organic humification. Reaction rates and influential factors were examined, and high-resolution mass spectrometry was used to identify possible products. Fluorides and partially fluorinated compounds were identified as likely products from PFOA and PFOS degradation, which were possibly formed via a combination of free radical decomposition, rearrangements and coupling processes. The findings suggest that PFOA and PFOS may be transformed during humification, and ECOHR can potentially be used for the remediation of these chemicals.

  17. The increase in fiber size in male rat gastrocnemius after chronic central leptin infusion is related to activation of insulin signaling.

    PubMed

    Burgos-Ramos, Emma; Canelles, Sandra; Rodríguez, Amaia; Frago, Laura M; Gómez-Ambrosi, Javier; Chowen, Julie A; Frühbeck, Gema; Argente, Jesús; Barrios, Vicente

    2018-07-15

    Insulin potentiates leptin effects on muscle accrual and glucose homeostasis. However, the relationship between leptin's central effects on peripheral insulin sensitivity and the associated structural changes remain unclear. We hypothesized that central leptin infusion modifies muscle size through activation of insulin signaling. Muscle insulin signaling, enzymes of fatty acid metabolism, mitochondrial respiratory chain complexes, proliferating cell nuclear antigen (PCNA) and fiber area were analyzed in the gastrocnemius of chronic central infused (L), pair-fed (PF) and control rats. PCNA-positive nuclei, fiber area, GLUT4 and glycogen levels and activation of Akt and mechanistic target of rapamycin were increased in L, with no changes in PF. Acetyl-CoA carboxylase-β mRNA levels and non-esterified fatty acid and triglyceride content were reduced and carnitine palmitoyltransferase-1b expression and mitochondrial complexes augmented in L. These results suggest that leptin promotes an increase in muscle size associated with improved insulin signaling favored by lipid profile. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Receptor for advanced glycation end products is targeted by FBXO10 for ubiquitination and degradation.

    PubMed

    Evankovich, John; Lear, Travis; Mckelvey, Alison; Dunn, Sarah; Londino, James; Liu, Yuan; Chen, Bill B; Mallampalli, Rama K

    2017-09-01

    The receptor for advanced glycation end products (RAGE) is a highly expressed cell membrane receptor serving to anchor lung epithelia to matrix components, and it also amplifies inflammatory signaling during acute lung injury. However, mechanisms that regulate its protein concentrations in cells remain largely unknown. Here we show that RAGE exhibits an extended life span in lung epithelia ( t ½ 6 h), is monoubiquitinated at K374, and is degraded in lysosomes. The RAGE ligand ODN2006, a synthetic oligodeoxynucleotide resembling pathogenic hypomethylated CpG DNA, promotes rapid lysosomal RAGE degradation through activation of protein kinase Cζ (PKCζ), which phosphorylates RAGE. PKCζ overexpression enhances RAGE degradation, while PKCζ knockdown stabilizes RAGE protein levels and prevents ODN2006-mediated degradation. We identify that RAGE is targeted by the ubiquitin E3 ligase subunit F-box protein O10 (FBXO10), which associates with RAGE to mediate its ubiquitination and degradation. FBXO10 depletion in cells stabilizes RAGE and is required for ODN2006-mediated degradation. These data suggest that modulation of regulators involved in ubiquitin-mediated disposal of RAGE might serve as unique molecular inputs directing RAGE cellular concentrations and downstream responses, which are critical in an array of inflammatory disorders, including acute lung injury.-Evankovich, J., Lear, T., Mckelvey, A., Dunn, S., Londino, J., Liu, Y., Chen, B. B., Mallampalli, R. K. Receptor for advanced glycation end products is targeted by FBXO10 for ubiquitination and degradation. © FASEB.

  19. Dysregulated miR-127-5p contributes to type II collagen degradation by targeting matrix metalloproteinase-13 in human intervertebral disc degeneration.

    PubMed

    Hua, Wen-Bin; Wu, Xing-Huo; Zhang, Yu-Kun; Song, Yu; Tu, Ji; Kang, Liang; Zhao, Kang-Cheng; Li, Shuai; Wang, Kun; Liu, Wei; Shao, Zeng-Wu; Yang, Shu-Hua; Yang, Cao

    2017-08-01

    Intervertebral disc degeneration (IDD) is a chronic disease associated with the degradation of extracellular matrix (ECM). Matrix metalloproteinase (MMP)-13 is a major enzyme that mediates the degradation of ECM components. MMP-13 has been predicted to be a potential target of miR-127-5p. However, the exact function of miR-127-5p in IDD is still unclear. We designed this study to evaluate the correlation between miR-127-5p level and the degeneration of human intervertebral discs and explore the potential mechanisms. miR-127-5p levels and MMP-13 mRNA levels were detected by quantitative real-time polymerase chain reaction (qPCR). To determine whether MMP-13 is a target of miR-127-5p, dual luciferase reporter assays were performed. miR-127-5p mimic and miR-127-5p inhibitor were used to overexpress or downregulate miR-127-5p expression in human NP cells, respectively. Small interfering RNA (siRNA) was used to knock down MMP-13 expression in human NP cells. Type II collagen expression in human NP cells was detected by qPCR, western blotting, and immunofluorescence staining. We confirmed that miR-127-5p was significantly downregulated in nucleus pulposus (NP) tissue of degenerative discs and its expression was inversely correlated with MMP-13 mRNA levels. We reveal that MMP-13 may act as a target of miR-127-5p. Expression of miR-127-5p was inversely correlated with type II collagen expression in human NP cells. Moreover, suppression of MMP-13 expression by siRNA blocked downstream signaling and increased type II collagen expression. Dysregulated miR-127-5p contributed to the degradation of type II collagen by targeting MMP-13 in human IDD. Our findings highlight that miR-127-5p may serve as a new therapeutic target in IDD. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  20. Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling.

    PubMed

    Su, Jiao; Zhang, Haijie; Jiang, Bingying; Zheng, Huzhi; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

    2011-11-15

    We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. A multi-substrate approach for functional metagenomics-based screening for (hemi)cellulases in two wheat straw-degrading microbial consortia unveils novel thermoalkaliphilic enzymes.

    PubMed

    Maruthamuthu, Mukil; Jiménez, Diego Javier; Stevens, Patricia; van Elsas, Jan Dirk

    2016-01-28

    Functional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). Most current methodologies used to screen for enzymes involved in plant biomass degradation are based on the use of single substrates. Moreover, highly diverse environments are used as metagenomic sources. However, such methods suffer from low hit rates of positive clones and hence the discovery of novel enzymatic activities from metagenomes has been hampered. Here, we constructed fosmid libraries from two wheat straw-degrading microbial consortia, denoted RWS (bred on untreated wheat straw) and TWS (bred on heat-treated wheat straw). Approximately 22,000 clones from each library were screened for (hemi)cellulose-degrading enzymes using a multi-chromogenic substrate approach. The screens yielded 71 positive clones for both libraries, giving hit rates of 1:440 and 1:1,047 for RWS and TWS, respectively. Seven clones (NT2-2, T5-5, NT18-17, T4-1, 10BT, NT18-21 and T17-2) were selected for sequence analyses. Their inserts revealed the presence of 18 genes encoding enzymes belonging to twelve different glycosyl hydrolase families (GH2, GH3, GH13, GH17, GH20, GH27, GH32, GH39, GH53, GH58, GH65 and GH109). These encompassed several carbohydrate-active gene clusters traceable mainly to Klebsiella related species. Detailed functional analyses showed that clone NT2-2 (containing a beta-galactosidase of ~116 kDa) had highest enzymatic activity at 55 °C and pH 9.0. Additionally, clone T5-5 (containing a beta-xylosidase of ~86 kDa) showed > 90% of enzymatic activity at 55 °C and pH 10.0. This study employed a high-throughput method for rapid screening of fosmid metagenomic libraries for (hemi)cellulose-degrading enzymes. The approach, consisting of screens on multi-substrates coupled to further analyses, revealed high hit rates, as compared with recent other studies. Two

  2. Interacting signal pathways control defense gene expression in Arabidopsis in response to cell wall-degrading enzymes from Erwinia carotovora.

    PubMed

    Norman-Setterblad, C; Vidal, S; Palva, E T

    2000-04-01

    We have characterized the role of salicylic acid (SA)-independent defense signaling in Arabidopsis thaliana in response to the plant pathogen Erwinia carotovora subsp. carotovora. Use of pathway-specific target genes as well as signal mutants allowed us to elucidate the role and interactions of ethylene, jasmonic acid (JA), and SA signal pathways in this response. Gene expression studies suggest a central role for both ethylene and JA pathways in the regulation of defense gene expression triggered by the pathogen or by plant cell wall-degrading enzymes (CF) secreted by the pathogen. Our results suggest that ethylene and JA act in concert in this regulation. In addition, CF triggers another, strictly JA-mediated response inhibited by ethylene and SA. SA does not appear to have a major role in activating defense gene expression in response to CF. However, SA may have a dual role in controlling CF-induced gene expression, by enhancing the expression of genes synergistically induced by ethylene and JA and repressing genes induced by JA alone.

  3. Simultaneous saccharification and viscosity reduction of cassava pulp using a multi-component starch- and cell-wall degrading enzyme for bioethanol production.

    PubMed

    Poonsrisawat, Aphisit; Paemanee, Atchara; Wanlapatit, Sittichoke; Piyachomkwan, Kuakoon; Eurwilaichitr, Lily; Champreda, Verawat

    2017-10-01

    In this study, an efficient ethanol production process using simultaneous saccharification and viscosity reduction of raw cassava pulp with no prior high temperature pre-gelatinization/liquefaction step was developed using a crude starch- and cell wall-degrading enzyme preparation from Aspergillus aculeatus BCC17849. Proteomic analysis revealed that the enzyme comprised a complex mixture of endo- and exo-acting amylases, cellulases, xylanases, and pectina ses belonging to various glycosyl hydrolase families. Enzymatic hydrolysis efficiency was dependent on the initial solid loading in the reaction. Reduction in mixture viscosity was observed with a rapid decrease in complex viscosity from 3785 to 0.45 Pa s with the enzyme dosage of 2.19 mg/g on a dried weight basis within the first 2 h, which resulted from partial destruction of the plant cell wall fiber and degradation of the released starch granules by the enzymes as shown by scanning electron microscopy. Saccharification of cassava pulp at an initial solid of 16% (w/v) in a bench-scale bioreactor resulted in 736.4 mg glucose/g, which is equivalent to 82.92% glucose yield based on the total starch and glucan in the substrate, after 96 h at 40 °C. Simultaneous saccharification and fermentation of cassava pulp by Saccharomyces cerevisiae with the uncooked enzymatic process led to a final ethanol concentration of 6.98% w/v, equivalent to 96.7% theoretical yield based on the total starch and cellulose content. The results demonstrated potential of the enzyme for low-energy processing of cassava pulp in biofuel industry.

  4. New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes.

    PubMed

    Danso, Dominik; Schmeisser, Christel; Chow, Jennifer; Zimmermann, Wolfgang; Wei, Ren; Leggewie, Christian; Li, Xiangzhen; Hazen, Terry; Streit, Wolfgang R

    2018-04-15

    Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and β-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria , Proteobacteria , and Bacteroidetes Within the Proteobacteria , the Betaproteobacteria , Deltaproteobacteria , and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria , as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil. IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with

  5. New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes

    PubMed Central

    Danso, Dominik; Schmeisser, Christel; Chow, Jennifer; Wei, Ren; Leggewie, Christian; Li, Xiangzhen

    2018-01-01

    ABSTRACT Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and β-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes. Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil. IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with

  6. Development and in vivo evaluation of an oral insulin-PEG delivery system.

    PubMed

    Calceti, P; Salmaso, S; Walker, G; Bernkop-Schnürch, A

    2004-07-01

    Insulin-monomethoxypoly(ethylene glycol) derivatives were obtained by preparation of mono- and di-terbutyl carbonate insulin derivatives, reaction of available protein amino groups with activated 750 Da PEG and, finally, amino group de-protection. This procedure allowed for obtaining high yield of insulin-1PEG and insulin-2PEG. In vivo studies carried out by subcutaneous injection into diabetic mice demonstrated that the two bioconjugates maintained the native biological activity. In vitro, PEGylation was found to enhance the hormone stability towards proteases. After 1 h incubation with elastase, native insulin, insulin-1PEG and insulin-2PEG undergo about 70, 30 and 10% degradation, respectively, while in the presence of pepsin protein degradation was 100, 70 and 50%, respectively. The attachment of low molecular weight PEG did not significantly (P >0.05) alter insulin permeation behavior across the intestinal mucosa. Insulin-1PEG was formulated into mucoadhesive tablets constituted by the thiolated polymer poly(acrylic acid)-cysteine. The therapeutic agent was sustained released from these tablets within 5 h. In vivo, by oral administration to diabetic mice, the glucose levels were found to decrease of about 40% since the third hour from administration and the biological activity was maintained up to 30 h. According to these results, the combination of PEGylated insulin with a thiolated polymer used as drug carrier matrix might be a promising strategy for oral insulin administration.

  7. Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

    PubMed

    Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Pentachlorophenol hydroxylase, a poorly functioning enzyme required for degradation of pentachlorophenol by Sphingobium chlorophenolicum

    PubMed Central

    Hlouchova, Klara; Rudolph, Johannes; Pietari, Jaana M.H.; Behlen, Linda S.; Copley, Shelley D.

    2014-01-01

    Several strains of Sphingobium chlorophenolicum have been isolated from soil that was heavily contaminated with pentachlorophenol (PCP), a toxic pesticide introduced in the 1930s. S. chlorophenolicum appears to have assembled a poorly functioning pathway for degradation of PCP by patching enzymes recruited via two independent horizontal gene transfer events into an existing metabolic pathway. Flux through the pathway is limited by PCP hydroxylase. PCP hydroxylase is a dimeric protein that belongs to the family of flavin-dependent phenol hydroxylases. In the presence of NADPH, PCP hydroxylase converts PCP to tetrachlorobenzoquinone (TCBQ). The kcat for PCP (0.024 s−1) is very low, suggesting that the enzyme is not well evolved for turnover of this substrate. Structure/activity studies reveal that substrate binding and activity are enhanced by a low pKa for the phenolic proton, increased hydrophobicity, and the presence of a substituent ortho to the hydroxyl group of the phenol. PCP hydroxylase exhibits substantial uncoupling; the C4a-hydroxyflavin intermediate, instead of hydroxylating the substrate, can decompose to produce H2O2 in a futile cycle that consumes NADPH. The extent of uncoupling varies from 0 – 100% with different substrates. Uncoupling is increased by the presence of bulky substituents in the 3-, 4-, or 5-position, and lessened by the presence of a chlorine in the ortho position. The effectiveness of PCP hydroxylase is additionally hindered by its promiscuous activity with TCHQ, a downstream metabolite in the degradation pathway. The conversion of TCHQ to TCBQ reverses flux through the pathway. Substantial uncoupling also occurs during the reaction with TCHQ. PMID:22482720

  9. An extract of Perilla stem inhibits Src homology phosphatase-1 (SHP)-1 and influences insulin signaling.

    PubMed

    Peng, Liu; Lei, Zhang; Xiao-na, Xie; Deli, Wang; Jing, Sun; Yong-sen, Wang; Zhi, Wang; Shu, Xing; Jun-feng, Ma; Wan-nan, Li; Xue-qi, Fu

    2015-03-01

    Protein tyrosine phosphatases (PTPs) are enzymes that catalyze protein tyrosine dephosphorylation of which Src homology phosphatase-1 (SHP-1) is one of the best-validated, a widely distributed intracellular tyrosine phosphatase that contains two SH2 domains. Down regulation of SHP-1 tyrosine phosphatases was significantly increased sensitivity to insulin in insulin signaling pathway. Through in vitro enzymatic reaction kinetics experiment, we found that the extract of Perilla stem was a potential inhibitor to δSHP-1, the catalytic domain of SHP-1 protein tyrosine phosphatase, and its IC(50) was 4ug/ml, and was more sensitive towards SHP-1than other PTPs, which indicated that SHP-1 might be a target of the extract of Perilla stem. It can strengthened the level of tyrosine phosphorylation of insulin receptor (IR) and extracellular signal-regulated protein kinase (ERK) in HepG2 cells, and then activated the insulin signaling pathway through inhibiting the protein phosphorylation of SHP-1. These results demonstrated that the extract of Perilla stem could play an important role for diabetes treatment through inhibiting the level of SHP-1 in insulin signaling pathway.

  10. Contribution of AmyA, an extracellular α-glucan degrading enzyme, to group A streptococcal host-pathogen interaction

    PubMed Central

    Shelburne, Samuel A.; Keith, David B.; Davenport, Michael T.; Beres, Stephen B.; Carroll, Ronan K.; Musser, James M.

    2010-01-01

    α-glucans such as starch and glycogen are abundant in the human oropharynx, the main site of group A Streptococcus (GAS) infection. However, the role in pathogenesis of GAS extracellular α-glucan binding and degrading enzymes is unknown. The serotype M1 GAS genome encodes two extracellular proteins putatively involved in α-glucan binding and degradation; pulA encodes a cell-wall anchored pullulanase and amyA encodes a freely secreted putative cyclomaltodextrin α-glucanotransferase. Genetic inactivation of amyA, but not pulA, abolished GAS α-glucan degradation. The ΔamyA strain had a slower rate of translocation across human pharyngeal epithelial cells. Consistent with this finding, the ΔamyA strain was less virulent following mouse mucosal challenge. Recombinant AmyA degraded α-glucans into β-cyclomaltodextrins that reduced pharyngeal cell transepithelial resistance, providing a physiologic explanation for the observed transepithelial migration phenotype. Higher amyA transcript levels were present in serotype M1 GAS strains causing invasive infection compared to strains causing pharyngitis. GAS proliferation in a defined α-glucan-containing medium was dependent on the presence of human salivary α-amylase. These data delineate the molecular mechanisms by which α-glucan degradation contributes to GAS host-pathogen interaction including how GAS employs human salivary α-amylase for its own metabolic benefit. PMID:19735442

  11. Amyloid-clearing proteins and their epigenetic regulation as a therapeutic target in Alzheimer’s disease

    PubMed Central

    Nalivaeva, Natalia N.; Belyaev, Nikolai D.; Kerridge, Caroline; Turner, Anthony J.

    2014-01-01

    Abnormal elevation of amyloid β-peptide (Aβ) levels in the brain is the primary trigger for neuronal cell death specific to Alzheimer’s disease (AD). It is now evident that Aβ levels in the brain are manipulable due to a dynamic equilibrium between its production from the amyloid precursor protein (APP) and removal by amyloid clearance proteins. Clearance can be either enzymic or non-enzymic (binding/transport proteins). Intriguingly several of the main amyloid-degrading enzymes (ADEs) are members of the M13 peptidase family (neprilysin (NEP), NEP2 and the endothelin converting enzymes (ECE-1 and -2)). A distinct metallopeptidase, insulin-degrading enzyme (IDE), also contributes to Aβ degradation in the brain. The ADE family currently embraces more than 20 members, both membrane-bound and soluble, and of differing cellular locations. NEP plays an important role in brain function terminating neuropeptide signals. Its decrease in specific brain areas with age or after hypoxia, ischaemia or stroke contribute significantly to the development of AD pathology. The recently discovered mechanism of epigenetic regulation of NEP (and other genes) by the APP intracellular domain (AICD) and its dependence on the cell type and APP isoform expression suggest possibilities for selective manipulation of NEP gene expression in neuronal cells. We have also observed that another amyloid-clearing protein, namely transthyretin (TTR), is also regulated in the neuronal cell by a mechanism similar to NEP. Dependence of amyloid clearance proteins on histone deacetylases and the ability of HDAC inhibitors to up-regulate their expression in the brain opens new avenues for developing preventive strategies in AD. PMID:25278875

  12. Subcellular Localization and Biochemical Comparison of Cytosolic and Secreted Cytokinin Dehydrogenase Enzymes from Maize

    USDA-ARS?s Scientific Manuscript database

    Cytokinin dehydrogenase (CKX, EC 1.5.99.12) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in cells of each plant. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a t...

  13. Streptozotocin-Induced Autophagy Reduces Intracellular Insulin in Insulinoma INS-1E Cells.

    PubMed

    Yoo, Yeong-Min; Park, Yung Chul

    2018-03-01

    Streptozotocin (STZ), a glucose analog, induces diabetes in experimental animals by inducing preferential cytotoxicity in pancreatic beta cells. We investigated whether STZ reduced the production of intracellular insulin through autophagy in insulinoma INS-1E cells. Typically, 2 mM STZ treatment for 24 h significantly decreased cell survival. STZ treatment led to significant decrease in phospho-AMP-activated protein kinase (p-AMPK) level; reduction in levels of phospho-protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1α (IRE1α); significant reduction in levels of p85α, p110, phospho-serine and threonine kinase/protein kinase B (p-Akt/PKB) (Ser473), phospho-extracellular-regulated kinase (p-ERK), and phospho-mammalian target of rapamycin (p-mTOR); increase in levels of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase; decrease in B-cell lymphoma 2 (Bcl-2) expression; increase in Bcl-2-associated X protein (Bax) expression; increase in levels of microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1; and reduction in production of intracellular insulin. These results suggest that insulin synthesis during STZ treatment involves autophagy in INS-1E cells and, subsequently, results in a decrease in intracellular production of insulin.

  14. Advances in the Engineering of the Gene Editing Enzymes and the Genomes: Understanding and Handling the Off-Target Effects of CRISPR/Cas9.

    PubMed

    Yin, Yufang; Wang, Qian; Xiao, Li; Wang, Fengjiao; Song, Zhuo; Zhou, Cuilan; Liu, Xuan; Xing, Chungen; He, Nongyue; Li, Kai; Feng, Yan; Zhang, Jia

    2018-03-01

    In the past decades, significant progresses have been achieved in genetic engineering of nucleases. Among the genetically engineered nucleases, zinc finger nucleases, transcription activator-like (TAL) effector nucleases, and CRIPSPR/Cas9 system form a new field of gene editing. The gene editing efficiency or targeting effect and the off-target effect are the two major determinant factors in evaluating the usefulness of a new enzyme. Engineering strategies in improving these gene editing enzymes, particularly in minimizing their off-target effects, are the focus of this paper. Examples of using these genetically engineered enzymes in genome modification are discussed in order to better understand the requirement of engineering efforts in obtaining more powerful and useful gene editing enzymes. In addition, the identification of naturally existed anti-Cas proteins has been employed in minimizing off-target effects. Considering the future application in human gene therapy, optimization of these well recognized gene editing enzymes and exploration of more novel enzymes are both required. Before people find an ideal gene editing system having virtually no off-target effect, technologies used to screen and identify off-target effects are of importance in clinical trials employing gene therapy.

  15. Insulin-Like Growth Factor System in Cancer: Novel Targeted Therapies

    PubMed Central

    Brahmkhatri, Varsha P.; Prasanna, Chinmayi; Atreya, Hanudatta S.

    2015-01-01

    Insulin-like growth factors (IGFs) are essential for growth and survival that suppress apoptosis and promote cell cycle progression, angiogenesis, and metastatic activities in various cancers. The IGFs actions are mediated through the IGF-1 receptor that is involved in cell transformation induced by tumour. These effects depend on the bioavailability of IGFs, which is regulated by IGF binding proteins (IGFBPs). We describe here the role of the IGF system in cancer, proposing new strategies targeting this system. We have attempted to expand the general viewpoint on IGF-1R, its inhibitors, potential limitations of IGF-1R, antibodies and tyrosine kinase inhibitors, and IGFBP actions. This review discusses the emerging view that blocking IGF via IGFBP is a better option than blocking IGF receptors. This can lead to the development of novel cancer therapies. PMID:25866791

  16. Microbial Activity and Silica Degradation in Rice Straw

    NASA Astrophysics Data System (ADS)

    Kim, Esther Jin-kyung

    Abundantly available agricultural residues like rice straw have the potential to be feedstocks for bioethanol production. Developing optimized conditions for rice straw deconstruction is a key step toward utilizing the biomass to its full potential. One challenge associated with conversion of rice straw to bioenergy is its high silica content as high silica erodes machinery. Another obstacle is the availability of enzymes that hydrolyze polymers in rice straw under industrially relevant conditions. Microbial communities that colonize compost may be a source of enzymes for bioconversion of lignocellulose to products because composting systems operate under thermophilic and high solids conditions that have been shown to be commercially relevant. Compost microbial communities enriched on rice straw could provide insight into a more targeted source of enzymes for the breakdown of rice straw polysaccharides and silica. Because rice straw is low in nitrogen it is important to understand the impact of nitrogen concentrations on the production of enzyme activity by the microbial community. This study aims to address this issue by developing a method to measure microbial silica-degrading activity and measure the effect of nitrogen amendment to rice straw on microbial activity and extracted enzyme activity during a high-solids, thermophilic incubation. An assay was developed to measure silica-degrading enzyme or silicase activity. This process included identifying methods of enzyme extraction from rice straw, identifying a model substrate for the assay, and optimizing measurement techniques. Rice straw incubations were conducted with five different levels of nitrogen added to the biomass. Microbial activity was measured by respiration and enzyme activity. A microbial community analysis was performed to understand the shift in community structure with different treatments. With increased levels of nitrogen, respiration and cellulose and hemicellulose degrading activity

  17. Docking analysis targeted to the whole enzyme: an application to the prediction of inhibition of PTP1B by thiomorpholine and thiazolyl derivatives.

    PubMed

    Ganou, C A; Eleftheriou, P Th; Theodosis-Nobelos, P; Fesatidou, M; Geronikaki, A A; Lialiaris, T; Rekka, E A

    2018-02-01

    PTP1b is a protein tyrosine phosphatase involved in the inactivation of insulin receptor. Since inhibition of PTP1b may prolong the action of the receptor, PTP1b has become a drug target for the treatment of type II diabetes. In the present study, prediction of inhibition using docking analysis targeted specifically to the active or allosteric site was performed on 87 compounds structurally belonging to 10 different groups. Two groups, consisting of 15 thiomorpholine and 10 thiazolyl derivatives exhibiting the best prediction results, were selected for in vitro evaluation. All thiomorpholines showed inhibitory action (with IC 50 = 4-45 μΜ, Ki = 2-23 μM), while only three thiazolyl derivatives showed low inhibition (best IC 50 = 18 μΜ, Ki = 9 μΜ). However, free binding energy (E) was in accordance with the IC 50 values only for some compounds. Docking analysis targeted to the whole enzyme revealed that the compounds exhibiting IC 50 values higher than expected could bind to other peripheral sites with lower free energy, E o , than when bound to the active/allosteric site. A prediction factor, E- (Σ Eo × 0.16), which takes into account lower energy binding to peripheral sites, was proposed and was found to correlate well with the IC 50 values following an asymmetrical sigmoidal equation with r 2 = 0.9692.

  18. Carbohydrate-to-insulin ratio is estimated from 300-400 divided by total daily insulin dose in type 1 diabetes patients who use the insulin pump.

    PubMed

    Kuroda, Akio; Yasuda, Tetsuyuki; Takahara, Mitsuyoshi; Sakamoto, Fumie; Kasami, Ryuichi; Miyashita, Kazuyuki; Yoshida, Sumiko; Kondo, Eri; Aihara, Ken-ichi; Endo, Itsuro; Matsuoka, Taka-aki; Kaneto, Hideaki; Matsumoto, Toshio; Shimomura, Iichiro; Matsuhisa, Munehide

    2012-11-01

    To optimize insulin dose using insulin pump, basal and bolus insulin doses are widely calculated from total daily insulin dose (TDD). It is recommended that total daily basal insulin dose (TBD) is 50% of TDD and that the carbohydrate-to-insulin ratio (CIR) equals 500 divided by TDD. We recently reported that basal insulin requirement is approximately 30% of TDD. We therefore investigated CIR after adjustment of the proper basal insulin rate. Forty-five Japanese patients with type 1 diabetes were investigated during several weeks of hospitalization. The patients were served standard diabetes meals (25-30 kcal/kg of ideal body weight). Each meal omission was done to confirm basal insulin rate. Target blood glucose level was set at 100 and 150 mg/dL before and 2 h after each meal, respectively. After the basal insulin rate was fixed and target blood glucose levels were achieved, TBD, CIR, TDD, and their products were determined. Mean (±SD) blood glucose levels before and 2 h after meals were 121±47 and 150±61 mg/dL, respectively. TDD was 31.5±9.0 U, and TBD was 27.0±6.5% of TDD. CIR×TDD of breakfast was significantly lower than those of lunch and supper (288±73 vs. 408±92 and 387±83, respectively; P<0.01). CIR has diurnal variance and is estimated from the formula CIR=300/TDD at breakfast or CIR=400/TDD at lunch and supper in type 1 diabetes patients. These results indicate that the insulin dose has been underestimated by using previously established calculations.

  19. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

    NASA Astrophysics Data System (ADS)

    Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

    2016-05-01

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike

  20. JNK: bridging the insulin signaling and inflammatory pathway.

    PubMed

    Liu, Gang; Rondinone, Cristina M

    2005-10-01

    Obesity and insulin resistance are strongly associated with systemic markers of inflammation and endoplasmic reticulum stress. c-Jun N-terminal kinases (JNK) are activated by inflammatory cytokines and have a key role in beta-cell apoptosis and in negative regulation of insulin signaling. JNK1-deficient mice are protected from diet-induced obesity and insulin resistance, while genetically obese mice with targeted mutations in JNK1 are leaner and have reduced insulin and blood glucose levels. These studies validate JNK as a link between inflammation and metabolic diseases and as a promising drug target. This review highlights recent advances in small-molecule inhibitors of JNK that have also been targeted for other diseases with an inflammatory component such as stroke, rheumatoid arthritis, and Alzheimer's and Parkinson's diseases.

  1. Sexual crossing of thermophilic fungus Myceliophthora heterothallica improved enzymatic degradation of sugar beet pulp.

    PubMed

    Aguilar-Pontes, Maria Victoria; Zhou, Miaomiao; van der Horst, Sjors; Theelen, Bart; de Vries, Ronald P; van den Brink, Joost

    2016-01-01

    Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Like most fungi, thermophilic Myceliophthora species therefore have a large set of enzymes targeting different linkages in plant polysaccharides. The majority of these enzymes have not been functionally characterized, and their role in plant biomass degradation is unknown. The biotechnological challenge is to select the right set of enzymes to efficiently degrade a particular biomass. This study describes a strategy using sexual crossing and screening with the thermophilic fungus Myceliophthora heterothallica to identify specific enzymes associated with improved sugar beet pulp saccharification. Two genetically diverse M. heterothallica strains CBS 203.75 and CBS 663.74 were used to generate progenies with improved growth on sugar beet pulp. One progeny, named SBP.F1.2.11, had a different genetic pattern from the parental strains and had improved saccharification activity after the growth on 3 % sugar beet pulp. The improved SBP saccharification was not explained by altered activities of the major (hemi-)cellulases. Exo-proteome analysis of progeny and parental strains after 7-day growth on sugar beet pulp showed that only 17 of the 133 secreted CAZy enzymes were more abundant in progeny SBP.F1.2.11. Particularly one enzyme belonging to the carbohydrate esterase family 5 (CE5) was more abundant in SBP.F1.2.11. This CE5-CBM1 enzyme, named as Axe1, was phylogenetically related to acetyl xylan esterases. Biochemical characterization of Axe1 confirmed de-acetylation activity with optimal activities at 75-85 °C and pH 5.5-6.0. Supplementing Axe1 to CBS 203.75 enzyme set improved release of xylose and glucose from sugar beet pulp. This study identified beneficial enzymes for sugar beet pulp saccharification by selecting progeny with improved growth on this particular substrate. Saccharification of sugar beet pulp was improved by supplementing enzyme mixtures with a previously

  2. The roles of ubiquitin modifying enzymes in neoplastic disease.

    PubMed

    Kumari, Nishi; Jaynes, Patrick William; Saei, Azad; Iyengar, Prasanna Vasudevan; Richard, John Lalith Charles; Eichhorn, Pieter Johan Adam

    2017-12-01

    The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFβ, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Targeted quantification of functional enzyme dynamics in environmental samples for microbially mediated biogeochemical processes: Targeted quantification of functional enzyme dynamics

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Minjing; Gao, Yuqian; Qian, Wei-Jun

    Microbially mediated biogeochemical processes are catalyzed by enzymes that control the transformation of carbon, nitrogen, and other elements in environment. The dynamic linkage between enzymes and biogeochemical species transformation has, however, rarely been investigated because of the lack of analytical approaches to efficiently and reliably quantify enzymes and their dynamics in soils and sediments. Herein, we developed a signature peptide-based technique for sensitively quantifying dissimilatory and assimilatory enzymes using nitrate-reducing enzymes in a hyporheic zone sediment as an example. Moreover, the measured changes in enzyme concentration were found to correlate with the nitrate reduction rate in a way different frommore » that inferred from biogeochemical models based on biomass or functional genes as surrogates for functional enzymes. This phenomenon has important implications for understanding and modeling the dynamics of microbial community functions and biogeochemical processes in environments. Our results also demonstrate the importance of enzyme quantification for the identification and interrogation of those biogeochemical processes with low metabolite concentrations as a result of faster enzyme-catalyzed consumption of metabolites than their production. The dynamic enzyme behaviors provide a basis for the development of enzyme-based models to describe the relationship between the microbial community and biogeochemical processes.« less

  4. Modulation of gonadotrophin induced steroidogenic enzymes in granulosa cells by d-chiroinositol.

    PubMed

    Sacchi, Sandro; Marinaro, Federica; Tondelli, Debora; Lui, Jessica; Xella, Susanna; Marsella, Tiziana; Tagliasacchi, Daniela; Argento, Cindy; Tirelli, Alessandra; Giulini, Simone; La Marca, Antonio

    2016-08-31

    d-chiroinositol (DCI) is a inositolphosphoglycan (IPG) involved in several cellular functions that control the glucose metabolism. DCI functions as second messenger in the insulin signaling pathway and it is considered an insulin sensitizer since deficiency in tissue availability of DCI were shown to cause insulin resistance (IR). Polycystic ovary syndrome (PCOS) is a pathological condition that is often accompanied with insulin resistance. DCI can positively affects several aspect of PCOS etiology decreasing the total and free testosterone, lowering blood pressure, improving the glucose metabolism and increasing the ovulation frequency. The purpose of this study was to evaluate the effects of DCI and insulin combined with gonadotrophins namely follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on key steroidogenic enzymes genes regulation, cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and cytochrome P450 side-chain cleavage (P450scc) in primary cultures of human granulosa cells (hGCs). We also investigated whether DCI, being an insulin-sensitizer would be able to counteract the expected stimulator activity of insulin on human granulosa cells (hGCs). The study was conducted on primary cultures of hGCs. Gene expression was evaluated by RT-qPCR method. Statistical analysis was performed applying student t-test, as appropriate (P < 0.05) set for statistical significance. DCI is able to reduce the gene expression of CYP19A1, P450scc and insulin-like growth factor 1 receptor (IGF-1R) in dose-response manner. The presence of DCI impaired the increased expression of steroidogenic enzyme genes generated by the insulin treatment in gonadotrophin-stimulated hGCs. Insulin acts as co-gonadotrophin increasing the expression of steroidogenic enzymes genes in gonadotrophin-stimulated granulosa cells. DCI is an insulin-sensitizer that counteracts this action by reducing the expression of the genes CYP19A1, P450scc and IGF-1R. The ability of DCI to

  5. Somatomedins inhibit protein degradation in muscle cell

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Roeder, R.A.; Blann, D.L.; Bauer, C.A.

    1986-03-01

    Protein degradation has been measured in cultures of L6 myotubes as the rate of release of trichloroacetic acid-soluble radioactivity after prelabeling cell protein with (/sup 3/H) leucine. Insulin-like growth factor-I (IGF-1), ovine somatomedin (oSM) and insulin (I), at concentrations from 10/sup -11/M to 10/sup -7/M (5 x 10/sup -7/M-oSM) were added at the beginning of a 4-hour degradation period to determine the effects of these hormones on inducible proteolysis occurring in serum-free media. In addition the effects of fetal bovine serum, at concentrations from 1% to 30%, on protein degradation were determined in parallel experiments to relate serum inhibition ofmore » proteolysis to somatomedin actions. Results from this study indicate the apparent half maximal inhibition of proteolysis (18%, 15%, 11%) occurred at .4nM-IGF-1, .6nM-oSM and 4nM-I, respectively. Thus protein degradation was approximately 10 times more sensitive to somatomedins than insulin. The half maximal inhibition of proteolysis (15%) observed with serum occurred at 7.7%. The magnitude of the response between IFG-I and serum (37% vs. 31%) was similar. These results are consistent with the hypothesis that somatomedins are important factors in regulating growth and development of muscle.« less

  6. Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets – “Sand out and Gold Stays”

    PubMed Central

    Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y.; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T.; Wang, Hong; Yang, Xiao-feng

    2016-01-01

    To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: 1) Histone enzymes are differentially expressed in cardiovascular, immune and other tissues; 2) Our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, histone methylation/demethylation are in the highest varieties; and 3) Histone enzymes are more downregulated than upregulated in metabolic diseases and Treg polarization/differentiation, but not in tumors. These results have demonstrated a new working model of “sand out and gold stays,” where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity. PMID:26746407

  7. NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL MAPPING OF THE GENES ENCODING BIPHENYL DIOXYGENASE, A MULTICOMPONENT POLYCHLORINATED-BIPHENYL-DEGRADING ENZYME IN PSEUDOMONAS STRAIN LB400

    EPA Science Inventory

    The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. ix open reading frames were identified, four of which are, homologous to the components of toluene dioxy...

  8. Impact of cell wall-degrading enzymes on water-holding capacity and solubility of dietary fibre in rye and wheat bran.

    PubMed

    Petersson, Karin; Nordlund, Emilia; Tornberg, Eva; Eliasson, Ann-Charlotte; Buchert, Johanna

    2013-03-15

    Rye and wheat bran were treated with several xylanases and endoglucanases, and the effects on physicochemical properties such as solubility, viscosity, water-holding capacity and particle size as well as the chemical composition of the soluble and insoluble fractions of the bran were studied. A large number of enzymes with well-defined activities were used. This enabled a comparison between enzymes of different origins and with different activities as well as a comparison between the effects of the enzymes on rye and wheat bran. The xylanases derived from Bacillus subtilis were the most effective in solubilising dietary fibre from wheat and rye bran. There was a tendency for a higher degree of degradation of the soluble or solubilised dietary fibre in rye bran than in wheat bran when treated with most of the enzymes. None of the enzymes increased the water-holding capacity of the bran or the viscosity of the aqueous phase. The content of insoluble material decreased as the dietary fibre was solubilised by the enzymes. The amount of material that may form a network to retain water in the system was thereby decreased. © 2012 Society of Chemical Industry.

  9. Purification and crystallization of Bacillus subtilis NrnA, a novel enzyme involved in nanoRNA degradation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nelersa, Claudiu M.; Schmier, Brad J.; Malhotra, Arun

    2012-05-08

    The final step in RNA degradation is the hydrolysis of RNA fragments five nucleotides or less in length (nanoRNA) to mononucleotides. In Escherichia coli this step is carried out by oligoribonuclease (Orn), a DEDD-family exoribonuclease that is conserved throughout eukaryotes. However, many bacteria lack Orn homologs, and an unrelated DHH-family phosphoesterase, NrnA, has recently been identified as one of the enzymes responsible for nanoRNA degradation in Bacillus subtilis. To understand its mechanism of action, B. subtilis NrnA was purified and crystallized at room temperature using the hanging-drop vapor-diffusion method with PEG 4000, PEG 3350 or PEG MME 2000 as precipitant.more » The crystals belonged to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 50.62, b = 121.3, c = 123.4 {angstrom}, {alpha} = 90, {beta} = 91.31, {gamma} = 90{sup o}.« less

  10. Chemical stability of insulin. 1. Hydrolytic degradation during storage of pharmaceutical preparations.

    PubMed

    Brange, J; Langkjaer, L; Havelund, S; Vølund, A

    1992-06-01

    Hydrolysis of insulin has been studied during storage of various preparations at different temperatures. Insulin deteriorates rapidly in acid solutions due to extensive deamidation at residue AsnA21. In neutral formulations deamidation takes place at residue AsnB3 at a substantially reduced rate under formation of a mixture of isoAsp and Asp derivatives. The rate of hydrolysis at B3 is independent of the strength of the preparation, and in most cases the species of insulin, but varies with storage temperature and formulation. Total transformation at B3 is considerably reduced when insulin is in the crystalline as compared to the amorphous or soluble state, indicating that formation of the rate-limiting cyclic imide decreases when the flexibility of the tertiary structure is reduced. Neutral solutions containing phenol showed reduced deamidation probably because of a stabilizing effect of phenol on the tertiary structure (alpha-helix formation) around the deamidating residue, resulting in a reduced probability for formation of the intermediate imide. The ratio of isoAsp/Asp derivative was independent of time and temperature, suggesting a pathway involving only intermediate imide formation, without any direct side-chain hydrolysis. However, increasing formation of Asp relative to isoAsp derivative was observed with decreasing flexibility of the insulin three-dimensional structure in the formulation. In certain crystalline suspensions a cleavage of the peptide bond A8-A9 was observed. Formation of this split product is species dependent: bovine greater than porcine greater than human insulin. The hydrolytic cleavage of the peptide backbone takes place only in preparations containing rhombohedral crystals in addition to free zinc ions.

  11. Challenges constraining insulin access in Nepal-a country with no local insulin production.

    PubMed

    Sharma, Abhishek; Bhandari, Parash Mani; Neupane, Dipika; Kaplan, Warren A; Mishra, Shiva Raj

    2018-05-01

    Nepal is facing an increasing burden of diabetes and relies almost entirely on insulin imported through India. We employed a modified version of the WHO/Health Action International standard survey to assess insulin availability and prices, along with qualitative interviews with insulin retailers (pharmacists) and wholesalers in the Kathmandu Valley, Nepal. The mean availability of the two human insulins listed on the 2011 Nepal Essential Medicine List were 14.3% and 42.85% in the surveyed private- and public-sector pharmacies, respectively, compared with the WHO target of 80% availability. The median consumer price of human insulin cartridges, analogue insulin cartridges and pens was, respectively, 2.1, 4.6 and 5.3 times that of human insulin vials (US$5.54). The insulin cartridges made in India were less expensive (p<0.001) than those made elsewhere. The lowest-paid worker would need to spend between 3 and 17 days' wages to purchase a monthly insulin supply out of pocket. Insulin access is limited in Kathmandu owing to low availability and the highly unaffordable price. Insulin access could improve with the government exploring additional suppliers, pooling insulin tenders, auditing insulin utilization and developing independent prescribing guidelines. Furthermore, there is a need to educate physicians and develop a consensus statement on insulin initiation to curb the growing analogue use and promote rational use.

  12. Microbial Enzyme Activity and Carbon Cycling in Grassland Soil Fractions

    NASA Astrophysics Data System (ADS)

    Allison, S. D.; Jastrow, J. D.

    2004-12-01

    Extracellular enzymes are necessary to degrade complex organic compounds present in soils. Using physical fractionation procedures, we tested whether old soil carbon is spatially isolated from degradative enzymes across a prairie restoration chronosequence in Illinois, USA. We found that carbon-degrading enzymes were abundant in all soil fractions, including macroaggregates, microaggregates, and the clay fraction, which contains carbon with a mean residence time of ~200 years. The activities of two cellulose-degrading enzymes and a chitin-degrading enzyme were 2-10 times greater in organic matter fractions than in bulk soil, consistent with the rapid turnover of these fractions. Polyphenol oxidase activity was 3 times greater in the clay fraction than in the bulk soil, despite very slow carbon turnover in this fraction. Changes in enzyme activity across the restoration chronosequence were small once adjusted for increases in soil carbon concentration, although polyphenol oxidase activity per unit carbon declined by 50% in native prairie versus cultivated soil. These results are consistent with a `two-pool' model of enzyme and carbon turnover in grassland soils. In light organic matter fractions, enzyme production and carbon turnover both occur rapidly. However, in mineral-dominated fractions, both enzymes and their carbon substrates are immobilized on mineral surfaces, leading to slow turnover. Soil carbon accumulation in the clay fraction and across the prairie restoration chronosequence probably reflects increasing physical isolation of enzymes and substrates on the molecular scale, rather than the micron to millimeter scale.

  13. PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

    PubMed Central

    Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.

    2002-01-01

    The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659

  14. Effects of microbial enzymes on starch and hemicellulose degradation in total mixed ration silages.

    PubMed

    Ning, Tingting; Wang, Huili; Zheng, Mingli; Niu, Dongze; Zuo, Sasa; Xu, Chuncheng

    2017-02-01

    This study investigated the association of enzyme-producing microbes and their enzymes with starch and hemicellulose degradation during fermentation of total mixed ration (TMR) silage. The TMRs were prepared with soybean curd residue, alfalfa hay (ATMR) or Leymus chinensis hay (LTMR), corn meal, soybean meal, vitamin-mineral supplements, and salt at a ratio of 25:40:30:4:0.5:0.5 on a dry matter basis. Laboratory-scale bag silos were randomly opened after 1, 3, 7, 14, 28, and 56 days of ensiling and subjected to analyses of fermentation quality, carbohydrates loss, microbial amylase and hemicellulase activities, succession of dominant amylolytic or hemicellulolytic microbes, and their microbial and enzymatic properties. Both ATMR and LTMR silages were well preserved, with low pH and high lactic acid concentrations. In addition to the substantial loss of water soluble carbohydrates, loss of starch and hemicellulose was also observed in both TMR silages with prolonged ensiling. The microbial amylase activity remained detectable throughout the ensiling in both TMR silages, whereas the microbial hemicellulase activity progressively decreased until it was inactive at day 14 post-ensiling in both TMR silages. During the early stage of fermentation, the main amylase-producing microbes were Bacillus amyloliquefaciens ( B. amyloliquefaciens ), B. cereus , B. licheniformis , and B. subtilis in ATMR silage and B. flexus , B. licheniformis , and Paenibacillus xylanexedens ( P. xylanexedens ) in LTMR silage, whereas Enterococcus faecium was closely associated with starch hydrolysis at the later stage of fermentation in both TMR silages. B. amyloliquefaciens , B. licheniformis , and B. subtilis and B. licheniformis , B. pumilus , and P. xylanexedens were the main source of microbial hemicellulase during the early stage of fermentation in ATMR and LTMR silages, respectively. The microbial amylase contributes to starch hydrolysis during the ensiling process in both TMR silages

  15. Effect of chloroquine on insulin and glucose homoeostasis in normal subjects and patients with non-insulin-dependent diabetes mellitus.

    PubMed Central

    Smith, G D; Amos, T A; Mahler, R; Peters, T J

    1987-01-01

    Plasma glucose, insulin, and C peptide concentrations were determined after an oral glucose load in normal subjects and in a group of patients with non-insulin-dependent diabetes mellitus before and during a short course of treatment with chloroquine. In the control group there was a small but significant reduction in fasting blood glucose concentration but overall glucose tolerance and hormone concentrations were unaffected. In contrast, the patients with non-insulin-dependent diabetes mellitus showed a significant improvement in their glucose tolerance, which paralleled the severity of their diabetes. This response seems to reflect decreased degradation of insulin rather than increased pancreatic output. These observations suggest that treatment with chloroquine or suitable analogues may be a new approach to the management of diabetes. PMID:3103729

  16. Molecular characterization of the enzymes involved in the degradation of a brominated aromatic herbicide.

    PubMed

    Chen, Kai; Huang, Linglong; Xu, Changfeng; Liu, Xiaomei; He, Jian; Zinder, Stephen H; Li, Shunpeng; Jiang, Jiandong

    2013-09-01

    Dehalogenation is the key step in the degradation of halogenated aromatics, while reductive dehalogenation is originally thought to rarely occur in aerobes. In this study, an aerobic strain of Comamonas sp. 7D-2 was shown to degrade the brominated aromatic herbicide bromoxynil completely and release two equivalents of bromides under aerobic conditions. The enzymes involved in the degradation of bromoxynil to 4-carboxy-2-hydroxymuconate-6-semialdehyde, including nitrilase, reductive dehalogenase (BhbA), 4-hydroxybenzoate 3-monooxygenase and protocatechuate 4,5-dioxygenase, were molecularly characterized. The novel dehalogenase BhbA was shown to be a complex of a respiration-linked reductive dehalogenase (RdhA) domain and a NAD(P)H-dependent oxidoreductase domain and to have key features of anaerobic respiratory RdhAs, including two predicted binding motifs for [4Fe-4S] clusters and a close association with a hydrophobic membrane protein (BhbB). BhbB was confirmed to anchor BhbA to the membrane. BhbA was partially purified and found to use NAD(P)H as electron donors. Full-length bhbA homologues were found almost exclusively in marine aerobic proteobacteria, suggesting that reductive dehalogenation occurs extensively in aerobes and that bhbA is horizontally transferred from marine microorganisms. The discovery of a functional reductive dehalogenase and ring-cleavage oxygenases in an aerobe opens up possibilities for basic research as well as the potential application for bioremediation. © 2013 John Wiley & Sons Ltd.

  17. Insulin degludec/insulin aspart combination for the treatment of type 1 and type 2 diabetes

    PubMed Central

    Dardano, Angela; Bianchi, Cristina; Del Prato, Stefano; Miccoli, Roberto

    2014-01-01

    Glycemic control remains the major therapeutic objective to prevent or delay the onset and progression of complications related to diabetes mellitus. Insulin therapy represents a cornerstone in the treatment of diabetes and has been used widely for achieving glycemic goals. Nevertheless, a large portion of the population with diabetes does not meet the internationally agreed glycemic targets. Moreover, insulin treatment, especially if intensive, may be associated with emergency room visits and hospitalization due to hypoglycemic events. Therefore, fear of hypoglycemia or hypoglycemic events represents the main barriers to the attainment of glycemic targets. The burden associated with multiple daily injections also remains a significant obstacle to initiating and maintaining insulin therapy. The most attractive insulin treatment approach should meet the patients’ preference, rather than demanding patients to change or adapt their lifestyle. Insulin degludec/insulin aspart (IDegAsp) is a new combination, formulated with ultra-long-acting insulin degludec and rapid-acting insulin aspart, with peculiar pharmacological features, clinical efficacy, safety, and tolerability. IDegAsp provides similar, noninferior glycemic control to a standard basal–bolus regimen in patients with type 1 diabetes mellitus, with additional benefits of significantly lower episodes of hypoglycemia (particularly nocturnal) and fewer daily insulin injections. Moreover, although treatment strategy and patients’ viewpoint are different in type 1 and type 2 diabetes, trial results suggest that IDegAsp may be an appropriate and reasonable option for initiating insulin therapy in patients with type 2 diabetes inadequately controlled on maximal doses of conventional oral agents. This paper will discuss the role of IDegAsp combination as a novel treatment option in diabetic patients. PMID:25143741

  18. Insulin Signaling in Type 2 Diabetes

    PubMed Central

    Brännmark, Cecilia; Nyman, Elin; Fagerholm, Siri; Bergenholm, Linnéa; Ekstrand, Eva-Maria; Cedersund, Gunnar; Strålfors, Peter

    2013-01-01

    Type 2 diabetes originates in an expanding adipose tissue that for unknown reasons becomes insulin resistant. Insulin resistance reflects impairments in insulin signaling, but mechanisms involved are unclear because current research is fragmented. We report a systems level mechanistic understanding of insulin resistance, using systems wide and internally consistent data from human adipocytes. Based on quantitative steady-state and dynamic time course data on signaling intermediaries, normally and in diabetes, we developed a dynamic mathematical model of insulin signaling. The model structure and parameters are identical in the normal and diabetic states of the model, except for three parameters that change in diabetes: (i) reduced concentration of insulin receptor, (ii) reduced concentration of insulin-regulated glucose transporter GLUT4, and (iii) changed feedback from mammalian target of rapamycin in complex with raptor (mTORC1). Modeling reveals that at the core of insulin resistance in human adipocytes is attenuation of a positive feedback from mTORC1 to the insulin receptor substrate-1, which explains reduced sensitivity and signal strength throughout the signaling network. Model simulations with inhibition of mTORC1 are comparable with experimental data on inhibition of mTORC1 using rapamycin in human adipocytes. We demonstrate the potential of the model for identification of drug targets, e.g. increasing the feedback restores insulin signaling, both at the cellular level and, using a multilevel model, at the whole body level. Our findings suggest that insulin resistance in an expanded adipose tissue results from cell growth restriction to prevent cell necrosis. PMID:23400783

  19. Insulin Resistance and Mitochondrial Dysfunction.

    PubMed

    Gonzalez-Franquesa, Alba; Patti, Mary-Elizabeth

    2017-01-01

    Insulin resistance precedes and predicts the onset of type 2 diabetes (T2D) in susceptible humans, underscoring its important role in the complex pathogenesis of this disease. Insulin resistance contributes to multiple tissue defects characteristic of T2D, including reduced insulin-stimulated glucose uptake in insulin-sensitive tissues, increased hepatic glucose production, increased lipolysis in adipose tissue, and altered insulin secretion. Studies of individuals with insulin resistance, both with established T2D and high-risk individuals, have consistently demonstrated a diverse array of defects in mitochondrial function (i.e., bioenergetics, biogenesis and dynamics). However, it remains uncertain whether mitochondrial dysfunction is primary (critical initiating defect) or secondary to the subtle derangements in glucose metabolism, insulin resistance, and defective insulin secretion present early in the course of disease development. In this chapter, we will present the evidence linking mitochondrial dysfunction and insulin resistance, and review the potential for mitochondrial targets as a therapeutic approach for T2D.

  20. Cell Wall-Degrading Enzymes Enlarge the Pore Size of Intervessel Pit Membranes in Healthy and Xylella fastidiosa-Infected Grapevines1[C][W][OA

    PubMed Central

    Pérez-Donoso, Alonso G.; Sun, Qiang; Roper, M. Caroline; Greve, L. Carl; Kirkpatrick, Bruce; Labavitch, John M.

    2010-01-01

    The pit membrane (PM) is a primary cell wall barrier that separates adjacent xylem water conduits, limiting the spread of xylem-localized pathogens and air embolisms from one conduit to the next. This paper provides a characterization of the size of the pores in the PMs of grapevine (Vitis vinifera). The PM porosity (PMP) of stems infected with the bacterium Xylella fastidiosa was compared with the PMP of healthy stems. Stems were infused with pressurized water and flow rates were determined; gold particles of known size were introduced with the water to assist in determining the size of PM pores. The effect of introducing trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), oligogalacturonides, and polygalacturonic acid into stems on water flux via the xylem was also measured. The possibility that cell wall-degrading enzymes could alter the pore sizes, thus facilitating the ability of X. fastidiosa to cross the PMs, was tested. Two cell wall-degrading enzymes likely to be produced by X. fastidiosa (polygalactuoronase and endo-1,4- β -glucanase) were infused into stems, and particle passage tests were performed to check for changes in PMP. Scanning electron microscopy of control and enzyme-infused stem segments revealed that the combination of enzymes opened holes in PMs, probably explaining enzyme impacts on PMP and how a small X. fastidiosa population, introduced into grapevines by insect vectors, can multiply and spread throughout the vine and cause Pierce's disease. PMID:20107028

  1. Increased production of biomass-degrading enzymes by double deletion of creA and creB genes involved in carbon catabolite repression in Aspergillus oryzae.

    PubMed

    Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

    2018-02-01

    In a previous study, we reported that a double gene deletion mutant for CreA and CreB, which constitute the regulatory machinery involved in carbon catabolite repression, exhibited improved production of α-amylase compared with the wild-type strain and single creA or creB deletion mutants in Aspergillus oryzae. Because A. oryzae can also produce biomass-degrading enzymes, such as xylolytic and cellulolytic enzymes, we examined the production levels of those enzymes in deletion mutants in this study. Xylanase and β-glucosidase activities in the wild-type were hardly detected in submerged culture containing xylose as the carbon source, whereas those enzyme activities were significantly increased in the single creA deletion (ΔcreA) and double creA and creB deletion (ΔcreAΔcreB) mutants. In particular, the ΔcreAΔcreB mutant exhibited >100-fold higher xylanase and β-glucosidase activities than the wild-type. Moreover, in solid-state culture, the β-glucosidase activity of the double deletion mutant was >7-fold higher than in the wild-type. These results suggested that deletion of both creA and creB genes could also efficiently improve the production levels of biomass-degrading enzymes in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Defining functional diversity for lignocellulose degradation in a microbial community using multi-omics studies.

    PubMed

    Alessi, Anna M; Bird, Susannah M; Oates, Nicola C; Li, Yi; Dowle, Adam A; Novotny, Etelvino H; deAzevedo, Eduardo R; Bennett, Joseph P; Polikarpov, Igor; Young, J Peter W; McQueen-Mason, Simon J; Bruce, Neil C

    2018-01-01

    Lignocellulose is one of the most abundant forms of fixed carbon in the biosphere. Current industrial approaches to the degradation of lignocellulose employ enzyme mixtures, usually from a single fungal species, which are only effective in hydrolyzing polysaccharides following biomass pre-treatments. While the enzymatic mechanisms of lignocellulose degradation have been characterized in detail in individual microbial species, the microbial communities that efficiently breakdown plant materials in nature are species rich and secrete a myriad of enzymes to perform "community-level" metabolism of lignocellulose. Single-species approaches are, therefore, likely to miss important aspects of lignocellulose degradation that will be central to optimizing commercial processes. Here, we investigated the microbial degradation of wheat straw in liquid cultures that had been inoculated with wheat straw compost. Samples taken at selected time points were subjected to multi-omics analysis with the aim of identifying new microbial mechanisms for lignocellulose degradation that could be applied in industrial pre-treatment of feedstocks. Phylogenetic composition of the community, based on sequenced bacterial and eukaryotic ribosomal genes, showed a gradual decrease in complexity and diversity over time due to microbial enrichment. Taxonomic affiliation of bacterial species showed dominance of Bacteroidetes and Proteobacteria and high relative abundance of genera Asticcacaulis , Leadbetterella and Truepera . The eukaryotic members of the community were enriched in peritrich ciliates from genus Telotrochidium that thrived in the liquid cultures compared to fungal species that were present in low abundance. A targeted metasecretome approach combined with metatranscriptomics analysis, identified 1127 proteins and showed the presence of numerous carbohydrate-active enzymes extracted from the biomass-bound fractions and from the culture supernatant. This revealed a wide array of

  3. Mapping the polysaccharide degradation potential of Aspergillus niger

    PubMed Central

    2012-01-01

    Background The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation. Results Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan. Conclusions The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger. PMID:22799883

  4. Mapping the polysaccharide degradation potential of Aspergillus niger.

    PubMed

    Andersen, Mikael R; Giese, Malene; de Vries, Ronald P; Nielsen, Jens

    2012-07-16

    The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation. Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan. The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger.

  5. Enzymes involved in the anaerobic degradation of ortho-phthalate by the nitrate-reducing bacterium Azoarcus sp. strain PA01.

    PubMed

    Junghare, Madan; Spiteller, Dieter; Schink, Bernhard

    2016-09-01

    The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium Azoarcus sp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins in o-phthalate-grown compared to benzoate-grown cells. The genes encoding o-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01. The o-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA-transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobic o-phthalate degradation. We propose that o-phthalate is first activated to o-phthalyl-CoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, and o-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. Results from in vitro enzyme assays with cell-free extracts of o-phthalate-grown cells demonstrated the formation of o-phthalyl-CoA from o-phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl-CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity for o-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o-phthalate whereas the putative o-phthalyl-CoA decarboxylase converted fluoro-o-phthalyl-CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygen-insensitive and inducible only after growth with o-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  6. Effect of Choline on the Composition and Degradation Enzyme of Extracellular Matrix of Mice Chondrocytes Exposed to Fluoride.

    PubMed

    Zhao, Yangfei; Hao, Jing; Wang, Jinming; Wang, Jundong

    2017-02-01

    Choline has been shown to mediate damage of the chondrocyte matrix and degradation enzymes of mice exposed to fluoride (F). To test the action of choline, pregnant mice were treated with differing amounts of F and choline. Newborn mice were weaned at 21 days after birth and treated with the same doses of F and choline as they mothers for 12 weeks. Using hematoxylin-eosin (HE) staining, real-time PCR (RT-PCR), and western blotting, changes in the structure of the cartilage, the expression of mRNA and protein related to proteoglycans (PG), and degradation enzymes were detected. The RT-PCR results show that the expression of the Aggrecan (Acan), transforming growth factor beta (TGF-β1), and Aggrecanases-1 gene were abnormal in the high fluoride (HiF) group, and treatments with choline reversed this phenomenon. The western blotting results show that the protein expression of Aggrecanases-1 was significantly increased in the HiF group (p < 0.01). These findings suggest that F can change the morphology of cartilage tissue, the gene expression of the Acan, TGF-β1, Aggrecanases-1, and the protein expression of the Acan, and that choline can attenuate the effect of F. This may provide the basis for the treatment and prevention of fluorosis.

  7. Effects of glucose, insulin and triiodothyroxine on leptin and leptin receptor expression and the effects of leptin on activities of enzymes related to glucose metabolism in grass carp (Ctenopharyngodon idella) hepatocytes.

    PubMed

    Lu, Rong-Hua; Zhou, Yi; Yuan, Xiao-Chen; Liang, Xu-Fang; Fang, Liu; Bai, Xiao-Li; Wang, Min; Zhao, Yu-Hua

    2015-08-01

    Leptin is an important regulator of appetite and energy expenditure in mammals, but its role in fish metabolism control is poorly understood. Our previous studies demonstrated that leptin has an effect on the regulation of food intake and energy expenditure as well as lipid metabolism (stimulation of lipolysis and inhibition of adipogenesis) in the grass carp Ctenopharyngodon idella. To further investigate the role of leptin in fish, the effects of glucose, insulin and triiodothyroxine (T3) on the expression levels of leptin and leptin receptor (Lepr) and the effects of leptin on the activities of critical glucose metabolism enzymes in grass carp hepatocytes were evaluated in the present study. Our data indicated that leptin gene expression was induced by glucose in a dose-dependent manner, while Lepr gene expression exhibited a biphasic change. A high dose of insulin (100 ng/mL) significantly up-regulated the expression of leptin and Lepr. Leptin expression was markedly up-regulated by a low concentration of T3 but inhibited by a high concentration of T3. T3 up-regulated Lepr expression in a dose-dependent manner. Together, these data suggest that leptin had a close relationship with three factors (glucose, insulin and T3) and might participate in the regulation of glucose metabolism in grass carp. In addition, we also found that leptin affected the activities of key enzymes that are involved in glucose metabolism, which might be mediated by insulin receptor substrate-phosphoinositol 3-kinase signaling.

  8. Leucoagaricus gongylophorus Produces Diverse Enzymes for the Degradation of Recalcitrant Plant Polymers in Leaf-Cutter Ant Fungus Gardens

    PubMed Central

    Aylward, Frank O.; Burnum-Johnson, Kristin E.; Tringe, Susannah G.; Teiling, Clotilde; Tremmel, Daniel M.; Moeller, Joseph A.; Scott, Jarrod J.; Barry, Kerrie W.; Piehowski, Paul D.; Nicora, Carrie D.; Malfatti, Stephanie A.; Monroe, Matthew E.; Purvine, Samuel O.; Goodwin, Lynne A.; Smith, Richard D.; Weinstock, George M.; Gerardo, Nicole M.; Suen, Garret; Lipton, Mary S.

    2013-01-01

    Plants represent a large reservoir of organic carbon comprised primarily of recalcitrant polymers that most metazoans are unable to deconstruct. Many herbivores gain access to nutrients in this material indirectly by associating with microbial symbionts, and leaf-cutter ants are a paradigmatic example. These ants use fresh foliar biomass as manure to cultivate gardens composed primarily of Leucoagaricus gongylophorus, a basidiomycetous fungus that produces specialized hyphal swellings that serve as a food source for the host ant colony. Although leaf-cutter ants are conspicuous herbivores that contribute substantially to carbon turnover in Neotropical ecosystems, the process through which plant biomass is degraded in their fungus gardens is not well understood. Here we present the first draft genome of L. gongylophorus, and, using genomic and metaproteomic tools, we investigate its role in lignocellulose degradation in the gardens of both Atta cephalotes and Acromyrmex echinatior leaf-cutter ants. We show that L. gongylophorus produces a diversity of lignocellulases in ant gardens and is likely the primary driver of plant biomass degradation in these ecosystems. We also show that this fungus produces distinct sets of lignocellulases throughout the different stages of biomass degradation, including numerous cellulases and laccases that likely play an important role in lignocellulose degradation. Our study provides a detailed analysis of plant biomass degradation in leaf-cutter ant fungus gardens and insight into the enzymes underlying the symbiosis between these dominant herbivores and their obligate fungal cultivar. PMID:23584789

  9. Original paper: Efficacy and safety analysis of insulin degludec/insulin aspart compared with biphasic insulin aspart 30: A phase 3, multicentre, international, open-label, randomised, treat-to-target trial in patients with type 2 diabetes fasting during Ramadan.

    PubMed

    Hassanein, Mohamed; Echtay, Akram Salim; Malek, Rachid; Omar, Mahomed; Shaikh, Shehla Sajid; Ekelund, Magnus; Kaplan, Kadriye; Kamaruddin, Nor Azmi

    2018-01-01

    To compare the efficacy and safety of insulin degludec/insulin aspart (IDegAsp) and biphasic insulin aspart 30 (BIAsp 30) before, during and after Ramadan in patients with type 2 diabetes mellitus (T2DM) who fasted during Ramadan. In this multinational, randomised, treat-to-target trial, patients with T2DM who intended to fast and were on basal, pre- or self-mixed insulin ± oral antidiabetic drugs for ≥90 days were randomised (1:1) to IDegAsp twice daily (BID) or BIAsp 30 BID. Treatment period included pre-Ramadan treatment initiation (with insulin titration for 8-20 weeks), Ramadan (4 weeks) and post-Ramadan (4 weeks). Insulin doses were reduced by 30-50% for the pre-dawn meal (suhur) on the first day of Ramadan, and readjusted to the pre-Ramadan levels at the end of Ramadan. Hypoglycaemia was analysed as overall (severe or plasma glucose <3.1 mmol/L [56 mg/dL]), nocturnal (00:01-05:59) or severe (requiring assistance of another person). During the treatment period, IDegAsp (n = 131) had significantly lower overall and nocturnal hypoglycaemia rates with similar glycaemic efficacy, versus BIAsp 30 (n = 132). During Ramadan, despite achieving significantly lower pre-iftar (meal at sunset) self-measured plasma glucose (estimated treatment difference: -0.54 mmol/L [-1.02; -0.07] 95% CI , p = .0247; post hoc) with similar overall glycaemic efficacy, IDegAsp showed significantly lower overall and nocturnal hypoglycaemia rates versus BIAsp 30. IDegAsp is a suitable therapeutic agent for patients who need insulin for sustained glucose control before, during and after Ramadan fasting, with a significantly lower risk of hypoglycaemia, versus BIAsp 30, an existing premixed insulin analogue. Copyright © 2017. Published by Elsevier B.V.

  10. An antennal carboxylesterase from Drosophila melanogaster, esterase 6, is a candidate odorant-degrading enzyme toward food odorants.

    PubMed

    Chertemps, Thomas; Younus, Faisal; Steiner, Claudia; Durand, Nicolas; Coppin, Chris W; Pandey, Gunjan; Oakeshott, John G; Maïbèche, Martine

    2015-01-01

    Reception of odorant molecules within insect olfactory organs involves several sequential steps, including their transport through the sensillar lymph, interaction with the respective sensory receptors, and subsequent inactivation. Odorant-degrading enzymes (ODEs) putatively play a role in signal dynamics by rapid degradation of odorants in the vicinity of the receptors, but this hypothesis is mainly supported by in vitro results. We have recently shown that an extracellular carboxylesterase, esterase-6 (EST-6), is involved in the physiological and behavioral dynamics of the response of Drosophila melanogaster to its volatile pheromone ester, cis-vaccenyl acetate. However, as the expression pattern of the Est-6 gene in the antennae is not restricted to the pheromone responding sensilla, we tested here if EST-6 could play a broader function in the antennae. We found that recombinant EST-6 is able to efficiently hydrolyse several volatile esters that would be emitted by its natural food in vitro. Electrophysiological comparisons of mutant Est-6 null flies and a control strain (on the same genetic background) showed that the dynamics of the antennal response to these compounds is influenced by EST-6, with the antennae of the null mutants showing prolonged activity in response to them. Antennal responses to the strongest odorant, pentyl acetate, were then studied in more detail, showing that the repolarization dynamics were modified even at low doses but without modification of the detection threshold. Behavioral choice experiments with pentyl acetate also showed differences between genotypes; attraction to this compound was observed at a lower dose among the null than control flies. As EST-6 is able to degrade various bioactive odorants emitted by food and plays a role in the response to these compounds, we hypothesize a role as an ODE for this enzyme toward food volatiles.

  11. Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

    PubMed

    Zhang, Meiling; Chekan, Jonathan R; Dodd, Dylan; Hong, Pei-Ying; Radlinski, Lauren; Revindran, Vanessa; Nair, Satish K; Mackie, Roderick I; Cann, Isaac

    2014-09-02

    Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

  12. Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

    PubMed

    de Lima-Morales, Daiana; Chaves-Moreno, Diego; Wos-Oxley, Melissa L; Jáuregui, Ruy; Vilchez-Vargas, Ramiro; Pieper, Dietmar H

    2016-01-01

    Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. The Effect of Insulin and Insulin-Like Growth Factors on Hippocampus- and Amygdala-Dependent Long-Term Memory Formation

    ERIC Educational Resources Information Center

    Stern, Sarah A.; Chen, Dillon Y.; Alberini, Cristina M.

    2014-01-01

    Recent work has reported that the insulin-like growth factor 2 (IGF2) promotes memory enhancement. Furthermore, impaired insulin or IGF1 functions have been suggested to play a role in the pathogenesis of neurodegeneration and cognitive impairments, hence implicating the insulin/IGF system as an important target for cognitive enhancement and/or…

  14. Coevolution and life cycle specialization of plant cell wall degrading enzymes in a hemibiotrophic pathogen.

    PubMed

    Brunner, Patrick C; Torriani, Stefano F F; Croll, Daniel; Stukenbrock, Eva H; McDonald, Bruce A

    2013-06-01

    Zymoseptoria tritici is an important fungal pathogen on wheat that originated in the Fertile Crescent. Its closely related sister species Z. pseudotritici and Z. ardabiliae infect wild grasses in the same region. This recently emerged host-pathogen system provides a rare opportunity to investigate the evolutionary processes shaping the genome of an emerging pathogen. Here, we investigate genetic signatures in plant cell wall degrading enzymes (PCWDEs) that are likely affected by or driving coevolution in plant-pathogen systems. We hypothesize four main evolutionary scenarios and combine comparative genomics, transcriptomics, and selection analyses to assign the majority of PCWDEs in Z. tritici to one of these scenarios. We found widespread differential transcription among different members of the same gene family, challenging the idea of functional redundancy and suggesting instead that specialized enzymatic activity occurs during different stages of the pathogen life cycle. We also find that natural selection has significantly affected at least 19 of the 48 identified PCWDEs. The majority of genes showed signatures of purifying selection, typical for the scenario of conserved substrate optimization. However, six genes showed diversifying selection that could be attributed to either host adaptation or host evasion. This study provides a powerful framework to better understand the roles played by different members of multigene families and to determine which genes are the most appropriate targets for wet laboratory experimentation, for example, to elucidate enzymatic function during relevant phases of a pathogen's life cycle.

  15. Testosterone treatment increases androgen receptor and aromatase gene expression in myotubes from patients with PCOS and controls, but does not induce insulin resistance.

    PubMed

    Eriksen, Mette Brandt; Glintborg, Dorte; Nielsen, Michael Friberg Bruun; Jakobsen, Marianne Antonius; Brusgaard, Klaus; Tan, Qihua; Gaster, Michael

    2014-09-05

    Polycystic ovary syndrome (PCOS) is associated with insulin resistance and increased risk of type 2 diabetes. Skeletal muscle is the major site of insulin mediated glucose disposal and the skeletal muscle tissue is capable to synthesize, convert and degrade androgens. Insulin sensitivity is conserved in cultured myotubes (in vitro) from patients with PCOS, but the effect of testosterone on this insulin sensitivity is unknown. We investigated the effect of 7days testosterone treatment (100nmol/l) on glucose transport and gene expression levels of hormone receptors and enzymes involved in the synthesis and conversion of testosterone (HSD17B1, HSD17B2, CYP19A1, SRD5A1-2, AR, ER-α, HSD17B6 and AKR1-3) in myotubes from ten patients with PCOS and ten matched controls. Testosterone treatment significantly increased aromatase and androgen receptor gene expression levels in patients and controls. Glucose transport in myotubes was comparable in patients with PCOS vs. controls and was unchanged by testosterone treatment (p=0.21 PCOS vs. controls). These results suggest that testosterone treatment of myotubes increases the aromatase and androgen receptor gene expression without affecting insulin sensitivity and if testosterone is implicated in muscular insulin resistance in PCOS, this is by and indirect mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Effect of Insulin Resistance on Monounsaturated Fatty Acid Levels: A Multi-cohort Non-targeted Metabolomics and Mendelian Randomization Study

    PubMed Central

    Ganna, Andrea; Brandmaier, Stefan; Broeckling, Corey D.; Prenni, Jessica E.; Wang-Sattler, Rui; Peters, Annette; Strauch, Konstantin; Meitinger, Thomas; Giedraitis, Vilmantas; Ärnlöv, Johan; Berne, Christian; Gieger, Christian; Ripatti, Samuli; Lind, Lars; Sundström, Johan; Ingelsson, Erik

    2016-01-01

    Insulin resistance (IR) and impaired insulin secretion contribute to type 2 diabetes and cardiovascular disease. Both are associated with changes in the circulating metabolome, but causal directions have been difficult to disentangle. We combined untargeted plasma metabolomics by liquid chromatography/mass spectrometry in three non-diabetic cohorts with Mendelian Randomization (MR) analysis to obtain new insights into early metabolic alterations in IR and impaired insulin secretion. In up to 910 elderly men we found associations of 52 metabolites with hyperinsulinemic-euglycemic clamp-measured IR and/or β-cell responsiveness (disposition index) during an oral glucose tolerance test. These implicated bile acid, glycerophospholipid and caffeine metabolism for IR and fatty acid biosynthesis for impaired insulin secretion. In MR analysis in two separate cohorts (n = 2,613) followed by replication in three independent studies profiled on different metabolomics platforms (n = 7,824 / 8,961 / 8,330), we discovered and replicated causal effects of IR on lower levels of palmitoleic acid and oleic acid. A trend for a causal effect of IR on higher levels of tyrosine reached significance only in meta-analysis. In one of the largest studies combining “gold standard” measures for insulin responsiveness with non-targeted metabolomics, we found distinct metabolic profiles related to IR or impaired insulin secretion. We speculate that the causal effects on monounsaturated fatty acid levels could explain parts of the raised cardiovascular disease risk in IR that is independent of diabetes development. PMID:27768686

  17. Branched-chain amino acids in metabolic signalling and insulin resistance

    PubMed Central

    Lynch, Christopher J.; Adams, Sean H.

    2015-01-01

    Branched-chain amino acids (BCAAs) are important nutrient signals that have direct and indirect effects. Frequently, BCAAs have been reported to mediate antiobesity effects, especially in rodent models. However, circulating levels of BCAAs tend to be increased in individuals with obesity and are associated with worse metabolic health and future insulin resistance or type 2 diabetes mellitus (T2DM). A hypothesized mechanism linking increased levels of BCAAs and T2DM involves leucine-mediated activation of the mammalian target of rapamycin complex 1 (mTORC1), which results in uncoupling of insulin signalling at an early stage. A BCAA dysmetabolism model proposes that the accumulation of mitotoxic metabolites (and not BCAAs per se) promotes β-cell mitochondrial dysfunction, stress signalling and apoptosis associated with T2DM. Alternatively, insulin resistance might promote aminoacidaemia by increasing the protein degradation that insulin normally suppresses, and/or by eliciting an impairment of efficient BCAA oxidative metabolism in some tissues. Whether and how impaired BCAA metabolism might occur in obesity is discussed in this Review. Research on the role of individual and model-dependent differences in BCAA metabolism is needed, as several genes (BCKDHA, PPM1K, IVD and KLF15) have been designated as candidate genes for obesity and/or T2DM in humans, and distinct phenotypes of tissue-specific branched chain ketoacid dehydrogenase complex activity have been detected in animal models of obesity and T2DM. PMID:25287287

  18. Branched-chain amino acids in metabolic signalling and insulin resistance.

    PubMed

    Lynch, Christopher J; Adams, Sean H

    2014-12-01

    Branched-chain amino acids (BCAAs) are important nutrient signals that have direct and indirect effects. Frequently, BCAAs have been reported to mediate antiobesity effects, especially in rodent models. However, circulating levels of BCAAs tend to be increased in individuals with obesity and are associated with worse metabolic health and future insulin resistance or type 2 diabetes mellitus (T2DM). A hypothesized mechanism linking increased levels of BCAAs and T2DM involves leucine-mediated activation of the mammalian target of rapamycin complex 1 (mTORC1), which results in uncoupling of insulin signalling at an early stage. A BCAA dysmetabolism model proposes that the accumulation of mitotoxic metabolites (and not BCAAs per se) promotes β-cell mitochondrial dysfunction, stress signalling and apoptosis associated with T2DM. Alternatively, insulin resistance might promote aminoacidaemia by increasing the protein degradation that insulin normally suppresses, and/or by eliciting an impairment of efficient BCAA oxidative metabolism in some tissues. Whether and how impaired BCAA metabolism might occur in obesity is discussed in this Review. Research on the role of individual and model-dependent differences in BCAA metabolism is needed, as several genes (BCKDHA, PPM1K, IVD and KLF15) have been designated as candidate genes for obesity and/or T2DM in humans, and distinct phenotypes of tissue-specific branched chain ketoacid dehydrogenase complex activity have been detected in animal models of obesity and T2DM.

  19. Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites.

    PubMed

    Perchat, Nadia; Saaidi, Pierre-Loïc; Darii, Ekaterina; Pellé, Christine; Petit, Jean-Louis; Besnard-Gonnet, Marielle; de Berardinis, Véronique; Dupont, Maeva; Gimbernat, Alexandra; Salanoubat, Marcel; Fischer, Cécile; Perret, Alain

    2018-05-08

    Trigonelline (TG; N- methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium Acinetobacter baylyi ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields ( Z )-2-(( N- methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into ( E )-2-(( N- methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available.

  20. Ex vivo 18O-labeling mass spectrometry identifies a peripheral amyloid β clearance pathway.

    PubMed

    Portelius, Erik; Mattsson, Niklas; Pannee, Josef; Zetterberg, Henrik; Gisslén, Magnus; Vanderstichele, Hugo; Gkanatsiou, Eleni; Crespi, Gabriela A N; Parker, Michael W; Miles, Luke A; Gobom, Johan; Blennow, Kaj

    2017-02-20

    Proteolytic degradation of amyloid β (Aβ) peptides has been intensely studied due to the central role of Aβ in Alzheimer's disease (AD) pathogenesis. While several enzymes have been shown to degrade Aβ peptides, the main pathway of Aβ degradation in vivo is unknown. Cerebrospinal fluid (CSF) Aβ42 is reduced in AD, reflecting aggregation and deposition in the brain, but low CSF Aβ42 is, for unknown reasons, also found in some inflammatory brain disorders such as bacterial meningitis. Using 18 O-labeling mass spectrometry and immune-affinity purification, we examined endogenous proteolytic processing of Aβ in human CSF. The Aβ peptide profile was stable in CSF samples from healthy controls but in CSF samples from patients with bacterial meningitis, showing increased leukocyte cell count, 18 O-labeling mass spectrometry identified proteolytic activities degrading Aβ into several short fragments, including abundant Aβ1-19 and 1-20. After antibiotic treatment, no degradation of Aβ was detected. In vitro experiments located the source of the proteolytic activity to blood components, including leukocytes and erythrocytes, with insulin-degrading enzyme as the likely protease. A recombinant version of the mid-domain anti-Aβ antibody solanezumab was found to inhibit insulin-degrading enzyme-mediated Aβ degradation. 18 O labeling-mass spectrometry can be used to detect endogenous proteolytic activity in human CSF. Using this technique, we found an enzymatic activity that was identified as insulin-degrading enzyme that cleaves Aβ in the mid-domain of the peptide, and could be inhibited by a recombinant version of the mid-domain anti-Aβ antibody solanezumab.

  1. Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

    PubMed

    Lakshmikumaran, M S; D'Ambrosio, E; Laimins, L A; Lin, D T; Furano, A V

    1985-09-01

    The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this.

  2. Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus.

    PubMed Central

    Lakshmikumaran, M S; D'Ambrosio, E; Laimins, L A; Lin, D T; Furano, A V

    1985-01-01

    The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this. Images PMID:3016521

  3. Free Fatty Acid-Induced PP2A Hyperactivity Selectively Impairs Hepatic Insulin Action on Glucose Metabolism

    PubMed Central

    Galbo, Thomas; Olsen, Grith Skytte; Quistorff, Bjørn; Nishimura, Erica

    2011-01-01

    In type 2 Diabetes (T2D) free fatty acids (FFAs) in plasma are increased and hepatic insulin resistance is “selective”, in the sense that the insulin-mediated decrease of glucose production is blunted while insulin's effect on stimulating lipogenesis is maintained. We investigated the molecular mechanisms underlying this pathogenic paradox. Primary rat hepatocytes were exposed to palmitate for twenty hours. To establish the physiological relevance of the in vitro findings, we also studied insulin-resistant Zucker Diabetic Fatty (ZDF) rats. While insulin-receptor phosphorylation was unaffected, activation of Akt and inactivation of the downstream targets Glycogen synthase kinase 3α (Gsk3α and Forkhead box O1 (FoxO1) was inhibited in palmitate-exposed cells. Accordingly, dose-response curves for insulin-mediated suppression of the FoxO1-induced gluconeogenic genes and for de novo glucose production were right shifted, and insulin-stimulated glucose oxidation and glycogen synthesis were impaired. In contrast, similar to findings in human T2D, the ability of insulin to induce triglyceride (TG) accumulation and transcription of the enzymes that catalyze de novo lipogenesis and TG assembly was unaffected. Insulin-induction of these genes could, however, be blocked by inhibition of the atypical PKCs (aPKCs). The activity of the Akt-inactivating Protein Phosphatase 2A (PP2A) was increased in the insulin-resistant cells. Furthermore, inhibition of PP2A by specific inhibitors increased insulin-stimulated activation of Akt and phosphorylation of FoxO1 and Gsk3α. Finally, PP2A mRNA levels were increased in liver, muscle and adipose tissue, while PP2A activity was increased in liver and muscle tissue in insulin-resistant ZDF rats. In conclusion, our findings indicate that FFAs may cause a selective impairment of insulin action upon hepatic glucose metabolism by increasing PP2A activity. PMID:22087313

  4. NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL MAPPING OF THE GENES ENCODING BIPHENYL DIOXYGENASE, A MULTICOM- PONENT POLYCHLORINATED-BIPHENYL-DEGRADING ENZYME IN PSEUDOMONAS STRAIN LB400

    EPA Science Inventory

    The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxy...

  5. Targeting of Several Glycolytic Enzymes Using RNA Interference Reveals Aldolase Affects Cancer Cell Proliferation through a Non-glycolytic Mechanism

    PubMed Central

    Lew, Carolyn Ritterson; Tolan, Dean R.

    2012-01-01

    In cancer, glucose uptake and glycolysis are increased regardless of the oxygen concentration in the cell, a phenomenon known as the Warburg effect. Several (but not all) glycolytic enzymes have been investigated as potential therapeutic targets for cancer treatment using RNAi. Here, four previously untargeted glycolytic enzymes, aldolase A, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase, and enolase 1, are targeted using RNAi in Ras-transformed NIH-3T3 cells. Of these enzymes, knockdown of aldolase causes the greatest effect, inhibiting cell proliferation by 90%. This defect is rescued by expression of exogenous aldolase. However, aldolase knockdown does not affect glycolytic flux or intracellular ATP concentration, indicating a non-metabolic cause for the cell proliferation defect. Furthermore, this defect could be rescued with an enzymatically dead aldolase variant that retains the known F-actin binding ability of aldolase. One possible model for how aldolase knockdown may inhibit transformed cell proliferation is through its disruption of actin-cytoskeleton dynamics in cell division. Consistent with this hypothesis, aldolase knockdown cells show increased multinucleation. These results are compared with other studies targeting glycolytic enzymes with RNAi in the context of cancer cell proliferation and suggest that aldolase may be a useful target in the treatment of cancer. PMID:23093405

  6. Degradative enzymes modulate airway responses to intravenous neurokinins A and B.

    PubMed

    Shore, S A; Drazen, J M

    1989-12-01

    We studied the effects of the neutral endopeptidase (NEP) inhibitor thiorphan (1.7 mg/kg iv) and the angiotensin-converting enzyme (ACE) inhibitor captopril (5.7 mg/kg iv) on airway responses to rapid intravenous infusions of neurokinin A (NKA) and neurokinin B (NKB) in anesthetized, mechanically ventilated guinea pigs. The dose of NKA required to decrease pulmonary conductance to 50% of its base-line value (ED50GL) was fivefold less (P less than 0.0001) in animals treated with thiorphan compared with controls. NKA1-8, a product resulting from cleavage of NKA by NEP, had no bronchoconstrictor activity. Similar results were obtained by using NKB as the bronchoconstricting agent. Captopril had no significant effect on airway responses to NKA or NKB. In contrast, both thiorphan and captopril decrease the ED50GL for substance P (SP). We also compared the relative bronchoconstrictor potency of NKA, NKB, and SP. In control animals, the rank order of ED50GL values was NKA much less than NKB = SP. NKA also caused a more prolonged bronchoconstriction than SP or NKB. Thiorphan had no effect on the rank order of bronchoconstrictor potency, but in animals treated with captopril, the rank order of ED50GL values was altered to NKA less than SP less than NKB. These results suggest that degradation of NKA and NKB by NEP but not by ACE is an important determinant of the bronchoconstriction induced by these peptides. The degradation by ACE of SP but not NKA or NKB influences the observed relative potency of the three tachykinins as bronchoactive agents.

  7. Insulin and Metformin Regulate Circulating and Adipose Tissue Chemerin

    PubMed Central

    Tan, Bee K.; Chen, Jing; Farhatullah, Syed; Adya, Raghu; Kaur, Jaspreet; Heutling, Dennis; Lewandowski, Krzysztof C.; O'Hare, J. Paul; Lehnert, Hendrik; Randeva, Harpal S.

    2009-01-01

    OBJECTIVE To assess chemerin levels and regulation in sera and adipose tissue from women with polycystic ovary syndrome (PCOS) and matched control subjects. RESEARCH DESIGN AND METHODS Real-time RT-PCR and Western blotting were used to assess mRNA and protein expression of chemerin. Serum chemerin was measured by enzyme-linked immunosorbent assay. We investigated the in vivo effects of insulin on serum chemerin levels via a prolonged insulin-glucose infusion. Ex vivo effects of insulin, metformin, and steroid hormones on adipose tissue chemerin protein production and secretion into conditioned media were assessed by Western blotting and enzyme-linked immunosorbent assay, respectively. RESULTS Serum chemerin, subcutaneous, and omental adipose tissue chemerin were significantly higher in women with PCOS (n = 14; P < 0.05, P < 0.01). Hyperinsulinemic induction in human subjects significantly increased serum chemerin levels (n = 6; P < 0.05, P < 0.01). In adipose tissue explants, insulin significantly increased (n = 6; P < 0.05, P < 0.01) whereas metformin significantly decreased (n = 6; P < 0.05, P < 0.01) chemerin protein production and secretion into conditioned media, respectively. After 6 months of metformin treatment, there was a significant decrease in serum chemerin (n = 21; P < 0.01). Importantly, changes in homeostasis model assessment–insulin resistance were predictive of changes in serum chemerin (P = 0.046). CONCLUSIONS Serum and adipose tissue chemerin levels are increased in women with PCOS and are upregulated by insulin. Metformin treatment decreases serum chemerin in these women. PMID:19502420

  8. Chemical stability of insulin. 5. Isolation, characterization and identification of insulin transformation products.

    PubMed

    Brange, J; Hallund, O; Sørensen, E

    1992-01-01

    During storage of insulin formulated for therapy, minor amounts of various degradation and covalent di- and polymerization products are formed [1-3]. The main chemical transformation products were isolated from aged preparations and characterized chemically and biologically. The most prominent products formed in neutral medium were identified as a mixture of deamidation products hydrolyzed at residue B3, namely isoAsp B3 and Asp B3 derivatives. A hydrolysis product formed only in crystals of insulin zinc suspensions containing a surplus of zinc ions in the supernatant was identified as an A8-A9 cleavage product. The small amounts of covalent insulin dimers (CID) formed in all formulations were shown to be a heterogenous mixture of 5-6 different CIDs with a composition dependent on the pharmaceutical formulation. The chemical characteristics of the CIDs indicate that they are formed through a transamidation reaction mainly between the B-chain N-terminal and one of the four amide side-chains of the A chain. GlnA15, AsnA18 and, in particular, AsnA21 participate in the formation of such isopeptide links between two insulin molecules. The covalent insulin-protamine products (CIPP) formed during storage of NPH preparations presumably originate from a similar reaction between the protamine N-terminal with an amide in insulin. Covalent polymerization products, mainly formed during storage of amorphously suspended insulin at higher temperature, were shown to be due to disulfide interactions. Biological in vivo potencies relative to native insulin were less than 2% for the split-(A8-A9)-product and for the covalent disulfide exchange polymers, 4% for the CIPP, approximately 15% for the CIDs, whereas the B3 derivatives exhibited full potency. Rabbit immunization experiments revealed that none of the insulin transformation products had significantly increased immunogenicity in rabbits.

  9. Microbial degradation of polyurethane, polyester polyurethanes and polyether polyurethanes.

    PubMed

    Nakajima-Kambe, T; Shigeno-Akutsu, Y; Nomura, N; Onuma, F; Nakahara, T

    1999-02-01

    Polyurethane (PUR) is a polymer derived from the condensation of polyisocyanate and polyol and it is widely used as a base material in various industries. PUR, in particular, polyester PUR, is known to be vulnerable to microbial attack. Recently, environmental pollution by plastic wastes has become a serious issue and polyester PUR had attracted attention because of its biodegradability. There are many reports on the degradation of polyester PUR by microorganisms, especially by fungi. Microbial degradation of polyester PUR is thought to be mainly due to the hydrolysis of ester bonds by esterases. Recently, polyester-PUR-degrading enzymes have been purified and their characteristics reported. Among them, a solid-polyester-PUR-degrading enzyme (PUR esterase) derived from Comamonas acidovorans TB-35 had unique characteristics. This enzyme has a hydrophobic PUR-surface-binding domain and a catalytic domain, and the surface-binding domain was considered as being essential for PUR degradation. This hydrophobic surface-binding domain is also observed in other solid-polyester-degrading enzymes such as poly(hydroxyalkanoate) (PHA) depolymerases. There was no significant homology between the amino acid sequence of PUR esterase and that of PHA depolymerases, except in the hydrophobic surface-binding region. Thus, PUR esterase and PHA depolymerase are probably different in terms of their evolutionary origin and it is possible that PUR esterases come to be classified as a new solid-polyester-degrading enzyme family.

  10. Degradation of PHLPP2 by KCTD17, via a Glucagon-Dependent Pathway, Promotes Hepatic Steatosis.

    PubMed

    Kim, KyeongJin; Ryu, Dongryeol; Dongiovanni, Paola; Ozcan, Lale; Nayak, Shruti; Ueberheide, Beatrix; Valenti, Luca; Auwerx, Johan; Pajvani, Utpal B

    2017-12-01

    Obesity-induced nonalcoholic fatty liver disease (NAFLD) develops, in part, via excess insulin-stimulated hepatic de novo lipogenesis, which increases, paradoxically, in patients with obesity-induced insulin resistance. Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) terminates insulin signaling by dephosphorylating Akt; levels of PHLPP2 are reduced in livers from obese mice. We investigated whether loss of hepatic PHLPP2 is sufficient to induce fatty liver in mice, mechanisms of PHLPP2 degradation in fatty liver, and expression of genes that regulate PHLPP2 in livers of patients with NAFLD. C57BL/6J mice (controls), obese db/db mice, and mice with liver-specific deletion of PHLPP2 (L-PHLPP2) fed either normal chow or high-fat diet (HFD) were analyzed for metabolic phenotypes, including glucose tolerance and hepatic steatosis. PHLPP2-deficient primary hepatocytes or CRISPR/Cas9-mediated PHLPP2-knockout hepatoma cells were analyzed for insulin signaling and gene expression. We performed mass spectrometry analyses of liver tissues from C57BL/6J mice transduced with Ad-HA-Flag-PHLPP2 to identify posttranslational modifications to PHLPP2 and proteins that interact with PHLPP2. We measured levels of mRNAs by quantitative reverse transcription polymerase chain reaction in liver biopsies from patients with varying degrees of hepatic steatosis. PHLPP2-knockout hepatoma cells and hepatocytes from L-PHLPP2 mice showed normal initiation of insulin signaling, but prolonged insulin action. Chow-fed L-PHLPP2 mice had normal glucose tolerance but hepatic steatosis. In HFD-fed C57BL/6J or db/db obese mice, endogenous PHLPP2 was degraded by glucagon and PKA-dependent phosphorylation of PHLPP2 (at Ser1119 and Ser1210), which led to PHLPP2 binding to potassium channel tetramerization domain containing 17 (KCTD17), a substrate-adaptor for Cul3-RING ubiquitin ligases. Levels of KCTD17 mRNA were increased in livers of HFD-fed C57BL/6J or db/db obese mice

  11. Oligogalacturonide-mediated induction of a gene involved in jasmonic acid synthesis in response to the cell-wall-degrading enzymes of the plant pathogen Erwinia carotovora.

    PubMed

    Norman, C; Vidal, S; Palva, E T

    1999-07-01

    Identification of Arabidopsis thaliana genes responsive to plant cell-wall-degrading enzymes of Erwinia carotovora subsp. carotovora led to the isolation of a cDNA clone with high sequence homology to the gene for allene oxide synthase, an enzyme involved in the biosynthesis of jasmonates. Expression of the corresponding gene was induced by the extracellular enzymes from this pathogen as well as by treatment with methyl jasmonate and short oligogalacturonides (OGAs). This suggests that OGAs are involved in the induction of the jasmonate pathway during plant defense response to E. carotovora subsp. carotovora attack.

  12. Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

    PubMed

    Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

    2015-01-01

    Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

  13. Influence of exogenous lead pollution on enzyme activities and organic matter degradation in the surface of river sediment.

    PubMed

    Huang, Danlian; Xu, Juanjuan; Zeng, Guangming; Lai, Cui; Yuan, Xingzhong; Luo, Xiangying; Wang, Cong; Xu, Piao; Huang, Chao

    2015-08-01

    As lead is one of the most hazardous heavy metals in river ecosystem, the influence of exogenous lead pollution on enzyme activities and organic matter degradation in the surface of river sediment with high moisture content were studied at laboratory scale. The dynamic changes of urease, catalase, protease activities, organic matter content, and exchangeable or ethylenediaminetetraacetic acid (EDTA)-extractable Pb concentration in sediment were monitored during different levels of exogenous lead infiltrating into sediment. At the early stage of incubation, the activities of catalase and protease were inhibited, whereas the urease activities were enhanced with different levels of exogenous lead. Organic matter content in polluted sediment with exogenous lead was lower than control and correlated with enzyme activities. In addition, the effects of lead on the three enzyme activities were strongly time-dependent and catalase activities showed lower significant difference (P < 0.05) than urease and protease. Correlations between catalase activities and EDTA-extractable Pb in the experiment were significantly negative. The present findings will improve the understandings about the ecotoxicological mechanisms in sediment.

  14. Lignocellulose degradation and enzyme production by Irpex lacteus CD2 during solid-state fermentation of corn stover.

    PubMed

    Xu, Chunyan; Ma, Fuying; Zhang, Xiaoyu

    2009-11-01

    The white rot fungus Irpex lacteus CD2 was incubated on corn stover under solid-state fermentation conditions for different durations, from 5 days up to 120 days. Lignocellulose component loss, enzyme production and Fe3+-reducing activity were studied. The average weight loss ranged from 1.7% to 60.5% during the period of 5-120 days. In contrast to lignin, hemicellulose and cellulose were degraded during the initial time period. After 15 days, 63.0% of hemicellulose was degraded. Cellulose was degraded the most during the first 10 days, and 17.2% was degraded after 10 days. Lignin was significantly degraded and modified, with acid insoluble lignin loss being nearly 80% after 60 days. That weight loss, which was lower than the total component loss, indicated that not all of the lost lignocellulose was converted to carbon dioxide and water, which was indicated by the increase in soluble reducing sugars and acid soluble lignin. Filter paper activity, which corresponds to total cellulase activity, peaked at day 5 and remained at a high level from 40 to 60 days. High hemicellulase activity appeared after 30 days. No ligninases activity was detected during the incipient stage of lignin removal and only low lignin peroxidase activity was detected after 25 days. Apparently, neither of the enzymatic peaks coincided well with the highest amount of component loss. Fe3+-reducing activity could be detected during all the decay periods, which might play an important role in lignin biodegradation by I. lacteus CD2.

  15. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

    PubMed

    Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

    2016-06-07

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.

  16. CNC-bZIP protein Nrf1-dependent regulation of glucose-stimulated insulin secretion.

    PubMed

    Zheng, Hongzhi; Fu, Jingqi; Xue, Peng; Zhao, Rui; Dong, Jian; Liu, Dianxin; Yamamoto, Masayuki; Tong, Qingchun; Teng, Weiping; Qu, Weidong; Zhang, Qiang; Andersen, Melvin E; Pi, Jingbo

    2015-04-01

    The inability of pancreatic β-cells to secrete sufficient insulin in response to glucose stimulation is a major contributing factor to the development of type 2 diabetes (T2D). We investigated both the in vitro and in vivo effects of deficiency of nuclear factor-erythroid 2-related factor 1 (Nrf1) in β-cells on β-cell function and glucose homeostasis. Silencing of Nrf1 in β-cells leads to a pre-T2D phenotype with disrupted glucose metabolism and impaired insulin secretion. Specifically, MIN6 β-cells with stable knockdown of Nrf1 (Nrf1-KD) and isolated islets from β-cell-specific Nrf1-knockout [Nrf1(b)-KO] mice displayed impaired glucose responsiveness, including elevated basal insulin release and decreased glucose-stimulated insulin secretion (GSIS). Nrf1(b)-KO mice exhibited severe fasting hyperinsulinemia, reduced GSIS, and glucose intolerance. Silencing of Nrf1 in MIN6 cells resulted in oxidative stress and altered glucose metabolism, with increases in both glucose uptake and aerobic glycolysis, which is associated with the elevated basal insulin release and reduced glucose responsiveness. The elevated glycolysis and reduced glucose responsiveness due to Nrf1 silencing likely result from altered expression of glucose metabolic enzymes, with induction of high-affinity hexokinase 1 and suppression of low-affinity glucokinase. Our study demonstrated a novel role of Nrf1 in regulating glucose metabolism and insulin secretion in β-cells and characterized Nrf1 as a key transcription factor that regulates the coupling of glycolysis and mitochondrial metabolism and GSIS. Nrf1 plays critical roles in regulating glucose metabolism, mitochondrial function, and insulin secretion, suggesting that Nrf1 may be a novel target to improve the function of insulin-secreting β-cells.

  17. Dual-mode lensless imaging device for digital enzyme linked immunosorbent assay

    NASA Astrophysics Data System (ADS)

    Sasagawa, Kiyotaka; Kim, Soo Heyon; Miyazawa, Kazuya; Takehara, Hironari; Noda, Toshihiko; Tokuda, Takashi; Iino, Ryota; Noji, Hiroyuki; Ohta, Jun

    2014-03-01

    Digital enzyme linked immunosorbent assay (ELISA) is an ultra-sensitive technology for detecting biomarkers and viruses etc. As a conventional ELISA technique, a target molecule is bonded to an antibody with an enzyme by antigen-antibody reaction. In this technology, a femto-liter droplet chamber array is used as reaction chambers. Due to its small volume, the concentration of fluorescent product by single enzyme can be sufficient for detection by a fluorescent microscopy. In this work, we demonstrate a miniaturized lensless imaging device for digital ELISA by using a custom image sensor. The pixel array of the sensor is coated with a 20 μm-thick yellow filter to eliminate excitation light at 470 nm and covered by a fiber optic plate (FOP) to protect the sensor without resolution degradation. The droplet chamber array formed on a 50μm-thick glass plate is directly placed on the FOP. In the digital ELISA, microbeads coated with antibody are loaded into the droplet chamber array, and the ratio of the fluorescent to the non-fluorescent chambers with the microbeads are observed. In the fluorescence imaging, the spatial resolution is degraded by the spreading through the glass plate because the fluorescence is irradiated omnidirectionally. This degradation is compensated by image processing and the resolution of ~35 μm was achieved. In the bright field imaging, the projected images of the beads with collimated illumination are observed. By varying the incident angle and image composition, microbeads were successfully imaged.

  18. A Computational Methodology to Overcome the Challenges Associated With the Search for Specific Enzyme Targets to Develop Drugs Against Leishmania major

    PubMed Central

    Catharina, Larissa; Lima, Carlyle Ribeiro; Franca, Alexander; Guimarães, Ana Carolina Ramos; Alves-Ferreira, Marcelo; Tuffery, Pierre; Derreumaux, Philippe; Carels, Nicolas

    2017-01-01

    We present an approach for detecting enzymes that are specific of Leishmania major compared with Homo sapiens and provide targets that may assist research in drug development. This approach is based on traditional techniques of sequence homology comparison by similarity search and Markov modeling; it integrates the characterization of enzymatic functionality, secondary and tertiary protein structures, protein domain architecture, and metabolic environment. From 67 enzymes represented by 42 enzymatic activities classified by AnEnPi (Analogous Enzymes Pipeline) as specific for L major compared with H sapiens, only 40 (23 Enzyme Commission [EC] numbers) could actually be considered as strictly specific of L major and 27 enzymes (19 EC numbers) were disregarded for having ambiguous homologies or analogies with H sapiens. Among the 40 strictly specific enzymes, we identified sterol 24-C-methyltransferase, pyruvate phosphate dikinase, trypanothione synthetase, and RNA-editing ligase as 4 essential enzymes for L major that may serve as targets for drug development. PMID:28638238

  19. A Computational Methodology to Overcome the Challenges Associated With the Search for Specific Enzyme Targets to Develop Drugs Against Leishmania major.

    PubMed

    Catharina, Larissa; Lima, Carlyle Ribeiro; Franca, Alexander; Guimarães, Ana Carolina Ramos; Alves-Ferreira, Marcelo; Tuffery, Pierre; Derreumaux, Philippe; Carels, Nicolas

    2017-01-01

    We present an approach for detecting enzymes that are specific of Leishmania major compared with Homo sapiens and provide targets that may assist research in drug development. This approach is based on traditional techniques of sequence homology comparison by similarity search and Markov modeling; it integrates the characterization of enzymatic functionality, secondary and tertiary protein structures, protein domain architecture, and metabolic environment. From 67 enzymes represented by 42 enzymatic activities classified by AnEnPi (Analogous Enzymes Pipeline) as specific for L major compared with H sapiens , only 40 (23 Enzyme Commission [EC] numbers) could actually be considered as strictly specific of L major and 27 enzymes (19 EC numbers) were disregarded for having ambiguous homologies or analogies with H sapiens . Among the 40 strictly specific enzymes, we identified sterol 24-C-methyltransferase, pyruvate phosphate dikinase, trypanothione synthetase, and RNA-editing ligase as 4 essential enzymes for L major that may serve as targets for drug development.

  20. A New Mechanism for β‐Lactamases: Class D Enzymes Degrade 1β‐Methyl Carbapenems through Lactone Formation

    PubMed Central

    Lohans, Christopher T.; van Groesen, Emma; Kumar, Kiran; Tooke, Catherine L.; Spencer, James; Paton, Robert S.; Brem, Jürgen

    2018-01-01

    Abstract β‐Lactamases threaten the clinical use of carbapenems, which are considered antibiotics of last resort. The classical mechanism of serine carbapenemase catalysis proceeds through hydrolysis of an acyl‐enzyme intermediate. We show that class D β‐lactamases also degrade clinically used 1β‐methyl‐substituted carbapenems through the unprecedented formation of a carbapenem‐derived β‐lactone. β‐Lactone formation results from nucleophilic attack of the carbapenem hydroxyethyl side chain on the ester carbonyl of the acyl‐enzyme intermediate. The carbapenem‐derived lactone products inhibit both serine β‐lactamases (particularly class D) and metallo‐β‐lactamases. These results define a new mechanism for the class D carbapenemases, in which a hydrolytic water molecule is not required. PMID:29236332