Suppressive effects of RXR agonist PA024 on adrenal CYP11B2 expression, aldosterone secretion and blood pressure.

PubMed

Suzuki, Dai; Saito-Hakoda, Akiko; Ito, Ryo; Shimizu, Kyoko; Parvin, Rehana; Shimada, Hiroki; Noro, Erika; Suzuki, Susumu; Fujiwara, Ikuma; Kagechika, Hiroyuki; Rainey, William E; Kure, Shigeo; Ito, Sadayoshi; Yokoyama, Atsushi; Sugawara, Akira

2017-01-01

The effects of retinoids on adrenal aldosterone synthase gene (CYP11B2) expression and aldosterone secretion are still unknown. We therefore examined the effects of nuclear retinoid X receptor (RXR) pan-agonist PA024 on CYP11B2 expression, aldosterone secretion and blood pressure, to elucidate its potential as a novel anti-hypertensive drug. We demonstrated that PA024 significantly suppressed angiotensin II (Ang II)-induced CYP11B2 mRNA expression, promoter activity and aldosterone secretion in human adrenocortical H295R cells. Human CYP11B2 promoter functional analyses using its deletion and point mutants indicated that the suppression of CYP11B2 promoter activity by PA024 was in the region from -1521 (full length) to -106 including the NBRE-1 and the Ad5 elements, and the Ad5 element may be mainly involved in the PA024-mediated suppression. PA024 also significantly suppressed the Ang II-induced mRNA expression of transcription factors NURR1 and NGFIB that bind to and activate the Ad5 element. NURR1 overexpression demonstrated that the decrease of NURR1 expression may contribute to the PA024-mediated suppression of CYP11B2 transcription. PA024 also suppressed the Ang II-induced mRNA expression of StAR, HSD3β2 and CYP21A2, a steroidogenic enzyme group involved in aldosterone biosynthesis. Additionally, the PA024-mediated CYP11B2 transcription suppression was shown to be exerted via RXRα. Moreover, the combination of PPARγ agonist pioglitazone and PA024 caused synergistic suppressive effects on CYP11B2 mRNA expression. Finally, PA024 treatment significantly lowered both the systolic and diastolic blood pressure in Tsukuba hypertensive mice (hRN8-12 x hAG2-5). Thus, RXR pan-agonist PA024 may be a candidate anti-hypertensive drugs that acts via the suppression of aldosterone synthesis and secretion.

Agreement Between the United States Secret Service and the Department of Defense Concerning Protection of the President and Other Officials

DTIC Science & Technology

1986-09-17

States Secret Service, in accordance with the responsibilities of the Secret Service as authorized by Section 3056, Title 18, U.S. Code; and Section 202 of...Title 3, U.S. Code. II. General Responsibilities Subject to the direction of the Secretary of the Treasury, the Secret Service is authorized to... the Secret Service, other than those persons or positions specifically identified under "II General Responsibilities" in this agreement, will be

Boron bridging of rhamnogalacturonan-II, monitored by gel electrophoresis, occurs during polysaccharide synthesis and secretion but not post-secretion

PubMed Central

Chormova, Dimitra; Messenger, David J; Fry, Stephen C

2014-01-01

The cell-wall pectic domain rhamnogalacturonan-II (RG-II) is cross-linked via borate diester bridges, which influence the expansion, thickness and porosity of the wall. Previously, little was known about the mechanism or subcellular site of this cross-linking. Using polyacrylamide gel electrophoresis (PAGE) to separate monomeric from dimeric (boron-bridged) RG-II, we confirmed that Pb2+ promotes H3BO3-dependent dimerisation in vitro. H3BO3 concentrations as high as 50 mm did not prevent cross-linking. For in-vivo experiments, we successfully cultured ‘Paul's Scarlet’ rose (Rosa sp.) cells in boron-free medium: their wall-bound pectin contained monomeric RG-II domains but no detectable dimers. Thus pectins containing RG-II domains can be held in the wall other than via boron bridges. Re-addition of H3BO3 to 3.3 μm triggered a gradual appearance of RG-II dimer over 24 h but without detectable loss of existing monomers, suggesting that only newly synthesised RG-II was amenable to boron bridging. In agreement with this, Rosa cultures whose polysaccharide biosynthetic machinery had been compromised (by carbon starvation, respiratory inhibitors, anaerobiosis, freezing or boiling) lost the ability to generate RG-II dimers. We conclude that RG-II normally becomes boron-bridged during synthesis or secretion but not post-secretion. Supporting this conclusion, exogenous [3H]RG-II was neither dimerised in the medium nor cross-linked to existing wall-associated RG-II domains when added to Rosa cultures. In conclusion, in cultured Rosa cells RG-II domains have a brief window of opportunity for boron-bridging intraprotoplasmically or during secretion, but secretion into the apoplast is a point of no return beyond which additional boron-bridging does not readily occur. PMID:24320597

Reduced IL-10 Production in Fetal Type II Epithelial Cells Exposed to Mechanical Stretch Is Mediated via Activation of IL-6-SOCS3 Signaling Pathway

PubMed Central

Hawwa, Renda L.; Huang, Zheping; Sharma, Surendra; Sanchez-Esteban, Juan

2013-01-01

An imbalance between pro-inflammatory and anti-inflammatory cytokines is a key factor in the lung injury of premature infants exposed to mechanical ventilation. Previous studies have shown that lung cells exposed to stretch produces reduced amounts of the anti-inflammatory cytokine IL-10. The objective of these studies was to analyze the signaling mechanisms responsible for the decreased IL-10 production in fetal type II cells exposed to mechanical stretch. Fetal mouse type II epithelial cells isolated at embryonic day 18 were exposed to 20% stretch to simulate lung injury. We show that IL-10 receptor gene expression increased with gestational age. Mechanical stretch decreased not only IL-10 receptor gene expression but also IL-10 secretion. In contrast, mechanical stretch increased release of IL-6. We then investigated IL-10 signaling pathway-associated proteins and found that in wild-type cells, mechanical stretch decreased activation of JAK1 and TYK2 and increased STAT3 and SOCS3 activation. However, opposite effects were found in cells isolated from IL-10 knockout mice. Reduction in IL-6 secretion by stretch was observed in cells isolated from IL-10 null mice. To support the idea that stretch-induced SOCS3 expression via IL-6 leads to reduced IL-10 expression, siRNA-mediated inhibition of SOCS3 restored IL-10 secretion in cells exposed to stretch and decreased IL-6 secretion. Taken together, these studies suggest that the inhibitory effect of mechanical stretch on IL-10 secretion is mediated via activation of IL-6-STAT3-SOCS3 signaling pathway. SOCS3 could be a therapeutic target to increase IL-10 production in lung cells exposed to mechanical injury. PMID:23527226

Reduced IL-10 production in fetal type II epithelial cells exposed to mechanical stretch is mediated via activation of IL-6-SOCS3 signaling pathway.

PubMed

Hokenson, Michael A; Wang, Yulian; Hawwa, Renda L; Huang, Zheping; Sharma, Surendra; Sanchez-Esteban, Juan

2013-01-01

An imbalance between pro-inflammatory and anti-inflammatory cytokines is a key factor in the lung injury of premature infants exposed to mechanical ventilation. Previous studies have shown that lung cells exposed to stretch produces reduced amounts of the anti-inflammatory cytokine IL-10. The objective of these studies was to analyze the signaling mechanisms responsible for the decreased IL-10 production in fetal type II cells exposed to mechanical stretch. Fetal mouse type II epithelial cells isolated at embryonic day 18 were exposed to 20% stretch to simulate lung injury. We show that IL-10 receptor gene expression increased with gestational age. Mechanical stretch decreased not only IL-10 receptor gene expression but also IL-10 secretion. In contrast, mechanical stretch increased release of IL-6. We then investigated IL-10 signaling pathway-associated proteins and found that in wild-type cells, mechanical stretch decreased activation of JAK1 and TYK2 and increased STAT3 and SOCS3 activation. However, opposite effects were found in cells isolated from IL-10 knockout mice. Reduction in IL-6 secretion by stretch was observed in cells isolated from IL-10 null mice. To support the idea that stretch-induced SOCS3 expression via IL-6 leads to reduced IL-10 expression, siRNA-mediated inhibition of SOCS3 restored IL-10 secretion in cells exposed to stretch and decreased IL-6 secretion. Taken together, these studies suggest that the inhibitory effect of mechanical stretch on IL-10 secretion is mediated via activation of IL-6-STAT3-SOCS3 signaling pathway. SOCS3 could be a therapeutic target to increase IL-10 production in lung cells exposed to mechanical injury.

Class I and class II major histocompatibility molecules play a role in bone marrow-derived macrophage development

NASA Technical Reports Server (NTRS)

Armstrong, J. W.; Simske, S. J.; Beharka, A. A.; Balch, S.; Luttges, M. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

Class I and class II major histocompatibility complex (MHC) molecules play significant roles in T cell development and immune function. We show that MHCI- and MHCII-deficient mice have low numbers of macrophage precursors and circulating monocytes, as well as abnormal bone marrow cell colony-stimulating factor type 1 secretion and bone composition. We suggest that MHCI and MHCII molecules play a significant role in macrophage development.

A Two-Year Randomized Trial of Interventions to Decrease Stress Hormone Vasopressin Production in Patients with Meniere's Disease-A Pilot Study.

PubMed

Kitahara, Tadashi; Okamoto, Hidehiko; Fukushima, Munehisa; Sakagami, Masaharu; Ito, Taeko; Yamashita, Akinori; Ota, Ichiro; Yamanaka, Toshiaki

2016-01-01

Meniere's disease, a common inner ear condition, has an incidence of 15-50 per 100,000. Because mental/physical stress and subsequent increase in the stress hormone vasopressin supposedly trigger Meniere's disease, we set a pilot study to seek new therapeutic interventions, namely management of vasopressin secretion, to treat this disease. We enrolled 297 definite Meniere's patients from 2010 to 2012 in a randomized-controlled and open-label trial, assigning Group-I (control) traditional oral medication, Group-II abundant water intake, Group-III tympanic ventilation tubes and Group-IV sleeping in darkness. Two hundred sixty-three patients completed the planned 2-year-follow-up, which included assessment of vertigo, hearing, plasma vasopressin concentrations and changes in stress/psychological factors. At 2 years, vertigo was completely controlled in 54.3% of patients in Group-I, 81.4% in Group-II, 84.1% in Group-III, and 80.0% in Group-IV (statistically I < II = III = IV). Hearing was improved in 7.1% of patients in Group-I, 35.7% in Group-II, 34.9% in Group-III, and 31.7% in Group-IV (statistically I < II = III = IV). Plasma vasopressin concentrations decreased more in Groups-II, -III, and -IV than in Groups-I (statistically I < II = III = IV), although patients' stress/psychological factors had not changed. Physicians have focused on stress management for Meniere's disease. However, avoidance of stress is unrealistic for patients who live in demanding social environments. Our findings in this pilot study suggest that interventions to decrease vasopressin secretion by abundant water intake, tympanic ventilation tubes and sleeping in darkness is feasible in treating Meniere's disease, even though these therapies did not alter reported mental/physical stress levels. ClinicalTrials.gov NCT01099046.

Primary hypogonadism in gonadotropin-releasing hormone II receptor knockdown boars

USDA-ARS?s Scientific Manuscript database

Paradoxically, the second mammalian GnRH isoform (GnRH-II) and its receptor (GnRHR-II) are not physiological regulators of gonadotropin secretion. Instead, our data suggests that both are abundantly produced in the porcine testis and mediate testosterone secretion, independent of luteinizing hormone...

Inadequate vitamin D status: does it contribute to the disorders comprising syndrome 'X'?

PubMed

Boucher, B J

1998-04-01

Environmental factors are important in the aetiology of glucose intolerance, type II diabetes and IHD. The lack of vitamin D, which is necessary for adequate insulin secretion, relates demographically to increased risk of myocardial infarction. These disorders are connected, degenerative vascular disease increasing with glucose intolerance and diabetes and, with its risk factors, comprising syndrome 'X'. Evidence is presented suggesting that vitamin D deficiency may be an avoidable risk factor for syndrome 'X', adding another preventative measure to current recommendations which are aimed at reducing the worldwide epidemic of these disorders. Experimentally, vitamin D deficiency progressively reduces insulin secretion; glucose intolerance follows and becomes irreversible. Relationships between vitamin D status, glucose tolerance and 30 min insulin secretion during oral glucose tolerance tests are reported in British Asians; insulin secretion, but not glycaemia, improving with short-term supplementation. Studies showing reduction in blood pressure and in risk of heart attack and diabetes with exercise (usually outdoor), rarely consider the role of vitamin D status. Glycaemia and insulin secretion in elderly European men, however, relate to vitamin D status, independent of season or physical activity. Prolonged supplementation can improve glycaemia. Hypertension improves with vitamin D treatment with or without initial deficiency. Vitamin D status and climate are reviewed as risk factors for myocardial infarction; the risk reducing with altitude despite increasing cold. Glycaemia and fibrinogenaemia improve with insulin secretion increases in summer. Variation in vitamin D requirements could arise from genetic differences in vitamin D processing since bone density can vary with vitamin D-receptor genotype. Vitamin D receptors are present in islet beta cells and we report insulin secretion in healthy Asians differing profoundly with the Apa I genotype, being independent of vitamin D status. Those at risk of vitamin D deficiency include the elderly, those living indoors or having a covered-up style of dress, especially dark-skinned immigrants, and pregnant women, and these are groups recognized as being at increased risk of diabetes.

Effect of gonadotropin-releasing hormone II receptor (GnRHR-II) knockdown on testosterone secretion in the boar

USDA-ARS?s Scientific Manuscript database

Unlike the classical gonadotropin-releasing hormone (GnRH-I), the second mammalian GnRH isoform (GnRH-II; His5, Trp7, Tyr8) is a poor stimulator of gonadotropin secretion. In addition, GnRH-II is ubiquitously expressed, with transcript levels highest in tissues outside of the brain. A receptor speci...

Activation of the sigma-1 receptor by haloperidol metabolites facilitates brain-derived neurotrophic factor secretion from human astroglia.

PubMed

Dalwadi, Dhwanil A; Kim, Seongcheol; Schetz, John A

2017-05-01

Glial cells play a critical role in neuronal support which includes the production and release of the neurotrophin brain-derived neurotrophic factor (BDNF). Activation of the sigma-1 receptor (S1R) has been shown to attenuate inflammatory stress-mediated brain injuries, and there is emerging evidence that this may involve a BDNF-dependent mechanism. In this report we studied S1R-mediated BDNF release from human astrocytic glial cells. Astrocytes express the S1R, which mediates BDNF release when stimulated with the prototypical S1R agonists 4-PPBP and (+)-SKF10047. This effect could be antagonized by a selective concentration of the S1R antagonist BD1063. Haloperidol is known to have high affinity interactions with the S1R, yet it was unable to facilitate BDNF release. Remarkably, however, two metabolites of haloperidol, haloperidol I and haloperidol II (reduced haloperidol), were discovered to facilitate BDNF secretion and this effect was antagonized by BD1063. Neither 4-PPBP, nor either of the haloperidol metabolites affected the level of BDNF mRNA as assessed by qPCR. These results demonstrate for the first time that haloperidol metabolites I and II facilitate the secretion of BDNF from astrocytes by acting as functionally selective S1R agonists. Copyright © 2017 Elsevier Ltd. All rights reserved.

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

PubMed Central

2010-01-01

Background Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein. PMID:20979600

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

PubMed

Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

2010-10-27

Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

Urea synthesis in patients with chronic pancreatitis: relation to glucagon secretion and dietary protein intake.

PubMed

Hamberg, O; Andersen, V; Sonne, J; Larsen, S; Vilstrup, H

2001-12-01

Up-regulation of urea synthesis by amino acids and dietary protein intake may be impaired in patients with chronic pancreatitis (CP) due to the reduced glucagon secretion. Conversely, urea synthesis may be increased as a result of the chronic inflammation. The aims of the study were to determine urea synthesis kinetics in CP patients in relation to glucagon secretion (study I) and during an increase in protein intake (study II). In study I, urea synthesis rate, calculated as urinary excretion rate corrected for accumulation in total body water and intestinal loss, was measured during infusion of alanine in 7 CP patients and 5 control subjects on spontaneous protein intake. The functional hepatic nitrogen clearance (FHNC), i.e. urea synthesis expressed independent of changes in plasma amino acid concentration, was calculated as the slope of the linear relation between urea synthesis rate and plasma alpha -amino nitrogen concentration. In study II, 6 of the patients of study I had urea synthesis and FHNC determined before and after a period of 14 days of supplementation with a protein-enriched liquid (dietary sequence randomized). Study I: Alanine infusion increased urea synthesis rate by a factor of 10 in the control subjects, and by a factor of 5 in the CP patients (P<0.01). FHNC was 31.9+/-2.4 l/h in the control subjects and 16.5+/-2.0 l/h (P<0.05) in the CP patients. The glucagon response to alanine infusion (AUC) was reduced by 75 % in the CP patients. The reduction in FHNC paralleled the reduced glucagon response (r(2)=0.55, P<0.01). Study II: The spontaneous protein intake was 0.75+/-0.14 g/(kg x day) and increased during the high protein period to 1.77+/-0.12 g/(kg x day). This increased alanine stimulated urea synthesis by a factor of 1.3 (P<0.05), FHNC from 13.5+/-2.6 l/h to 19.4+/-3.1 l/h (P<0.01), and the glucagon response to alanine infusion (AUC) by a factor of 1.8 (P<0.05). Urea synthesis rate and FHNC are markedly reduced in CP patients. This is associated with, and probably a result of, impaired glucagon secretion, and predicts a lower than normal postprandial hepatic loss of amino nitrogen. An increase in dietary protein intake increases alanine stimulated urea synthesis and FHNC by a mechanism that involves an increase in glucagon. This indicates that the low FHNC during spontaneous protein intake included an adaptation to the low protein intake, effectuated by a further decrease in glucagon secretion. Copyright 2001 Harcourt Publishers Ltd.

The growth hormone-insulin-like growth factor axis in glycogen storage disease type 1: evidence of different growth patterns and insulin-like growth factor levels in patients with glycogen storage disease type 1a and 1b.

PubMed

Melis, Daniela; Pivonello, Rosario; Parenti, Giancarlo; Della Casa, Roberto; Salerno, Mariacarolina; Balivo, Francesca; Piccolo, Pasquale; Di Somma, Carolina; Colao, Annamaria; Andria, Generoso

2010-04-01

To investigate the growth hormone (GH)-insulin-like growth factor (IGF) system in patients with glycogen storage disease type 1 (GSD1). This was a prospective, case-control study. Ten patients with GSD1a and 7 patients with GSD1b who were given dietary treatment and 34 sex-, age-, body mass index-, and pubertal stage-matched control subjects entered the study. Auxological parameters were correlated with circulating GH, either at basal or after growth hormone releasing hormone plus arginine test, insulin-like growth factors (IGF-I and IGF-II), and anti-pituitary antibodies (APA). Short stature was detected in 10.0% of patients with GSD1a, 42.9% of patients with GSD1b (P = .02), and none of the control subjects. Serum IGF-I levels were lower in patients with GSD1b (P = .0001). An impaired GH secretion was found in 40% of patients with GSD1a (P = .008), 57.1% of patients with GSD1b (P = .006), and none of the control subjects. Short stature was demonstrated in 3 of 4 patients with GSD1b and GH deficiency. The prevalence of APA was significantly higher in patients with GSD1b than in patients with GSD1a (P = .02) and control subjects (P = .03). The GH response to the provocative test inversely correlated with the presence of APA (P = .003). Compared with levels in control subjects, serum IGF-II and insulin levels were higher in both groups of patients, in whom IGF-II levels directly correlated with height SD scores (P = .003). Patients with GSD1a have an impaired GH secretion associated with reference range serum IGF-I levels and normal stature, whereas in patients with GSD1b, the impaired GH secretion, probably because of the presence of APA, was associated with reduced IGF-I levels and increased prevalence of short stature. The increased IGF-II levels, probably caused by increased insulin levels, in patients with GSD1 are presumably responsible for the improved growth pattern observed in patients receiving strict dietary treatment. Copyright 2010 Mosby, Inc. All rights reserved.

The role of intrinsic disorder and dynamics in the assembly and function of the type II secretion system.

PubMed

Gu, Shuang; Shevchik, Vladimir E; Shaw, Rosie; Pickersgill, Richard W; Garnett, James A

2017-10-01

Many Gram-negative commensal and pathogenic bacteria use a type II secretion system (T2SS) to transport proteins out of the cell. These exported proteins or substrates play a major role in toxin delivery, maintaining biofilms, replication in the host and subversion of host immune responses to infection. We review the current structural and functional work on this system and argue that intrinsically disordered regions and protein dynamics are central for assembly, exo-protein recognition, and secretion competence of the T2SS. The central role of intrinsic disorder-order transitions in these processes may be a particular feature of type II secretion. Copyright © 2017 Elsevier B.V. All rights reserved.

Phenolic Compounds from Fermented Berry Beverages Modulated Gene and Protein Expression To Increase Insulin Secretion from Pancreatic β-Cells in Vitro.

PubMed

Johnson, Michelle H; de Mejia, Elvira Gonzalez

2016-03-30

Berries are a rich source of bioactive phenolic compounds that are able to bind and inhibit the enzyme dipeptidyl peptidase-IV (DPP-IV), a current target for type-2 diabetes therapy. The objectives were to determine the role of berry phenolic compounds to modulate incretin-cleaving DPP-IV and its substrate glucagon-like peptide-1 (GLP-1), insulin secretion from pancreatic β-cells, and genes and proteins involved in the insulin secretion pathway using cell culture. Anthocyanins (ANC) from 50% blueberry-50% blackberry (Blu-Bla) and 100% blackberry (Bla) fermented beverages at 50 μM cyanidin-3-glucoside equivalents increased (p < 0.05) glucose-stimulated insulin secretion from pancreatic β-cells (iNS-1E) both when applied directly and following simulated absorption through Caco-2 cells (by 233 and 100 μIU insulin/mL, respectively). ANC 50%Blu-Bla and ANC 100%Bla upregulated the gene for incretin hormone GLP-1 (fold-change 3.0 ± 1.4 and 2.0 ± 0.3, respectively) and genes in the insulin secretory pathway including insulin-like growth factor 1 receptor (iGF1R, 2.3 ± 0.6 and 1.6 ± 0.3, respectively), and increased (p < 0.05) the protein expression of insulin-like growth factor 2 (IGF-II), insulin-like growth factor binding proteins (IGFBP-2 and 3), and vascular endothelial growth factor (VEGF) in iNS-1E cells. Taken together, anthocyanins, predominantly delphinidin-3-arabinoside, from fermented berry beverages have the potential to modulate DPP-IV and its substrate GLP-1, to increase insulin secretion, and to upregulate expression of mRNA of insulin-receptor associated genes and proteins in pancreatic β-cells.

Distinct Differentiation Programs Triggered by IL-6 and LPS in Teleost IgM(+) B Cells in The Absence of Germinal Centers.

PubMed

Abós, Beatriz; Wang, Tiehui; Castro, Rosario; Granja, Aitor G; Leal, Esther; Havixbeck, Jeffrey; Luque, Alfonso; Barreda, Daniel R; Secombes, Chris J; Tafalla, Carolina

2016-08-02

Although originally identified as a B cell differentiation factor, it is now known that mammalian interleukin-6 (IL-6) only regulates B cells committed to plasma cells in response to T-dependent (TD) antigens within germinal centers (GCs). Even though adaptive immunity is present in teleost fish, these species lack lymph nodes and GCs. Thus, the aim of the present study was to establish the role of trout IL-6 on B cells, comparing its effects to those induced by bacterial lipopolysaccharide (LPS). We demonstrate that the effects of teleost IL-6 on naïve spleen B cells include proliferation, activation of NF-κB, increased IgM secretion, up-regulation of Blimp1 transcription and decreased MHC-II surface expression that point to trout IL-6 as a differentiation factor for IgM antibody-secreting cells (ASCs). However, LPS induced the secretion of IgM without up-regulating Blimp1, driving the cells towards an intermediate activation state in which antigen presenting mechanisms are elicited together with antibody secretion and expression of pro-inflammatory genes. Our results reveal that, in trout, IL-6 is a differentiation factor for B cells, stimulating IgM responses in the absence of follicular structures, and suggest that it was after follicular structures appeared that this cytokine evolved to modulate TD responses within the GC.

External Loops at the C Terminus of Erwinia chrysanthemi Pectate Lyase C Are Required for Species-Specific Secretion through the Out Type II Pathway

PubMed Central

Lindeberg, Magdalen; Boyd, Carol M.; Keen, Noel T.; Collmer, Alan

1998-01-01

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pel1. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning. PMID:9515910

Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor.

PubMed Central

Cheng, S Y; Huang, H J; Nagane, M; Ji, X D; Wang, D; Shih, C C; Arap, W; Huang, C M; Cavenee, W K

1996-01-01

The development of new capillary networks from the normal microvasculature of the host appears to be required for growth of solid tumors. Tumor cells influence this process by producing both inhibitors and positive effectors of angiogenesis. Among the latter, the vascular endothelial growth factor (VEGF) has assumed prime candidacy as a major positive physiological effector. Here, we have directly tested this hypothesis in the brain tumor, glioblastoma multiforme, one of the most highly vascularized human cancers. We introduced an antisense VEGF expression construct into glioblastoma cells and found that (i) VEGF mRNA and protein levels were markedly reduced, (ii) the modified cells did not secrete sufficient factors so as to be chemoattractive for primary human microvascular endothelial cells, (iii) the modified cells were not able to sustain tumor growth in immunodeficient animals, and (iv) the density of in vivo blood vessel formation was reduced in direct relation to the reduction of VEGF secretion and tumor formation. Moreover, revertant cells that recovered the ability to secrete VEGF regained each of these tumorigenic properties. These results suggest that VEGF plays a major angiogenic role in glioblastoma. Images Fig. 1 Fig. 4 PMID:8710899

Adipocyte-Derived Hormone Leptin Is a Direct Regulator of Aldosterone Secretion, Which Promotes Endothelial Dysfunction and Cardiac Fibrosis.

PubMed

Huby, Anne-Cécile; Antonova, Galina; Groenendyk, Jake; Gomez-Sanchez, Celso E; Bollag, Wendy B; Filosa, Jessica A; Belin de Chantemèle, Eric J

2015-12-01

In obesity, the excessive synthesis of aldosterone contributes to the development and progression of metabolic and cardiovascular dysfunctions. Obesity-induced hyperaldosteronism is independent of the known regulators of aldosterone secretion, but reliant on unidentified adipocyte-derived factors. We hypothesized that the adipokine leptin is a direct regulator of aldosterone synthase (CYP11B2) expression and aldosterone release and promotes cardiovascular dysfunction via aldosterone-dependent mechanisms. Immunostaining of human adrenal cross-sections and adrenocortical cells revealed that adrenocortical cells coexpress CYP11B2 and leptin receptors. Measurements of adrenal CYP11B2 expression and plasma aldosterone levels showed that increases in endogenous (obesity) or exogenous (infusion) leptin dose-dependently raised CYP11B2 expression and aldosterone without elevating plasma angiotensin II, potassium or corticosterone. Neither angiotensin II receptors blockade nor α and β adrenergic receptors inhibition blunted leptin-induced aldosterone secretion. Identical results were obtained in cultured adrenocortical cells. Enhanced leptin signaling elevated CYP11B2 expression and plasma aldosterone, whereas deficiency in leptin or leptin receptors blunted obesity-induced increases in CYP11B2 and aldosterone, ruling out a role for obesity per se. Leptin increased intracellular calcium, elevated calmodulin and calmodulin-kinase II expression, whereas calcium chelation blunted leptin-mediated increases in CYP11B2, in adrenocortical cells. Mineralocorticoid receptor blockade blunted leptin-induced endothelial dysfunction and increases in cardiac fibrotic markers. Leptin is a newly described regulator of aldosterone synthesis that acts directly on adrenal glomerulosa cells to increase CYP11B2 expression and enhance aldosterone production via calcium-dependent mechanisms. Furthermore, leptin-mediated aldosterone secretion contributes to cardiovascular disease by promoting endothelial dysfunction and the expression of profibrotic markers in the heart. © 2015 American Heart Association, Inc.

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

DOE PAGES

Xu, Qi; Knoshaug, Eric P.; Wang, Wei; ...

2017-07-24

Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose twomore » prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. Lastly, the effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well.« less

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

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

Xu, Qi; Knoshaug, Eric P.; Wang, Wei

Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose twomore » prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. Lastly, the effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well.« less

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi.

PubMed

Xu, Qi; Knoshaug, Eric P; Wang, Wei; Alahuhta, Markus; Baker, John O; Yang, Shihui; Vander Wall, Todd; Decker, Stephen R; Himmel, Michael E; Zhang, Min; Wei, Hui

2017-07-24

Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. The effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well.

Acinetobacter baumannii Is Dependent on the Type II Secretion System and Its Substrate LipA for Lipid Utilization and In Vivo Fitness

PubMed Central

Johnson, Tanya L.; Waack, Ursula; Smith, Sara; Mobley, Harry

2015-01-01

ABSTRACT Gram-negative bacteria express a number of sophisticated secretion systems to transport virulence factors across the cell envelope, including the type II secretion (T2S) system. Genes for the T2S components GspC through GspN and PilD are conserved among isolates of Acinetobacter baumannii, an increasingly common nosocomial pathogen that is developing multidrug resistance at an alarming rate. In contrast to most species, however, the T2S genes are dispersed throughout the genome rather than linked into one or two operons. Despite this unique genetic organization, we show here that the A. baumannii T2S system is functional. Deletion of gspD or gspE in A. baumannii ATCC 17978 results in loss of secretion of LipA, a lipase that breaks down long-chain fatty acids. Due to a lack of extracellular lipase, the gspD mutant, the gspE mutant, and a lipA deletion strain are incapable of growth on long-chain fatty acids as a sole source of carbon, while their growth characteristics are indistinguishable from those of the wild-type strain in nutrient-rich broth. Genetic inactivation of the T2S system and its substrate, LipA, also has a negative impact on in vivo fitness in a neutropenic murine model for bacteremia. Both the gspD and lipA mutants are outcompeted by the wild-type strain as judged by their reduced numbers in spleen and liver following intravenous coinoculation. Collectively, our findings suggest that the T2S system plays a hitherto-unrecognized role in in vivo survival of A. baumannii by transporting a lipase that may contribute to fatty acid metabolism. IMPORTANCE Infections by multidrug-resistant Acinetobacter baumannii are a growing health concern worldwide, underscoring the need for a better understanding of the molecular mechanisms by which this pathogen causes disease. In this study, we demonstrated that A. baumannii expresses a functional type II secretion (T2S) system that is responsible for secretion of LipA, an extracellular lipase required for utilization of exogenously added lipids. The T2S system and the secreted lipase support in vivo colonization and thus contribute to the pathogenic potential of A. baumannii. PMID:26668261

Chronic inhibition of glycogen synthase kinase-3 protects against rotenone-induced cell death in human neuron-like cells by increasing BDNF secretion.

PubMed

Giménez-Cassina, Alfredo; Lim, Filip; Díaz-Nido, Javier

2012-12-07

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. Likewise, activation of glycogen synthase kinase-3 (GSK-3) has been proposed to play an important role in neurodegeneration. This multifunctional protein kinase is involved in a number of cellular functions and we previously showed that chronic inhibition of GSK-3 protects neuronal cells against mitochondrial dysfunction-elicited cell death, through a mechanism involving increased glucose metabolism and the translocation of hexokinase II (HKII) to mitochondria. Here, we sought to gain deeper insight into the molecular basis of this neuroprotection. We found that chronic inhibition of GSK-3, either genetically or pharmacologically, elicited a marked increase in brain-derived neurotrophic factor (BDNF) secretion, which in turn conferred resistance to mitochondrial dysfunction through subcellular re-distribution of HKII. These results define a molecular pathway through which chronic inhibition of GSK-3 may protect neuronal cells from death. Moreover, they highlight the potential benefits of enhanced neurotrophic factor secretion as a therapeutic approach to treat neurodegenerative diseases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Regulation of intracellular trafficking and secretion of adiponectin by myosin II.

PubMed

Bedi, Deepa; Dennis, John C; Morrison, Edward E; Braden, Tim D; Judd, Robert L

2017-08-19

Adiponectin is a protein secreted by white adipocytes that plays an important role in insulin action, energy homeostasis and the development of atherosclerosis. The intracellular localization and trafficking of GLUT4 and leptin in adipocytes has been well studied, but little is known regarding the intracellular trafficking of adiponectin. Recent studies have demonstrated that constitutive adiponectin secretion is dependent on PIP2 levels and the integrity of cortical F-actin. Non-muscle myosin II is an actin-based motor that is associated with membrane vesicles and participates in vesicular trafficking in mammalian cells. Therefore, we investigated the role of myosin II in the trafficking and secretion of adiponectin in 3T3-L1 adipocytes. Confocal microscopy revealed that myosin IIA and IIB were dispersed throughout the cytoplasm of the adipocyte. Both myosin isoforms were localized in the Golgi/TGN region as evidenced by colocalization with the cis-Golgi marker, p115 and the trans-Golgi marker, γ-adaptin. Inhibition of myosin II activity by blebbistatin or actin depolymerization by latrunculin B dispersed myosin IIA and IIB towards the periphery while significantly inhibiting adiponectin secretion. Therefore, the constitutive trafficking and secretion of adiponectin in 3T3-L1 adipocytes occurs by an actin-dependent mechanism that involves the actin-based motors, myosin IIA and IIB. Copyright © 2017 Elsevier Inc. All rights reserved.

Purification and partial characterization of PfHRP-II protein of Plasmodium falciparum.

PubMed

Ghimire, Prakash; Samantaray, J C; Mirdha, B R; Patra, A K; Panda, A K

2003-12-01

The human malarial parasite Plasmodium falciparum secretes various intra-and extra-cellular proteins during its asexual life cycle in human RBC. Histidine rich protein-II (HRP-II) is one of the most prominent proteins, found to be secreted by P. falciparum throughout the asexual cycle with the peak during mature schizont stage of the parasite development in human IRBC. The high histidine content (35% of the total amino acids in protein) of this protein suggested the potential to bind divalent metal ions. We have demonstrated by metal chelate chromatography, an extraordinary capacity of HRP-II to bind nickel ions (Ni++) and employed this characteristic to purify the extra-cellular HRP-II protein secreted by P. falciparum from culture supernatant. The identity of the purified protein was verified by the relative molecular weight on SDS-PAGE, by reacting with polyclonal antibodies directed against it using Western blot technique.

Autosomal Dominant Growth Hormone Deficiency (Type II).

PubMed

Alatzoglou, Kyriaki S; Kular, Dalvir; Dattani, Mehul T

2015-06-01

Isolated growth hormone deficiency (IGHD) is the commonest pituitary hormone deficiency resulting from congenital or acquired causes, although for most patients its etiology remains unknown. Among the known factors, heterozygous mutations in the growth hormone gene (GH1) lead to the autosomal dominant form of GHD, also known as type II GHD. In many cohorts this is the commonest form of congenital isolated GHD and is mainly caused by mutations that affect the correct splicing of GH-1. These mutations cause skipping of the third exon and lead to the production of a 17.5-kDa GH isoform that exerts a dominant negative effect on the secretion of the wild type GH. The identification of these mutations has clinical implications for the management of patients, as there is a well-documented correlation between the severity of the phenotype and the increased expression of the 17.5-kDa isoform. Patients with type II GHD have a variable height deficit and severity of GHD and may develop additional pituitary hormone defiencies over time, including ACTH, TSH and gonadotropin deficiencies. Therefore, their lifelong follow-up is recommended. Detailed studies on the effect of heterozygous GH1 mutations on the trafficking, secretion and action of growth hormone can elucidate their mechanism on a cellular level and may influence future treatment options for GHD type II.

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

Solheim, B.A.

Three families of termites have the ability to produce a sticky secretion that envelopes and immobilizes the enemy. In the family Termitidae the secretion contains the diterpenoid hydrocarbons, kempene I and kempene II. The molecular structure of kempene II from the termite, Nasutitermes kempae, is described in detail. Another species of termite, Cubitermes umbratus, contained the diterpenoid hydrocarbon biflora-4,10-19,15-triene in the secretion and this compound is described. Studies were also conducted on the mucous secretion of the pedal gland of the marine nudibranch, Tidachiella diomedea. Tridachione, a substituted ..gamma..-pyrone, was isolated in the pure state and its molecular structure ismore » described in detail. (HLW)« less

Ovarian factors inhibit and fetal factors stimulate the secretion of rat placental lactogen.

PubMed

Robertson, M C; Owens, R E; McCoshen, J A; Friesen, H G

1984-01-01

Removal of fetuses at day 14 of gestation (Ftx14) in the pregnant rat leads to a marked suppression of serum levels of rat placental lactogen (rPL-II). One might attribute this to compromised placental growth in the absence of a fetus. However, if ovariectomy and fetectomy (Ftx14 Ovx14) are carried out at the same time, a great increase in serum rPL-II levels is seen. This occurs despite a significant decrease in placental weight. When Ftx14 was performed on day 14 and Ovx was delayed 1, 2, or 3 days, the expected large increase in serum rPL-II was progressively attenuated compared to that seen when Ftx and Ovx were carried out simultaneously. Daily administration of 17 beta-estradiol (4 micrograms/rat X day) to Ftx14 Ovx14 pregnant rats resulted in a significant suppression of rPL-II and elevation of rPRL levels, a reversal of what is seen for these hormones in untreated Ftx14 Ovx14 animals. To test whether 17 beta-estradiol was acting through rat PRL (rPRL), serum levels of rPRL were elevated in Ftx13 Ovx13 animals with pimozide (0.6 mg/kg), a dopamine receptor blocker. There was no effect of this treatment on rPL-II levels. In late pregnancy (day 17) serum rPL-II levels remained high after removal of half the fetuses (1/2 Ftx), compared to the rapid fall in pregnant rats in which all fetuses were removed (Ftx17). Serum levels were also elevated in 1/2 Ftx animals compared to those in which half of the fetuses and placentas were removed by hemi-hysterectomy, suggesting that the increase in rPL-II levels in 1/2 Ftx animals was due to a stimulatory effect of the remaining fetuses on all of the placentas. These results indicate that the presence of the fetus is necessary for the normally observed increase in rPL-II levels in late pregnancy. In conclusion, fetal stimulators and ovarian inhibitors influence rPL-II secretion.

Arginase-II Promotes Tumor Necrosis Factor-α Release From Pancreatic Acinar Cells Causing β-Cell Apoptosis in Aging.

PubMed

Xiong, Yuyan; Yepuri, Gautham; Necetin, Sevil; Montani, Jean-Pierre; Ming, Xiu-Fen; Yang, Zhihong

2017-06-01

Aging is associated with glucose intolerance. Arginase-II (Arg-II), the type-II L -arginine-ureahydrolase, is highly expressed in pancreas. However, its role in regulation of pancreatic β-cell function is not known. Here we show that female (not male) mice deficient in Arg-II (Arg-II -/- ) are protected from age-associated glucose intolerance and reveal greater glucose induced-insulin release, larger islet size and β-cell mass, and more proliferative and less apoptotic β-cells compared with the age-matched wild-type (WT) controls. Moreover, Arg-II is mainly expressed in acinar cells and is upregulated with aging, which enhances p38 mitogen-activated protein kinase (p38 MAPK) activation and release of tumor necrosis factor-α (TNF-α). Accordingly, conditioned medium of isolated acinar cells from old WT (not Arg-II -/- ) mice contains higher TNF-α levels than the young mice and stimulates β-cell apoptosis and dysfunction, which are prevented by a neutralizing anti-TNF-α antibody. In acinar cells, our study demonstrates an age-associated Arg-II upregulation, which promotes TNF-α release through p38 MAPK leading to β-cell apoptosis, insufficient insulin secretion, and glucose intolerance in female rather than male mice. © 2017 by the American Diabetes Association.

EcpA, an extracellular protease, is a specific virulence factor required by Xanthomonas oryzae pv. oryzicola but not by X. oryzae pv. oryzae in rice

USDA-ARS?s Scientific Manuscript database

Previously, twelve protease-deficient mutants of Xanthomonas oryzae pv. oryzicola (Xoc) RS105 strain were recovered from a Tn5-tagged mutant library. In the current study, the Tn5 insertion site in each mutant was mapped. Mutations in genes encoding components of the type II secretion apparatus, cAM...

Atrial natriuretic peptide stimulates salt secretion by shark rectal gland by releasing VIP

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

Silva, P.; Stoff, J.S.; Solomon, R.J.

1987-01-01

Salt secretion by the isolated perfused rectal gland of the spiny dogfish shark, Squalus acanthias, is stimulated by synthetic rat atrial natriuretic peptide (ANP II) as well as extracts of shark heart, but not by 8-bromo-cyclic guanosine 5'-monophosphate. Cardiac peptides have no effect on isolated rectal gland cells or perfused tubules, suggesting that stimulation requires an intact gland. The stimulation of secretion by ANP II is eliminated by maneuvers that block neurotransmitter release. Cardiac peptides stimulate the release of vasoactive intestinal peptide (VIP), known to be present in rectal glands nerves, into the venous effluent of perfused glands in parallelmore » with their stimulation of salt secretion, but the release of VIP induced by ANP II is prevented by perfusion with procaine. VIP was measured by radioimmunoassay. Cardiac peptides thus appear to regulate rectal gland secretion by releasing VIP from neural stores within the gland. It is possible that other physiological effects of these hormones might be explained by an action to enhanced local release of neurotransmitters.« less

The Potassium Channel, Kir3.4 Participates in Angiotensin II-Stimulated Aldosterone Production by a Human Adrenocortical Cell Line

PubMed Central

Oki, Kenji; Plonczynski, Maria W.; Lam, Milay Luis; Gomez-Sanchez, Elise P.

2012-01-01

Angiotensin II (A-II) regulation of aldosterone secretion is initiated by inducing cell membrane depolarization, thereby increasing intracellular calcium and activating the calcium calmodulin/calmodulin kinase cascade. Mutations in the selectivity filter of the KCNJ5 gene coding for inward rectifying potassium channel (Kir)3.4 has been found in about one third of aldosterone-producing adenomas. These mutations result in loss of selectivity of the inward rectifying current for potassium, which causes membrane depolarization and opening of calcium channels and activation of the calcium calmodulin/calmodulin kinase cascade and results in an increase in aldosterone secretion. In this study we show that A-II and a calcium ionophore down-regulate the expression of KCNJ5 mRNA and protein. Activation of Kir3.4 by naringin inhibits A-II-stimulated membrane voltage and aldosterone secretion. Overexpression of KCNJ5 in the HAC15 cells using a lentivirus resulted in a decrease in membrane voltage, intracellular calcium, expression of steroidogenic acute regulatory protein, 3-β-hydroxysteroid dehydrogenase 3B2, cytochrome P450 11B1 and cytochrome P450 11B2 mRNA, and aldosterone synthesis. In conclusion, A-II appears to stimulate aldosterone secretion by depolarizing the membrane acting in part through the regulation of the expression and activity of Kir3.4. PMID:22798349

RGS2 is regulated by angiotensin II and functions as a negative feedback of aldosterone production in H295R human adrenocortical cells.

PubMed

Romero, Damian G; Plonczynski, Maria W; Gomez-Sanchez, Elise P; Yanes, Licy L; Gomez-Sanchez, Celso E

2006-08-01

Regulator of G protein signaling (RGS) proteins interact with Galpha-subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. Angiotensin (Ang) II interacts with its G protein-coupled receptor in zona glomerulosa adrenal cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. We studied Ang II-mediated regulation of RGS2, the role of RGS2 in steroidogenesis, and the intracellular signal events involved in H295R human adrenal cells. We report that both H295R cells and human adrenal gland express RGS2 mRNA. In H295R cells, Ang II caused a rapid and transient increase in RGS2 mRNA levels quantified by real-time RT-PCR. Ang II effects were mimicked by calcium ionophore A23187 and blocked by calcium channel blocker nifedipine. Ang II effects also were blocked by calmodulin antagonists (W-7 and calmidazolium) and calcium/calmodulin-dependent kinase antagonist KN-93. RGS2 overexpression by retroviral infection in H295R cells caused a decrease in Ang II-stimulated aldosterone secretion but did not modify cortisol secretion. In reporter assays, RGS2 decreased Ang II-mediated aldosterone synthase up-regulation. These results suggest that Ang II up-regulates RGS2 mRNA by the calcium/calmodulin-dependent kinase pathway in H295R cells. RGS2 overexpression specifically decreases aldosterone secretion through a decrease in Ang II-mediated aldosterone synthase-induced expression. In conclusion, RGS2 expression is induced by Ang II to terminate the intracellular signaling cascade generated by Ang II. RGS2 alterations in expression levels or functionality could be implicated in deregulations of Ang II signaling and abnormal aldosterone secretion by the adrenal gland.

Losartan counteracts the effects of cardiomyocyte swelling on glucose uptake and insulin receptor substrate-1 levels.

PubMed

Gerena, Yamil; Lozada, Janice Griselle; Collazo, Bryan Jael; Méndez-Álvarez, Jarold; Méndez-Estrada, Jennifer; De Mello, Walmor C

2017-10-01

A growing body of evidence demonstrates an association between Angiotensin II (Ang II) receptor blockers (ARBs) and enhanced glucose metabolism during ischemic heart disease. Despite these encouraging results, the mechanisms responsible for these effects during ischemia remain poorly understood. In this study we investigated the influence of losartan, an AT1 receptor blocker, and secreted Ang II (sAng II) on glucose uptake and insulin receptor substrate (IRS-1) levels during cardiomyocyte swelling. H9c2 cells were differentiated to cardiac muscle and the levels of myogenin, Myosin Light Chain (MLC), and membrane AT1 receptors were measured using flow cytometry. Intracellular Ang II (iAng II) was overexpressed in differentiated cardiomyocytes and swelling was induced after incubation with hypotonic solution for 40min. Glucose uptake and IRS-1 levels were monitored by flow cytometry using 2-NBDG fluorescent glucose (10μM) or an anti-IRS-1 monoclonal antibody in the presence or absence of losartan (10 -7 M). Secreted Angiotensin II was quantified from the medium using a specific Ang II-EIA kit. To evaluate the relationship between sAng II and losartan effects on glucose uptake, transfected cells were pretreated with the drug for 24h and then exposed to hypotonic solution in the presence or absence of the secreted peptide. The results indicate that: (1) swelling of transfected cardiomyocytes decreased glucose uptake and induced the secretion of Ang II to the extracellular medium; (2) losartan antagonized the effects of swelling on glucose uptake and IRS-1 levels in transfected cardiomyocytes; (3) the effects of losartan on glucose uptake were observed during swelling only in the presence of sAng II in the culture medium. Our study demonstrates that both losartan and sAng II have essential roles in glucose metabolism during cardiomyocyte swelling. Copyright © 2017 Elsevier Inc. All rights reserved.

75 FR 77783 - Designation of National Security Positions

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-14

... requiring eligibility for access to Top Secret or ``Q'' classified information; (ii) Positions involving... at the Top Secret or ``Q'' level; (xix) Positions working with significant life-critical/mission... Sensitive information, requiring involvement in Top Secret Special Access Programs (SAP), or positions which...

Jamming dynamics of stretch-induced surfactant release by alveolar type II cells

PubMed Central

Majumdar, Arnab; Arold, Stephen P.; Bartolák-Suki, Erzsébet; Parameswaran, Harikrishnan

2012-01-01

Secretion of pulmonary surfactant by alveolar epithelial type II cells is vital for the reduction of interfacial surface tension, thus preventing lung collapse. To study secretion dynamics, rat alveolar epithelial type II cells were cultured on elastic membranes and cyclically stretched. The amounts of phosphatidylcholine, the primary lipid component of surfactant, inside and outside the cells, were measured using radiolabeled choline. During and immediately after stretch, cells secreted less surfactant than unstretched cells; however, stretched cells secreted significantly more surfactant than unstretched cells after an extended lag period. We developed a model based on the hypothesis that stretching leads to jamming of surfactant traffic escaping the cell, similar to vehicular traffic jams. In the model, stretch increases surfactant transport from the interior to the exterior of the cell. This transport is mediated by a surface layer with a finite capacity due to the limited number of fusion pores through which secretion occurs. When the amount of surfactant in the surface layer approaches this capacity, interference among lamellar bodies carrying surfactant reduces the rate of secretion, effectively creating a jam. When the stretch stops, the jam takes an extended time to clear, and subsequently the amount of secreted surfactant increases. We solved the model analytically and show that its dynamics are consistent with experimental observations, implying that surfactant secretion is a fundamentally nonlinear process with memory representing collective behavior at the level of single cells. Our results thus highlight the importance of a jamming dynamics in stretch-induced cellular secretory processes. PMID:22033531

Treatments for diabetes mellitus type II: New perspectives regarding the possible role of calcium and cAMP interaction.

PubMed

Carvalho, Diego Soares; de Almeida, Alexandre Aparecido; Borges, Aurélio Ferreira; Campos, Vannucci

2018-07-05

Diabetes mellitus (DM) is among the top ten causes of death worldwide. It is considered to be one of the major global epidemics of the 21st century, with a significant impact on public health budgets. DM is a metabolic disorder with multiple etiologies. Its pathophysiology is marked by dysfunction of pancreatic β-cells which compromises the synthesis and secretion of insulin along with resistance to insulin action in peripheral tissues (muscle and adipose). Subjects presenting insulin resistance in DM type 2 often also exhibit increased insulin secretion and hyperinsulinemia. Insulin secretion is controlled by several factors such as nutrients, hormones, and neural factors. Exocytosis of insulin granules has, as its main stimulus, increased intracellular calcium ([Ca +2 ]i) and it is further amplified by cyclic AMP (cAMP). In the event of this hyperfunction, it is very common for β-cells to go into exhaustion leading to failure or death. Several animal studies have demonstrated pleiotropic effects of L-type Ca 2+ channel blockers (CCBs). In animal models of obesity and diabetes, treatment with CCBs promoted restoration of insulin secretion, glycemic control, and reduction of pancreatic β-cell apoptosis. In addition, hypertensive individuals treated with CCBs presented a lower incidence of DM when compared with other antihypertensive agents. In this review, we propose that pharmacological manipulation of the Ca 2+ /cAMP interaction system could lead to important targets for pharmacological improvement of insulin secretion in DM type 2. Copyright © 2018 Elsevier B.V. All rights reserved.

Serum from Chronic Hepatitis B Patients Promotes Growth and Proliferation via the IGF-II/IGF-IR/MEK/ERK Signaling Pathway in Hepatocellular Carcinoma Cells.

PubMed

Ji, Yuanyuan; Wang, Zhidong; Chen, Haiyan; Zhang, Lei; Zhuo, Fei; Yang, Qingqing

2018-05-09

Chronic hepatitis B virus (HBV) infection (CHB) plays a central role in the etiology of hepatocellular carcinoma (HCC). Emerging evidence implicates insulin-like growth factor (IGF)-II as a major risk factor for the growth and development of HCC. However, the relationship between HBV infection and IGF-II functions remains to be elucidated. Levels of circulating IGF-II and IGF-I receptor (IGF-IR) in healthy donors (HDs) and CHB patients were tested by ELISA. Human HCC cell lines (HepG-2, SMMC-7721, MHCC97-H) were incubated with serum from HDs and CHB patients at various concentrations for 24, 48, and 72 h. MTT and plate colony formation assays, BrdU ELISA, ELISA, small-interfering RNA (siRNA) transfection, quantitative real-time PCR, and western blot were applied to assess the functional and molecular mechanisms in HCC cell lines. Serum levels of IGF-II and IGF-IR were significantly higher in CHB patients than in HDs. Additionally, serum from CHB patients directly induced cell growth, proliferation, IGF-II secretion, and HDGF-related protein-2 (HRP-2) and nuclear protein 1 (NUPR1) mRNA and protein expression in HCC cells. Moreover, serum from CHB patients increased IGF-II-induced cell growth, proliferation, and HRP-2 and NUPR1 mRNA and protein expression in HCC cells. Blockade of IGF-IR clearly inhibited the above effects. Most importantly, interference with IGF-II function markedly repressed the cell proliferation and HRP-2 and NUPR1 mRNA and protein expression induced by serum from CHB patients. Furthermore, serum from CHB patients induced ERK phosphorylation via IGF-IR, with the MEK inhibitor PD98059 significantly decreasing CHB patient serum-induced IGF-II secretion, cell proliferation, and HRP-2 and NUPR1 mRNA and protein expression. Serum from CHB patients increases cell growth and proliferation and enhances HRP-2 and NUPR1 expression in HCC cells via the IGF-II/IGF-IR/MEK/ERK signaling pathway. These findings help to explain the molecular mechanisms underlying HBV-related HCC and may lead to the development of effective therapies. © 2018 The Author(s). Published by S. Karger AG, Basel.

Innate immunity in the vagina (Part II): Anti-HIV activity and antiviral content of human vaginal secretions.

PubMed

Patel, Mickey V; Ghosh, Mimi; Fahey, John V; Ochsenbauer, Christina; Rossoll, Richard M; Wira, Charles R

2014-07-01

Whether the concentrations of antiviral proteins, and anti-HIV activity, within human vaginal secretions change across the menstrual cycle is unknown. Using a menstrual cup, vaginal secretions from pre-menopausal women were recovered at the proliferative (d6-8), mid-cycle (d13-15), and secretory (d21-23) stages of the menstrual cycle. Antiviral protein concentration was determined by ELISA, and anti-HIV activity assessed using the TZM-bl reporter cell line. CCL20, RANTES, elafin, HBD2, SDF-1α, and IL-8 levels were detectable in the secretions. Vaginal secretions had anti-HIV activity against specific clade B strains of HIV, with significant inhibition of IIIB and increased infectivity of transmitted/founder CH077.t. No significant differences in either antiviral protein concentration or anti-HIV activity with respect to menstrual cycle stage were measured, but marked differences were observed in both parameters over the course of the cycle between different women and in consecutive cycles from the same woman. The vagina contains a complement of antiviral proteins. The variation in anti-HIV activity demonstrates that immune protection in the vagina is not constant. Intra- and interindividual variations suggest that factors in addition to sex hormones influence antiviral protection. Lastly, the menstrual cup is a new model for recovering undiluted vaginal secretions from women throughout their reproductive life. © 2014 John Wiley & Sons Ltd.

Solution Structure of Homology Region (HR) Domain of Type II Secretion System*

PubMed Central

Gu, Shuang; Kelly, Geoff; Wang, Xiaohui; Frenkiel, Tom; Shevchik, Vladimir E.; Pickersgill, Richard W.

2012-01-01

The type II secretion system of Gram-negative bacteria is important for bacterial pathogenesis and survival; it is composed of 12 mostly multimeric core proteins, which build a sophisticated secretion machine spanning both bacterial membranes. OutC is the core component of the inner membrane subcomplex thought to be involved in both recognition of substrate and interaction with the outer membrane secretin OutD. Here, we report the solution structure of the HR domain of OutC and explore its interaction with the secretin. The HR domain adopts a β-sandwich-like fold consisting of two β-sheets each composed of three anti-parallel β-strands. This structure is strikingly similar to the periplasmic region of PilP, an inner membrane lipoprotein from the type IV pilus system highlighting the common evolutionary origin of these two systems and showing that all the core components of the type II secretion system have a structural or sequence ortholog within the type IV pili system. The HR domain is shown to interact with the N0 domain of the secretin. The importance of this interaction is explored in the context of the functional secretion system. PMID:22253442

Dose and dose rate effects of whole-body gamma-irradiation: II. Hematological variables and cytokines

NASA Technical Reports Server (NTRS)

Gridley, D. S.; Pecaut, M. J.; Miller, G. M.; Moyers, M. F.; Nelson, G. A.

2001-01-01

The goal of part II of this study was to evaluate the effects of gamma-radiation on circulating blood cells, functional characteristics of splenocytes, and cytokine expression after whole-body irradiation at varying total doses and at low- and high-dose-rates (LDR, HDR). Young adult C57BL/6 mice (n = 75) were irradiated with either 1 cGy/min or 80 cGy/min photons from a 60Co source to cumulative doses of 0.5, 1.5, and 3.0 Gy. The animals were euthanized at 4 days post-exposure for in vitro assays. Significant dose- (but not dose-rate-) dependent decreases were observed in erythrocyte and blood leukocyte counts, hemoglobin, hematocrit, lipopolysaccharide (LPS)-induced 3H-thymidine incorporation, and interleukin-2 (IL-2) secretion by activated spleen cells when compared to sham-irradiated controls (p < 0.05). Basal proliferation of leukocytes in the blood and spleen increased significantly with increasing dose (p < 0.05). Significant dose rate effects were observed only in thrombocyte counts. Plasma levels of transforming growth factor-beta 1 (TGF-beta 1) and splenocyte secretion of tumor necrosis factor-alpha (TNF-alpha) were not affected by either the dose or dose rate of radiation. The data demonstrate that the responses of blood and spleen were largely dependent upon the total dose of radiation employed and that an 80-fold difference in the dose rate was not a significant factor in the great majority of measurements.

Inositol 1,4,5-trisphosphate receptor type II (InsP3R-II) is reduced in obese mice, but metabolic homeostasis is preserved in mice lacking InsP3R-II

PubMed Central

Feriod, Colleen N.; Nguyen, Lily; Jurczak, Michael J.; Kruglov, Emma A.; Nathanson, Michael H.; Shulman, Gerald I.; Bennett, Anton M.

2014-01-01

Inositol 1,4,5-trisphosphate receptor type II (InsP3R-II) is the most prevalent isoform of the InsP3R in hepatocytes and is concentrated under the canalicular membrane, where it plays an important role in bile secretion. We hypothesized that altered calcium (Ca2+) signaling may be involved in metabolic dysfunction, as InsP3R-mediated Ca2+ signals have been implicated in the regulation of hepatic glucose homeostasis. Here, we find that InsP3R-II, but not InsP3R-I, is reduced in the livers of obese mice. In our investigation of the functional consequences of InsP3R-II deficiency, we found that organic anion secretion at the canalicular membrane and Ca2+ signals were impaired. However, mice lacking InsP3R-II showed no deficits in energy balance, glucose production, glucose tolerance, or susceptibility to hepatic steatosis. Thus, our results suggest that reduced InsP3R-II expression is not sufficient to account for any disruptions in metabolic homeostasis that are observed in mouse models of obesity. We conclude that metabolic homeostasis is maintained independently of InsP3R-II. Loss of InsP3R-II does impair secretion of bile components; therefore, we suggest that conditions of obesity would lead to a decrease in this Ca2+-sensitive process. PMID:25315698

Role of the EGF-Related Growth Factor cripto in Murine Mammary Tumorigenesis

DTIC Science & Technology

2000-10-01

necessary for emergency repair or overhaul or (ii) is a release or disclosure of technical data (other than detailed manufacturing or process data) to, or...constructs that express secreted processed NODAL protein in transfected mammalian cells. We found that processing of unmodified NODAL protein is...NODAL processing (Constam and Robertson, 1999). As shown in our 1999 report, cleaved mature Nodal protein can be produced in the culture supernatants

Development of a quantitative assay amenable for high-throughput screening to target the type II secretion system for new treatments against plant-pathogenic bacteria.

PubMed

Tran, Nini; Zielke, Ryszard A; Vining, Oliver B; Azevedo, Mark D; Armstrong, Donald J; Banowetz, Gary M; McPhail, Kerry L; Sikora, Aleksandra E

2013-09-01

Plant-pathogenic bacteria are the causative agents of diseases in important agricultural crops and ornamental plants. The severe economic burden of these diseases requires seeking new approaches for their control, particularly because phytopathogenic bacteria are often resistant to available treatments. The type II secretion (T2S) system is a key virulence factor used by major groups of phytopathogenic bacteria. The T2S machinery transports many hydrolytic enzymes responsible for degradation of the plant cell wall, thus enabling successful colonization and dissemination of the bacteria in the plant host. The genetic inactivation of the T2S system leads to loss of virulence, which strongly suggests that targeting the T2S could enable new treatments against plant-pathogenic bacteria. Accordingly, we have designed and optimized an assay to identify small-molecule inhibitors of the T2S system. This assay uses a double parametric output: measurement of bacterial growth and the enzymatic activity of cellulase, which is secreted via the T2S pathway in our model organism Dickeya dadantii. The assay was evaluated by screening natural extracts, culture filtrates isolated from rhizosphere bacteria, and a collection of pharmaceutically active compounds in LOPAC(1280). The calculated Z' values of 0.63, 0.63, and 0.58, respectively, strongly suggest that the assay is applicable for a high-throughput screening platform.

Urinary retention and syndrome of inappropriate antidiuretic hormone secretion (SIADH) secondary to impacted gravid uterus.

PubMed

Irani, M; Fisher, N; Mor, A; Bensinger, G

2016-06-01

Urinary retention is an emergency that rarely occurs during pregnancy. Previous case reports have suggested multiple risk factors that can cause the gravid uterus to become impacted in the pelvis leading to lower bladder or urethral compression with subsequent urinary retention. However, no cases of urinary obstruction in a pregnancy that was complicated with severe electrolyte imbalance have been reported. To our knowledge, we report the first case of a 31-year-old woman presenting at 8 weeks' gestation with acute urinary retention caused by a retroflexed, retroverted uterus with a 6-cm posterior uterine fibroid leading to syndrome of inappropriate antidiuretic hormone secretion and severe hyponatremia requiring intensive care unit admission. The cornerstones of effective management of urinary retention should include: (i) urgent bladder catheterization; (ii) assessment of sodium levels to rule out syndrome of inappropriate antidiuretic hormone secretion, and prompt treatment before neurological damage occurs; (iii) reduction of the impacted uterus; and (iv) monitoring for post-obstructive diuresis. © 2016 Japan Society of Obstetrics and Gynecology.

The role of growth factors in maintenance of stemness in bone marrow-derived mesenchymal stem cells

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

Eom, Young Woo; Oh, Ji-Eun; Lee, Jong In

2014-02-28

Highlights: • Expression of FGF-2, FGF-4, EGF, and HGF decreased during long-term culture of BMSCs. • Loss of growth factors induced autophagy, senescence and decrease of stemness. • FGF-2 increased proliferation potential via AKT and ERK activation in BMSCs. • FGF-2 suppressed LC3-II expression and down-regulated senescence of BMSCs. • HGF was important in maintenance of the differentiation potential of BMSCs. - Abstract: Mesenchymal stem cells (MSCs) are an active topic of research in regenerative medicine due to their ability to secrete a variety of growth factors and cytokines that promote healing of damaged tissues and organs. In addition, thesemore » secreted growth factors and cytokines have been shown to exert an autocrine effect by regulating MSC proliferation and differentiation. We found that expression of EGF, FGF-4 and HGF were down-regulated during serial passage of bone marrow-derived mesenchymal stem cells (BMSCs). Proliferation and differentiation potentials of BMSCs treated with these growth factors for 2 months were evaluated and compared to BMSCs treated with FGF-2, which increased proliferation of BMSCs. FGF-2 and -4 increased proliferation potentials at high levels, about 76- and 26-fold, respectively, for 2 months, while EGF and HGF increased proliferation of BMSCs by less than 2.8-fold. Interestingly, differentiation potential, especially adipogenesis, was maintained only by HGF treatment. Treatment with FGF-2 rapidly induced activation of AKT and later induced ERK activation. The basal level of phosphorylated ERK increased during serial passage of BMSCs treated with FGF-2. The expression of LC3-II, an autophagy marker, was gradually increased and the population of senescent cells was increased dramatically at passage 7 in non-treated controls. But FGF-2 and FGF-4 suppressed LC3-II expression and down-regulated senescent cells during long-term (i.e. 2 month) cultures. Taken together, depletion of growth factors during serial passage could induce autophagy, senescence and down-regulation of stemness (proliferation via FGF-2/-4 and differentiation via HGF) through suppression of AKT and ERK signaling.« less

Structural and functional dissection reveals distinct roles of Ca2+-binding sites in the giant adhesin SiiE of Salmonella enterica

PubMed Central

Klingl, Stefan; Sandmann, Achim; Taccardi, Nicola; Sticht, Heinrich; Muller, Yves A.; Hensel, Michael

2017-01-01

The giant non-fimbrial adhesin SiiE of Salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. SiiE is a 595 kDa protein composed of 53 repetitive bacterial immunoglobulin (BIg) domains and the only known substrate of the SPI4-encoded type 1 secretion system (T1SS). The crystal structure of BIg50-52 of SiiE revealed two distinct Ca2+-binding sites per BIg domain formed by conserved aspartate or glutamate residues. In a mutational analysis Ca2+-binding sites were disrupted by aspartate to serine exchange at various positions in the BIg domains of SiiE. Amounts of secreted SiiE diminish with a decreasing number of intact Ca2+-binding sites. BIg domains of SiiE contain distinct Ca2+-binding sites, with type I sites being similar to other T1SS-secreted proteins and type II sites newly identified in SiiE. We functionally and structurally dissected the roles of type I and type II Ca2+-binding sites in SiiE, as well as the importance of Ca2+-binding sites in various positions of SiiE. Type I Ca2+-binding sites were critical for efficient secretion of SiiE and a decreasing number of type I sites correlated with reduced secretion. Type II sites were less important for secretion, stability and surface expression of SiiE, however integrity of type II sites in the C-terminal portion was required for the function of SiiE in mediating adhesion and invasion. PMID:28558023

Pseudomonas aeruginosa LasB protease impairs innate immunity in mice and humans by targeting a lung epithelial cystic fibrosis transmembrane regulator–IL-6–antimicrobial–repair pathway

PubMed Central

Saint-Criq, Vinciane; Villeret, Bérengère; Bastaert, Fabien; Kheir, Saadé; Hatton, Aurélie; Cazes, Aurélie; Xing, Zhou; Sermet-Gaudelus, Isabelle; Garcia-Verdugo, Ignacio; Edelman, Aleksander

2018-01-01

Background Pseudomonas aeruginosa lung infections are a huge problem in ventilator-associated pneumonia, cystic fibrosis (CF) and in chronic obstructive pulmonary disease (COPD) exacerbations. This bacterium secretes virulence factors that may subvert host innate immunity. Objective We evaluated the effect of P. aeruginosa elastase LasB, an important virulence factor secreted by the type II secretion system, on ion transport, innate immune responses and epithelial repair, both in vitro and in vivo. Methods Wild-type (WT) or cystic fibrosis transmembrane conductance regulator (CFTR)-mutated epithelial cells (cell lines and primary cells from patients) were treated with WT or ΔLasB pseudomonas aeruginosa O1 (PAO1) secretomes. The effect of LasB and PAO1 infection was also assessed in vivo in murine models. Results We showed that LasB was the most abundant protein in WT PAO1 secretomes and that it decreased epithelial CFTR expression and activity. In airway epithelial cell lines and primary bronchial epithelial cells, LasB degraded the immune mediators interleukin (IL)-6 and trappin-2, an important epithelial-derived antimicrobial molecule. We further showed that an IL-6/STAT3 signalling pathway was downregulated by LasB, resulting in inhibition of epithelial cell repair. In mice, intranasally instillated LasB induced significant weight loss, inflammation, injury and death. By contrast, we showed that overexpression of IL-6 and trappin-2 protected mice against WT-PAO1-induced death, by upregulating IL-17/IL-22 antimicrobial and repair pathways. Conclusions Our data demonstrate that PAO1 LasB is a major P. aeruginosa secreted factor that modulates ion transport, immune response and tissue repair. Targeting this virulence factor or upregulating protective factors such as IL-6 or antimicrobial molecules such as trappin-2 could be beneficial in P. aeruginosa-infected individuals. PMID:28790180

Small-Conductance Ca2+-Activated Potassium Channels Negatively Regulate Aldosterone Secretion in Human Adrenocortical Cells.

PubMed

Yang, Tingting; Zhang, Hai-Liang; Liang, Qingnan; Shi, Yingtang; Mei, Yan-Ai; Barrett, Paula Q; Hu, Changlong

2016-09-01

Aldosterone, which plays a key role in maintaining water and electrolyte balance, is produced by zona glomerulosa cells of the adrenal cortex. Autonomous overproduction of aldosterone from zona glomerulosa cells causes primary hyperaldosteronism. Recent clinical studies have highlighted the pathological role of the KCNJ5 potassium channel in primary hyperaldosteronism. Our objective was to determine whether small-conductance Ca(2+)-activated potassium (SK) channels may also regulate aldosterone secretion in human adrenocortical cells. We found that apamin, the prototypic inhibitor of SK channels, decreased membrane voltage, raised intracellular Ca(2+) and dose dependently increased aldosterone secretion from human adrenocortical H295R cells. By contrast, 1-Ethyl-2-benzimidazolinone, an agonist of SK channels, antagonized apamin's action and decreased aldosterone secretion. Commensurate with an increase in aldosterone production, apamin increased mRNA expression of steroidogenic acute regulatory protein and aldosterone synthase that control the early and late rate-limiting steps in aldosterone biosynthesis, respectively. In addition, apamin increased angiotensin II-stimulated aldosterone secretion, whereas 1-Ethyl-2-benzimidazolinone suppressed both angiotensin II- and high K(+)-stimulated production of aldosterone in H295R cells. These findings were supported by apamin-modulation of basal and angiotensin II-stimulated aldosterone secretion from acutely prepared slices of human adrenals. We conclude that SK channel activity negatively regulates aldosterone secretion in human adrenocortical cells. Genetic association studies are necessary to determine whether mutations in SK channel subtype 2 genes may also drive aldosterone excess in primary hyperaldosteronism. © 2016 American Heart Association, Inc.

Expression of MIF and CD74 in leukemic cell lines: correlation to DR expression destiny.

PubMed

Georgouli, Mirella; Papadimitriou, Lina; Glymenaki, Maria; Patsaki, Valia; Athanassakis, Irene

2016-06-01

Invariant chain (Ii) or CD74 is a non-polymorphic glycoprotein, which apart from its role as a chaperone dedicated to MHCII molecules, is known to be a high-affinity receptor for macrophage migration inhibitory factor (MIF). The present study aimed to define the roles of CD74 and MIF in the immune surveillance escape process. Towards this direction, the cell lines HL-60, Raji, K562 and primary pre-B leukemic cells were examined for expression and secretion of MIF. Flow cytometry analysis detected high levels of MIF and intracellular/membrane CD74 expression in all leukemic cells tested, while MIF secretion was shown to be inversely proportional to intracellular HLA-DR (DR) expression. In the MHCII-negative cells, IFN-γ increased MIF expression and induced its secretion in HL-60 and K562 cells, respectively. In K562 cells, CD74 (Iip33Iip35) was shown to co-precipitate with HLA-DOβ (DOβ), inhibiting thus MIF or DR binding. Induced expression of DOα in K562 (DOα-DOβ+) cells in different transfection combinations decreased MIF expression and secretion, while increasing surface DR expression. Thus, MIF could indeed be part of the antigen presentation process.

Pluripotency and a secretion mechanism of Drosophila transglutaminase.

PubMed

Shibata, Toshio; Kawabata, Shun-Ichiro

2018-03-01

Transglutaminase (TG) catalyses the formation of an isopeptide bond between glutamine and lysine residues and amine incorporation into specific glutamine residues. TG is conserved in all metazoans and functions both intracellularly and extracellularly. Here we review the existing knowledge of Drosophila TG with an emphasis on its pluripotency: Drosophila TG (i) plays a key role in cuticular morphogenesis, haemolymph coagulation, and entrapment against invading pathogens, (ii) suppresses the immune deficiency pathway to enable immune tolerance against commensal bacteria through the incorporation of polyamines into the nuclear factor-κB-like transcription factor Relish as well as through the protein-protein cross-linking of Relish, (iii) forms a physical matrix in the gut through cross-linking of chitin-binding proteins and (iv) is involved in the maintenance of homeostasis in microbiota in the gut. Moreover, we review the evidence that TG-A, one of alternative splicing-derived isoforms of Drosophila TG, is secreted through an endoplasmic reticulum/Golgi-independent pathway involving exosomes and fatty acylations.

Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila

EPA Science Inventory

Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrop...

Molecular Characterization of a Functional Type VI Secretion System from a Clinical Isolate of Aeromonas hydrophilia

EPA Science Inventory

Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrop...

Role of adipose secreted factors and kisspeptin in the metabolic control of gonadotropin secretion and puberty

USDA-ARS?s Scientific Manuscript database

Factors secreted by adipose tissue continue to be discovered. Evidence indicates a strong link between neural influences and adipocyte expression and secretion of a wide array of cytokines, neurotrophic factors, growth factors, binding proteins, and neuropeptides. These “adipokines” are linked to im...

Effects of taurodihydrofusidate, a bile salt analogue, on bile formation and biliary lipid secretion in the rhesus monkey.

PubMed Central

Beaudoin, M; Carey, M C; Small, D M

1975-01-01

Bile salts play a major role in bile formation and biliary lipid secretion. Sodium taurodihydrofusidate (TDHF), a derivative of the antibiotic fusidic acid, closely resembles bile salts in terms of structure, micellar characteristics, and capacity ot solubilize otherwise insolbule lipids. We have therefore studied the biliary secretion of this bile salt analogue and its influence on bile formation and biliary lipid secretion in primates. Alert, unanesthetized female rhesus monkeys prepared with a total biliary fistula were allowed to reach a steady bile salt secretion rate before each study. In three animals (group I),[14C]TDHF was infused intravenously. Most of the compound was secreted rapidly in bile chemically unchanged. The biliary secretion of this drug produced a twofold increase in bile flow; however, the bile salt output was markedly reduced during the infusion. In spite of this reduction, the phospholipid output remained essentially unchanged whereas the cholesterol output increased almost twofold. In five other animals (group II), the effect of TDHF on the bile salt secretion was further investigated by an intravenous infusion of [14C]taurocholate followed by a combined infusion of [14C]taurocholate and TDHF. When TDHF was added to the infusate, a reduction in the [14C]taurocholate output and a progressive rise in the plasma [14C]taurocholate concentration were observed in each animal. An analysis of the data in both groups indicates that (a) the most likely explanation to account for the decreased bile salt output is that the bile salt analogue, TDHF, interfered with bile salt secretion into the biliary canaliculi; (b) TDHF induces a greater secretion of biliary water than was observed with bile salts, an effect consistent with a stimulation of the bile salt-independent canalicular flow; (c) at similar 3alpha-hydroxysteroid secretion rates TDHF caused a significant increase in cholesterol secretion compared to that induced by bile salt. This finding suggests that TDHF affects cholesterol metabolism or secretion in a way distinct from bile salts. Thus, the solubilization of biliary lipids in mixed micelles, although essential, is only one of the factors which determine their secretion into bile. PMID:811689

Immunity in the Vagina (Part II): Anti-HIV Activity and Antiviral Content of Human Vaginal Secretions

PubMed Central

Patel, Mickey V.; Ghosh, Mimi; Fahey, John V.; Ochsenbauer, Christina; Rossoll, Richard M.; Wira, Charles R.

2015-01-01

Problem Whether the concentrations of antiviral proteins, and anti-HIV activity, within human vaginal secretions changes across the menstrual cycle is unknown. Method of Study Using a menstrual cup, vaginal secretions from premenopausal women were recovered at the proliferative (d6–8), mid-cycle (d13–15) and secretory (d21–23) stages of the menstrual cycle. Antiviral protein concentration was determined by ELISA, and anti-HIV activity assessed using the TZM-bl reporter cell line. Results CCL20, RANTES, elafin, HBD2, SDF-1α and IL-8 levels were detectable in the secretions. Vaginal secretions had anti-HIV activity against specific clade B strains of HIV, with significant inhibition of IIIB and increased infectivity of transmitted/founder CH077.t. No significant differences in either antiviral protein concentration or anti-HIV activity with respect to menstrual cycle stage were measured, but marked differences were observed in both parameters over the course of the cycle between different women, and in consecutive cycles from the same woman. Conclusion The vagina contains a complement of antiviral proteins. The variation in anti-HIV activity demonstrates that immune protection in the vagina is not constant. Intra- and inter-individual variations suggest that factors in addition to sex hormones influence antiviral protection. Lastly, the menstrual cup is a new model for recovering undiluted vaginal secretions from women throughout their reproductive life. PMID:24806967

Neonatal hyperthyroidism impairs epinephrine-provoked secretion of nerve growth factor and epidermal growth factor in mouse saliva.

PubMed

Lakshmanan, J; Landel, C P

1986-07-01

We examined long-term effects of neonatal hyperthyroidism on salivary secretions of nerve growth factor and epidermal growth factor in male and female mice at the age of 31 days. Hyperthyroidism was induced by thyroxine (T4) injections (0.4 microgram/g body weight/day) during days 0-6. Littermate control mice were treated with vehicle. T4 treatment did not alter the amounts of protein secreted into saliva but hormone administration induced alteration in the types of protein secreted. T4 treatment decreased the contents of both nerve growth factor and epidermal growth factor secreted into the saliva. A Sephadex G-200 column chromatographic profile revealed the presence of two distinct nerve growth factor immunoreactive peaks, while epidermal growth factor immunoreactivity predominantly eluted as a single low molecular weight form. T4 treatment did not alter the molecular nature of their secretion, but the treatment decreased their contents. These results indicate an impairment in salivary secretion of nerve growth factor and epidermal growth factor long after T4 treatment has been discontinued.

LPS-Challenged TNFα Production, Prostaglandin Secretion, and TNFα/TNFRs Expression in the Endometrium of Domestic Cats in Estrus or Diestrus, and in Cats with Pyometra or Receiving Medroxyprogesterone Acetate

PubMed Central

Jursza, Ewelina; Szóstek, Anna Z.; Kowalewski, Mariusz P.; Boos, Alois; Okuda, Kiyoshi; Siemieniuch, Marta J.

2014-01-01

Progesterone (P4) derivatives which are commonly used to block the cyclicity of domestic cats disturb the endocrine balance in the endometrium. The aims of this study were (i) to examine whether lipopolysaccharide (LPS) is responsible for enhancement of tumor necrosis factor-α (TNFα) secretion by the feline endometrial epithelial and stromal cells in vitro, (ii) to know whether immunolocalization of TNFα/TNFR1 and TNFR2 differs in cats at estrus or diestrus, receiving medroxyprogesterone acetate and suffering from pyometra, and (iii) to determine if TNFα-challenged prostaglandin secretion is stopped by prostaglandin synthases inhibitors. A total of 37 domestic adult cats in estrus or diestrus, receiving octane medroxyprogesterone or having clinical symptoms of pyometra, were enrolled in this study. The results obtained showed a distinct increase in LPS-challenged TNFα secretion in endometrial epithelial, but not stromal cells. TNFα augmented PG secretion was blocked by phospholipase A2 (PLA2) and cyclooxygeanase-2 (COX-2), but not by mitogen-activated protein kinase (MAPK) inhibitor. TNFα/TNFR1 and 2 protein expressions were limited mostly to the surface and glandular epithelium. TNFα/TNFRs protein was upregulated in the inflammatory uterus and hence may be involved in development of pathologic changes in the endometrial glands in cats receiving exogenous P4 as a hormonal contraceptive. PMID:25028529

Synthesis, characterization, molecular modeling and biological activity of metal complexes derived from (E)-N'-(furan-2-ylmethylene)morpholine-4-carbothiohydrazide

NASA Astrophysics Data System (ADS)

El-Samanody, El-Sayed A.; Emam, Sanaa M.; Emara, Esam M.

2017-10-01

A new series of some biologically active Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) complexes was synthesized from the novel thiosemicarbazone ligand; (E)-N'-(furan-2-ylmethylene)morpholine-4-carbothiohydrazide (HL). Elemental, spectral, thermal analyses, magnetic susceptibility and molar conductivity measurements were used to elucidate the structure of separated compounds. The data prove that the ligand reacts with all metal ions in a neutral thione form. The electrolytic tetra-coordinate Cu(II); Zn(II) complexes (5, 6; 10) bind through the thione sulfur, furfural oxygen and azomethine nitrogen atoms of the ligand (NSO type) to construct fused five membered rings. However, the rest non-electrolyte octahedral complexes chelate via the furfural oxygen and azomethine nitrogen atoms of the ligand (NO type). Molecular modeling was conducted for the ligand and two representative complexes (1, 5) in order to substantiate their chemical structures. Thermal analyses are compatible with molecular modeling studies to support the proposed thermal decomposition pathways of metal complexes which start with the rupture of the long and weak N-NH bond. The thermal stability of metal complexes varies according to the number of solvents of crystallization, ionic radii and steric effect of anions. The ESR spectra of Cu(II) complexes are compatible with a primarily (dx2-y2)1 ground state with axial symmetry. The ligand and its Co(II); Cu(II); Cd(II) complexes (1; 5, 8; 11) along with their mixtures with metaldehyde were screened in vitro for their molluscicidal activity against Eobania vermiculata. Combination with metaldehyde enhances the toxicity effect of the tested compounds through reducing the period required for mortality and increasing the percentage of mortality after 24 h of treatments. The tested compounds gathered with metaldehyde are strongly affecting on the activity of ACP and ALP enzymes and TP content which are very important factors in the mucous secretion of Eobania vermiculata. These effects lead to excess mucous secretion, causing dryness and death for the snails.

A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins.

PubMed

Tejeda-Dominguez, Farid; Huerta-Cantillo, Jazmin; Chavez-Dueñas, Lucia; Navarro-Garcia, Fernando

2017-03-28

The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia , require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens. IMPORTANCE The type 3 secretion system is essential for injection of virulence factors, which are delivered directly into the cytosol of the host cells for usurping and subverting host processes. Recent studies have shown that these effectors proteins indeed travel inside an "injectisome" conduit through a single step of translocation by connecting the bacterium and host cell cytoplasms. However, all findings are not compatible with this model. For example, both YopH, a protein detected on the surface of Yersinia , and EspC, an autotransporter protein secreted by enteropathogenic E. coli , require a functional T3SS for host cell translocation. Both proteins have an intermediate extracellular step before their T3SS-dependent translocation. Here, we show an alternative delivery mechanism for these extracellularly secreted virulence factors that are then incorporated into the T3SS to enter the cells; this novel mechanism coexists with but diverges from the canonical injection model that involves the passage of the protein inside the injectisome. Copyright © 2017 Tejeda-Dominguez et al.

Effect of basic fibroblast growth factor and transforming growth factor β1 on the healing of reconstructed dura by carbon dioxide laser soldering in minipigs.

PubMed

Zhong, Hong-liang; Wang, Zhen-min; Yang, Zhi-jun; Zhao, Fu; Wang, Bo; Wang, Zhong-cheng; Liu, Pi-nan

2012-02-01

Carbon dioxide (CO2) laser soldering is an alternative technique for tissue bonding. Basic fibroblast growth factor (bFGF) and transforming growth factor β(1) (TGFβ(1)) are two key factors for wound healing. This study was performed to demonstrate the efficacy of CO2 laser soldering for dural reconstruction and the effect of bFGF and TGFβ(1) on healing. In Part I, 10 minipigs were randomized into two equal groups. Dural defects were reconstructed by conventional fibrin glue bonding (group I(a)) or CO2 laser soldering (group I(b)). The reconstructed dura was subjected to burst pressure (BP) measurement and immunohistochemical staining after 1 week. In Part II, 36 minipigs were randomized into three equal groups. Dural reconstruction was achieved by CO2 laser soldering. Exogenous bFGF (group II(b)) or TGFβ(1) (group II(c)) was administered while group II(a) served as a control group. The specimens were subjected to BP measurement after 1, 2, 3, and 4 weeks, respectively. In Part I, the dura specimens displayed positive staining of only bFGF in group I(a) and of both bFGF and TGFβ(1) in group I(b). Group I(b) showed higher BP than group I(a) ((98.00 ± 21.41) mmHg vs. (70.80 ± 15.09) mmHg, respectively; P < 0.05). In Part II, BP of group II(c) was significantly higher than that of group II(a) (P < 0.01). The BP of group II(a) trended toward stabilization after 3 weeks of growth, while that of groups II(b) and II(c) trended toward stabilization after 2 weeks of growth. CO2 laser soldering is a reliable technique for dural reconstruction. The superior healing of dural reconstruction by CO2 laser soldering may be related to higher expression of bFGF and TGFβ(1), and CO2 lasers may stimulate their secretion. Exogenous bFGF or TGFβ(1) may improve healing by shortening the wound healing time, and exogenous TGFβ(1) may improve the tensile strength.

Kidney tubular-cell secretion of osteoblast growth factor is increased by kaempferol: a scientific basis for "the kidney controlling the bone" theory of Chinese medicine.

PubMed

Long, Mian; Li, Shun-xiang; Xiao, Jiang-feng; Wang, Jian; Lozanoff, Scott; Zhang, Zhi-guang; Luft, Benjamin J; Johnson, Francis

2014-09-01

To study, at the cytological level, the basic concept of Chinese medicine that "the Kidney (Shen) controls the bone". Kaempferol was isolated form Rhizoma Drynariae (Gu Sui Bu, GSB) and at several concentrations was incubated with opossum kidney (OK) cells, osteoblasts (MC3T3 E1) and human fibroblasts (HF) at cell concentrations of 2×10(4)/mL. Opossum kidney cell-conditioned culture media with kaempferol at 70 nmol/L (70kaeOKM) and without kaempferol (0OKM) were used to stimulate MC3T3 E1 and HF proliferation. The bone morphological protein receptors I and II (BMPR I and II) in OK cells were identified by immune-fluorescence staining and Western blot analysis. Kaempferol was found to increase OK cell growth (P<0.05), but alone did not promote MC3T3 E1 or HF cell proliferation. However, although OKM by itself increased MC3T3 E1 growth by 198% (P<0.01), the 70kaeOKM further increased the growth of these cells by an additional 127% (P<0.01). It indicates that the kidney cell generates a previously unknown osteoblast growth factor (OGF) and kaempferol increases kidney cell secretion of OGF. Neither of these media had any significant effect on HF growth. Kaempferol also was found to increase the level of the BMPR II in OK cells. This lends strong support to the original idea that the Kidney has a significant influence over bone-formation, as suggested by some long-standing Chinese medical beliefs, kaempferol may also serve to stimulate kidney repair and indirectly stimulate bone formation.

Effects of recombinant human keratinocyte growth factor on surfactant, plasma, and liver phospholipid homeostasis in hyperoxic neonatal rats.

PubMed

Raith, Marco; Schaal, Katharina; Koslowski, Roland; Fehrenbach, Heinz; Poets, Christian F; Schleicher, Erwin; Bernhard, Wolfgang

2012-04-01

Respiratory distress and bronchopulmonary dysplasia (BPD) are major problems in preterm infants that are often addressed by glucocorticoid treatment and increased oxygen supply, causing catabolic and injurious side effects. Recombinant human keratinocyte growth factor (rhKGF) is noncatabolic and antiapoptotic and increases surfactant pools in immature lungs. Despite its usefulness in injured neonatal lungs, the mechanisms of improved surfactant homeostasis in vivo and systemic effects on lipid homeostasis are unknown. We therefore exposed newborn rats to 85% vs. 21% oxygen and treated them systemically with rhKGF for 48 h before death at 7 days. We determined type II pneumocyte (PN-II) proliferation, surfactant protein (SP) mRNA expression, and the pulmonary metabolism of individual phosphatidylcholine (PC) species using [D(9)-methyl]choline and tandem mass spectrometry. In addition, we assessed liver and plasma lipid metabolism, addressing PC synthesis de novo, the liver-specific phosphatidylethanolamine methyl transferase (PEMT) pathway, and triglyceride concentrations. rhKGF was found to maintain PN-II proliferation and increased SP-B/C expression and surfactant PC in both normoxic and hyperoxic lungs. We found increased total PC together with decreased [D(9)-methyl]choline enrichment, suggesting decreased turnover rather than increased secretion and synthesis as the underlying mechanism. In the liver, rhKGF increased PC synthesis, both de novo and via PEMT, underlining the organotypic differences of rhKGF actions on lipid metabolism. rhKGF increased the hepatic secretion of newly synthesized polyunsaturated PC, indicating improved systemic supply with choline and essential fatty acids. We suggest that rhKGF has potential as a therapeutic agent in neonates by improving pulmonary and systemic PC homeostasis.

Significant hyperkalemia and hyponatremia secondary to telmisartan/hydrochlorothiazide treatment.

PubMed

Cakir, Mehtap

2010-12-01

The renin-angiotensin-aldosterone system (RAAS) has crucial importance in maintaining blood pressure; thus blockade of RAAS is an effective antihypertensive treatment choice. The final step in RAAS stimulation is aldosterone secretion by angiotensin II, which leads to increased renal tubular sodium absorption and potassium secretion. Angiotensin II receptor blockers (ARBs) allow blockade of RAAS by blocking binding of angiotensin II to the AT(1) receptors. There are several fixed-dose combinations of ARBs with hydrochlorothiazide in the market, providing antihypertensive therapies with complimentary mechanisms of action. With such combinations, while ARB inhibits the vasoconstricting action and aldosterone-secreting effects of angiotensin II, hydrochlorothiazide affects the renal tubular mechanisms of electrolyte reabsorption and directly increases excretion of sodium and chloride in the distal tubule, and promotes water excretion. Also, hypokalemia, which may be triggered by increased urinary potassium loss induced by hydrochlorothiazide, is opposed by ARB use and hence ARB/hydrochlorothiazide combination is known to be safe in terms of potassium imbalance. In this case report, significant hyperkalemia and hyponatremia related to telmisartan/hydrochlorothiazide use in a diabetic patient has been presented.

Brucella abortus down-regulates MHC class II by the IL-6-dependent inhibition of CIITA through the downmodulation of IFN regulatory factor-1 (IRF-1).

PubMed

Velásquez, Lis N; Milillo, M Ayelén; Delpino, M Victoria; Trotta, Aldana; Fernández, Pablo; Pozner, Roberto G; Lang, Roland; Balboa, Luciana; Giambartolomei, Guillermo H; Barrionuevo, Paula

2017-03-01

Brucella abortus is an intracellular pathogen capable of surviving inside of macrophages. The success of B. abortus as a chronic pathogen relies on its ability to orchestrate different strategies to evade the adaptive CD4 + T cell responses that it elicits. Previously, we demonstrated that B. abortus inhibits the IFN-γ-induced surface expression of MHC class II (MHC-II) molecules on human monocytes, and this phenomenon correlated with a reduction in antigen presentation. However, the molecular mechanisms, whereby B. abortus is able to down-regulate the expression of MHC-II, remained to be elucidated. In this study, we demonstrated that B. abortus infection inhibits the IFN-γ-induced transcription of MHC-II, transactivator (CIITA) and MHC-II genes. Accordingly, we observed that the synthesis of MHC-II proteins was also diminished. B. abortus was not only able to reduce the expression of mature MHC-II, but it also inhibited the expression of invariant chain (Ii)-associated immature MHC-II molecules. Outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, diminished the expression of MHC-II and CIITA transcripts to the same extent as B. abortus infection. IL-6 contributes to these down-regulatory phenomena. In addition, B. abortus and its lipoproteins, through IL-6 secretion, induced the transcription of the negative regulators of IFN-γ signaling, suppressor of cytokine signaling (SOCS)-1 and -3, without interfering with STAT1 activation. Yet, B. abortus lipoproteins via IL-6 inhibit the expression of IFN regulatory factor 1 (IRF-1), a critical regulatory transcription factor for CIITA induction. Overall, these results indicate that B. abortus inhibits the expression of MHC-II molecules at very early points in their synthesis and in this way, may prevent recognition by T cells establishing a chronic infection. © Society for Leukocyte Biology.

32 CFR 2800.6 - Delegation of classification and declassification authority.

Code of Federal Regulations, 2010 CFR

2010-07-01

.... 12065: (i) Staff Security Officer/Top Secret Control Officer. (ii) Assistant Staff Security Officer/Assistant Top Secret Control Officer. ... to originally classify and declassify information as “SECRET” and/or “CONFIDENTIAL”: (a) Chief of...

The role of extrinsic and intrinsic factors in the evolution of the control of pulmonary surfactant maturation during development in the amniotes.

PubMed

Sullivan, Lucy C; Orgeig, Sandra; Daniels, Christopher B

2003-01-01

Pulmonary surfactant is a mixture of lipids and proteins that is secreted by alveolar Type II cells. It reduces alveolar surface tension and hence the work of breathing. Despite the tremendous diversity of lung structures amongst the vertebrates, the composition of surfactant is highly conserved. Conserved elements of the surfactant system amongst distantly related species are likely to be crucial factors for successful lung development. Understanding the mechanisms by which the surfactant system becomes operational in animals with dramatically different birthing strategies and in distantly related species will provide important information about the role of the surfactant system in the commencement of air breathing and the processes regulating surfactant maturation and secretion. In mammals, the embryonic maturation of the surfactant system is controlled by a host of factors, including glucocorticoids, thyroid hormones, and autonomic neurotransmitters. Here we review the mechanisms controlling the maturation of surfactant production, including birthing strategy, phylogeny, lung structure, and posthatching environment. Using four species of egg-laying amniote (chicken, dragon lizard, sea turtle, and crocodile) previously described in detail and the large amount of information available for mammals, we examine the hypothesis that the control of surfactant production is dependent on glucocorticoids (dexamethasone [Dex]), thyroid hormones (T3), and autonomic neurotransmitters (epinephrine and carbachol). We also examine whether the overall intrinsic pattern of the control of surfactant maturation is conserved throughout the vertebrate radiation and then how the environment (extrinsic factors) may account for the observed differences in the patterns of development. We also discuss the utility of a coculture system of embryonic Type II cells and fibroblasts to determine the evolutionary pattern behind the control of surfactant and to demonstrate that the surfactant system matures under multihormonal control. We demonstrate that Dex and T3 are stimulators of surfactant production during embryonic development, but they lose their efficacy closer to hatching or birth. Epinephrine stimulates surfactant secretion beyond 75% of development and also after hatching or birth. Carbachol stimulates surfactant secretion in the bearded dragon and saltwater crocodile but not in the sea turtle, chicken, or mammals. It is likely that the differences in control of surfactant development are likely to be primarily related to metabolic activity and the duration of incubation (i.e., the "speed" of development). Moreover, the hormones examined appear important in promoting development and therefore appear conserved within the amniotes. However, the autonomic neurotransmitters induced different responses in different species. Hence, some factors are crucial for the proper maturation of the surfactant system, whereas others vary throughout evolution without being detrimental to the overall function of the system.

Granulopoietic Growth Factor Secretion in Ovarian Carcinoma as a Mechanism for the Emergence of Immune Suppressive Myeloid Subsets

DTIC Science & Technology

2014-08-01

levels will be determined and tracked biweekly during the course of tumor growth. Age/ gender -matched non-tumor-bearing mice will serve as controls for...parameter flow cytometry, we compared 21 ovarian cancer patients reflecting stages II - IV to 22 gender - race- and age-matched controls. All patient...Sitkovsky, M. A2A adenosine receptors protect tumors from anti- tumor T cells. (*equal authorship ) Proc. Natl. Acad. Sci. USA 103: 13132-13137, 2006

Transgenic mice overexpressing insulin-like growth factor-II in β cells develop type 2 diabetes

PubMed Central

Devedjian, Jean-Christophe; George, Monica; Casellas, Alba; Pujol, Anna; Visa, Joana; Pelegrín, Mireia; Gros, Laurent; Bosch, Fatima

2000-01-01

During embryonic development, insulin-like growth factor-II (IGF-II) participates in the regulation of islet growth and differentiation. We generated transgenic mice (C57BL6/SJL) expressing IGF-II in β cells under control of the rat Insulin I promoter in order to study the role of islet hyperplasia and hyperinsulinemia in the development of type 2 diabetes. In contrast to islets from control mice, islets from transgenic mice displayed high levels of IGF-II mRNA and protein. Pancreases from transgenic mice showed an increase in β-cell mass (about 3-fold) and in insulin mRNA levels. However, the organization of cells within transgenic islets was disrupted, with glucagon-producing cells randomly distributed throughout the core. We also observed enhanced glucose-stimulated insulin secretion and glucose utilization in islets from transgenic mice. These mice displayed hyperinsulinemia, mild hyperglycemia, and altered glucose and insulin tolerance tests, and about 30% of these animals developed overt diabetes when fed a high-fat diet. Furthermore, transgenic mice obtained from the N1 backcross to C57KsJ mice showed high islet hyperplasia and insulin resistance, but they also developed fatty liver and obesity. These results indicate that local overexpression of IGF-II in islets might lead to type 2 diabetes and that islet hyperplasia and hypersecretion of insulin might occur early in the pathogenesis of this disease. PMID:10727441

Protein-Phospholipid Interactions in Nonclassical Protein Secretion: Problem and Methods of Study

PubMed Central

Prudovsky, Igor; Kumar, Thallapuranam Krishnaswamy Suresh; Sterling, Sarah; Neivandt, David

2013-01-01

Extracellular proteins devoid of signal peptides use nonclassical secretion mechanisms for their export. These mechanisms are independent of the endoplasmic reticulum and Golgi. Some nonclassically released proteins, particularly fibroblast growth factors (FGF) 1 and 2, are exported as a result of their direct translocation through the cell membrane. This process requires specific interactions of released proteins with membrane phospholipids. In this review written by a cell biologist, a structural biologist and two membrane engineers, we discuss the following subjects: (i) Phenomenon of nonclassical protein release and its biological significance; (ii) Composition of the FGF1 multiprotein release complex (MRC); (iii) The relationship between FGF1 export and acidic phospholipid externalization; (iv) Interactions of FGF1 MRC components with acidic phospholipids; (v) Methods to study the transmembrane translocation of proteins; (vi) Membrane models to study nonclassical protein release. PMID:23396106

Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium.

PubMed

Michel, Gérard P F; Aguzzi, Anthony; Ball, Geneviève; Soscia, Chantal; Bleves, Sophie; Voulhoux, Romé

2011-07-01

Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

Integration of Golgi trafficking and growth factor signaling by the lipid phosphatase SAC1

PubMed Central

Blagoveshchenskaya, Anastasia; Cheong, Fei Ying; Rohde, Holger M.; Glover, Greta; Knödler, Andreas; Nicolson, Teresa; Boehmelt, Guido; Mayinger, Peter

2008-01-01

When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I–mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor–induced Golgi-to-ER shuttling of SAC1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery. PMID:18299350

Postprandial oxytocin secretion is associated with severity of anxiety and depressive symptoms in anorexia nervosa

PubMed Central

Lawson, Elizabeth A.; Holsen, Laura M.; Santin, McKale; DeSanti, Rebecca; Meenaghan, Erinne; Eddy, Kamryn T.; Herzog, David B.; Goldstein, Jill M.; Klibanski, Anne

2013-01-01

Objective Anorexia nervosa, a psychiatric disorder characterized by self-induced starvation, is associated with endocrine dysfunction and comorbid anxiety and depression. Animal data suggest that oxytocin may have anxiolytic and antidepressant effects. We have reported increased postprandial oxytocin levels in women with active anorexia nervosa (AN), and decreased levels in weight-recovered women with anorexia nervosa (ANWR) compared to healthy controls (HC). A meal may represent a significant source of stress in patients with disordered eating. We therefore investigated the association between post-prandial oxytocin secretion and symptoms of anxiety and depression in anorexia nervosa. Method We performed a cross-sectional study of 35 women (13 AN, 9 ANWR and 13 HC). Serum oxytocin and cortisol and plasma leptin levels were measured fasting and 30, 60, and 120min after a standardized mixed meal. The area under the curve (AUC), and for oxytocin, postprandial nadir and peak levels were determined. Anxiety and depressive symptoms were assessed using the Spielberger State-Trait Anxiety Inventory (STAI) and Beck Depression Inventory II (BDI-II). Results In women with anorexia nervosa, oxytocin AUC and post-prandial nadir and peak levels were positively associated with STAI scores. Oxytocin AUC and nadir levels were positively associated with BDI-II scores. After controlling for cortisol AUC, most relationships remained significant. After controlling for leptin AUC, all of the relationships remained significant. Oxytocin secretion explained up to 51% of the variance in STAI trait and 24% of BDI-II scores. Conclusions Abnormal post-prandial oxytocin secretion in women with anorexia nervosa is associated with increased symptoms of anxiety and depression. This may represent an adaptive response of oxytocin secretion to food-related symptoms of anxiety and depression. PMID:23759466

Testis composition and steroidogenic protein abundance in GnRH-II receptor knockdown boars

USDA-ARS?s Scientific Manuscript database

Testosterone, secreted from Leydig cells, is classically stimulated by luteinizing hormone (LH) from the anterior pituitary gland, but an LH-independent mechanism of testosterone production has also been identified in the boar. Gonadotropin-releasing hormone II (GnRH-II) and its receptor (GnRHR-II) ...

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines

PubMed Central

Nascimento, Rafael; Gouran, Hossein; Chakraborty, Sandeep; Gillespie, Hyrum W.; Almeida-Souza, Hebréia O.; Tu, Aye; Rao, Basuthkar J.; Feldstein, Paul A.; Bruening, George; Goulart, Luiz R.; Dandekar, Abhaya M.

2016-01-01

Pierce’s disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms. PMID:26753904

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines.

PubMed

Nascimento, Rafael; Gouran, Hossein; Chakraborty, Sandeep; Gillespie, Hyrum W; Almeida-Souza, Hebréia O; Tu, Aye; Rao, Basuthkar J; Feldstein, Paul A; Bruening, George; Goulart, Luiz R; Dandekar, Abhaya M

2016-01-12

Pierce's disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms.

Increase in Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) Had a Strong Impact on the Development of Type 2 Diabetes in Japanese Individuals with Impaired Insulin Secretion: The Saku Study

PubMed Central

Morimoto, Akiko; Tatsumi, Yukako; Soyano, Fumie; Miyamatsu, Naomi; Sonoda, Nao; Godai, Kayo; Ohno, Yuko; Noda, Mitsuhiko; Deura, Kijyo

2014-01-01

Our aim was to assess the impact of increase in homeostasis model assessment of insulin resistance (HOMA-IR) on the development of type 2 diabetes in Japanese individuals with impaired insulin secretion (IIS). This study included 2,209 participants aged 30–69 without diabetes at baseline who underwent comprehensive medical check-ups between April 2006 and March 2007 at Saku Central Hospital. Participants were classified into eight groups according to the combination of baseline IIS status (non-IIS and IIS) and category of HOMA-IR change between the baseline and follow-up examinations (decrease, no change/small increase, moderate increase, and large increase). Type 2 diabetes was determined from fasting and 2 h post-load plasma glucose concentrations at the follow-up examination between April 2009 and March 2011. At baseline, 669 individuals (30.3%) were classified as having IIS. At follow-up, 74 individuals developed type 2 diabetes. After adjusting for confounding factors including baseline HOMA-IR values, the multivariable-adjusted odds ratios (95% confidence intervals) for type 2 diabetes in the non-IIS with a decrease (mean change in HOMA-IR: −0.47), non-IIS with a moderate increase (mean change in HOMA-IR: 0.28), non-IIS with a large increase (mean change in HOMA-IR: 0.83), IIS with a decrease (mean change in HOMA-IR: −0.36), IIS with no change/small increase (mean change in HOMA-IR: 0.08), IIS with a moderate increase (mean change in HOMA-IR: 0.27), and IIS with a large increase (mean change in HOMA-IR: 0.73) groups, relative to the non-IIS with no change/small increase (mean change in HOMA-IR: 0.08) group were 0.23 (0.04, 1.11), 1.22 (0.26, 5.72), 2.01 (0.70, 6.46), 1.37 (0.32, 4.28), 3.60 (0.83, 15.57), 5.24 (1.34, 20.52), and 7.01 (1.75, 24.18), respectively. Moderate and large increases in HOMA-IR had a strong impact on the development of type 2 diabetes among individuals with IIS in this Japanese population. PMID:25166121

Increase in homeostasis model assessment of insulin resistance (HOMA-IR) had a strong impact on the development of type 2 diabetes in Japanese individuals with impaired insulin secretion: the Saku study.

PubMed

Morimoto, Akiko; Tatsumi, Yukako; Soyano, Fumie; Miyamatsu, Naomi; Sonoda, Nao; Godai, Kayo; Ohno, Yuko; Noda, Mitsuhiko; Deura, Kijyo

2014-01-01

Our aim was to assess the impact of increase in homeostasis model assessment of insulin resistance (HOMA-IR) on the development of type 2 diabetes in Japanese individuals with impaired insulin secretion (IIS). This study included 2,209 participants aged 30-69 without diabetes at baseline who underwent comprehensive medical check-ups between April 2006 and March 2007 at Saku Central Hospital. Participants were classified into eight groups according to the combination of baseline IIS status (non-IIS and IIS) and category of HOMA-IR change between the baseline and follow-up examinations (decrease, no change/small increase, moderate increase, and large increase). Type 2 diabetes was determined from fasting and 2 h post-load plasma glucose concentrations at the follow-up examination between April 2009 and March 2011. At baseline, 669 individuals (30.3%) were classified as having IIS. At follow-up, 74 individuals developed type 2 diabetes. After adjusting for confounding factors including baseline HOMA-IR values, the multivariable-adjusted odds ratios (95% confidence intervals) for type 2 diabetes in the non-IIS with a decrease (mean change in HOMA-IR: -0.47), non-IIS with a moderate increase (mean change in HOMA-IR: 0.28), non-IIS with a large increase (mean change in HOMA-IR: 0.83), IIS with a decrease (mean change in HOMA-IR: -0.36), IIS with no change/small increase (mean change in HOMA-IR: 0.08), IIS with a moderate increase (mean change in HOMA-IR: 0.27), and IIS with a large increase (mean change in HOMA-IR: 0.73) groups, relative to the non-IIS with no change/small increase (mean change in HOMA-IR: 0.08) group were 0.23 (0.04, 1.11), 1.22 (0.26, 5.72), 2.01 (0.70, 6.46), 1.37 (0.32, 4.28), 3.60 (0.83, 15.57), 5.24 (1.34, 20.52), and 7.01 (1.75, 24.18), respectively. Moderate and large increases in HOMA-IR had a strong impact on the development of type 2 diabetes among individuals with IIS in this Japanese population.

Adsorption of biometals to monosodium titanate in biological environments

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

HOBBS, D.T.; MESSER, R. L. W.; LEWIS, J. B.

2005-06-06

Monosodium titanate (MST) is an inorganic sorbent/ion exchanger developed for the removal of radionuclides from nuclear wastes. We investigated the ability of MST to bind Cd(II), Hg(II), or Au(III) to establish the utility of MST for applications in environmental decontamination or medical therapy (drug delivery). Adsorption isotherms for MST were determined at pH 7-7.5 in water or phosphate-buffered saline. The extent of metal binding was determined spectroscopically by measuring the concentrations of the metals in solution before and after contact with the MST. Cytotoxic responses to MST were assessed using THP1 monocytes and succinate dehydrogenase activity. Monocytic activation by MSTmore » was assessed by TNF{alpha} secretion (ELISA) with or without lipopolysaccharide (LPS) activation. MST sorbed Cd(II), Hg(II), and Au(III) under conditions similar to that in physiological systems. MST exhibited the highest affinity for Cd(II) followed by Hg(II) and Au (III). MST (up to 100 mg/L) exhibited only minor (< 25% suppression of succinate dehydrogenase) cytotoxicity and did not trigger TNF{alpha} secretion nor modulate LPS-induced TNF{alpha} secretion from monocytes. MST exhibits high affinity for biometals with no significant biological liabilities in these introductory studies. MST deserves further scrutiny as a substance with the capacity to decontaminate biological environments or deliver metals in a controlled fashion.« less

Extracellular and intracellular cleavages of proBDNF required at two distinct stages of late-phase LTP

NASA Astrophysics Data System (ADS)

Pang, Petti T.; Nagappan, Guhan; Guo, Wei; Lu, Bai

2016-05-01

Although late-phase long-term potentiation (L-LTP) is implicated in long-term memory, its molecular mechanisms are largely unknown. Here we provide evidence that L-LTP can be divided into two stages: an induction stage (I) and a maintenance stage (II). Both stages require mature brain-derived neurotrophic factor (mBDNF), but involve distinct underlying mechanisms. Stage I requires secretion of existing proBDNF followed by extracellular cleavage by tPA/plasmin. Stage II depends on newly synthesized BDNF. Surprisingly, mBDNF at stage II is derived from intracellular cleavage of proBDNF by furin/PC1. Moreover, stage I involves BDNF-TrkB signaling mainly through MAP kinase, whereas all three signaling pathways (phospholipase C-γ, PI3 kinase, and MAP kinase) are required for the maintenance of L-LTP at stage II. These results reveal the molecular basis for two temporally distinct stages in L-LTP, and provide insights on how BDNF modulates this long-lasting synaptic alternation at two critical time windows.

The manifold phospholipases A of Legionella pneumophila - identification, export, regulation, and their link to bacterial virulence.

PubMed

Banerji, Sangeeta; Aurass, Philipp; Flieger, Antje

2008-04-01

The intracellular lung pathogen Legionella pneumophila expresses secreted and cell-associated phospholipase A (PLA) and lysophospholipase A (LPLA) activities belonging to at least three enzyme families. The first family consists of three secreted PLA and LPLA activities displaying the amino acid signature motif 'GDSL'; PlaA, PlaC and PlaD. The second group contains the cell-associated and very potent PLA/LPLA, PlaB. The third group, the patatin-like proteins, comprises 11 members. One patatin-like protein, PatA/VipD, shows LPLA and PLA activities and interferes with vesicular trafficking when expressed in yeast and therefore is possibly involved in the intracellular infection process. Likewise, members of the first two phospholipase families have roles in bacterial virulence because phospholipases are important virulence factors that have been shown to promote bacterial survival, spread and host cell modification/damage. The GDSL enzyme PlaA detoxifies cytolytic lysophospholipids, and PlaB shows contact-dependent haemolytic activity. PlaC acylates cholesterol, a lipid present in eukaryotic hosts but not in the bacterium. Many of the L. pneumophila PLAs are exported by the type II Lsp or the type IVB Dot/Icm secretion systems involved in virulence factor export. Moreover, the regulation of lipolytic activities depends on the transcriptional regulators LetA/S and RpoS, inducing the expression of virulence traits, and on posttranscriptional activators like the zinc metalloprotease ProA.

Differential effect of inhibitors of oligosaccharide processing on the secretion of thyrotropin from dispersed rodent pituitary cells.

PubMed

Stannard, B S; Gesundheit, N; Thotakura, N R; Gyves, P W; Ronin, C; Weintraub, B D

1989-12-15

We examined the effect of various inhibitors of oligosaccharide processing on the content and secretion of newly synthesized thyroid-stimulating hormone (TSH) from dispersed hypothyroid rodent pituitary cells. 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin, both inhibitors of glucosidases I and II, decreased intracellular TSH (to 60-76% of control) and secreted TSH (to 60-63% of control) after a 1-hour incubation (pulse) with [35S]methionine and an 8-hour incubation (chase) in isotope-free media. In contrast, deoxymannojirimycin and swainsonine, inhibitors of mannosidase I and II, respectively, increased both intracellular TSH (to 267-309% of control) and secreted TSH (to 192% of control) at 8 hours. TSH oligosaccharides synthesized in the presence of these glucosidase and mannosidase inhibitors were largely sensitive to endo-beta-N-acetylglucosaminidase H (endo H), confirming inhibition of processing. Despite differences in oligosaccharide structure, the in vitro bioactivities of these secreted TSH isoforms were nearly identical. These data confirm and extend previous work performed with 1-deoxynojirimycin suggesting that glucosylated high mannose forms of TSH are more susceptible to intracellular degradation. The novel finding that deoxymannojirimycin and swainsonine increase secreted and total TSH above control levels suggests that non-glucosylated high mannose forms as well as hybrid-type oligosaccharides may facilitate secretion and direct TSH away from a natural degradation pathway.

Vibrio parahaemolyticus Inhibition of Rho Family GTPase Activation Requires a Functional Chromosome I Type III Secretion System▿

PubMed Central

Casselli, Timothy; Lynch, Tarah; Southward, Carolyn M.; Jones, Bryan W.; DeVinney, Rebekah

2008-01-01

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis; however, its virulence mechanisms are not well understood. The identification of type III secreted proteins has provided candidate virulence factors whose functions are still being elucidated. Genotypic strain variability contributes a level of complexity to understanding the role of different virulence factors. The ability of V. parahaemolyticus to inhibit Rho family GTPases and cause cytoskeletal disruption was examined with HeLa cells. After HeLa cells were infected, intracellular Rho activation was inhibited in response to external stimuli. In vitro activation of Rho, Rac, and Cdc42 isolated from infected HeLa cell lysates was also inhibited, indicating that the bacteria were specifically targeting GTPase activation. The inhibition of Rho family GTPase activation was retained for clinical and environmental isolates of V. parahaemolyticus and was dependent on a functional chromosome I type III secretion system (CI-T3SS). GTPase inhibition was independent of hemolytic toxin genotype and the chromasome II (CII)-T3SS. Rho inhibition was accompanied by a shift in the total actin pool to its monomeric form. These phenotypes were abrogated in a mutant strain lacking the CI-T3S effector Vp1686, suggesting that the inhibiting actin polymerization may be a downstream effect of Vp1686-dependent GTPase inhibition. Although Vp1686 has been previously characterized as a potential virulence factor in macrophages, our findings reveal an effect on cultured HeLa cells. The ability to inhibit Rho family GTPases independently of the CII-T3SS and the hemolytic toxins may provide insight into the mechanisms of virulence used by strains lacking these virulence factors. PMID:18347050

Quantification of a Non-conventional Protein Secretion: The Low-Molecular-Weight FGF-2 Example.

PubMed

Arcondéguy, Tania; Touriol, Christian; Lacazette, Eric

2016-01-01

Quantification of secreted factors is most often measured with enzyme-linked immunosorbent assay (ELISA), Western Blot, or more recently with antibody arrays. However, some of these, like low-molecular-weight fibroblast growth factor-2 (LMW FGF-2; the 18 kDa form), exemplify a set of secreted but almost non-diffusible molecular actors. It has been proposed that phosphorylated FGF-2 is secreted via a non-vesicular mechanism and that heparan sulfate proteoglycans function as extracellular reservoir but also as actors for its secretion. Heparan sulfate is a linear sulfated polysaccharide present on proteoglycans found in the extracellular matrix or anchored in the plasma membrane (syndecan). Moreover the LMW FGF-2 secretion appears to be activated upon FGF-1 treatment. In order to estimate quantification of such factor export across the plasma membrane, technical approaches are presented (evaluation of LMW FGF-2: (1) secretion, (2) extracellular matrix reservoir, and (3) secretion modulation by surrounding factors) and the importance of such procedures in the comprehension of the biology of these growth factors is underlined.

Testicular gonadotropin-releasing hormone II receptor (GnRHR-II) knockdown constitutively impairs diurnal testosterone secretion in the boar

USDA-ARS?s Scientific Manuscript database

The second mammalian GnRH isoform (GnRH-II) and its specific receptor (GnRHR-II) are highly expressed in the testis, suggesting an important role in testis biology. Gene coding errors prevent the production of GnRH-II and GnRHR-II in many species, but both genes are functional in swine. We have demo...

Biological Mechanisms Underlying the Ultraviolet Radiation-Induced Formation of Skin Wrinkling and Sagging II: Over-Expression of Neprilysin Plays an Essential Role

PubMed Central

Imokawa, Genji; Nakajima, Hiroaki; Ishida, Koichi

2015-01-01

Our previous studies strongly indicated that the up-regulated activity of skin fibroblast-derived elastase plays a pivotal role in wrinkling and/or sagging of the skin via the impairment of elastic fiber configuration and the subsequent loss of skin elasticity. Fortunately, we succeeded in identifying human skin fibroblast-derived elastase as a previously known enzyme, neprilysin or neutral endopeptidase (NEP). We have also characterized epithelial-mesenchymal paracrine cytokine interactions between UVB-exposed-keratinocytes and dermal fibroblasts and found that interleukin-1α and granulocyte macrophage colony stimulatory factor (GM-CSF) are intrinsic cytokines secreted by UVB-exposed keratinocytes that stimulate the expression of neprilysin by fibroblasts. On the other hand, direct UVA exposure of human fibroblasts significantly stimulates the secretion of IL-6 and also elicits a significant increase in the gene expression of matrix metallo-protease(MMP)-1 as well as neprilysin (to a lesser extent), which is followed by distinct increases in their protein and enzymatic activity levels. Direct UVA exposure of human keratinocytes also stimulates the secretion of IL-6, IL-8 and GM-CSF but not of IL-1 and endothelin-1. These findings suggest that GM-CSF secreted by UVA-exposed keratinocytes as well as IL-6 secreted by UVA-exposed dermal fibroblasts play important and additional roles in UVA-induced sagging and wrinkling by up-regulation of neprilysin and MMP-1, respectively, in dermal fibroblasts. PMID:25856676

GnRHR-II knockdown swine have constitutively lower serum testosterone concentrations, impaired senstitivity to GnRH analogues and reduced semen quality

USDA-ARS?s Scientific Manuscript database

The second mammalian GnRH isoform (GnRH-II) and its specific receptor (GnRHR-II) are abundantly produced within swine testes. GnRHR-II localizes to porcine Leydig cells and exogenous GnRH-II treatment robustly stimulates testosterone production in vivo, despite minimal secretion of luteinizing hormo...

Pro-tumorigenic effects of transforming growth factor beta 1 in canine osteosarcoma.

PubMed

Portela, R F; Fadl-Alla, B A; Pondenis, H C; Byrum, M L; Garrett, L D; Wycislo, K L; Borst, L B; Fan, T M

2014-01-01

Transforming growth factor beta 1 (TGFβ1) is a pleiotropic cytokine that contributes to reparative skeletal remodeling by inducing osteoblast proliferation, migration, and angiogenesis. Organic bone matrix is the largest bodily reservoir for latent TGFβ1, and active osteoblasts express cognate receptors for TGFβ1 (TGFβRI and TGFβRII). During malignant osteolysis, TGFβ1 is liberated from eroded bone matrix and promotes local progression of osteotropic solid tumors by its mitogenic and prosurvival activities. Canine osteosarcoma (OS) cells will possess TGFβ1 signaling machinery. Blockade of TGFβ1 signaling will attenuate pro-tumorigenic activities in OS cells. Naturally occurring primary OS samples will express cognate TGFβ1 receptors; and in dogs with OS, focal malignant osteolysis will contribute to circulating TGFβ1 concentrations. Thirty-three dogs with appendicular OS. Expression of TGFβ1 and its cognate receptors, as well as the biologic effects of TGFβ1 blockade, was characterized in OS cells. Ten spontaneous OS samples were characterized for TGFβRI/II expressions by immunohistochemistry. In 33 dogs with OS, plasma TGFβ1 concentrations were quantified and correlated with bone resorption. Canine OS cells secrete TGFβ1, express cognate receptors, and TGFβ1 signaling blockade decreases proliferation, migration, and vascular endothelial growth factor secretion. Naturally occurring OS samples abundantly and uniformly express TGFβRI/II, and in OS-bearing dogs, circulating TGFβ1 concentrations correlate with urine N-telopeptide excretion. Canine OS cells possess TGFβ1 signaling machinery, potentially allowing for the establishment of an autocrine and paracrine pro-tumorigenic signaling loop. As such, TGFβ1 inhibitors might impede localized OS progression in dogs. Copyright © 2014 by the American College of Veterinary Internal Medicine.

Differential effects of LPS, IFN-gamma and TNF alpha on the secretion of lysozyme by individual human mononuclear phagocytes: relationship to cell maturity.

PubMed Central

Lewis, C E; McCarthy, S P; Lorenzen, J; McGee, J O

1990-01-01

Human mononuclear phagocytes can be activated to perform a variety of complex functions by exposure to the immunomodulators, lipopolysaccharide (LPS), interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF alpha). Although such activation often involves the release of various cytokines by monocytes and macrophages, little is known of the effects of such signals on their secretion of lysozyme (LZM). In this study, a reverse haemolytic plaque assay for LZM secretion is coupled with immunocytochemistry for the pan macrophage (CD68) marker, EBM/11. This enabled the direct effects of LPS, IFN-gamma and TNF alpha on the secretion of LZM by individual, immunoidentified human mononuclear phagocytes to be investigated. The overall secretion of this peptide by populations of freshly isolated or 3-day cultured monocytes was augmented by exposure for 6 hr to bacterial LPS, recombinant human IFN-gamma or recombinant human TNF alpha. Extension of the culture period for monocytes from 3 to 7 days prior to use in the assay resulted in higher levels of LZM secretion, which could be further increased by TNF alpha but not by LPS or IFN-gamma. Individual peritoneal macrophages activated by inflammation in vivo were uniform in their augmented LZM responses to TNF alpha, but a small subpopulation of human peritoneal macrophages, which may represent younger 'inflammatory' exudate macrophages, was seen to be preferentially responsive to the LZM-stimulating effects of LPS and IFN-gamma. These studies suggest that (i) secretion of LZM by human mononuclear phagocytes can be regulated by LPS and IFN-gamma, although the effects of these agents may be dependent upon the state of maturation and/or differentiation of the cells, and (ii) TNF alpha is a potent stimulant of LZM secretion by monocytes and macrophages irrespective of cell maturity. Images Figure 1 Figure 1 PMID:2107146

Salmonella-secreted Virulence Factors

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

Heffron, Fred; Niemann, George; Yoon, Hyunjin

In this short review we discuss secreted virulence factors of Salmonella, which directly affect Salmonella interaction with its host. Salmonella secretes protein to subvert host defenses but also, as discussed, to reduce virulence thereby permitting the bacteria to persist longer and more successfully disperse. The type III secretion system (TTSS) is the best known and well studied of the mechanisms that enable secretion from the bacterial cytoplasm to the host cell cytoplasm. Other secretion systems include outer membrane vesicles, which are present in all Gram-negative bacteria examined to date, two-partner secretion, and type VI secretion will also be addressed. Excellentmore » reviews of Salmonella secreted effectors have focused on themes such as actin rearrangements, vesicular trafficking, ubiquitination, and the activities of the virulence factors themselves. This short review is based on S. Typhimurium infection of mice because it is a model of typhoid like disease in humans. We have organized effectors in terms of events that happen during the infection cycle and how secreted effectors may be involved.« less

Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations: How bacterial secretion systems bias Brownian motion for efficient translocation of macromolecules.

PubMed

Hepp, Christof; Maier, Berenike

2017-10-01

Secretion systems enable bacteria to import and secrete large macromolecules including DNA and proteins. While most components of these systems have been identified, the molecular mechanisms of macromolecular transport remain poorly understood. Recent findings suggest that various bacterial secretion systems make use of the translocation ratchet mechanism for transporting polymers across the cell envelope. Translocation ratchets are powered by chemical potential differences generated by concentration gradients of ions or molecules that are specific to the respective secretion systems. Bacteria employ these potential differences for biasing Brownian motion of the macromolecules within the conduits of the secretion systems. Candidates for this mechanism include DNA import by the type II secretion/type IV pilus system, DNA export by the type IV secretion system, and protein export by the type I secretion system. Here, we propose that these three secretion systems employ different molecular implementations of the translocation ratchet mechanism. © 2017 The Authors. BioEssays Published by WILEY Periodicals, Inc.

Risk of developing pneumonia is enhanced by the combined traits of fluoroquinolone resistance and type III secretion virulence in respiratory isolates of Pseudomonas aeruginosa.

PubMed

Sullivan, Eva; Bensman, Joyce; Lou, Mimi; Agnello, Melissa; Shriner, Kimberly; Wong-Beringer, Annie

2014-01-01

To determine the differential association of host characteristics, antimicrobial resistance, and type III secretion system virulence of Pseudomonas aeruginosa isolates with respiratory syndromes in hospitalized adult patients. Retrospective, cohort study. Community teaching hospital. Two hundred eighteen consecutive adult patients with respiratory culture positive for P. aeruginosa between January 2005 to January 2010. Medical charts were reviewed to obtain demographic, laboratory, radiographic, and clinical information. Isolates were assayed by polymerase chain reaction for genes encoding the type III secretion system effectors (ExoU, ExoS, and PcrV) and for strain relatedness using randomly amplified polymorphic DNA analysis. Levofloxacin susceptibility was determined by broth microdilution. Patients were grouped by colonization, bronchitis, or pneumonia and were compared for differential risk of developing the clinical syndrome with respect to host and microbial characteristics. Half of the study cohort (54%, 117 of 218) had pneumonia, 32% (70 of 218) had bronchitis, and 14% (31 of 218) had colonization; in-hospital mortality was 35%, 11%, and 0%, respectively. Host factors strongly associated with pneumonia development were residence in long-term care facility, healthcare-associated acquisition of P. aeruginosa, higher Acute Physiology and Chronic Health Evaluation II score, presence of enteral feeding tube, mechanical ventilation, and recent history of pneumonia. Fluoroquinolone-resistant (57% vs 34%, 16%; p < 0.0001) and multidrug-resistant (36% vs 26%, 7%; p = 0.0045) strains were more likely to cause pneumonia than bronchitis or colonization, respectively. Analysis of host and microbial factors in a multivariate regression model yielded the combined traits of fluoroquinolone resistance and gene encoding the type III secretion system ExoU effector in P. aeruginosa as the single most significant predictor of pneumonia development. These results suggest that fluoroquinolone-resistant phenotype in a type III secretion system exoU strain background contributes toward the pathogenesis of P. aeruginosa in pneumonia.

GnRH-II and its receptor are critical regulators of testicular steroidogenesis in swine

USDA-ARS?s Scientific Manuscript database

The second mammalian form of GnRH (GnRH-II) and its receptor (GnRHR-II) are produced in one livestock species, the pig. However, the interaction of GnRH-II with its receptor does not stimulate gonadotropin secretion. Instead, both are abundantly produced in the gonads and have been implicated in aut...

Knockdown of the GnRH-II receptor in the porcine testis impairs the biosynthesis of 10 gonadal steroids

USDA-ARS?s Scientific Manuscript database

The second mammalian GnRH isoform (GnRH-II) and its cognate receptor (GnRHR-II) are poor modulators of gonadotropin secretion in swine. However, both are abundantly produced within the porcine testis suggesting an autocrine/paracrine role. Within the boar testis, GnRHR-II immunolocalizes to the plas...

Insulin-like growth factor-I (lGF-l): safety and efficacy.

PubMed

Laron, Zvi

2004-11-01

Insulin-like growth factor I (IGF-I) is a peptide synthesized mainly in the liver by stimulation by pituitary growth hormone (GH). It circulates almost entirely bound to its binding proteins. It is the anabolic effector hormone of GH. It is the only treatment in states of GH resistance such as Laron syndrome and blocking antibodies to human GH. As it suppresses insulin and GH secretion it has been used in states of insulin resistance including Type II diabetes mellitus. IGF-I is administered by once or twice daily injections. Adverse effects are mostly caused by overdosage. The usual daily dose in children ranges from 100-200 microg/kg.

Regulators of G-protein signaling 4 in adrenal gland: localization, regulation, and role in aldosterone secretion.

PubMed

Romero, Damian G; Zhou, Ming Yi; Yanes, Licy L; Plonczynski, Maria W; Washington, Tanganika R; Gomez-Sanchez, Celso E; Gomez-Sanchez, Elise P

2007-08-01

Regulators of G-protein signaling (RGS proteins) interact with Galpha subunits of heterotrimeric G-proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G-protein-coupled receptor (GPCR)-ligand interaction. Angiotensin II (Ang II) interacts with its GPCR in adrenal zona glomerulosa cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. On screening for adrenal zona glomerulosa-specific genes, we found that RGS4 was exclusively localized in the zona glomerulosa of the rat adrenal cortex. We studied RGS4 expression and regulation in the rat adrenal gland, including the signaling pathways involved, as well as the role of RGS4 in steroidogenesis in human adrenocortical H295R cells. We reported that RGS4 mRNA expression in the rat adrenal gland was restricted to the adrenal zonal glomerulosa and upregulated by low-salt diet and Ang II infusion in rat adrenal glands in vivo. In H295R cells, Ang II caused a rapid and transient increase in RGS4 mRNA levels mediated by the calcium/calmodulin/calmodulin-dependent protein kinase and protein kinase C pathways. RGS4 overexpression by retroviral infection in H295R cells decreased Ang II-stimulated aldosterone secretion. In reporter assays, RGS4 decreased Ang II-mediated aldosterone synthase upregulation. In summary, RGS4 is an adrenal gland zona glomerulosa-specific gene that is upregulated by aldosterone secretagogues, in vivo and in vitro, and functions as a negative feedback of Ang II-triggered intracellular signaling. Alterations in RGS4 expression levels or functions may be involved in deregulations of Ang II signaling and abnormal aldosterone secretion.

CD4+ T cell-mediated rejection of MHC class II-positive tumor cells is dependent on antigen secretion and indirect presentation on host APCs.

PubMed

Haabeth, Ole Audun Werner; Fauskanger, Marte; Manzke, Melanie; Lundin, Katrin U; Corthay, Alexandre; Bogen, Bjarne; Tveita, Anders Aune

2018-05-11

Tumor-specific CD4+ T cells have been shown to mediate efficient anti-tumor immune responses against cancer. Such responses can occur through direct binding to MHC class II (MHC II)-expressing tumor cells or indirectly via activation of professional antigen-presenting cells (APC) that take up and present the tumor antigen. We have previously shown that CD4+ T cells reactive against an epitope within the Ig light chain variable region of a murine B cell lymphoma can reject established tumors. Given the presence of MHC II molecules at the surface of lymphoma cells, we investigated whether MHC II-restricted antigen presentation on tumor cells alone was required for rejection. Variants of the A20 B lymphoma cell line that either secreted or intracellularly retained different versions of the tumor-specific antigen revealed that antigen secretion by the MHC II-expressing tumor cells was essential both for the priming and effector phase of CD4+ T cell-driven anti-tumor immune responses. Consistent with this, genetic ablation of MHC II in tumor cells, both in the case of B lymphoma and B16 melanoma, did not preclude rejection of tumors by tumor antigen-specific CD4+ T cells in vivo. These findings demonstrate that MHC class II expression on tumor cells themselves is not required for CD4+ T cell-mediated rejection, and that indirect display on host APC is sufficient for effective tumor elimination. These results support the importance of tumor-infiltrating APC as mediators of tumor cell killing by CD4+ T cells. Copyright ©2018, American Association for Cancer Research.

Gonadotropin-releasing hormone II receptor (GnRHR-II) knockdown reduces testis size and decreases testosterone secretion during pubertal development in swine

USDA-ARS?s Scientific Manuscript database

The second mammalian isoform of gonadotropin-releasing hormone (GnRH-II) functions quite differently from the classical form (GnRH-I) as it is a poor stimulator of gonadotropin release. Unlike most species, a functional GnRHR-II has been identified in swine. Our laboratory detected GnRHR-IIs on Leyd...

Evolving concepts on regulation and function of renin in distal nephron

PubMed Central

Prieto, Minolfa C.; Gonzalez, Alexis A.

2012-01-01

Sustained stimulation of the intrarenal/intratubular renin–angiotensin system in a setting of elevated arterial pressure elicits renal vasoconstriction, increased sodium reabsorption, proliferation, fibrosis, and eventual renal injury. Activation of luminal AT1 receptors in proximal and distal nephron segments by local Ang II formation stimulates various transport systems. Augmented angiotensinogen (AGT) production by proximal tubule cells increases AGT secretion contributing to increased proximal Ang II levels and leading to spillover of AGT into the distal nephron segments, as reflected by increased urinary AGT excretion. The increased distal delivery of AGT provides substrate for renin, which is expressed in principal cells of the collecting tubule and collecting ducts, and is also stimulated by AT1 receptor activation. Renin and prorenin are secreted into the tubular lumen and act on the AGT delivered from the proximal tubule to form more Ang I. The catalytic actions of renin and or prorenin may be enhanced by binding to prorenin receptors on the intercalated cells or soluble prorenin receptor secreted into the tubular fluid. There is also increased luminal angiotensin converting enzyme in collecting ducts facilitating Ang II formation leading to stimulation of sodium reabsorption via sodium channel and sodium/chloride co-transporter. Thus, increased collecting duct renin contributes to Ang II-dependent hypertension by augmenting distal nephron intra-tubular Ang II formation leading to sustained stimulation of sodium reabsorption and progression of hypertension. PMID:22990760

Stromal Cells Positively and Negatively Modulate the Growth of Cancer Cells: Stimulation via the PGE2-TNFα-IL-6 Pathway and Inhibition via Secreted GAPDH-E-Cadherin Interaction

PubMed Central

Kawada, Manabu; Inoue, Hiroyuki; Ohba, Shun-ichi; Yoshida, Junjiro; Masuda, Tohru; Yamasaki, Manabu; Usami, Ihomi; Sakamoto, Shuichi; Abe, Hikaru; Watanabe, Takumi; Yamori, Takao; Shibasaki, Masakatsu; Nomoto, Akio

2015-01-01

Fibroblast-like stromal cells modulate cancer cells through secreted factors and adhesion, but those factors are not fully understood. Here, we have identified critical stromal factors that modulate cancer growth positively and negatively. Using a cell co-culture system, we found that gastric stromal cells secreted IL-6 as a growth and survival factor for gastric cancer cells. Moreover, gastric cancer cells secreted PGE2 and TNFα that stimulated IL-6 secretion by the stromal cells. Furthermore, we found that stromal cells secreted glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Extracellular GAPDH, or its N-terminal domain, inhibited gastric cancer cell growth, a finding confirmed in other cell systems. GAPDH bound to E-cadherin and downregulated the mTOR-p70S6 kinase pathway. These results demonstrate that stromal cells could regulate cancer cell growth through the balance of these secreted factors. We propose that negative regulation of cancer growth using GAPDH could be a new anti-cancer strategy. PMID:25785838

Bacterial Superantigens Promote Acute Nasopharyngeal Infection by Streptococcus pyogenes in a Human MHC Class II-Dependent Manner

PubMed Central

Kasper, Katherine J.; Zeppa, Joseph J.; Wakabayashi, Adrienne T.; Xu, Stacey X.; Mazzuca, Delfina M.; Welch, Ian; Baroja, Miren L.; Kotb, Malak; Cairns, Ewa; Cleary, P. Patrick; Haeryfar, S. M. Mansour; McCormick, John K.

2014-01-01

Establishing the genetic determinants of niche adaptation by microbial pathogens to specific hosts is important for the management and control of infectious disease. Streptococcus pyogenes is a globally prominent human-specific bacterial pathogen that secretes superantigens (SAgs) as ‘trademark’ virulence factors. SAgs function to force the activation of T lymphocytes through direct binding to lateral surfaces of T cell receptors and class II major histocompatibility complex (MHC-II) molecules. S. pyogenes invariably encodes multiple SAgs, often within putative mobile genetic elements, and although SAgs are documented virulence factors for diseases such as scarlet fever and the streptococcal toxic shock syndrome (STSS), how these exotoxins contribute to the fitness and evolution of S. pyogenes is unknown. Here we show that acute infection in the nasopharynx is dependent upon both bacterial SAgs and host MHC-II molecules. S. pyogenes was rapidly cleared from the nasal cavity of wild-type C57BL/6 (B6) mice, whereas infection was enhanced up to ∼10,000-fold in B6 mice that express human MHC-II. This phenotype required the SpeA superantigen, and vaccination with an MHC –II binding mutant toxoid of SpeA dramatically inhibited infection. Our findings indicate that streptococcal SAgs are critical for the establishment of nasopharyngeal infection, thus providing an explanation as to why S. pyogenes produces these potent toxins. This work also highlights that SAg redundancy exists to avoid host anti-SAg humoral immune responses and to potentially overcome host MHC-II polymorphisms. PMID:24875883

Effect of a Histone Deacetylases Inhibitor of IL-18 and TNF-Alpha Secretion in Vitro.

PubMed

Dobreva, Zlatka Georgieva; Grigorov, Boncho Grigorov; Stanilova, Spaska Angelova

2018-02-15

Interleukin-18 (IL-18) and Tumor Necrosis Factor-alpha (TNF-α) are proinflammatory cytokines that increased the development of Th1 immune response, but have a different type of regulation of the gene expression. Whereas TNF-α has an inducible expression, IL-18 is translated as an inactive protein and required proteolytic cleavage by Casp-1 in inflammasome complexes. To investigate the effect of the histone deacetylases inhibitor Suberoylanilide Hydroxamic Acid (SAHA) on the gene expression and secretion of both cytokines, IL-18 and TNF-α, according to their contribution to the cancer development and anticancer immunity. Isolated peripheral blood mononuclear cells (PBMC) were stimulated with LPS and C3bgp with or without SAHA. Cytokine production was assessed by ELISA at 6 and 24h. IL-18 and TNF-α secretion was significantly increased at 6h and 24h in response to stimulation. TNF-α production from stimulated PBMC was downregulated by SAHA at 6 and 24h. Treatment with SAHA does not inhibit the secretion of IL-18 significantly either at 6 or 24h of stimulation. The inhibition of histone deacetylases by SAHA does not influence the inflammasome-dependent production of immunologically active IL-18. In contrast, the production of proinflammatory TNF-α in cultures was mediated by the activity of HDAC class I and class II enzymes.

Mechanisms of Hepatocyte Growth Factor Activation in Cancer Tissues

PubMed Central

Kawaguchi, Makiko; Kataoka, Hiroaki

2014-01-01

Hepatocyte growth factor/scatter factor (HGF/SF) plays critical roles in cancer progression through its specific receptor, MET. HGF/SF is usually synthesized and secreted as an inactive proform (pro-HGF/SF) by stromal cells, such as fibroblasts. Several serine proteases are reported to convert pro-HGF/SF to mature HGF/SF and among these, HGF activator (HGFA) and matriptase are the most potent activators. Increased activities of both proteases have been observed in various cancers. HGFA is synthesized mainly by the liver and secreted as an inactive pro-form. In cancer tissues, pro-HGFA is likely activated by thrombin and/or human kallikrein 1-related peptidase (KLK)-4 and KLK-5. Matriptase is a type II transmembrane serine protease that is expressed by most epithelial cells and is also synthesized as an inactive zymogen. Matriptase activation is likely to be mediated by autoactivation or by other trypsin-like proteases. Recent studies revealed that matriptase autoactivation is promoted by an acidic environment. Given the mildly acidic extracellular environment of solid tumors, matriptase activation may, thus, be accelerated in the tumor microenvironment. HGFA and matriptase activities are regulated by HGFA inhibitor (HAI)-1 (HAI-1) and/or HAI-2 in the pericellular microenvironment. HAIs may have an important role in cancer cell biology by regulating HGF/SF-activating proteases. PMID:25268161

Effect of brain-derived neurotrophic factor (BDNF) on sperm quality of normozoospermic men.

PubMed

Safari, Hassan; Khanlarkhani, Neda; Sobhani, Aligholi; Najafi, Atefeh; Amidi, Fardin

2017-07-05

The neurotrophin family of proteins and their receptors act as important proliferative and pro-survival factors in differentiation of nerve cells and are thought to play key roles in the development of reproductive tissues and normal function of spermatozoa. The objective of the present study was to evaluate the effect of Brain-Derived Neurotrophic Factor (BDNF) on the sperm viability and motility, lipid peroxidation (LPO), mitochondrial activity and concentration of leptin, nitric oxide (NO) and insulin in normozoospermic men. Semen samples from 20 normozoospermic men were divided into three groups: (i) control, (ii) BDNF and (iii) BDNF + K252a. BDNF and K252a were added in the dose of 0.133 and 0.1 nM, respectively. Viability was assessed by eosin-nigrosin staining technique, and motility was observed by microscopy. NO concentration and mitochondrial activity were measured with flow cytometry, and LPO was analyzed using enzyme-linked immunosorbent assay (ELISA) kits. Results showed that exogenous BDNF at 0.133 nM could significantly (p < 0.05) influence viability, motility, NO concentration, mitochondrial activity and LPO content. Secretions of insulin and leptin by human sperm were increased in cells exposed to the exogenous BDNF, whereas viability, mitochondrial activity and insulin and leptin secretions were decreased in cells exposed to the K252.

The role of Shewanella oneidensis MR-1 outer surface structures in extracellular electron transfer

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

Bouhenni, Rachida; Vora, Gary J.; Biffinger, Justin C.

2010-04-20

Shewanella oneidensis is a facultative anaerobe that uses more than 14 different terminal electron acceptors for respiration. These include metal oxides and hydroxyoxides, and toxic metals such as uranium and chromium. Mutants deficient in metal reduction were isolated using the mariner transposon derivative, minihimar RB1. These included mutants with transposon insertions in the prepilin peptidase and type II secretion system genes. All mutants were deficient in Fe(III) and Mn(IV) reduction, and exhibited slow growth when DMSO was used as the electron acceptor. The genome sequence of S. oneidensis contains one prepilin peptidase gene, pilD. A similar prepilin peptidase that maymore » function in the processing of type II secretion prepilins was not found. Single and multiple chromosomal deletions of four putative type IV pilin genes did not affect Fe(III) and Mn(IV) reduction. These results indicate that PilD in S. oneidensis is responsible for processing both type IV and type II secretion prepilin proteins. Type IV pili do not appear to be required for Fe(III) and Mn(IV) reduction.« less

Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb.

PubMed

Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

2016-08-12

Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb*

PubMed Central

Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

2016-01-01

Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. PMID:27342778

Neural control of salivary glands in ixodid ticks.

PubMed

Šimo, Ladislav; Zitňan, Dušan; Park, Yoonseong

2012-04-01

Studies of tick salivary glands (SGs) and their components have produced a number of interesting discoveries over the last four decades. However, the precise neural and physiological mechanisms controlling SG secretion remain enigmatic. Major studies of SG control have identified and characterized many pharmacological and biological compounds that activate salivary secretion, including dopamine (DA), octopamine, γ-aminobutyric acid (GABA), ergot alkaloids, pilocarpine (PC), and their pharmacological relatives. Specifically, DA has shown the most robust activities in various tick species, and its effect on downstream actions in the SGs has been extensively studied. Our recent work on a SG dopamine receptor has aided new interpretations of previous pharmacological studies and provided new concepts for SG control mechanisms. Furthermore, our recent studies have suggested that multiple neuropeptides are involved in SG control. Myoinhibitory peptide (MIP) and SIFamide have been identified in the neural projections reaching the basal cells of acini types II and III. Pigment-dispersing factor (PDF)-immunoreactive neural projections reach type II acini, and RFamide- and tachykinin-immunoreactive projections reach the SG ducts, but the chemical nature of the latter three immunoreactive substances are unidentified yet. Here, we briefly review previous pharmacological studies and provide a revised summary of SG control mechanisms in ticks. Copyright © 2011 Elsevier Ltd. All rights reserved.

The impact of reduced gastric acid secretion on dissolution of salts of weak bases in the fasted upper gastrointestinal lumen: Data in biorelevant media and in human aspirates.

PubMed

Litou, Chara; Vertzoni, Maria; Xu, Wei; Kesisoglou, Filippos; Reppas, Christos

2017-06-01

To propose media for simulating the intragastric environment under reduced gastric acid secretion in the fasted state at three levels of simulation of the gastric environment and evaluate their usefulness in evaluating the intragastric dissolution of salts of weak bases. To evaluate the importance of bicarbonate buffer in biorelevant in vitro dissolution testing when using Level II biorelevant media simulating the environment in the fasted upper small intestine, regardless of gastric acid secretions. Media for simulating the hypochlorhydric and achlorhydric conditions in stomach were proposed using phosphates, maleates and bicarbonates buffers. The impact of bicarbonates in Level II biorelevant media simulating the environment in upper small intestine was evaluated so that pH and bulk buffer capacity were maintained. Dissolution data were collected using two model compounds, pioglitazone hydrochloride and semifumarate cocrystal of Compound B, and the mini-paddle dissolution apparatus in biorelevant media and in human aspirates. Simulated gastric fluids proposed in this study were in line with pH, buffer capacity, pepsin content, total bile salt/lecithin content and osmolality of the fasted stomach under partial and under complete inhibition of gastric acid secretion. Fluids simulating the conditions under partial inhibition of acid secretion were useful in simulating concentrations of both model compounds in gastric aspirates. Bicarbonates in Level III biorelevant gastric media and in Level II biorelevant media simulating the composition in the upper intestinal lumen did not improve simulation of concentrations in human aspirates. Level III biorelevant media for simulating the intragastric environment under hypochlorhydric conditions were proposed and their usefulness in the evaluation of concentrations of two model salts of weak bases in gastric aspirates was shown. Level II biorelevant media for simulating the environment in upper intestinal lumen led to underestimation of concentrations in aspirates, even when bicarbonate buffer was used. Copyright © 2017 Elsevier B.V. All rights reserved.

Postprandial oxytocin secretion is associated with severity of anxiety and depressive symptoms in anorexia nervosa.

PubMed

Lawson, Elizabeth A; Holsen, Laura M; Santin, McKale; DeSanti, Rebecca; Meenaghan, Erinne; Eddy, Kamryn T; Herzog, David B; Goldstein, Jill M; Klibanski, Anne

2013-05-01

Anorexia nervosa, a psychiatric disorder characterized by self-induced starvation, is associated with endocrine dysfunction and comorbid anxiety and depression. Animal data suggest that oxytocin may have anxiolytic and antidepressant effects. We have reported increased postprandial oxytocin levels in women with active anorexia nervosa and decreased levels in weight-recovered women with anorexia nervosa compared to healthy controls. A meal may represent a significant source of stress in patients with disordered eating. We therefore investigated the association between postprandial oxytocin secretion and symptoms of anxiety and depression in anorexia nervosa. We performed a cross-sectional study of 35 women (13 women with active anorexia nervosa, 9 with weight-recovered anorexia nervosa, and 13 healthy controls). Anorexia nervosa was diagnosed according to DSM-IV-TR criteria. Serum oxytocin and cortisol and plasma leptin levels were measured fasting and 30, 60, and 120 minutes after a standardized mixed meal. The area under the curve (AUC) and, for oxytocin, postprandial nadir and peak levels were determined. Anxiety and depressive symptoms were assessed using the Spielberger State-Trait Anxiety Inventory (STAI) and Beck Depression Inventory II (BDI-II). The study was conducted from January 2009 to March 2011. In women with anorexia nervosa, oxytocin AUC and postprandial nadir and peak levels were positively associated with STAI trait and STAI premeal and postmeal state scores. Oxytocin AUC and nadir levels were positively associated with BDI-II scores. After controlling for cortisol AUC, all of the relationships remained significant. After controlling for leptin AUC, most of the relationships remained significant. Oxytocin secretion explained up to 51% of the variance in STAI trait and 24% of the variance in BDI-II scores. Abnormal postprandial oxytocin secretion in women with anorexia nervosa is associated with increased symptoms of anxiety and depression. This link may represent an adaptive response of oxytocin secretion to food-related symptoms of anxiety and depression. © Copyright 2013 Physicians Postgraduate Press, Inc.

Tissue-specific expression of transgenic secreted ACE in vasculature can restore normal kidney functions, but not blood pressure, of Ace-/- mice.

PubMed

Chattopadhyay, Saurabh; Kessler, Sean P; Colucci, Juliana Almada; Yamashita, Michifumi; Senanayake, Preenie deS; Sen, Ganes C

2014-01-01

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE.

Tissue-Specific Expression of Transgenic Secreted ACE in Vasculature Can Restore Normal Kidney Functions, but Not Blood Pressure, of Ace-/- Mice

PubMed Central

Chattopadhyay, Saurabh; Kessler, Sean P.; Colucci, Juliana Almada; Yamashita, Michifumi; Senanayake, Preenie deS; Sen, Ganes C.

2014-01-01

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE. PMID:24475296

Active and separate secretion of fiber and penton base during the early phase of Ad2 or Ad5 infection

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

Yan, Yuhua; Zhang, Bo; Hou, Weihong

Fiber and penton base overproduced in adenovirus (Ad) infected cells can be secreted prior to progeny release and thereby regulate progeny spread. We aimed to investigate the mechanisms of fiber and penton base secretion in Ad2- or Ad5-infected A549 cells. Our flow cytometry analyses detected abundant surface fiber molecules, but little penton base molecules at 12 h post infection. Immunogold staining combined with transmission electron microscopic analyses revealed separate, non-co-localized release of fiber and penton base in the proximity of the plasma membrane. Depolymerization of microtubule and actin cytoskeletons, and inhibition of Rock kinase and myosin II activity together demonstratedmore » cytoskeletal network-dependent fiber secretion. Inhibition of intracellular calcium [Ca{sup 2+}]{sub i} signaling caused diminished fiber secretion, which was associated with diminished progeny production. Thus, fiber and penton base are actively and separately secreted during the early stages of Ad2 or Ad5 infection, their secretion may play important role in Ad life cycle. - Highlights: •Excessive production of structural proteins is common to viral infection, which may regulate the host-virus equilibrium and the spreading of viruses. •The adenovirus (Ad) structural proteins, fiber and penton base, are respectively important for Ad binding to its receptor and subsequent internalization in host cells. In Ad infected cells, these two structural proteins are excessively produced. •The mechanisms underlying the release of fiber and penton base molecules at the early phase of Ad infection is yet poorly understood. •Our studies show that in Ad5 or Ad2 infected A549 cells, fiber and penton base molecules are actively and separately secreted. •Fiber secretion is dependent on cytoskeleton-mediated protein traffic. •Inhibition of myosin II motor and Ca{sup 2+} signaling activity significantly diminishes fiber secretion. •These findings could contribute to our understanding of Ad spread in human populations.« less

Factors secreted from dental pulp stem cells show multifaceted benefits for treating experimental rheumatoid arthritis.

PubMed

Ishikawa, Jun; Takahashi, Nobunori; Matsumoto, Takuya; Yoshioka, Yutaka; Yamamoto, Noriyuki; Nishikawa, Masaya; Hibi, Hideharu; Ishigro, Naoki; Ueda, Minoru; Furukawa, Koichi; Yamamoto, Akihito

2016-02-01

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial hyperplasia and chronic inflammation, which lead to the progressive destruction of cartilage and bone in the joints. Numerous studies have reported that administrations of various types of MSCs improve arthritis symptoms in animal models, by paracrine mechanisms. However, the therapeutic effects of the secreted factors alone, without the cell graft, have been uncertain. Here, we show that a single intravenous administration of serum-free conditioned medium (CM) from human deciduous dental pulp stem cells (SHED-CM) into anti-collagen type II antibody-induced arthritis (CAIA), a mouse model of rheumatoid arthritis (RA), markedly improved the arthritis symptoms and joint destruction. The therapeutic efficacy of SHED-CM was associated with an induction of anti-inflammatory M2 macrophages in the CAIA joints and the abrogation of RANKL expression. SHED-CM specifically depleted of an M2 macrophage inducer, the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9), exhibited a reduced ability to induce M2-related gene expression and attenuate CAIA. SHED-CM also inhibited the RANKL-induced osteoclastogenesis in vitro. Collectively, our findings suggest that SHED-CM provides multifaceted therapeutic effects for treating CAIA, including the ED-Siglec-9-dependent induction of M2 macrophage polarization and inhibition of osteoclastogenesis. Thus, SHED-CM may represent a novel anti-inflammatory and reparative therapy for RA. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Brady, Robert T.; Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin; Advanced Materials and BioEngineering Research Centre

Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferationmore » and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting secretome between osteocytes and osteoblasts.« less

The Effect of α-Mating Factor Secretion Signal Mutations on Recombinant Protein Expression in Pichia pastoris

PubMed Central

Lin-Cereghino, Geoff P.; Stark, Carolyn M.; Kim, Daniel; Chang, Jennifer; Shaheen, Nadia; Poerwanto, Hansel; Agari, Kimiko; Moua, Pachai; Low, Lauren K.; Tran, Namphuong; Huang, Amy D.; Nattestad, Maria; Oshiro, Kristin T.; Chang, John William; Chavan, Archana; Tsai, Jerry W.; Lin-Cereghino, Joan

2013-01-01

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins are initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future. PMID:23454485

Complement Evasion by Pathogenic Leptospira.

PubMed

Fraga, Tatiana Rodrigues; Isaac, Lourdes; Barbosa, Angela Silva

2016-01-01

Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira . Pathogenic microorganisms, notably those which reach the blood circulation such as Leptospira , have evolved multiple strategies to escape the host complement system, which is important for innate and acquired immunity. Leptospira avoid complement-mediated killing through: (i) recruitment of host complement regulators; (ii) acquisition of host proteases that cleave complement proteins on the bacterial surface; and, (iii) secretion of proteases that inactivate complement proteins in the Leptospira surroundings. The recruitment of host soluble complement regulatory proteins includes the acquisition of Factor H (FH) and FH-like-1 (alternative pathway), C4b-binding protein (C4BP) (classical and lectin pathways), and vitronectin (Vn) (terminal pathway). Once bound to the leptospiral surface, FH and C4BP retain cofactor activity of Factor I in the cleavage of C3b and C4b, respectively. Vn acquisition by leptospires may result in terminal pathway inhibition by blocking C9 polymerization. The second evasion mechanism lies in plasminogen (PLG) binding to the leptospiral surface. In the presence of host activators, PLG is converted to enzymatically active plasmin, which is able to degrade C3b, C4b, and C5 at the surface of the pathogen. A third strategy used by leptospires to escape from complement system is the active secretion of proteases. Pathogenic, but not saprophytic leptospires, are able to secrete metalloproteases that cleave C3 (central complement molecule), Factor B (alternative pathway), and C4 and C2 (classical and lectin pathways). The purpose of this review is to fully explore these complement evasion mechanisms, which act together to favor Leptospira survival and multiplication in the host.

Complement Evasion by Pathogenic Leptospira

PubMed Central

Fraga, Tatiana Rodrigues; Isaac, Lourdes; Barbosa, Angela Silva

2016-01-01

Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira. Pathogenic microorganisms, notably those which reach the blood circulation such as Leptospira, have evolved multiple strategies to escape the host complement system, which is important for innate and acquired immunity. Leptospira avoid complement-mediated killing through: (i) recruitment of host complement regulators; (ii) acquisition of host proteases that cleave complement proteins on the bacterial surface; and, (iii) secretion of proteases that inactivate complement proteins in the Leptospira surroundings. The recruitment of host soluble complement regulatory proteins includes the acquisition of Factor H (FH) and FH-like-1 (alternative pathway), C4b-binding protein (C4BP) (classical and lectin pathways), and vitronectin (Vn) (terminal pathway). Once bound to the leptospiral surface, FH and C4BP retain cofactor activity of Factor I in the cleavage of C3b and C4b, respectively. Vn acquisition by leptospires may result in terminal pathway inhibition by blocking C9 polymerization. The second evasion mechanism lies in plasminogen (PLG) binding to the leptospiral surface. In the presence of host activators, PLG is converted to enzymatically active plasmin, which is able to degrade C3b, C4b, and C5 at the surface of the pathogen. A third strategy used by leptospires to escape from complement system is the active secretion of proteases. Pathogenic, but not saprophytic leptospires, are able to secrete metalloproteases that cleave C3 (central complement molecule), Factor B (alternative pathway), and C4 and C2 (classical and lectin pathways). The purpose of this review is to fully explore these complement evasion mechanisms, which act together to favor Leptospira survival and multiplication in the host. PMID:28066433

Distal Potassium Handling Based On Flow Modulation of Maxi-K Channel Activity

PubMed Central

Rodan, Aylin R.; Huang, Chou-Long

2011-01-01

Purpose of review Studies on the mechanisms of distal K+ secretion have highlighted the importance of the renal outer-medullary K+ (ROMK) and maxi-K channels. This review considers several human disorders characterized by hypo- and hyperkalemia, as well as mouse models of these disorders, and the mechanisms by which ROMK and maxi-K may be dysregulated. Recent findings Analysis of knockout mice lacking ROMK, a model for type II Bartter’s syndrome, has shown a role for maxi-K in distal K+ secretion. Knockout mice lacking either the α or β1 subunits of maxi-K also show deficits in flow-dependent K+ secretion. Analysis of transgenic and knock-in mouse models of pseudohypoaldsoteronism type II (PHA2), in which mutant forms of with-no-lysine kinase 4 (WNK4) are expressed, suggests ways in which ROMK and maxi-K may be dysregulated to result in hyperkalemia. Modeling studies also provide insights into the role of Na+ delivery versus flow in K+ secretion. Summary The importance of both ROMK and maxi-K to distal K+ secretion is now well-established, but the relative role each of these two channels plays in normal and diseased states has not been definitively established. Analysis of human and animal model data can generate hypotheses for future experiments. PMID:19448535

Biogenesis of the Saccharomyces cerevisiae Pheromone a-Factor, from Yeast Mating to Human Disease

PubMed Central

Barrowman, Jemima

2012-01-01

Summary: The mating pheromone a-factor secreted by Saccharomyces cerevisiae is a farnesylated and carboxylmethylated peptide and is unusually hydrophobic compared to other extracellular signaling molecules. Mature a-factor is derived from a precursor with a C-terminal CAAX motif that directs a series of posttranslational reactions, including prenylation, endoproteolysis, and carboxylmethylation. Historically, a-factor has served as a valuable model for the discovery and functional analysis of CAAX-processing enzymes. In this review, we discuss the three modules comprising the a-factor biogenesis pathway: (i) the C-terminal CAAX-processing steps carried out by Ram1/Ram2, Ste24 or Rce1, and Ste14; (ii) two sequential N-terminal cleavage steps, mediated by Ste24 and Axl1; and (iii) export by a nonclassical mechanism, mediated by the ATP binding cassette (ABC) transporter Ste6. The small size and hydrophobicity of a-factor present both challenges and advantages for biochemical analysis, as discussed here. The enzymes involved in a-factor biogenesis are conserved from yeasts to mammals. Notably, studies of the zinc metalloprotease Ste24 in S. cerevisiae led to the discovery of its mammalian homolog ZMPSTE24, which cleaves the prenylated C-terminal tail of the nuclear scaffold protein lamin A. Mutations that alter ZMPSTE24 processing of lamin A in humans cause the premature-aging disease progeria and related progeroid disorders. Intriguingly, recent evidence suggests that the entire a-factor pathway, including all three biogenesis modules, may be used to produce a prenylated, secreted signaling molecule involved in germ cell migration in Drosophila. Thus, additional prenylated signaling molecules resembling a-factor, with as-yet-unknown roles in metazoan biology, may await discovery. PMID:22933563

Human Leukocyte Antigen and Interleukin 2, 10 and 12p40 Cytokine Responses to Measles: Is There Evidence of the HLA Effect?

PubMed Central

Ovsyannikova, Inna G.; Ryan, Jenna E.; Jacobson, Robert M.; Vierkant, Robert A.; Pankratz, V. Shane; Poland, Gregory A.

2007-01-01

HLA class I and class II associations were examined in relation to measles virus-specific cytokine responses in 339 healthy children who had received two doses of live attenuated measles vaccine. Multivariate linear regression modeling analysis revealed suggestions of associations between the expression of DPA1*0201 (p=0.03) and DPA1*0202 (p=0.09) alleles and interleukin-2 (IL-2) cytokine production (global p-value 0.06). Importantly, cytokine production and DQB1 allele associations (global p-value 0.04) revealed that the alleles with the strongest association with IL-10 secretion were DQB1*0302 (p=0.02), DQB1*0303 (p=0.07) and DQB1*0502 (p=0.06). Measles-specific IL-10 secretion associations approached significance with DRB1 and DQA1 loci (both global p-values 0.08). Specifically, suggestive associations were found between DRB1*0701 (p=0.07), DRB1*1103 (p=0.06), DRB1*1302 (p=0.08), DRB1*1303 (p=0.06), DQA1*0101 (p=0.08), and DQA1*0201 (p=0.04) alleles and measles-induced IL-10 secretion. Further, suggestive association was observed between specific DQA1*0505 (p=0.002) alleles and measles-specific IL-12p40 secretion (global p-value 0.09) indicating that cytokine responses to measles antigens are predominantly influenced by HLA class II genes. We found no associations between any of the alleles of HLA A, B, and Cw loci and cytokine secretion. These novel findings suggest that HLA class II genes may influence the level of cytokine production in the adaptive immune responses to measles vaccine. PMID:17234427

NCI-H295R, a human adrenal cortex-derived cell line, expresses purinergic receptors linked to Ca²⁺-mobilization/influx and cortisol secretion.

PubMed

Nishi, Haruhisa; Arai, Hirokazu; Momiyama, Toshihiko

2013-01-01

Purinergic receptor expression and involvement in steroidogenesis were examined in NCI-H295R (H295R), a human adrenal cortex cell line which expresses all the key enzymes necessary for steroidogenesis. mRNA/protein for multiple P1 (A(2A) and A(2B)), P2X (P2X₅ and P2X₇), and P2Y (P2Y₁, P2Y₂, P2Y₆, P2Y₁₂, P2Y₁₃, and P2Y₁₄) purinergic receptors were detected in H295R. 2MeS-ATP (10-1000 µM), a P2Y₁ agonist, induced glucocorticoid (GC) secretion in a dose-dependent manner, while other extracellular purine/pyrimidine agonists (1-1000 µM) had no distinct effect on GC secretion. Extracellular purines, even non-steroidogenic ones, induced Ca²⁺-mobilization in the cells, independently of the extracellular Ca²⁺ concentration. Increases in intracellular Ca²⁺ concentration induced by extracellular purine agonists were transient, except when induced by ATP or 2MeS-ATP. Angiotensin II (AngII: 100 nM) and dibutyryl-cyclic AMP (db-cAMP: 500 µM) induced both GC secretion and Ca²⁺-mobilization in the presence of extracellular Ca²⁺ (1.2 mM). GC secretion by AngII was reduced by nifedipine (10-100 µM); whereas the Ca²⁺ channel blocker did not inhibit GC secretion by 2MeS-ATP. Thapsigargin followed by extracellular Ca²⁺ exposure induced Ca²⁺-influx in H295R, and the cells expressed mRNA/protein of the component molecules for store-operated calcium entry (SOCE): transient receptor C (TRPC) channels, calcium release-activated calcium channel protein 1 (Orai-1), and the stromal interaction molecule 1 (STIM1). In P2Y₁-knockdown, 2MeS-ATP-induced GC secretion was significantly inhibited. These results suggest that H295R expresses a functional P2Y₁ purinergic receptor for intracellular Ca²⁺-mobilization, and that P2Y₁ is linked to SOCE-activation, leading to Ca²⁺-influx which might be necessary for glucocorticoid secretion.

A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi.

PubMed

Login, Frédéric H; Fries, Markus; Wang, Xiaohui; Pickersgill, Richard W; Shevchik, Vladimir E

2010-05-01

The type II secretion system (T2SS) is widely exploited by proteobacteria to secrete enzymes and toxins involved in bacterial survival and pathogenesis. The outer membrane pore formed by the secretin OutD and the inner membrane protein OutC are two key components of the secretion complex, involved in secretion specificity. Here, we show that the periplasmic regions of OutC and OutD interact directly and map the interaction site of OutC to a 20-residue peptide named OutCsip (secretin interacting peptide, residues 139-158). This peptide interacts in vitro with two distinct sites of the periplasmic region of OutD, one located on the N0 subdomain and another overlapping the N2-N3' subdomains. The two interaction sites of OutD have different modes of binding to OutCsip. A single substitution, V143S, located within OutCsip prevents its interaction with one of the two binding sites of OutD and fully inactivates the T2SS. We show that the N0 subdomain of OutD interacts also with a second binding site within OutC located in the region proximal to the transmembrane segment. We suggest that successive interactions between these distinct regions of OutC and OutD may have functional importance in switching the secretion machine.

Insulin-like growth factor-I receptor activity is essential for Kaposi's sarcoma growth and survival.

PubMed

Catrina, S-B; Lewitt, M; Massambu, C; Dricu, A; Grünler, J; Axelson, M; Biberfeld, P; Brismar, K

2005-04-25

Kaposi's sarcoma (KS) is a highly vascular tumour and is the most common neoplasm associated with human immunodeficiency virus (HIV-1) infection. Growth factors, in particular vascular endothelial growth factor (VEGF), have been shown to play an important role in its development. The role of insulin-like growth factors (IGFs) in the pathophysiology of different tumours led us to evaluate the role of IGF system in KS. The IGF-I receptors (IGF-IR) were identified by immunohistochemistry in biopsies taken from patients with different AIDS/HIV-related KS stages and on KSIMM cells (an established KS-derived cell line). Insulin-like growth factor-I is a growth factor for KSIMM cells with a maximum increase of 3H-thymidine incorporation of 130 +/- 27.6% (P < 0.05) similar to that induced by VEGF and with which it is additive (281 +/- 13%) (P < 0.05). Moreover, specific blockade of the receptor (either by alpha IR3 antibody or by picropodophyllin, a recently described selective IGF-IR tyrosine phosphorylation inhibitor) induced KSIMM apoptosis, suggesting that IGF-IR agonists (IGF-I and -II) mediate antiapoptotic signals for these cells. We were able to identify an autocrine loop essential for KSIMM cell survival in which IGF-II is the IGF-IR agonist secreted by the cells. In conclusion, IGF-I pathway inhibition is a promising therapeutical approach for KS tumours.

Renal denervation attenuates aldosterone expression and associated cardiovascular pathophysiology in angiotensin II-induced hypertension.

PubMed

Hong, Mo-Na; Li, Xiao-Dong; Chen, Dong-Rui; Ruan, Cheng-Chao; Xu, Jian-Zhong; Chen, Jing; Wu, Yong-Jie; Ma, Yu; Zhu, Ding-Liang; Gao, Ping-Jin

2016-10-18

The sympathetic nervous system interacts with the renin-angiotensin-aldosterone system (RAAS) contributing to cardiovascular diseases. In this study, we sought to determine if renal denervation (RDN) inhibits aldosterone expression and associated cardiovascular pathophysiological changes in angiotensin II (Ang II)-induced hypertension. Bilateral RDN or SHAM operation was performed before chronic 14-day Ang II subcutaneous infusion (200ng/kg/min) in male Sprague-Dawley rats. Bilateral RDN blunted Ang II-induced hypertension and ameliorated the mesenteric vascular dysfunction. Cardiovascular hypertrophy in response to Ang II was significantly attenuated by RDN as shown by histopathology and transthoracic echocardiography. Moreover, Ang II-induced vascular and myocardial inflammation and fibrosis were suppressed by RDN with concurrent decrease in fibronectin and collagen deposition, macrophage infiltration, and MCP-1 expression. Interestingly, RDN also inhibited Ang II-induced aldosterone expression in the plasma, kidney and heart. This was associated with the reduction of calcitonin gene-related peptide (CGRP) in the adrenal gland. Ang II promoted aldosterone secretion which was partly attenuated by CGRP in the adrenocortical cell line, suggesting a protective role of CGRP in this model. Activation of transforming growth factor-β (TGF-β)/Smad and mitogen-activated protein kinases (MAPKs) signaling pathway was both inhibited by RDN especially in the heart. These results suggest that the regulation of the renal sympathetic nerve in Ang II-induced hypertension and associated cardiovascular pathophysiological changes is likely mediated by aldosterone, with CGRP involvement.

In vitro studies on immunotoxic potential of Orange II in splenocytes.

PubMed

Yadav, Ashish; Kumar, Arvind; Dwivedi, Premendra Dhar; Tripathi, Anurag; Das, Mukul

2012-02-05

Orange II, an azo dye, is not permitted in food preparations, but high levels of the dye have been detected in different food commodities. Though there are reports on the toxicity of Orange II but knowledge based on the immunomodulatory properties of Orange II is scanty. The present investigation was undertaken to study the in vitro immunotoxic potential of Orange II in splenocytes. Splenocytes were isolated, cultured and subjected to immunophenotypic analysis, mixed lymphocyte reaction (MLR) assay or stimulated with lipopolysaccharide (LPS) or concanavalin A (Con A) for 72 h. The supernatant was collected for cytokine assays. Orange II showed cytotoxic effects at 100-1000μg/ml concentrations and 50μg/ml was determined as the highest non-cytotoxic dose. Orange II at the non-cytotoxic dose (50μg/ml) significantly altered the relative distribution of T and B-cells, MLR response and the mitogen induced proliferative response of T-cells and B-cells. Consistent with the hypo-responsiveness of the T and B-lymphocytes, Orange II induced a concomitant decline in the secretion of cytokines IL-2, IL-4, IL-6, IFN-γ, TNF-α and IL-17. On the contrary, there was an increase in the production of IL-10, an anti-inflammatory regulatory cytokine, which may be one of the causative factor for immunosuppressive property of Orange II. These results suggest that non-cytotoxic dose of Orange II may have immunomodulatory effects. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Renal denervation attenuates aldosterone expression and associated cardiovascular pathophysiology in angiotensin II-induced hypertension

PubMed Central

Chen, Dong-Rui; Ruan, Cheng-Chao; Xu, Jian-Zhong; Chen, Jing; Wu, Yong-Jie; Ma, Yu; Zhu, Ding-Liang; Gao, Ping-Jin

2016-01-01

The sympathetic nervous system interacts with the renin-angiotensin-aldosterone system (RAAS) contributing to cardiovascular diseases. In this study, we sought to determine if renal denervation (RDN) inhibits aldosterone expression and associated cardiovascular pathophysiological changes in angiotensin II (Ang II)-induced hypertension. Bilateral RDN or SHAM operation was performed before chronic 14-day Ang II subcutaneous infusion (200ng/kg/min) in male Sprague-Dawley rats. Bilateral RDN blunted Ang II-induced hypertension and ameliorated the mesenteric vascular dysfunction. Cardiovascular hypertrophy in response to Ang II was significantly attenuated by RDN as shown by histopathology and transthoracic echocardiography. Moreover, Ang II-induced vascular and myocardial inflammation and fibrosis were suppressed by RDN with concurrent decrease in fibronectin and collagen deposition, macrophage infiltration, and MCP-1 expression. Interestingly, RDN also inhibited Ang II-induced aldosterone expression in the plasma, kidney and heart. This was associated with the reduction of calcitonin gene-related peptide (CGRP) in the adrenal gland. Ang II promoted aldosterone secretion which was partly attenuated by CGRP in the adrenocortical cell line, suggesting a protective role of CGRP in this model. Activation of transforming growth factor-β (TGF-β)/Smad and mitogen-activated protein kinases (MAPKs) signaling pathway was both inhibited by RDN especially in the heart. These results suggest that the regulation of the renal sympathetic nerve in Ang II-induced hypertension and associated cardiovascular pathophysiological changes is likely mediated by aldosterone, with CGRP involvement. PMID:27661131

Pavlovian Conditioning of Rat Mucosal Mast Cells to Secrete Rat Mast Cell Protease II

NASA Astrophysics Data System (ADS)

MacQueen, Glenda; Marshall, Jean; Perdue, Mary; Siegel, Shepard; Bienenstock, John

1989-01-01

Antigen (egg albumin) injections, which stimulate mucosal mast cells to secrete mediators, were paired with an audiovisual cue. After reexposure to the audiovisual cue, a mediator (rat mast cell protease II) was measured with a sensitive and specific assay. Animals reexposed to only the audiovisual cue released a quantity of protease not significantly different from animals reexposed to both the cue and the antigen; these groups released significantly more protease than animals that had received the cue and antigen in a noncontingent manner. The results support a role for the central nervous system as a functional effector of mast cell function in the allergic state.

Alamandine reduces leptin expression through the c-Src/p38 MAP kinase pathway in adipose tissue.

PubMed

Uchiyama, Tsuyoshi; Okajima, Fumikazu; Mogi, Chihiro; Tobo, Ayaka; Tomono, Shoichi; Sato, Koichi

2017-01-01

Obesity is associated with an increased risk of diabetes mellitus, hypertension, and renal dysfunction. Angiotensin 1-7 and alamandine are heptameric renin angiotensin system peptide hormones. Further, alamandine levels increase with renal dysfunction. In the cardiovascular system, angiotensin 1-7 and alamandine produce similar improvements and counterbalance angiotensin II in regulating vascular function. We aimed to determine whether the effect of alamandine on leptin expression and secretion in adipocytes was similar to that of angiotensin 1-7. We studied isolated peri-renal visceral adipose tissue and peri-renal isolated visceral adipocytes from male Wistar rats. Angiotensin II from 0.01 to 10nM had no effect on leptin expression. Angiotensin 1-7 (1 nM) increased leptin secretion and expression, whereas alamandine (1 nM) decreased leptin secretion and expression in adipose tissue and isolated adipocytes and reduced blood leptin levels in vivo. These effects were mediated by Gq, c-Src, p38 mitogen-activated protein, and IκB activation. Additionally, alamandine induced nitric oxide expression via inducible nitric oxidase synthase and plasminogen activator inhibitor 1 expression in adipose tissue and isolated adipocytes. Angiotensin 1-7 and alamandine produced opposing effects on leptin expression and secretion in adipose tissue. This result suggests that the action of Mas (angiotensin 1-7 receptor) and Mas-related G-protein coupled receptor D in adipocytes exhibited opposing actions similar to angiotensin II type 1 and type 2 receptors.

Social and cognitive factors associated with children’s secret-keeping for a parent

PubMed Central

Gordon, Heidi M.; Lyon, Thomas D.; Lee, Kang

2014-01-01

This study examined children’s secret-keeping for a parent and its relationship to trust, theory of mind, secrecy endorsement, and executive functioning (EF). Children (N = 107) between 4 and 12 years of age participated in a procedure wherein parents broke a toy and asked children to promise secrecy. Responses to open-ended and direct questions were examined. Overall, secret-keeping increased with age and promising to keep the secret was related to fewer disclosures in open-ended questioning. Children who kept the secret in direct questioning exhibited greater trust and better parental ratings of EF than children who disclosed the secret. Findings highlight the importance of both social and cognitive factors in secret-keeping development. PMID:25291258

Identification of secreted bacterial proteins by noncanonical amino acid tagging

PubMed Central

Mahdavi, Alborz; Szychowski, Janek; Ngo, John T.; Sweredoski, Michael J.; Graham, Robert L. J.; Hess, Sonja; Schneewind, Olaf; Mazmanian, Sarkis K.; Tirrell, David A.

2014-01-01

Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy. PMID:24347637

Calcium-dependent mitochondrial cAMP production enhances aldosterone secretion.

PubMed

Katona, Dávid; Rajki, Anikó; Di Benedetto, Giulietta; Pozzan, Tullio; Spät, András

2015-09-05

Glomerulosa cells secrete aldosterone in response to agonists coupled to Ca(2+) increases such as angiotensin II and corticotrophin, coupled to a cAMP dependent pathway. A recently recognized interaction between Ca(2+) and cAMP is the Ca(2+)-induced cAMP formation in the mitochondrial matrix. Here we describe that soluble adenylyl cyclase (sAC) is expressed in H295R adrenocortical cells. Mitochondrial cAMP formation, monitored with a mitochondria-targeted fluorescent sensor (4mtH30), is enhanced by HCO3(-) and the Ca(2+) mobilizing agonist angiotensin II. The effect of angiotensin II is inhibited by 2-OHE, an inhibitor of sAC, and by RNA interference of sAC, but enhanced by an inhibitor of phosphodiesterase PDE2A. Heterologous expression of the Ca(2+) binding protein S100G within the mitochondrial matrix attenuates angiotensin II-induced mitochondrial cAMP formation. Inhibition and knockdown of sAC significantly reduce angiotensin II-induced aldosterone production. These data provide the first evidence for a cell-specific functional role of mitochondrial cAMP. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Skeletal muscle hypertrophy and regeneration: interplay between the myogenic regulatory factors (MRFs) and insulin-like growth factors (IGFs) pathways.

PubMed

Zanou, Nadège; Gailly, Philippe

2013-11-01

Adult skeletal muscle can regenerate in response to muscle damage. This ability is conferred by the presence of myogenic stem cells called satellite cells. In response to stimuli such as injury or exercise, these cells become activated and express myogenic regulatory factors (MRFs), i.e., transcription factors of the myogenic lineage including Myf5, MyoD, myogenin, and Mrf4 to proliferate and differentiate into myofibers. The MRF family of proteins controls the transcription of important muscle-specific proteins such as myosin heavy chain and muscle creatine kinase. Different growth factors are secreted during muscle repair among which insulin-like growth factors (IGFs) are the only ones that promote both muscle cell proliferation and differentiation and that play a key role in muscle regeneration and hypertrophy. Different isoforms of IGFs are expressed during muscle repair: IGF-IEa, IGF-IEb, or IGF-IEc (also known as mechano growth factor, MGF) and IGF-II. MGF is expressed first and is observed in satellite cells and in proliferating myoblasts whereas IGF-Ia and IGF-II expression occurs at the state of muscle fiber formation. Interestingly, several studies report the induction of MRFs in response to IGFs stimulation. Inversely, IGFs expression may also be regulated by MRFs. Various mechanisms are proposed to support these interactions. In this review, we describe the general process of muscle hypertrophy and regeneration and decipher the interactions between the two groups of factors involved in the process.

Cryptography; An Introductory Bibliography of Books and Periodical Articles.

DTIC Science & Technology

1982-06-05

Martin’s H25 Pr., c1976. D810 Johnson, Brian. The Secret War, N.Y., NY: Methuen, c1978. $2J6 D810 Jones, Reginald Victor. The Wizard War: British...Park Pr., c1976. UB290 Langie, Andre. Cryptography, London, Eng.: Constable & L3 Co., Ltd., c1922. D810 Lawson, Don. The Secret World War II, N.Y., NY...Writing, N.Y., NY: W.W. Norton & Co., c1943. Z6724 Smith, Myron J., Jr. The Secret Wars, Vol. 1: Intelligence, I7 Propaganda and Psychological Warfare

Production and action of cytokines in space

NASA Technical Reports Server (NTRS)

Chapes, Stephen K.; Morrison, Dennis R.; Guikema, James A.; Lewis, Marian L.; Spooner, Brian S.

1994-01-01

B6MP102 cells, a continuously cultured murine bone marrow macrophage cell line, were tested for secretion of tumor necrosis factor-alpha and Interleukin-1 during space flight. We found that B6MP102 cells secreted more tumor necrosis factor-alpha and interleukin-1 when stimulated in space with lipopolysaccharide than controls similarly stimulated on earth. This compared to increased secretion of interferon-beta and -gamma by lymphocytes that was measured on the same shuttle flights. Although space flight enhanced B6MP102 secretion of tumor necrosis factor-alpha, an experiment on a subsequent space flight (STS-50) found that cellular cytotoxicity, mediated by tumor necrosis factor-alpha, was inhibited.

Thrombospondin-1 protects against pathogen-induced lung injury by limiting extracellular matrix proteolysis

PubMed Central

Qu, Yanyan; Olonisakin, Tolani; Bain, William; Zupetic, Jill; Brown, Rebecca; Hulver, Mei; Xiong, Zeyu; Shanks, Robert M.Q.; Bomberger, Jennifer M.; Cooper, Vaughn S.; Zegans, Michael E.; Han, Jongyoon; Pilewski, Joseph; Ray, Anuradha; Ray, Prabir; Lee, Janet S.

2018-01-01

Acute lung injury is characterized by excessive extracellular matrix proteolysis and neutrophilic inflammation. A major risk factor for lung injury is bacterial pneumonia. However, host factors that protect against pathogen-induced and host-sustained proteolytic injury following infection are poorly understood. Pseudomonas aeruginosa (PA) is a major cause of nosocomial pneumonia and secretes proteases to amplify tissue injury. We show that thrombospondin-1 (TSP-1), a matricellular glycoprotein released during inflammation, dose-dependently inhibits PA metalloendoprotease LasB, a virulence factor. TSP-1–deficient (Thbs1–/–) mice show reduced survival, impaired host defense, and increased lung permeability with exaggerated neutrophil activation following acute intrapulmonary PA infection. Administration of TSP-1 from platelets corrects the impaired host defense and aberrant injury in Thbs1–/– mice. Although TSP-1 is cleaved into 2 fragments by PA, TSP-1 substantially inhibits Pseudomonas elastolytic activity. Administration of LasB inhibitor, genetic disabling of the PA type II secretion system, or functional deletion of LasB improves host defense and neutrophilic inflammation in mice. Moreover, TSP-1 provides an additional line of defense by directly subduing host-derived proteolysis, with dose-dependent inhibition of neutrophil elastase from airway neutrophils of mechanically ventilated critically ill patients. Thus, a host matricellular protein provides dual levels of protection against pathogen-initiated and host-sustained proteolytic injury following microbial trigger. PMID:29415890

The CpxRA two-component system contributes to Legionella pneumophila virulence.

PubMed

Tanner, Jennifer R; Li, Laam; Faucher, Sébastien P; Brassinga, Ann Karen C

2016-06-01

The bacterium Legionella pneumophila is capable of intracellular replication within freshwater protozoa as well as human macrophages, the latter of which results in the serious pneumonia Legionnaires' disease. A primary factor involved in these host cell interactions is the Dot/Icm Type IV secretion system responsible for translocating effector proteins needed to establish and maintain the bacterial replicative niche. Several regulatory factors have been identified to control the expression of the Dot/Icm system and effectors, one of which is the CpxRA two-component system, suggesting essentiality for virulence. In this study, we generated cpxR, cpxA and cpxRA in-frame null mutant strains to further delineate the role of the CpxRA system in bacterial survival and virulence. We found that cpxR is essential for intracellular replication within Acanthamoeba castellanii, but not in U937-derived macrophages. Transcriptome analysis revealed that CpxRA regulates a large number of virulence-associated proteins including Dot/Icm effectors as well as Type II secreted substrates. Furthermore, the cpxR and cpxRA mutant strains were more sodium resistant than the parental strain Lp02, and cpxRA expression reaches maximal levels during postexponential phase. Taken together, our findings suggest the CpxRA system is a key contributor to L. pneumophila virulence in protozoa via virulence factor regulation. © 2016 John Wiley & Sons Ltd.

The anti-fibrotic effects of mesenchymal stem cells on irradiated lungs via stimulating endogenous secretion of HGF and PGE2

PubMed Central

Dong, Li-Hua; Jiang, Yi-Yao; Liu, Yong-Jun; Cui, Shuang; Xia, Cheng-Cheng; Qu, Chao; Jiang, Xin; Qu, Ya-Qin; Chang, Peng-Yu; Liu, Feng

2015-01-01

Radiation-induced pulmonary fibrosis is a common disease and has a poor prognosis owing to the progressive breakdown of gas exchange regions in the lung. Recently, a novel strategy of administering mesenchymal stem cells for pulmonary fibrosis has achieved high therapeutic efficacy. In the present study, we attempted to use human adipose tissue-derived mesenchymal stem cells to prevent disease in Sprague-Dawley rats that received semi-thoracic irradiation (15 Gy). To investigate the specific roles of mesenchymal stem cells in ameliorating radiation-induced pulmonary fibrosis, we treated control groups of irradiated rats with human skin fibroblasts or phosphate-buffered saline. After mesenchymal stem cells were infused, host secretions of hepatocyte growth factor (HGF) and prostaglandin E2 (PGE2) were elevated compared with those of the controls. In contrast, tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta1 (TGF-β1) levels were decreased after infusion of mesenchymal stem cells. Consequently, the architecture of the irradiated lungs was preserved without marked activation of fibroblasts or collagen deposition within the injured sites. Moreover, mesenchymal stem cells were able to prevent the irradiated type II alveolar epithelial cells from undergoing epithelial-mesenchymal transition. Collectively, these data confirmed that mesenchymal stem cells have the potential to limit pulmonary fibrosis after exposure to ionising irradiation. PMID:25736907

Tumor Secreted Autocrine Motility Factor (AMF): Causal Role in an Animal Model of Cachexia

DTIC Science & Technology

2005-08-01

AD Award Number: DAMD17-02-1-0586 TITLE: Tumor Secreted Autocrine Motility Factor ( AMF ): Causal Role in an Animal Model of Cachexia PRINCIPAL...5a. CONTRACT NUMBER Tumor Secreted Autocrine Motility Factor ( AMF ): Causal Role in an Animal Model of Cachexia 5b. GRANT NUMBER DAM D1 7-02-1-0586 5c...quality of life and postpone mortality. We proposed that autocrine motility factor ( AMF ) is released into the bloodstream from cancer sites and

The Effect of Solar Irradiated Vibrio cholerae on the Secretion of Pro-Inflammatory Cytokines and Chemokines by the JAWS II Dendritic Cell Line In Vitro

PubMed Central

Ssemakalu, Cornelius Cano; Ubomba-Jaswa, Eunice; Motaung, Keolebogile Shirley; Pillay, Michael

2015-01-01

The use of solar irradiation to sterilize water prior to its consumption has resulted in the reduction of water related illnesses in waterborne disease endemic communities worldwide. Currently, research on solar water disinfection (SODIS) has been directed towards understanding the underlying mechanisms through which solar irradiation inactivates the culturability of microorganisms in water, enhancement of the disinfection process, and the health impact of SODIS water consumption. However, the immunological consequences of SODIS water consumption have not been explored. In this study, we investigated the effect that solar irradiated V. cholerae may have had on the secretion of cytokines and chemokines by the JAWS II dendritic cell line in vitro. The JAWS II dendritic cell line was stimulated with the different strains of V. cholerae that had been: (i) prepared in PBS, (ii) inactivated through a combination of heat and chemical, (iii) solar irradiated, and (iv) non-solar irradiated, in bottled water. As controls, LPS (1 μg/ml) and CTB (1 μg/ml) were used as stimulants. After 48 hours of stimulation the tissue culture media from each treatment was qualitatively and quantitatively analysed for the presence of IL-1α, IL-1β, IL-6, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, MIP-1α, MIP-1β, MIP-2, RANTES, TNF-α, IL-23 and IL-27. Results showed that solar irradiated cultures of V. cholerae induced dendritic cells to secrete significant (p<0.05) levels of pro-inflammatory cytokines in comparison to the unstimulated dendritic cells. Furthermore, the amount of pro-inflammatory cytokines secreted by the dendritic cells in response to solar irradiated cultures of V. cholerae was not as high as observed in treatments involving non-solar irradiated cultures of V. cholerae or LPS. Our results suggest that solar irradiated microorganisms are capable of inducing the secretion of pro-inflammatory cytokines and chemokines. This novel finding is key towards understanding the possible immunological consequences of consuming SODIS treated water. PMID:26066787

Influence of Flavonoids on Mechanism of Modulation of Insulin Secretion.

PubMed

Soares, Juliana Mikaelly Dias; Pereira Leal, Ana Ediléia Barbosa; Silva, Juliane Cabral; Almeida, Jackson R G S; de Oliveira, Helinando Pequeno

2017-01-01

The development of alternatives for insulin secretion control in vivo or in vitro represents an important aspect to be investigated. In this direction, natural products have been progressively explored with this aim. In particular, flavonoids are potential candidates to act as insulin secretagogue. To study the influence of flavonoid on overall modulation mechanisms of insulin secretion. The research was conducted in the following databases and platforms: PubMed, Scopus, ISI Web of Knowledge, SciELO, LILACS, and ScienceDirect, and the MeSH terms used for the search were flavonoids, flavones, islets of Langerhans, and insulin-secreting cells. Twelve articles were included and represent the basis of discussion on mechanisms of insulin secretion of flavonoids. Papers in ISI Web of Knowledge were in number of 1, Scopus 44, PubMed 264, ScienceDirect 511, and no papers from LILACS and SciELO databases. According to the literature, the majority of flavonoid subclasses can modulate insulin secretion through several pathways, in an indication that corresponding molecule is a potential candidate for active materials to be applied in the treatment of diabetes. The action of natural products on insulin secretion represents an important investigation topic due to their importance in the diabetes controlIn addition to their typical antioxidant properties, flavonoids contribute to the insulin secretionThe modulation of insulin secretion is induced by flavonoids according to different mechanisms. Abbreviations used: K ATP channels: ATP-sensitive K + channels, GLUT4: Glucose transporter 4, ERK1/2: Extracellular signal-regulated protein kinases 1 and 2, L-VDCCs: L-type voltage-dependent Ca +2 channels, GLUT1: Glucose transporter 1, AMPK: Adenosine monophosphate-activated protein kinase, PTP1B: Protein tyrosine phosphatase 1B, GLUT2: Glucose transporter 2, cAMP: Cyclic adenosine monophosphate, PKA: Protein kinase A, PTK: Protein tyrosine kinase, CaMK II: Ca 2+ /calmodulin-dependent protein kinase II, GSIS: Glucose-stimulated insulin secretion, Insig-1: Insulin-induced gene 1, IRS-2: Insulin receptor substrate 2, PDX-1: Pancreatic and duodenal homeobox 1, SREBP-1c: Sterol regulatory element binding protein-1c, DMC: Dihydroxy-6'-methoxy-3',5'-dimethylchalcone, GLP-1: Glucagon-like peptide-1, GLP-1R: Glucagon-like peptide 1 receptor.

Altered Plasma Profile of Antioxidant Proteins as an Early Correlate of Pancreatic β Cell Dysfunction*

PubMed Central

Kuo, Taiyi; Kim-Muller, Ja Young; McGraw, Timothy E.; Accili, Domenico

2016-01-01

Insulin resistance and β cell dysfunction contribute to the pathogenesis of type 2 diabetes. Unlike insulin resistance, β cell dysfunction remains difficult to predict and monitor, because of the inaccessibility of the endocrine pancreas, the integrated relationship with insulin sensitivity, and the paracrine effects of incretins. The goal of our study was to survey the plasma response to a metabolic challenge in order to identify factors predictive of β cell dysfunction. To this end, we combined (i) the power of unbiased iTRAQ (isobaric tag for relative and absolute quantification) mass spectrometry with (ii) direct sampling of the portal vein following an intravenous glucose/arginine challenge (IVGATT) in (iii) mice with a genetic β cell defect. By so doing, we excluded the effects of peripheral insulin sensitivity as well as those of incretins on β cells, and focused on the first phase of insulin secretion to capture the early pathophysiology of β cell dysfunction. We compared plasma protein profiles with ex vivo islet secretome and transcriptome analyses. We detected changes to 418 plasma proteins in vivo, and detected changes to 262 proteins ex vivo. The impairment of insulin secretion was associated with greater overall changes in the plasma response to IVGATT, possibly reflecting metabolic instability. Reduced levels of proteins regulating redox state and neuronal stress markers, as well as increased levels of coagulation factors, antedated the loss of insulin secretion in diabetic mice. These results suggest that a reduced complement of antioxidants in response to a mixed secretagogue challenge is an early correlate of future β cell failure. PMID:26917725

Visualization of the post-Golgi vesicle-mediated transportation of TGF-β receptor II by quasi-TIRFM.

PubMed

Luo, Wangxi; Xia, Tie; Xu, Li; Chen, Ye-Guang; Fang, Xiaohong

2014-10-01

Transforming growth factor β receptor II (Tβ RII) is synthesized in the cytoplasm and then transported to the plasma membrane of cells to fulfil its signalling duty. Here, we applied live-cell fluorescence imaging techniques, in particular quasi-total internal reflection fluorescence microscopy, to imaging fluorescent protein-tagged Tβ RII and monitoring its secretion process. We observed punctuate-like Tβ RII-containing post-Golgi vesicles formed in MCF7 cells. Single-particle tracking showed that these vesicles travelled along the microtubules at an average speed of 0.51 μm/s. When stimulated by TGF-β ligand, these receptor-containing vesicles intended to move towards the plasma membrane. We also identified several factors that could inhibit the formation of such post-Golgi vesicles. Although the inhibitory mechanisms still remain unknown, the observed characteristics of Tβ RII-containing vesicles provide new information on intracellular Tβ RII transportation. It also renders Tβ RII a good model system for studying post-Golgi vesicle-trafficking and protein transportation. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression and Secretion of TNF-α in Mouse Taste Buds: A Novel Function of a Specific Subset of Type II Taste Cells

PubMed Central

Feng, Pu; Zhao, Hang; Chai, Jinghua; Huang, Liquan; Wang, Hong

2012-01-01

Taste buds are chemosensory structures widely distributed on the surface of the oral cavity and larynx. Taste cells, exposed to the oral environment, face great challenges in defense against potential pathogens. While immune cells, such as T-cells and macrophages, are rarely found in taste buds, high levels of expression of some immune-response-associated molecules are observed in taste buds. Yet, the cellular origins of these immune molecules such as cytokines in taste buds remain to be determined. Here, we show that a specific subset of taste cells selectively expresses high levels of the inflammatory cytokine tumor necrosis factor-α (TNF-α). Based on immuno-colocalization experiments using taste-cell-type markers, the TNF-α-producing cells are predominantly type II taste cells expressing the taste receptor T1R3. These cells can rapidly increase TNF-α production and secretion upon inflammatory challenges, both in vivo and in vitro. The lipopolysaccharide (LPS)-induced TNF-α expression in taste cells was completely eliminated in TLR2−/−/TLR4−/− double-gene-knockout mice, which confirms that the induction of TNF-α in taste buds by LPS is mediated through TLR signaling pathways. The taste-cell-produced TNF-α may contribute to local immune surveillance, as well as regulate taste sensation under normal and pathological conditions. PMID:22905218

Expression and secretion of TNF-α in mouse taste buds: a novel function of a specific subset of type II taste cells.

PubMed

Feng, Pu; Zhao, Hang; Chai, Jinghua; Huang, Liquan; Wang, Hong

2012-01-01

Taste buds are chemosensory structures widely distributed on the surface of the oral cavity and larynx. Taste cells, exposed to the oral environment, face great challenges in defense against potential pathogens. While immune cells, such as T-cells and macrophages, are rarely found in taste buds, high levels of expression of some immune-response-associated molecules are observed in taste buds. Yet, the cellular origins of these immune molecules such as cytokines in taste buds remain to be determined. Here, we show that a specific subset of taste cells selectively expresses high levels of the inflammatory cytokine tumor necrosis factor-α (TNF-α). Based on immuno-colocalization experiments using taste-cell-type markers, the TNF-α-producing cells are predominantly type II taste cells expressing the taste receptor T1R3. These cells can rapidly increase TNF-α production and secretion upon inflammatory challenges, both in vivo and in vitro. The lipopolysaccharide (LPS)-induced TNF-α expression in taste cells was completely eliminated in TLR2(-/-)/TLR4(-/-) double-gene-knockout mice, which confirms that the induction of TNF-α in taste buds by LPS is mediated through TLR signaling pathways. The taste-cell-produced TNF-α may contribute to local immune surveillance, as well as regulate taste sensation under normal and pathological conditions.

Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS

PubMed Central

Dunstan, Rhys A.; Heinz, Eva; Wijeyewickrema, Lakshmi C.; Pike, Robert N.; Purcell, Anthony W.; Evans, Timothy J.; Praszkier, Judyta; Robins-Browne, Roy M.; Strugnell, Richard A.; Korotkov, Konstantin V.; Lithgow, Trevor

2013-01-01

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS. PMID:23326233

Genetic selection for temperament affects behaviour and the secretion of adrenal and reproductive hormones in sheep subjected to stress.

PubMed

Hawken, P A R; Luckins, N; Tilbrook, A; Fiol, C; Martin, G B; Blache, D

2013-01-01

We investigated the effect of genetic selection for temperament on the way that stressors affect the behaviour and the adrenal and reproductive axes of sheep. We tested three hypotheses: (i) isolation would increase cortisol secretion and decrease luteinising hormone (LH) secretion more in nervous sheep than in calm sheep; (ii) isolation combined with simulated human presence would increase cortisol secretion and decrease LH secretion more in nervous sheep than in calm sheep and (iii) isolation combined with stressors that were not specific to the selection process (i.e. non-selection stressors) would increase cortisol secretion and decrease LH secretion equally in calm and nervous sheep. Isolation alone increased cortisol secretion and decreased LH secretion in nervous sheep but not in calm sheep. Compared to calm sheep, nervous sheep were more agitated during the first 2 h of isolation but not during the second 2 h of isolation. Exposure to non-selection stressors increased cortisol secretion, decreased LH pulse amplitude and the mean plasma concentrations of LH in both calm and nervous sheep. We conclude that genetic selection for temperament affects the behavioural expression of the stress response and the secretion of adrenal and reproductive hormones during isolation, but has less impact on their reactivity to non-selection stressors.

Microencapsulated equine mesenchymal stromal cells promote cutaneous wound healing in vitro.

PubMed

Bussche, Leen; Harman, Rebecca M; Syracuse, Bethany A; Plante, Eric L; Lu, Yen-Chun; Curtis, Theresa M; Ma, Minglin; Van de Walle, Gerlinde R

2015-04-11

The prevalence of impaired cutaneous wound healing is high and treatment is difficult and often ineffective, leading to negative social and economic impacts for our society. Innovative treatments to improve cutaneous wound healing by promoting complete tissue regeneration are therefore urgently needed. Mesenchymal stromal cells (MSCs) have been reported to provide paracrine signals that promote wound healing, but (i) how they exert their effects on target cells is unclear and (ii) a suitable delivery system to supply these MSC-derived secreted factors in a controlled and safe way is unavailable. The present study was designed to provide answers to these questions by using the horse as a translational model. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC-derived conditioned medium (CM), containing all factors secreted by MSCs, on equine dermal fibroblasts, a cell type critical for successful wound healing, and (ii) explore the potential of microencapsulated equine MSCs to deliver CM to wounded cells in vitro. MSCs were isolated from the peripheral blood of healthy horses. Equine dermal fibroblasts from the NBL-6 (horse dermal fibroblast cell) line were wounded in vitro, and cell migration and expression levels of genes involved in wound healing were evaluated after treatment with MSC-CM or NBL-6-CM. These assays were repeated by using the CM collected from MSCs encapsulated in core-shell hydrogel microcapsules. Our salient findings were that equine MSC-derived CM stimulated the migration of equine dermal fibroblasts and increased their expression level of genes that positively contribute to wound healing. In addition, we found that equine MSCs packaged in core-shell hydrogel microcapsules had similar effects on equine dermal fibroblast migration and gene expression, indicating that microencapsulation of MSCs does not interfere with the release of bioactive factors. Our results demonstrate that the use of CM from MSCs might be a promising new therapy for impaired cutaneous wounds and that encapsulation may be a suitable way to effectively deliver CM to wounded cells in vivo.

The semantics of secrecy: young children's classification of secret content.

PubMed

Anagnostaki, Lida; Wright, Michael J; Bourchier-Sutton, Alison J

2010-01-01

The authors explored whether young children can distinguish potential secrets from nonsecrets by their content, as can older children, adolescents, and adults. Ninety children, 4, 5, and 6 years old, rated the secrecy of items from an adult-validated list of personal information about an age- and gender-appropriate puppet. Two factors of the children's data corresponded to the adult categories of nonsecrets and secrets, and a third factor corresponded to surprises. All ages rated surprises as significantly more secret than nonsecret items; however, the surprise items contained linguistic cues to secrecy. A tendency to rate nonsecrets as secret decreased with age, but only the 6-year-olds rated secrets other than surprises as significantly more secret than nonsecrets. Thus, children acquire the implicit rules defining secret content from a somewhat later age than that reported for the cognitive or behavioral capacities for secrecy.

Norepinephrine is coreleased with serotonin in mouse taste buds.

PubMed

Huang, Yijen A; Maruyama, Yutaka; Roper, Stephen D

2008-12-03

ATP and serotonin (5-HT) are neurotransmitters secreted from taste bud receptor (type II) and presynaptic (type III) cells, respectively. Norepinephrine (NE) has also been proposed to be a neurotransmitter or paracrine hormone in taste buds. Yet, to date, the specific stimulus for NE release in taste buds is not well understood, and the identity of the taste cells that secrete NE is not known. Chinese hamster ovary cells were transfected with alpha(1A) adrenoceptors and loaded with fura-2 ("biosensors") to detect NE secreted from isolated mouse taste buds and taste cells. Biosensors responded to low concentrations of NE (>or=10 nm) with a reliable fura-2 signal. NE biosensors did not respond to stimulation with KCl or taste compounds. However, we recorded robust responses from NE biosensors when they were positioned against mouse circumvallate taste buds and the taste buds were stimulated with KCl (50 mm) or a mixture of taste compounds (cycloheximide, 10 microm; saccharin, 2 mm; denatonium, 1 mm; SC45647, 100 microm). NE biosensor responses evoked by stimulating taste buds were reversibly blocked by prazosin, an alpha(1A) receptor antagonist. Together, these findings indicate that taste bud cells secrete NE when they are stimulated. We isolated individual taste bud cells to identify the origin of NE release. NE was secreted only from presynaptic (type III) taste cells and not receptor (type II) cells. Stimulus-evoked NE release depended on Ca(2+) in the bathing medium. Using dual biosensors (sensitive to 5-HT and NE), we found all presynaptic cells secrete 5-HT and 33% corelease NE with 5-HT.

Computational and Experimental Analysis of the Secretome of Methylococcus capsulatus (Bath)

PubMed Central

Indrelid, Stine; Mathiesen, Geir; Jacobsen, Morten; Lea, Tor; Kleiveland, Charlotte R.

2014-01-01

The Gram-negative methanotroph Methylococcus capsulatus (Bath) was recently demonstrated to abrogate inflammation in a murine model of inflammatory bowel disease, suggesting interactions with cells involved in maintaining mucosal homeostasis and emphasizing the importance of understanding the many properties of M. capsulatus. Secreted proteins determine how bacteria may interact with their environment, and a comprehensive knowledge of such proteins is therefore vital to understand bacterial physiology and behavior. The aim of this study was to systematically analyze protein secretion in M. capsulatus (Bath) by identifying the secretion systems present and the respective secreted substrates. Computational analysis revealed that in addition to previously recognized type II secretion systems and a type VII secretion system, a type Vb (two-partner) secretion system and putative type I secretion systems are present in M. capsulatus (Bath). In silico analysis suggests that the diverse secretion systems in M.capsulatus transport proteins likely to be involved in adhesion, colonization, nutrient acquisition and homeostasis maintenance. Results of the computational analysis was verified and extended by an experimental approach showing that in addition an uncharacterized protein and putative moonlighting proteins are released to the medium during exponential growth of M. capsulatus (Bath). PMID:25479164

Characterization of Shikonin Derivative Secretion in Lithospermum erythrorhizon Hairy Roots as a Model of Lipid-Soluble Metabolite Secretion from Plants

PubMed Central

Tatsumi, Kanade; Yano, Mariko; Kaminade, Kenta; Sugiyama, Akifumi; Sato, Mayuko; Toyooka, Kiminori; Aoyama, Takashi; Sato, Fumihiko; Yazaki, Kazufumi

2016-01-01

Shikonin derivatives are specialized lipophilic metabolites, secreted in abundant amounts from the root epidermal cells of Lithospermum erythrorhizon. Because they have anti-microbial activities, these compounds, which are derivatives of red naphthoquinone, are thought to serve as a chemical barrier for plant roots. The mechanism by which they are secreted from cells is, however, largely unknown. The shikonin production system in L. erythrorhizon is an excellent model for studying the mechanism by which lipophilic compounds are secreted from plant cells, because of the abundant amounts of these compounds produced by L. erythrorhizon, the 0 to 100% inducibility of their production, the light-specific inhibition of production, and the visibility of these products as red pigments. To date, many factors regulating shikonin biosynthesis have been identified, but no mechanism that regulates shikonin secretion without inhibiting biosynthesis has been detected. This study showed that inhibitors of membrane traffic strongly inhibit shikonin secretion without inhibiting shikonin production, suggesting that the secretion of shikonin derivatives into the apoplast utilizes pathways common to the ADP-ribosylation factor/guanine nucleotide exchange factor (ARF/GEF) system and actin filament polymerization, at least in part. These findings provide clues about the machinery involved in secreting lipid-soluble metabolites from cells. PMID:27507975

Characterization of Shikonin Derivative Secretion in Lithospermum erythrorhizon Hairy Roots as a Model of Lipid-Soluble Metabolite Secretion from Plants.

PubMed

Tatsumi, Kanade; Yano, Mariko; Kaminade, Kenta; Sugiyama, Akifumi; Sato, Mayuko; Toyooka, Kiminori; Aoyama, Takashi; Sato, Fumihiko; Yazaki, Kazufumi

2016-01-01

Shikonin derivatives are specialized lipophilic metabolites, secreted in abundant amounts from the root epidermal cells of Lithospermum erythrorhizon. Because they have anti-microbial activities, these compounds, which are derivatives of red naphthoquinone, are thought to serve as a chemical barrier for plant roots. The mechanism by which they are secreted from cells is, however, largely unknown. The shikonin production system in L. erythrorhizon is an excellent model for studying the mechanism by which lipophilic compounds are secreted from plant cells, because of the abundant amounts of these compounds produced by L. erythrorhizon, the 0 to 100% inducibility of their production, the light-specific inhibition of production, and the visibility of these products as red pigments. To date, many factors regulating shikonin biosynthesis have been identified, but no mechanism that regulates shikonin secretion without inhibiting biosynthesis has been detected. This study showed that inhibitors of membrane traffic strongly inhibit shikonin secretion without inhibiting shikonin production, suggesting that the secretion of shikonin derivatives into the apoplast utilizes pathways common to the ADP-ribosylation factor/guanine nucleotide exchange factor (ARF/GEF) system and actin filament polymerization, at least in part. These findings provide clues about the machinery involved in secreting lipid-soluble metabolites from cells.

Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids.

PubMed

Murphy, Kaitlin C; Whitehead, Jacklyn; Zhou, Dejie; Ho, Steve S; Leach, J Kent

2017-12-01

Mesenchymal stem cells (MSCs) secrete endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE 2 ) that promote angiogenesis, modulate the inflammatory microenvironment, and stimulate wound repair, and MSC spheroids secrete more trophic factors than dissociated, individual MSCs. Compared to injection of cells alone, transplantation of MSCs in a biomaterial can enhance their wound healing potential by localizing cells at the defect site and upregulating trophic factor secretion. To capitalize on the therapeutic potential of spheroids, we engineered a fibrin gel delivery vehicle to simultaneously enhance the proangiogenic and anti-inflammatory potential of entrapped human MSC spheroids. We used multifactorial statistical analysis to determine the interaction between four input variables derived from fibrin gel synthesis on four output variables (gel stiffness, gel contraction, and secretion of VEGF and PGE 2 ). Manipulation of the four input variables tuned fibrin gel biophysical properties to promote the simultaneous secretion of VEGF and PGE 2 by entrapped MSC spheroids while maintaining overall gel integrity. MSC spheroids in stiffer gels secreted the most VEGF, while PGE 2 secretion was highest in more compliant gels. Simultaneous VEGF and PGE 2 secretion was greatest using hydrogels with intermediate mechanical properties, as small increases in stiffness increased VEGF secretion while maintaining PGE 2 secretion by entrapped spheroids. The fibrin gel formulation predicted to simultaneously increase VEGF and PGE 2 secretion stimulated endothelial cell proliferation, enhanced macrophage polarization, and promoted angiogenesis when used to treat a wounded three-dimensional human skin equivalent. These data demonstrate that a statistical approach is an effective strategy to formulate fibrin gel formulations that enhance the wound healing potential of human MSCs. Mesenchymal stem cells (MSCs) are under investigation for wound healing applications due to their secretion of bioactive factors that enhance granulation tissue formation, blood vessel ingrowth, and reduce inflammation. However, the effectiveness of cell-based therapies is reduced due to poor engraftment and high rates of cell death when transplanted into harsh environments characteristic of large wounds. Compared to dissociated cells, MSCs exhibit increased overall function when aggregated into three-dimensional spheroids, and transplantation of cells using biomaterials is one strategy for guiding cell function in the defect site. The present study demonstrates that the biophysical properties of fibrin hydrogels, designed for use as a cell carrier, can be engineered to dictate the secretion of bioactive factors by entrapped MSC spheroids. This strategy enables MSCs to contribute to wound healing by synergistically promoting neovascularization and modulating the inflammatory milieu. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Kinetics of tumor necrosis factor production by photodynamic-therapy-activated macrophages

NASA Astrophysics Data System (ADS)

Pass, Harvey I.; Evans, Steven; Perry, Roger; Matthews, Wilbert

1990-07-01

The ability of photodynamic therapy (PDT) to activate macrophages and produce cytokines, specifically tumor necrosis factor (TNF), is unknown. Three day thioglycolate elicited macrophages were incubated with 25 ug/mi Photofrin II (P11) for 2 hour, after which they were subjected to 630 nm light with fluences of 0-1800 J/m. The amount of TNF produced in the system as well as macrophage viability was measured 1, 3, 6, and 18 hours after POT. The level of TNF produced by the macrophages was significantly elevated over control levels 6 hours after POT and the absolute level of tumor necrosis factor production was influenced by the treatment energy and the resulting macrophage cytotoxicity. These data suggest that POT therapy induced cytotoxicity in vivo may be amplified by macrophage stimulation to secrete cytokines and these cytokines may also participate in other direct/indirect photodynamic therapy effects, i.e. immunosuppression, vascular effects.

Effect of Melanotan-II on Brain Fos Immunoreactivity and Oxytocin Neuronal Activity and Secretion in Rats.

PubMed

Paiva, L; Sabatier, N; Leng, G; Ludwig, M

2017-02-01

Melanocortins stimulate the central oxytocin systems that are involved in regulating social behaviours. Alterations in central oxytocin have been linked to neurological disorders such as autism, and melanocortins have been proposed for therapeutic treatment. In the present study, we investigated how systemic administration of melanotan-II (MT-II), a melanocortin agonist, affects oxytocin neuronal activity and secretion in rats. The results obtained show that i.v., but not intranasal, administration of MT-II markedly induced Fos expression in magnocellular neurones of the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus, and this response was attenuated by prior i.c.v. administration of the melanocortin antagonist, SHU-9119. Electrophysiological recordings from identified magnocellular neurones of the SON showed that i.v. administration of MT-II increased the firing rate in oxytocin neurones but did not trigger somatodendritic oxytocin release within the SON as measured by microdialysis. Our data suggest that, after i.v., but not intranasal, administration of MT-II, the activity of magnocellular neurones of the SON is increased. Because previous studies showed that SON oxytocin neurones are inhibited in response to direct application of melanocortin agonists, the actions of i.v. MT-II are likely to be mediated at least partly indirectly, possibly by activation of inputs from the caudal brainstem, where MT-II also increased Fos expression. © 2016 British Society for Neuroendocrinology.

Acute and Chronic Regulation of Aldosterone Production

PubMed Central

Hattangady, Namita; Olala, Lawrence; Bollag, Wendy B.; Rainey, William E.

2011-01-01

Aldosterone is the major mineralocorticoid synthesized by the adrenal. Secretion of aldosterone is regulated tightly by the adrenocortical glomerulosa cells due to the selective expression of CYP11B2 in the outermost zone, the zona glomerulosa. Aldosterone is largely responsible for regulation of systemic blood pressure through the absorption of electrolytes and water under the regulation of certain specific agonists. Angiotensin II (Ang II), potassium (K+) and adrenocorticotropin (ACTH) are the main physiological agonists which regulate aldosterone secretion. The mechanisms involved in this process may be regulated minutes after a stimulus (acutely) through increased expression and phosphorylation of the steroidogenic acute regulatory (StAR) protein, over hours to days (chronically) by increased expression of the enzymes involved in the synthesis of aldosterone, particularly aldosterone synthase (CYP11B2). Imbalance in any of these processes may lead to several aldosterone excess disorders. In this review we attempt to summarize the key molecular events involved in and specifically attributed to the acute and chronic phases of aldosterone secretion. PMID:21839803

Social and cognitive factors associated with children's secret-keeping for a parent.

PubMed

Gordon, Heidi M; Lyon, Thomas D; Lee, Kang

2014-01-01

This study examined children's secret-keeping for a parent and its relation to trust, theory of mind, secrecy endorsement, and executive functioning (EF). Children (N = 107) between 4 and 12 years of age participated in a procedure wherein parents broke a toy and asked children to promise secrecy. Responses to open-ended and direct questions were examined. Overall, secret-keeping increased with age and promising to keep the secret was related to fewer disclosures in open-ended questioning. Children who kept the secret in direct questioning exhibited greater trust and better parental ratings of EF than children who disclosed the secret. Findings highlight the importance of both social and cognitive factors in secret-keeping development. © 2014 The Authors. Child Development © 2014 Society for Research in Child Development, Inc.

The type II secretion system is essential for erythrocyte lysis and gut colonization by the leech digestive tract symbiont Aeromonas veronii.

PubMed

Maltz, Michele; Graf, Joerg

2011-01-01

Hemolysin and the type II secretion system (T2SS) have been shown to be important for virulence in many pathogens, but very few studies have shown their importance in beneficial microbes. Here, we investigated the importance of the type II secretion pathway in the beneficial digestive-tract association of Aeromonas veronii and the medicinal leech Hirudo verbana and revealed a critical role for the hemolysis of erythrocytes. A mutant with a miniTn5 insertion in exeM, which is involved in forming the inner membrane platform in the T2SS, was isolated by screening mutants for loss of hemolysis on blood agar plates. A hemolysis assay was used to quantify the mutant's deficiency in lysing sheep erythrocytes and revealed a 99.9% decrease compared to the parent strain. The importance of the T2SS in the colonization of the symbiotic host was assessed. Colonization assays revealed that the T2SS is critical for initial colonization of the leech gut. The defect was tied to the loss of hemolysin production by performing a colonization assay with blood containing lysed erythrocytes. This restored the colonization defect in the mutant. Complementation of the mutant using the promoter region and exeMN revealed that the T2SS is responsible for secreting hemolysin into the extracellular space and that both the T2SS and hemolysin export by the T2SS are critical for initial establishment of A. veronii in the leech gut.

How NaCl raises blood pressure: a new paradigm for the pathogenesis of salt-dependent hypertension

PubMed Central

Leenen, Frans H. H.; Chen, Ling; Golovina, Vera A.; Hamlyn, John M.; Pallone, Thomas L.; Van Huysse, James W.; Zhang, Jin; Wier, W. Gil

2012-01-01

Excess dietary salt is a major cause of hypertension. Nevertheless, the specific mechanisms by which salt increases arterial constriction and peripheral vascular resistance, and thereby raises blood pressure (BP), are poorly understood. Here we summarize recent evidence that defines specific molecular links between Na+ and the elevated vascular resistance that directly produces high BP. In this new paradigm, high dietary salt raises cerebrospinal fluid [Na+]. This leads, via the Na+-sensing circumventricular organs of the brain, to increased sympathetic nerve activity (SNA), a major trigger of vasoconstriction. Plasma levels of endogenous ouabain (EO), the Na+ pump ligand, also become elevated. Remarkably, high cerebrospinal fluid [Na+]-evoked, locally secreted (hypothalamic) EO participates in a pathway that mediates the sustained increase in SNA. This hypothalamic signaling chain includes aldosterone, epithelial Na+ channels, EO, ouabain-sensitive α2 Na+ pumps, and angiotensin II (ANG II). The EO increases (e.g.) hypothalamic ANG-II type-1 receptor and NADPH oxidase and decreases neuronal nitric oxide synthase protein expression. The aldosterone-epithelial Na+ channel-EO-α2 Na+ pump-ANG-II pathway modulates the activity of brain cardiovascular control centers that regulate the BP set point and induce sustained changes in SNA. In the periphery, the EO secreted by the adrenal cortex directly enhances vasoconstriction via an EO-α2 Na+ pump-Na+/Ca2+ exchanger-Ca2+ signaling pathway. Circulating EO also activates an EO-α2 Na+ pump-Src kinase signaling cascade. This increases the expression of the Na+/Ca2+ exchanger-transient receptor potential cation channel Ca2+ signaling pathway in arterial smooth muscle but decreases the expression of endothelial vasodilator mechanisms. Additionally, EO is a growth factor and may directly participate in the arterial structural remodeling and lumen narrowing that is frequently observed in established hypertension. These several central and peripheral mechanisms are coordinated, in part by EO, to effect and maintain the salt-induced elevation of BP. PMID:22058154

Secretion of apolipoproteins A-I and B by HepG2 cells: regulation by substrates and metabolic inhibitors.

PubMed

Kempen, H J; Imbach, A P; Giller, T; Neumann, W J; Hennes, U; Nakada, N

1995-08-01

It was the aim of this study to i) compare the effects of glucose and other hexoses with that of oleate on secretion of apolipoproteins (apos) A-I and B by HepG2 cells, and ii) document the effect of various metabolic inhibitors on the secretion of these apos in the absence or presence of extra glucose/oleate. i) The addition of 10 mM glucose increased secretion of apoA-I and apoB, as measured by enzyme immunoassay, by about 60% when cells were incubated for 48 h in DMEM + 10% fetal calf serum. The addition of extra glucose also increased the mRNA levels for these apos. Increased radioactivity was also found in these apolipoproteins by immunoprecipitation after metabolic labeling with [35S]methionine for 48 h. However, in a pulse-chase experiment (15 min labeling, 2 h chase), glucose was found to increase apoA-I synthesis but not apoB synthesis. More labeled apoB appeared in the medium during the chase because glucose inhibited its intracellular degradation. The effect of glucose on secretion of these apos could be mimicked by fructose and mannose but not by 6-deoxyglucose, showing that the hexoses must enter the cells and be phosphorylated. In contrast, the addition of 0.5 mM oleate had a weak inhibitory effect on secretion of apoA-I whereas it increased the secretion of apoB by more than twofold. The combination of 10 mM glucose and 0.5 mM oleate had no greater effect than glucose alone on apoA-I secretion but increased apoB secretion by fourfold. ii) Inhibiting glycolysis (by glucosamine) lowered secretion of both apoA-I and apoB, while inhibiting lipogenesis (using 8-Br-cyclic AMP or 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA)) did not affect apoA-I secretion but clearly decreased that of apoB. However, the inhibitory effect of TOFA on apoB secretion was much smaller in the presence of 0.5 mM oleate instead of extra glucose. Actinomycin-D and cycloheximide strongly suppressed the stimulatory effect of glucose on secretion of both apolipoproteins. Actinomycin-D also suppressed basal secretion of apoA-I but surprisingly stimulated that of apoB. These observations indicate that in HepG2 cells secretion of apoA-I is strongly dependent on ongoing protein synthesis and can be boosted by glucose, whereas that of apoB is primarily driven by internal (via lipogenesis from glucose) or external supply of fatty acyl-residues.

Characterization of a human antigen specific helper factor

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

Richardson, B.

1986-03-01

While antigen (Ag) specific helper factors have been characterized in mice, similar molecules have not been identified in humans. To characterize human antigen specific helper molecules, an IL-2 dependent tetanus toxoid (T.T.) reactive T cell line was fused with a 6-thioguanine resistant CEM line, and hybrids selected in medium containing hypoxanthine and azaserine. Hybrids were screened by culturing the cells with /sup 35/S-Met then reacting the supernatants with T.T. or hepatitis vaccine immobilized on nitrocellulose. One hybrid, TT6BA-O, was identified which secreted a Met-containing molecule which bound T.T. but not hepatitis vaccine. Supernatants from TT6BA-O, but not the parent CEMmore » line, when added to autologous peripheral blood mononuclear cells (PBMC's) stimulated secretion of T.T. specific antibodies (Abs). Specificity controls demonstrated that TT6BA-O supernatant did not induce antibodies to diphtheria toxoid, hepatitis vaccine or pneumococcal polysaccharide, and total immunoglobulin (lg) synthesis was minimally increased. In contrast, pokeweed mitogen stimulated significant lg synthesis as well as Ab's to pneumococcal polysaccharide and T.T. TT6BA-O supernatant induced anti-T.T.Ab's in autologous PBMC's but not PBMC's from 3 unrelated donors, suggesting that the activity of the helper factor is restricted, possibly by the MHC. The molecular weight of the helper factor was estimated at 100,000-150,000 by Sephacryl S-300 chromatography. Finally, the helper factor could be demonstrated to bind and elute from sephorose-immobilized T.T. and anti-DR antisera, but not anti-lg antisera or the T40/25 monoclonal antibody, which binds a nonpolymorphic determinant on the human T cell receptor. These results demonstrate that human Ag specific helper factors exist, bind antigen and bear class II MHC determinants.« less

Cytokine Response of Cultured Skeletal Muscle Cells Stimulated with Proinflammatory Factors Depends on Differentiation Stage

PubMed Central

Podbregar, Matej; Lainscak, Mitja; Prelovsek, Oja; Mars, Tomaz

2013-01-01

Myoblast proliferation and myotube formation are critical early events in skeletal muscle regeneration. The attending inflammation and cytokine signaling are involved in regulation of skeletal muscle cell proliferation and differentiation. Secretion of muscle-derived cytokines upon exposure to inflammatory factors may depend on the differentiation stage of regenerating muscle cells. Cultured human myoblasts and myotubes were exposed to 24-hour treatment with tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS). Secretion of interleukin 6 (IL-6), a major muscle-derived cytokine, and interleukin 1 (IL-1), an important regulator of inflammatory response, was measured 24 hours after termination of TNF-α or LPS treatment. Myoblasts pretreated with TNF-α or LPS displayed robustly increased IL-6 secretion during the 24-hour period after removal of treatments, while IL-1 secretion remained unaltered. IL-6 secretion was also increased in myotubes, but the response was less pronounced compared with myoblasts. In contrast to myoblasts, IL-1 secretion was markedly stimulated in LPS-pretreated myotubes. We demonstrate that preceding exposure to inflammatory factors stimulates a prolonged upregulation of muscle-derived IL-6 and/or IL-1 in cultured skeletal muscle cells. Our findings also indicate that cytokine response to inflammatory factors in regenerating skeletal muscle partially depends on the differentiation stage of myogenic cells. PMID:23509435

Reply to ``Comment II on `Quantum secret sharing based on reusable Greenberger-Horne-Zeilinger states as secure carriers' ''

NASA Astrophysics Data System (ADS)

Karimipour, V.

2006-07-01

In the preceding Comment [Jian-Zhong Du, Su-Juan Qin, Qiao-Yan Wen, and Fu-Chen Zhu, Phys. Rev. A 74, 016301 (2006)], it has been shown that in a quantum secret sharing protocol proposed in [S. Bagherinezhad and V. Karimipour, Phys. Rev. A 67, 044302 (2003)], one of the receivers can cheat by splitting the entanglement of the carrier and intercepting the secret, without being detected. In this reply we show that a simple modification of the protocol prevents the receivers from this kind of cheating.

Female effect in sheep. II. Role of volatile substances from the sexually receptive female; implication of the sense of smell.

PubMed

Gonzalez, R; Levy, F; Orgeur, P; Poindron, P; Signoret, J P

1991-01-01

The importance of olfactory cues in inducing luteinizing hormone and testosterone secretion as a response to stimulation by sexually receptive ewes has been tested. In sexually experienced rams, olfactory stimulation with urine, wool and vaginal secretions from sexually receptive females placed in a mask did not induce an endocrine response. The female-induced secretion of LH and testosterone was similar in anosmic, sham-operated and in control rams. These results show that olfactory cues are not necessary in the mediation of interindividual stimulation of endocrine response in the sexually experienced ram.

Comparison of different signal peptides for secretion of heterologous proteins in fission yeast

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

Kjaerulff, Soren; Jensen, Martin Roland

2005-10-28

In the fission yeast Schizosaccharomyces pombe, there are relatively few signal peptides available and most reports of their activity have not been comparative. Using sequence information from the S. pombe genome database we have identified three putative signal peptides, designated Cpy, Amy and Dpp, and compared their ability to support secretion of green fluorescent protein (GFP). In the comparison we also included the two well-described secretion signals derived from the precursors of, respectively, the Saccharomyces cerevisiae {alpha}-factor and the S. pombe P-factor. The capability of the tested signal peptides to direct secretion of GFP varied greatly. The {alpha}-factor signal didmore » not confer secretion to GFP and all the produced GFP was trapped intracellular. In contrast, the Cpy signal peptide supported efficient secretion of GFP with yields approximating 10 mg/L. We also found that the use of an attenuated version of the S. cerevisiae URA3 marker substantially increases vector copy number and expression yield in fission yeast.« less

Yarrowia lipolytica vesicle-mediated protein transport pathways

PubMed Central

Swennen, Dominique; Beckerich, Jean-Marie

2007-01-01

Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y. lipolytica proteins are closer to animal ones, whereas they are only 13% in the case of S. cerevisiae. Conclusion These results provide further support for the idea, previously noted about the endoplasmic reticulum translocation pathway, that Y. lipolytica is more representative of vesicular secretion of animals and other fungi than is S. cerevisiae. PMID:17997821

Nicotine promotes vascular endothelial growth factor secretion by human trophoblast cells under hypoxic conditions and improves the proliferation and tube formation capacity of human umbilical endothelial cells.

PubMed

Zhao, Hongbo; Wu, Lanxiang; Wang, Yahui; Zhou, Jiayi; Li, Ruixia; Zhou, Jiabing; Wang, Zehua; Xu, Congjian

2017-04-01

Pre-eclampsia, characterized as defective uteroplacental vascularization, remains the major cause of maternal and fetal mortality and morbidity. Previous epidemiological studies demonstrated that cigarette smoking reduced the risk of pre-eclampsia. However, the molecular mechanism remains elusive. In the present study, it is demonstrated that a low dose of nicotine decreased soluble vascular endothelial growth factor receptor 1 (sFlt1) secretion in human trophoblast cells under hypoxic conditions. Nicotine was then observed to promote vascular endothelial growth factor (VEGF) secretion by reducing sFlt1 secretion and increasing VEGF mRNA transcription. Further data showed that nicotine enhanced hypoxia-mediated hypoxia-inducible factor-1α (HIF-1α) expression and HIF-1α small interfering RNA abrogated nicotine-induced VEGF secretion, indicating that HIF-1α may be responsible for nicotine-mediated VEGF transcription under hypoxic conditions. Moreover, conditioned medium from human trophoblast cells treated with nicotine under hypoxic conditions promoted the proliferation and tube formation capacity of human umbilical endothelial cells (HUVEC) by promoting VEGF secretion. These findings indicate that nicotine may promote VEGF secretion in human trophoblast cells under hypoxic conditions by reducing sFlt1 secretion and up-regulating VEGF transcription and improve the proliferation and tube formation of HUVEC cells, which may contribute to elucidate the protective effect of cigarette smoking against pre-eclampsia. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Exposure to 60% oxygen promotes migration and upregulates angiogenesis factor secretion in breast cancer cells.

PubMed

Crowley, Peter D; Stuttgen, Vivian; O'Carroll, Emma; Ash, Simon A; Buggy, Donal J; Gallagher, Helen C

2017-01-01

Peri-operative factors, including anaesthetic drugs and techniques, may affect cancer cell biology and clinical recurrence. In breast cancer cells, we demonstrated that sevoflurane promotes migration and angiogenesis in high fractional oxygen but not in air. Follow-up analysis of the peri-operative oxygen fraction trial found an association between high inspired oxygen during cancer surgery and reduced tumor-free survival. Here we evaluated effects of acute, high oxygen exposure on breast cancer cell viability, migration and secretion of angiogenesis factors in vitro . MDA-MB-231 and MCF-7 breast cancer cells were exposed to 21%, 30%, 60%, or 80% v/v O 2 for 3 hours. Cell viability at 24 hours was determined by MTT and migration at 24 hours with the Oris™ Cell Migration Assay. Secretion of angiogenesis factors at 24 hours was measured via membrane-based immunoarray. Exposure to 30%, 60% or 80% oxygen did not affect cell viability. Migration of MDA-MB-231 and MCF-7 cells was increased by 60% oxygen ( P = 0.012 and P = 0.007, respectively) while 30% oxygen increased migration in MCF-7 cells ( P = 0.011). These effects were reversed by dimethyloxaloylglycine. In MDA-MB-231 cells high fractional oxygen increased secretion of angiogenesis factors monocyte chemotactic protein 1, regulated on activation normal T-cell expressed and vascular endothelial growth factor. In MCF-7 cells, interleukin-8, angiogenin and vascular endothelial growth factor secretion was significantly increased by high fractional oxygen. High oxygen exposure stimulates migration and secretion of angiogenesis factors in breast cancer cells in vitro .

A Continuum of Renin-Independent Aldosteronism in Normotension

PubMed Central

Baudrand, Rene; Guarda, Francisco J.; Fardella, Carlos; Hundemer, Gregory; Brown, Jenifer; Williams, Gordon; Vaidya, Anand

2017-01-01

Primary aldosteronism (PA) is a severe form of autonomous aldosteronism. Milder forms of autonomous and renin-independent aldosteronism may be common, even in normotension. We characterized aldosterone secretion in 210 normotensives who had suppressed plasma renin activity (PRA<1.0 ng/mL/h), completed an oral sodium suppression test, received an infusion of angiotensin II (AngII), and had measurements of blood pressure (BP) and renal plasma flow (RPF). Continuous associations between urinary aldosterone excretion rate (AER), renin, and potassium handling were investigated. Severe autonomous aldosterone secretion that was consistent with confirmed PA was defined based on accepted criteria of an AER >12 mcg/24h with urinary sodium excretion >200 mmol/24h. Across the population, there were strong and significant associations between higher AER and higher urinary potassium excretion, higher AngII-stimulated aldosterone, and lower PRA, suggesting a continuum of renin-independent aldosteronism and mineralocorticoid receptor activity. Autonomous aldosterone secretion that fulfilled confirmatory criteria for PA was detected in 29 participants (14%). Normotensives with evidence suggestive of confirmed PA had higher 24h urinary AER (20.2±12.2 vs. 6.2±2.9 mcg/24h, P<0.001) as expected, but also higher AngII-stimulated aldosterone (12.4±8.6 vs. 6.6±4.3 ng/dL, P<0.001) and lower 24h urinary sodium-to-potassium excretion (2.69±0.65 vs. 3.69±1.50 mmol/mmol, P=0.001); however, there were no differences in age, aldosterone-to-renin ratio, BP, or RPF between the two groups. These findings indicate a continuum of renin-independent aldosteronism and mineralocorticoid receptor activity in normotension that ranges from subtle to overtly dysregulated and autonomous. Longitudinal studies are needed to determine whether this spectrum of autonomous aldosterone secretion contributes to hypertension and cardiovascular disease. PMID:28289182

Overexpression of angiotensin II type 2 receptor promotes apoptosis and impairs insulin secretion in rat insulinoma cells.

PubMed

Liu, Min; Jing, Danqing; Wang, Yan; Liu, Yu; Yin, Shinan

2015-02-01

Angiotensin II (Ang II), the major effector hormone of renin-angiotensin system, acts as a promoter of insulin resistance and diabetes mellitus type 2 pathogenesis. Activation of Ang II type 2 receptor (AT2R) has been examined as a potential therapeutic strategy. However, there are conflicting findings regarding the role of AT2R. In the current study, we evaluated the effects of overexpressing AT2R by viral vector transduction on the apoptosis and function of pancreatic β-islet cells. The rat insulinoma cell line, INS-1, was transduced with a recombinant adenoviral vector expressing AT2R (Ad-G-AT2R-EGFP). AT2R overexpression resulted in significantly reduced cell viability and subsequently impaired glucose-stimulated insulin secretion (GSIS) function in INS-1 cells. Down-regulated expressions of GSIS pathway components, insulin, glucose transporter 2, and glucokinase were associated with AT2R overexpression. Further analysis determined that overexpression of AT2R induced G1-phase cell cycle arrest and Ang II-independent apoptotic cell death as indicated by increased Annexin V staining. To understand the apoptosis signaling triggered by AT2R overexpression, levels of caspase proteins were measured. Overexpression of AT2R significantly induced caspase-8, caspase-9, and caspase-3 cleavage, and decreased Bcl-2, pAkt, and pERK expression levels. AT2R-induced cell apoptosis was successfully blocked by the caspase inhibitor Z-VAD-FMK. Our findings suggested that AT2R overexpression triggers the apoptosis of INS-1 cells and dysfunction in insulin secretion. In conclusion, more careful design and consideration are required when applying AT2R-related therapies in treating diabetes.

Acetylcholine is released from taste cells, enhancing taste signalling

PubMed Central

Dando, Robin; Roper, Stephen D

2012-01-01

Acetylcholine (ACh), a candidate neurotransmitter that has been implicated in taste buds, elicits calcium mobilization in Receptor (Type II) taste cells. Using RT-PCR analysis and pharmacological interventions, we demonstrate that the muscarinic acetylcholine receptor M3 mediates these actions. Applying ACh enhanced both taste-evoked Ca2+ responses and taste-evoked afferent neurotransmitter (ATP) secretion from taste Receptor cells. Blocking muscarinic receptors depressed taste-evoked responses in Receptor cells, suggesting that ACh is normally released from taste cells during taste stimulation. ACh biosensors confirmed that, indeed, taste Receptor cells secrete acetylcholine during gustatory stimulation. Genetic deletion of muscarinic receptors resulted in significantly diminished ATP secretion from taste buds. The data demonstrate a new role for acetylcholine as a taste bud transmitter. Our results imply specifically that ACh is an autocrine transmitter secreted by taste Receptor cells during gustatory stimulation, enhancing taste-evoked responses and afferent transmitter secretion. PMID:22570381

Type II secretion system of Pseudomonas aeruginosa: in vivo evidence of a significant role in death due to lung infection.

PubMed

Jyot, Jeevan; Balloy, Viviane; Jouvion, Gregory; Verma, Amrisha; Touqui, Lhousseine; Huerre, Michel; Chignard, Michel; Ramphal, Reuben

2011-05-15

The role of toxins secreted by the type II secretion system (T2SS) of Pseudomonas aeruginosa during lung infection has been uncertain despite decades of research. Using a model of pneumonia in Toll-like receptor (TLR) 2,4(-/-) mice, we reexamined the role of the T2SS system. Flagellin-deficient mutants of P. aeruginosa, with mutations in the T2SS and/or T3SS, were used to infect mice. Mice were followed up for survival, with some killed at different intervals to study bacterial clearance, inflammatory responses, and lung pathology. Strains carrying either secretion system were lethal for mice. Double mutants were avirulent. The T3SS(+) strains killed mice within a day, and the T2SS(+) strains killed them later. Mice infected with a strain that had only the T2SS were unable to eradicate the organism from the lungs, whereas those infected with a T2SS-T3SS double deletion were able to clear this mutant. Death caused by the T2SS(+) strain was accompanied by a >50-fold increase in bacterial counts and higher numbers of viable intracellular bacteria. The T2SS of P. aeruginosa may play a role in death from pneumonia, but its action is delayed. These data suggest that antitoxin strategies against this organism will require measures against the toxins secreted by both T2SS and T3SS.

Macrophage Control of Phagocytosed Mycobacteria Is Increased by Factors Secreted by Alveolar Epithelial Cells through Nitric Oxide Independent Mechanisms

PubMed Central

Freidl, Raphaela; Fernández, Carmen

2014-01-01

Tissue-resident macrophages are heterogeneous with tissue-specific and niche-specific functions. Thus, simplified models of macrophage activation do not explain the extent of heterogeneity seen in vivo. We focus here on the respiratory tract and ask whether factors secreted by alveolar epithelial cells (AEC) can influence the functionality of resident pulmonary macrophages (PuM). We have previously reported that factors secreted by AEC increase control of intracellular growth of BCG in macrophages. In the current study, we also aimed to investigate possible mechanisms by which AEC-derived factors increase intracellular control of BCG in both primary murine interstitial macrophages, and bone marrow-derived macrophages and characterize further the effect of these factors on macrophage differentiation. We show that; a) in contrast to other macrophage types, IFN-γ did not increase intracellular growth control of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG) by interstitial pulmonary macrophages although the same macrophages could be activated by factors secreted by AEC; b) the lack of response of pulmonary macrophages to IFN-γ was apparently regulated by suppressor of cytokine signaling (SOCS)1; c) AEC-derived factors did not induce pro-inflammatory pathways induced by IFN-γ e.g. expression of inducible nitric oxide synthase (iNOS), secretion of nitric oxide (NO), or IL-12, d) in contrast to IFN-γ, intracellular bacterial destruction induced by AEC-derived factors was not dependent on iNOS transcription and NO production. Collectively, our data show that PuM were restricted in inflammatory responses mediated by IFN-γ through SOCS1 and that factors secreted by AEC- enhanced the microbicidal capacities of macrophages by iNOS independent mechanisms. PMID:25089618

A new transgenic rice line exhibiting enhanced ferric iron reduction and phytosiderophore production confers tolerance to low iron availability in calcareous soil.

PubMed

Masuda, Hiroshi; Shimochi, Erika; Hamada, Tatsuro; Senoura, Takeshi; Kobayashi, Takanori; Aung, May Sann; Ishimaru, Yasuhiro; Ogo, Yuko; Nakanishi, Hiromi; Nishizawa, Naoko K

2017-01-01

Iron (Fe) deficiency is a critical agricultural problem, especially in calcareous soil, which is distributed worldwide. Rice plants take up Fe(II) from soil through a OsIRT1 transporter (Strategy I-related system) and also take up Fe(III) via a phytosiderophore-based system (Strategy II system). However, rice plants are susceptible to low-Fe conditions because they have low Fe(III) reduction activity and low-level phytosiderophore secretion. Previously, we produced transgenic rice plants expressing a mutationally reconstructed yeast ferric chelate reductase, refre1/372, under the control of the OsIRT1 promoter. This transgenic rice line exhibited higher Fe(III) chelate reductase activity and tolerance to Fe deficiency. In addition, we produced transgenic rice overexpressing the Fe deficiency-inducible transcription factor, OsIRO2, which regulates the expression of various genes involved in the strategy II Fe(III) uptake system, including OsNAS1, OsNAAT1, OsDMAS1, OsYSL15, and TOM1. This transgenic rice exhibited improved phytosiderophore secretion ability and tolerance to Fe deficiency. In the present research, transgenic rice plants that possess both the OsIRT1 promoter-refre1/372 and the 35S promoter-OsIRO2 (RI lines) were produced to enhance both Strategy I Fe(II) reductase ability and Strategy II phytosiderophore productivity. RI lines exhibited enhanced tolerance to Fe-deficient conditions at the early and middle-late stages of growth in calcareous soil, compared to both the non-transgenic line and lines harboring either OsIRT1 promoter-refre1/372 or 35S promoter-OsIRO2 alone. RI lines also exhibited a 9-fold higher yield than the non-transgenic line. Moreover, we successfully produced Fe-deficiency-tolerant Tachisugata rice, which is a high-biomass variety used as fodder. Collectively, our results demonstrate that combined enhancement of two Fe uptake systems in rice is highly effective in conferring tolerance to low Fe availability in calcareous soil.

Dissection of the Human Multipotent Adult Progenitor Cell Secretome by Proteomic Analysis

PubMed Central

van't Hof, Wouter; Newell, Laura F.; Reddy, Ashok; Wilmarth, Phillip A.; David, Larry L.; Raber, Amy; Bogaerts, Annelies; Pinxteren, Jef; Deans, Robert J.; Maziarz, Richard T.

2013-01-01

Multipotent adult progenitor cells (MAPCs) are adult adherent stromal stem cells currently being assessed in acute graft versus host disease clinical trials with demonstrated immunomodulatory capabilities and the potential to ameliorate detrimental autoimmune and inflammation-related processes. Our previous studies documented that MAPCs secrete factors that play a role in regulating T-cell activity. Here we expand our studies using a proteomics approach to characterize and quantify MAPC secretome components secreted over 72 hours in vitro under steady-state conditions and in the presence of the inflammatory triggers interferon-γ and lipopolysaccharide, or a tolerogenic CD74 ligand, RTL1000. MAPCs differentially responded to each of the tested stimuli, secreting molecules that regulate the biological activity of the extracellular matrix (ECM), including proteins that make up the ECM itself, proteins that regulate its construction/deconstruction, and proteins that serve to attach and detach growth factors from ECM components for redistribution upon appropriate stimulation. MAPCs secreted a wide array of proteases, some detectable in their zymogen forms. MAPCs also secreted protease inhibitors that would regulate protease activity. MAPCs secreted chemokines and cytokines that could provide molecular guidance cues to various cell types, including neutrophils, macrophages, and T cells. In addition, MAPCs secreted factors involved in maintenance of a homeostatic environment, regulating such diverse programs as innate immunity, angiogenesis/angiostasis, targeted delivery of growth factors, and the matrix-metalloprotease cascade. PMID:23981727

Oleic acid and peanut oil high in oleic acid reverse the inhibitory effect of insulin production of the inflammatory cytokine TNF-alpha both in vitro and in vivo systems.

PubMed

Vassiliou, Evros K; Gonzalez, Andres; Garcia, Carlos; Tadros, James H; Chakraborty, Goutam; Toney, Jeffrey H

2009-06-26

Chronic inflammation is a key player in pathogenesis. The inflammatory cytokine, tumor necrosis factor-alpha is a well known inflammatory protein, and has been a therapeutic target for the treatment of diseases such as Rheumatoid Arthritis and Crohn's Disease. Obesity is a well known risk factor for developing non-insulin dependent diabetes melitus. Adipose tissue has been shown to produce tumor necrosis factor-alpha, which has the ability to reduce insulin secretion and induce insulin resistance. Based on these observations, we sought to investigate the impact of unsaturated fatty acids such as oleic acid in the presence of TNF-alpha in terms of insulin production, the molecular mechanisms involved and the in vivo effect of a diet high in oleic acid on a mouse model of type II diabetes, KKAy. The rat pancreatic beta cell line INS-1 was used as a cell biological model since it exhibits glucose dependent insulin secretion. Insulin production assessment was carried out using enzyme linked immunosorbent assay and cAMP quantification with competitive ELISA. Viability of TNF-alpha and oleic acid treated cells was evaluated using flow cytometry. PPAR-gamma translocation was assessed using a PPRE based ELISA system. In vivo studies were carried out on adult male KKAy mice and glucose levels were measured with a glucometer. Oleic acid and peanut oil high in oleic acid were able to enhance insulin production in INS-1. TNF-alpha inhibited insulin production but pre-treatment with oleic acid reversed this inhibitory effect. The viability status of INS-1 cells treated with TNF-alpha and oleic acid was not affected. Translocation of the peroxisome proliferator- activated receptor transcription factor to the nucleus was elevated in oleic acid treated cells. Finally, type II diabetic mice that were administered a high oleic acid diet derived from peanut oil, had decreased glucose levels compared to animals administered a high fat diet with no oleic acid. Oleic acid was found to be effective in reversing the inhibitory effect in insulin production of the inflammatory cytokine TNF-alpha. This finding is consistent with the reported therapeutic characteristics of other monounsaturated and polyunsaturated fatty acids. Furthermore, a diet high in oleic acid, which can be easily achieved through consumption of peanuts and olive oil, can have a beneficial effect in type II diabetes and ultimately reverse the negative effects of inflammatory cytokines observed in obesity and non insulin dependent diabetes mellitus.

Oleic acid and peanut oil high in oleic acid reverse the inhibitory effect of insulin production of the inflammatory cytokine TNF-α both in vitro and in vivo systems

PubMed Central

Vassiliou, Evros K; Gonzalez, Andres; Garcia, Carlos; Tadros, James H; Chakraborty, Goutam; Toney, Jeffrey H

2009-01-01

Background Chronic inflammation is a key player in pathogenesis. The inflammatory cytokine, tumor necrosis factor-alpha is a well known inflammatory protein, and has been a therapeutic target for the treatment of diseases such as Rheumatoid Arthritis and Crohn's Disease. Obesity is a well known risk factor for developing non-insulin dependent diabetes melitus. Adipose tissue has been shown to produce tumor necrosis factor-alpha, which has the ability to reduce insulin secretion and induce insulin resistance. Based on these observations, we sought to investigate the impact of unsaturated fatty acids such as oleic acid in the presence of TNF-α in terms of insulin production, the molecular mechanisms involved and the in vivo effect of a diet high in oleic acid on a mouse model of type II diabetes, KKAy. Methods The rat pancreatic beta cell line INS-1 was used as a cell biological model since it exhibits glucose dependent insulin secretion. Insulin production assessment was carried out using enzyme linked immunosorbent assay and cAMP quantification with competitive ELISA. Viability of TNF-α and oleic acid treated cells was evaluated using flow cytometry. PPAR-γ translocation was assessed using a PPRE based ELISA system. In vivo studies were carried out on adult male KKAy mice and glucose levels were measured with a glucometer. Results Oleic acid and peanut oil high in oleic acid were able to enhance insulin production in INS-1. TNF-α inhibited insulin production but pre-treatment with oleic acid reversed this inhibitory effect. The viability status of INS-1 cells treated with TNF-α and oleic acid was not affected. Translocation of the peroxisome proliferator- activated receptor transcription factor to the nucleus was elevated in oleic acid treated cells. Finally, type II diabetic mice that were administered a high oleic acid diet derived from peanut oil, had decreased glucose levels compared to animals administered a high fat diet with no oleic acid. Conclusion Oleic acid was found to be effective in reversing the inhibitory effect in insulin production of the inflammatory cytokine TNF-α. This finding is consistent with the reported therapeutic characteristics of other monounsaturated and polyunsaturated fatty acids. Furthermore, a diet high in oleic acid, which can be easily achieved through consumption of peanuts and olive oil, can have a beneficial effect in type II diabetes and ultimately reverse the negative effects of inflammatory cytokines observed in obesity and non insulin dependent diabetes mellitus. PMID:19558671

High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

PubMed Central

Santiago, Jon-Jon; McNaughton, Leslie J.; Koleini, Navid; Ma, Xin; Bestvater, Brian; Nickel, Barbara E.; Fandrich, Robert R.; Wigle, Jeffrey T.; Freed, Darren H.; Arora, Rakesh C.; Kardami, Elissavet

2014-01-01

Fibroblast growth factor 2 (FGF-2) is a multifunctional protein synthesized as high (Hi-) and low (Lo-) molecular weight isoforms. Studies using rodent models showed that Hi- and Lo-FGF-2 exert distinct biological activities: after myocardial infarction, rat Lo-FGF-2, but not Hi-FGF-2, promoted sustained cardioprotection and angiogenesis, while Hi-FGF-2, but not Lo-FGF-2, promoted myocardial hypertrophy and reduced contractile function. Because there is no information regarding Hi-FGF-2 in human myocardium, we undertook to investigate expression, regulation, secretion and potential tissue remodeling-associated activities of human cardiac (atrial) Hi-FGF-2. Human patient-derived atrial tissue extracts, as well as pericardial fluid, contained Hi-FGF-2 isoforms, comprising, respectively, 53%(±20 SD) and 68% (±25 SD) of total FGF-2, assessed by western blotting. Human atrial tissue-derived primary myofibroblasts (hMFs) expressed and secreted predominantly Hi-FGF-2, at about 80% of total. Angiotensin II (Ang II) up-regulated Hi-FGF-2 in hMFs, via activation of both type 1 and type 2 Ang II receptors; the ERK pathway; and matrix metalloprotease-2. Treatment of hMFs with neutralizing antibodies selective for human Hi-FGF-2 (neu-AbHi-FGF-2) reduced accumulation of proteins associated with fibroblast-to-myofibroblast conversion and fibrosis, including α-smooth muscle actin, extra-domain A fibronectin, and procollagen. Stimulation of hMFs with recombinant human Hi-FGF-2 was significantly more potent than Lo-FGF-2 in upregulating inflammation-associated proteins such as pro-interleukin-1β and plasminogen-activator-inhibitor-1. Culture media conditioned by hMFs promoted cardiomyocyte hypertrophy, an effect that was prevented by neu-AbHi-FGF-2 in vitro. In conclusion, we have documented that Hi-FGF-2 represents a substantial fraction of FGF-2 in human cardiac (atrial) tissue and in pericardial fluid, and have shown that human Hi-FGF-2, unlike Lo-FGF-2, promotes deleterious (pro-fibrotic, pro-inflammatory, and pro-hypertrophic) responses in vitro. Selective targeting of Hi-FGF-2 production may, therefore, reduce pathological remodelling in the human heart. PMID:24827991

High molecular weight fibroblast growth factor-2 in the human heart is a potential target for prevention of cardiac remodeling.

PubMed

Santiago, Jon-Jon; McNaughton, Leslie J; Koleini, Navid; Ma, Xin; Bestvater, Brian; Nickel, Barbara E; Fandrich, Robert R; Wigle, Jeffrey T; Freed, Darren H; Arora, Rakesh C; Kardami, Elissavet

2014-01-01

Fibroblast growth factor 2 (FGF-2) is a multifunctional protein synthesized as high (Hi-) and low (Lo-) molecular weight isoforms. Studies using rodent models showed that Hi- and Lo-FGF-2 exert distinct biological activities: after myocardial infarction, rat Lo-FGF-2, but not Hi-FGF-2, promoted sustained cardioprotection and angiogenesis, while Hi-FGF-2, but not Lo-FGF-2, promoted myocardial hypertrophy and reduced contractile function. Because there is no information regarding Hi-FGF-2 in human myocardium, we undertook to investigate expression, regulation, secretion and potential tissue remodeling-associated activities of human cardiac (atrial) Hi-FGF-2. Human patient-derived atrial tissue extracts, as well as pericardial fluid, contained Hi-FGF-2 isoforms, comprising, respectively, 53%(±20 SD) and 68% (±25 SD) of total FGF-2, assessed by western blotting. Human atrial tissue-derived primary myofibroblasts (hMFs) expressed and secreted predominantly Hi-FGF-2, at about 80% of total. Angiotensin II (Ang II) up-regulated Hi-FGF-2 in hMFs, via activation of both type 1 and type 2 Ang II receptors; the ERK pathway; and matrix metalloprotease-2. Treatment of hMFs with neutralizing antibodies selective for human Hi-FGF-2 (neu-AbHi-FGF-2) reduced accumulation of proteins associated with fibroblast-to-myofibroblast conversion and fibrosis, including α-smooth muscle actin, extra-domain A fibronectin, and procollagen. Stimulation of hMFs with recombinant human Hi-FGF-2 was significantly more potent than Lo-FGF-2 in upregulating inflammation-associated proteins such as pro-interleukin-1β and plasminogen-activator-inhibitor-1. Culture media conditioned by hMFs promoted cardiomyocyte hypertrophy, an effect that was prevented by neu-AbHi-FGF-2 in vitro. In conclusion, we have documented that Hi-FGF-2 represents a substantial fraction of FGF-2 in human cardiac (atrial) tissue and in pericardial fluid, and have shown that human Hi-FGF-2, unlike Lo-FGF-2, promotes deleterious (pro-fibrotic, pro-inflammatory, and pro-hypertrophic) responses in vitro. Selective targeting of Hi-FGF-2 production may, therefore, reduce pathological remodelling in the human heart.

[Factors causing damage and destruction of beta-cells of the islets of Langerhans in the pancreas].

PubMed

Anděl, Michal; Němcová, Vlasta; Pavlíková, Nela; Urbanová, Jana; Cecháková, Marie; Havlová, Andrea; Straková, Radka; Večeřová, Livia; Mandys, Václav; Kovář, Jan; Heneberg, Petr; Trnka, Jan; Polák, Jan

2014-09-01

Insulin secretion in patients with manifested diabetes mellitus tends to disappear months to decades after the diagnosis, which is a clear sign of a gradual loss of pancreatic islet beta-cells. In our sample of 30 type 2 diabetic patients, whose disease manifested between 30 and 45 years of age, about a half have retained or even increased insulin secretion 30 years later, while the other half exhibit a much diminished or lost insulin secretion. Factors that can damage or destroy beta-cells can be divided into the following groups: Metabolic factors: hyperglycemia and glucotoxicity, lipotoxicity, hypoxia, reactive oxygen species; Pharmacological factors: antimicrobial medication pentamidine, SSRI antidepressants; Factors related to impaired insulin secretion: MODY type diabetes; Environmental toxic factors: rat poison Vacor, streptozotocin, polychlorinated and polybrominated hydrocarbons; Disorders of the exocrine pancreas: tumor infiltration, fibrous infiltration, chronic pancreatitis, cystic fibrosis; Infections, inflammation, autoimmunity, viral factors: Coxsackie viruses, H1N1 influenza, enteroviruses. We are currently working on finding other factors leading to beta-cell damage, studying their effect on apoptosis and necrosis and looking for possible protective factors to prevent this damage. We our increasing knowledge about the mechanisms of beta-cell damage and destruction we come ever closer to suggest measures for their prevention. In this review we offer a brief and simplified summary of some of the findings related to this area.Key words: pancreatic islet beta-cells of Langerhans - factors damaging or destroying beta-cells - insulin secretion.

Maintaining K+ balance on the low-Na+, high-K+ diet

PubMed Central

Cornelius, Ryan J.; Wang, Bangchen; Wang-France, Jun

2016-01-01

A low-Na+, high-K+ diet (LNaHK) is considered a healthier alternative to the “Western” high-Na+ diet. Because the mechanism for K+ secretion involves Na+ reabsorptive exchange for secreted K+ in the distal nephron, it is not understood how K+ is eliminated with such low Na+ intake. Animals on a LNaHK diet produce an alkaline load, high urinary flows, and markedly elevated plasma ANG II and aldosterone levels to maintain their K+ balance. Recent studies have revealed a potential mechanism involving the actions of alkalosis, urinary flow, elevated ANG II, and aldosterone on two types of K+ channels, renal outer medullary K+ and large-conductance K+ channels, located in principal and intercalated cells. Here, we review these recent advances. PMID:26739887

Molecular Mechanisms of Insulin Secretion and Insulin Action.

ERIC Educational Resources Information Center

Flatt, Peter R.; Bailey, Clifford J.

1991-01-01

Information and current ideas on the factors regulating insulin secretion, the mechanisms underlying the secretion and biological actions of insulin, and the main characteristics of diabetes mellitus are presented. (Author)

Immune profiling of plasma and cervical secretions using recycling immunoaffinity chromatography.

PubMed

Castle, Philip E; Phillips, Terry M; Hildesheim, Allan; Herrero, Rolando; Bratti, M Concepcion; Rodríguez, Ana Cecilia; Morera, Lidia Ana; Pfeiffer, Ruth; Hutchinson, Martha L; Pinto, Ligia A; Schiffman, Mark

2003-12-01

Small volumes of cervical secretions have limited measurements of immunity at the cervix, which may be important to studies of human papillomavirus (HPV). We report the use of recycling immunoaffinity chromatography to efficiently study immune profiles in cervical secretions. Frozen pairs of plasma and cervical secretions (collected on ophthalmic sponges) were selected randomly from women with normal cervical cytology (n = 50) participating in a natural history study of HPV in Guanacaste, Costa Rica. Single 25- micro l aliquots of plasma and (diluted) cervical secretions were assayed for interleukin (IL) -1 beta, -2, -4, -6, -8, -10, -12, -13, -15, IFN-alpha, -beta, -gamma, tumor necrosis factor-alpha, -beta, RANTES (regulated on activation normal T-cell express and secreted), MCP-1 (monocyte chemoattractant protein), -2, -3, macrophage inflammatory protein-1 alpha, -1 beta (regulated on activation normal T-cell express and secreted), macrophage colony-stimulating factor, IgG, IgA, and cyclooxygenase 2. All of the specimens were tested as blind replicates, and refrozen plasma was retested 4 months later. To evaluate the reproducibility of the repeat measurements and to examine the correlation between plasma and cervical secretions, we calculated kappa values with 95% confidence intervals among categorized analyte values and Spearman correlation coefficients (rho) among detectable, continuous analyte values. Measurements of all of the analytes in either plasma or cervical secretions were highly reproducible, with all of the kappa > or = 0.78 (70% above 0.90), and all of the rho > or = 0.88 (96% above 0.90). Only IL-1 beta (kappa = 0.60 and rho = 0.82) and IL-6 (kappa = 0.50 and rho = 0.78) levels were strongly correlated between plasma and cervical secretions. IFN-gamma, tumor necrosis factor-beta, RANTES, MCP-1, MCP -2, macrophage inflammatory protein-1 alpha, and macrophage colony-stimulating factor levels were especially poorly correlated between plasma and cervical secretions (kappa < or = 0.25 and rho < or = 0.25). We conclude that recycling immunoaffinity chromatography is a reproducible method of measuring immune profiles from biological specimens, and immune profiles are not well correlated between plasma and cervical secretions, perhaps necessitating cervical collections to study cervix-specific immunity in HPV natural history studies.

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

Swanner, E. D.; Bayer, T.; Wu, W.

In this study, we couple iron isotope analysis to microscopic and mineralogical investigation of iron speciation during circumneutral Fe(II) oxidation and Fe(III) precipitation with photosynthetically produced oxygen. In the presence of the cyanobacterium Synechococcus PCC 7002, aqueous Fe(II) (Fe(II) aq) is oxidized and precipitated as amorphous Fe(III) oxyhydroxide minerals (iron precipitates, Fe ppt), with distinct isotopic fractionation (ε 56Fe) values determined from fitting the δ 56Fe(II) aq (1.79‰ and 2.15‰) and the δ 56Fe ppt (2.44‰ and 2.98‰) data trends from two replicate experiments. Additional Fe(II) and Fe(III) phases were detected using microscopy and chemical extractions and likely represent Fe(II)more » and Fe(III) sorbed to minerals and cells. The iron desorbed with sodium acetate (FeNaAc) yielded heavier δ 56Fe compositions than Fe(II) aq. Modeling of the fractionation during Fe(III) sorption to cells and Fe(II) sorption to Feppt, combined with equilibration of sorbed iron and with Fe(II) aq using published fractionation factors, is consistent with our resulting δ 56FeNaAc. The δ 56Fe ppt data trend is inconsistent with complete equilibrium exchange with Fe(II)aq. Because of this and our detection of microbially excreted organics (e.g., exopolysaccharides) coating Feppt in our microscopic analysis, we suggest that electron and atom exchange is partially suppressed in this system by biologically produced organics. These results indicate that cyanobacteria influence the fate and composition of iron in sunlit environments via their role in Fe(II) oxidation through O 2 production, the capacity of their cell surfaces to sorb iron, and the interaction of secreted organics with Fe(III) minerals.« less

Excessive fluoride induces endoplasmic reticulum stress and interferes enamel proteinases secretion.

PubMed

Wei, Wei; Gao, Yanhui; Wang, Cheng; Zhao, Lijun; Sun, Dianjun

2013-06-01

Protein retention in the enamel layer during tooth formation is well known to be associated with dental fluorosis but the underlying mechanism is unclear. The functions of the endoplasmic reticulum (ER) correlate directly with secreted protein metabolism. We used an ameloblast-derived cell line to determine whether excessive amounts of fluoride cause ER stress, and whether this interferes with the secretion of enamel matrix proteinases. ER stress activates a signaling network called the unfolded protein response (UPR). Here, we used real-time RT-PCR and immunofluorescence to study the effect of fluoride on the expression, translation, and secretion of UPR transcription factors in ameloblast-like cells. Measurement of both the gene and protein expression of UPR transcription factors indicated that high-dose fluoride increases the expression of UPR transcription factors in a dose-dependent manner. We also used ELISA to detect and quantify the enamel proteinases secreted by ameloblasts. We found a corresponding decrease in extracellular secretion of the enamel proteinases matrix metalloproteinase-20 and kallikrein-4, after exposure to fluoride. Furthermore, correlation analysis indicated that the expression of UPR transcription factors showed a strong inverse correlation with that of enamel proteinases. The results suggest that high-dose fluoride initiates an ER stress response in ameloblasts and induces the UPR, which interferes with the synthesis and secretion of enamel proteinases. Taken together, these results suggest that excessive ingestion of fluoride during tooth formation can decrease the secretion of proteinases, thus causing protein retention in the enamel layer, indicating that the ER stress response may be responsible for dental fluorosis. Copyright © 2011 Wiley Periodicals, Inc.

Macrophage cell lines derived from major histocompatibility complex II-negative mice

NASA Technical Reports Server (NTRS)

Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1998-01-01

Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

Indagation of serum and salivary reactive oxygen metabolite and cortisol levels in chronic periodontitis and stress-induced chronic periodontitis patients.

PubMed

Sudhakar, Uma; Thyagarajan, Ramakrishnan; Jeyapal, Bhagyameena; Jagadeesh, Sushuruthi; Jayakumar, Parvathee

2017-01-01

Periodontal disease is not a conventional bacterial infection but is an inflammatory disease initiated by immune response against a group of microorganisms in susceptible hosts. There are many intriguing researches that unfold the secrets of chronic periodontitis. The current researches in chronic periodontitis are directed toward an approach that respects the scientific relationship between the various risk factors, the genetic factors, and the progression of the disease. This study aims to evaluate the cortisol and reactive oxygen metabolites (ROM) concentration in serum and to find out their association in periodontal health and disease. In this study, totally thirty patients have been taken and divided into two groups of chronic periodontitis (Group I) and stress-induced chronic periodontitis (Group II) and evaluated the correlation between the ROM and cortisol levels in them. This is the first study, where both the levels of ROM and cortisol are checked in the serum and saliva. The analysis is done to check the association between them. The data were statistically analyzed using software program (SPSSV 16), Pearson correlation, and paired t -test. Comparison of the mean ROM levels in Group I and Group II showed that mean ROM level in Group II is highly significant than Group I. Our study suggests that stress can have a role in the progression of periodontal disease by increasing the cortisol and ROM levels.

Production of insulin-like growth factor binding proteins by small-cell lung cancer cell lines

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

Jaques, G.; Kiefer, P.; Rotsch, M.

1989-10-01

Conditioned serum-free media (CM) from small-cell lung cancer (SCLC) cell lines were examined for the presence of insulin-like growth-factor-binding proteins (IGF-BP). 6/9 SCLC cell lines secreted binding proteins with high affinity for IGFs. When ({sup 125}I)IGF-1 or ({sup 125}I)IGF-II was incubated with the CMs, complexes of tracer with proteins could be demonstrated by gel filtration, by precipitation with polyethylenglycol, and after adsorption of unbound tracer with activated charcoal. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF-I and IGF-II. Cross-linking of ({sup 125}I)IGF-I or ({sup 125}I)IGF-II to the CMs followed by sodium dodecylmore » sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions revealed the presence of IGF-BPs with molecular masses in the range of 24-32 kDa. Northern blot hybridization with an IGF-BP cDNA probe encoding a low-molecular-weight IGF-BP from a human placenta cDNA library and Western blot analysis with a corresponding polyclonal antibody showed no expression of this gene. These data demonstrate that SCLC cell lines release IGF-BPs in culture supernatants, which differ from IGF-BPs detected in liver and placenta. These IGF-BPs might be important mediators in the autocrine/paracrine growth regulation of IGFs in SCLC.« less

Differential effects of caffeine on hair shaft elongation, matrix and outer root sheath keratinocyte proliferation, and transforming growth factor-β2/insulin-like growth factor-1-mediated regulation of the hair cycle in male and female human hair follicles in vitro.

PubMed

Fischer, T W; Herczeg-Lisztes, E; Funk, W; Zillikens, D; Bíró, T; Paus, R

2014-11-01

Caffeine reportedly counteracts the suppression of hair shaft production by testosterone in organ-cultured male human hair follicles (HFs). We aimed to investigate the impact of caffeine (i) on additional key hair growth parameters, (ii) on major hair growth regulatory factors and (iii) on male vs. female HFs in the presence of testosterone. Microdissected male and female human scalp HFs were treated in serum-free organ culture for 120 h with testosterone alone (0·5 μg mL(-1)) or in combination with caffeine (0·005-0·0005%). The following effects on hair shaft elongation were evaluated by quantitative (immuno)histomorphometry: HF cycling (anagen-catagen transition); hair matrix keratinocyte proliferation; expression of a key catagen inducer, transforming growth factor (TGF)-β2; and expression of the anagen-prolonging insulin-like growth factor (IGF)-1. Caffeine effects were further investigated in human outer root sheath keratinocytes (ORSKs). Caffeine enhanced hair shaft elongation, prolonged anagen duration and stimulated hair matrix keratinocyte proliferation. Female HFs showed higher sensitivity to caffeine than male HFs. Caffeine counteracted testosterone-enhanced TGF-β2 protein expression in male HFs. In female HFs, testosterone failed to induce TGF-β2 expression, while caffeine reduced it. In male and female HFs, caffeine enhanced IGF-1 protein expression. In ORSKs, caffeine stimulated cell proliferation, inhibited apoptosis/necrosis, and upregulated IGF-1 gene expression and protein secretion, while TGF-β2 protein secretion was downregulated. This study reveals new growth-promoting effects of caffeine on human hair follicles in subjects of both sexes at different levels (molecular, cellular and organ). © 2014 British Association of Dermatologists.

Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.

PubMed

Zeelenberg, Ingrid S; Ostrowski, Matias; Krumeich, Sophie; Bobrie, Angélique; Jancic, Carolina; Boissonnas, Alexandre; Delcayre, Alain; Le Pecq, Jean-Bernard; Combadière, Béhazine; Amigorena, Sebastian; Théry, Clotilde

2008-02-15

Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter.

PubMed

Byun, Hyunjong; Park, Jiyeon; Kim, Sun Chang; Ahn, Jung Hoon

2017-12-01

Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

[Cephalic phase of insulin secretion and the variety of the food in rat feed].

PubMed

Louis-Sylvestre, J; Le Magnen, J

1983-01-01

Immunologically reactive insulin levels were determined in freely-moving normal rats offered three different test-meals. In test I, they were offered their normal diet for 4.5 min. In test II, they first ate the same diet for the same amount of time, then a cookie for 6 min, and finally they had free access to lard. In test III, the normal diet was followed by a synthetic sweetener and then by vaseline. It was shown that in a varied meal (tests II and III), the ingestion of each new food was immediately followed by a peak of insulin secretion superimposed on normal postprandial hyperinsulinemia. This resulted in an overall increase in insulinemia over the 15-min period measured. It is well known that in a varied meal more food is consumed. Thus, it may be that when a varied meal is offered, final satiety is postponed. This could be due to sensorially-triggered peaks of insulin secretion which would explain why the "cafeteria diet" induces hyperphagia and obesity.

Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines.

PubMed

Adalsteinsson, Viktor A; Tahirova, Narmin; Tallapragada, Naren; Yao, Xiaosai; Campion, Liam; Angelini, Alessandro; Douce, Thomas B; Huang, Cindy; Bowman, Brittany; Williamson, Christina A; Kwon, Douglas S; Wittrup, K Dane; Love, J Christopher

2013-10-01

Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via the CXCR1 and CXCR2 receptors, influencing tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.

Neurotrophin Signaling Is Required for Glucose-Induced Insulin Secretion.

PubMed

Houtz, Jessica; Borden, Philip; Ceasrine, Alexis; Minichiello, Liliana; Kuruvilla, Rejji

2016-11-07

Insulin secretion by pancreatic islet β cells is critical for glucose homeostasis, and a blunted β cell secretory response is an early deficit in type 2 diabetes. Here, we uncover a regulatory mechanism by which glucose recruits vascular-derived neurotrophins to control insulin secretion. Nerve growth factor (NGF), a classical trophic factor for nerve cells, is expressed in pancreatic vasculature while its TrkA receptor is localized to islet β cells. High glucose rapidly enhances NGF secretion and increases TrkA phosphorylation in mouse and human islets. Tissue-specific deletion of NGF or TrkA, or acute disruption of TrkA signaling, impairs glucose tolerance and insulin secretion in mice. We show that internalized TrkA receptors promote insulin granule exocytosis via F-actin reorganization. Furthermore, NGF treatment augments glucose-induced insulin secretion in human islets. These findings reveal a non-neuronal role for neurotrophins and identify a new regulatory pathway in insulin secretion that can be targeted to ameliorate β cell dysfunction. Copyright © 2016 Elsevier Inc. All rights reserved.

Renal hemodynamics and renin-angiotensin system activity in humans with multifocal renal artery fibromuscular dysplasia.

PubMed

van Twist, Daan J L; Houben, Alphons J H M; de Haan, Michiel W; de Leeuw, Peter W; Kroon, Abraham A

2016-06-01

Fibromuscular dysplasia (FMD) is the second most common cause of renovascular hypertension. Nonetheless, knowledge on the renal microvasculature and renin-angiotensin system (RAS) activity in kidneys with FMD is scarce. Given the fairly good results of revascularization, we hypothesized that the renal microvasculature and RAS are relatively spared in kidneys with FMD. In 58 hypertensive patients with multifocal renal artery FMD (off medication) and 116 matched controls with essential hypertension, we measured renal blood flow (Xenon washout method) per kidney and drew blood samples from the aorta and both renal veins to determine renin secretion and glomerular filtration rate per kidney. We found that renal blood flow and glomerular filtration rate in FMD were comparable to those in controls. Although systemic renin levels were somewhat higher in FMD, renal renin secretion was not elevated. Moreover, in patients with unilateral FMD, no differences between the affected and unaffected kidney were observed with regard to renal blood flow, glomerular filtration rate, or renin secretion. In men, renin levels and renin secretion were higher as compared with women. The renal blood flow response to RAS modulation (by intrarenal infusion of angiotensin II, angiotensin-(1-7), an angiotensin II type 1 receptor blocker, or a nitric oxide synthase blocker) was also comparable between FMD and controls. Renal blood flow, glomerular filtration, and the response to vasoactive substances in kidneys with multifocal FMD are comparable to patients with essential hypertension, suggesting that microvascular function is relatively spared. Renin secretion was not increased and the response to RAS modulation was not affected in kidneys with FMD.

Structural characterization of the α-mating factor prepro-peptide for secretion of recombinant proteins in Pichia pastoris.

PubMed

Chahal, Sabreen; Wei, Peter; Moua, Pachai; Park, Sung Pil James; Kwon, Janet; Patel, Arth; Vu, Anthony T; Catolico, Jason A; Tsai, Yu Fang Tina; Shaheen, Nadia; Chu, Tiffany T; Tam, Vivian; Khan, Zill-E-Huma; Joo, Hyun Henry; Xue, Liang; Lin-Cereghino, Joan; Tsai, Jerry W; Lin-Cereghino, Geoff P

2017-01-20

The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion. Copyright © 2016 Elsevier B.V. All rights reserved.

Type II Secretion Substrates of Legionella pneumophila Translocate Out of the Pathogen-Occupied Vacuole via a Semipermeable Membrane.

PubMed

Truchan, Hilary K; Christman, Harry D; White, Richard C; Rutledge, Nakisha S; Cianciotto, Nicholas P

2017-06-20

Legionella pneumophila replicates in macrophages in a host-derived phagosome, termed the Legionella- containing vacuole (LCV). While the translocation of type IV secretion (T4S) effectors into the macrophage cytosol is well established, the location of type II secretion (T2S) substrates in the infected host cell is unknown. Here, we show that the T2S substrate ProA, a metalloprotease, translocates into the cytosol of human macrophages, where it associates with the LCV membrane (LCVM). Translocation is detected as early as 10 h postinoculation (p.i.), which is approximately the midpoint of the intracellular life cycle. However, it is detected as early as 6 h p.i. if ProA is hyperexpressed, indicating that translocation depends on the timing of ProA expression and that any other factors necessary for translocation are in place by that time point. Translocation occurs with all L. pneumophila strains tested and in amoebae, natural hosts for L. pneumophila It was absent in murine bone marrow-derived macrophages and murine macrophage cell lines. The ChiA chitinase also associated with the cytoplasmic face of the LCVM at 6 h p.i. and in a T2S-dependent manner. Galectin-3 and galectin-8, eukaryotic proteins whose localization is influenced by damage to host membranes, appeared within the LCV of infected human but not murine macrophages beginning at 6 h p.i. Thus, we hypothesize that ProA and ChiA are first secreted into the vacuolar lumen by the activity of the T2S and subsequently traffic into the macrophage cytosol via a novel mechanism that involves a semipermeable LCVM. IMPORTANCE Infection of macrophages and amoebae plays a central role in the pathogenesis of L. pneumophila , the agent of Legionnaires' disease. We have previously demonstrated that the T2S system of L. pneumophila greatly contributes to intracellular infection. However, the location of T2S substrates within the infected host cell is unknown. This report presents the first evidence of a L. pneumophila T2S substrate in the host cell cytosol and, therefore, the first evidence of a non-T4S effector trafficking out of the LCV. We also provide the first indication that the LCV is not completely intact but is instead semipermeable and that this occurs in human but not murine macrophages. Given this permeability, we hypothesize that other T2S substrates and LCV lumenal contents can escape into the host cell cytosol. Thus, these substrates may represent a battery of previously unidentified effectors that can interact with host factors and contribute to intracellular infection by L. pneumophila . Copyright © 2017 Truchan et al.

Regulation of cell growth by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor beta.

PubMed

Okuyama, H; Shimahara, Y; Kawada, N; Seki, S; Kristensen, D B; Yoshizato, K; Uyama, N; Yamaoka, Y

2001-07-27

Redox-regulated processes are important elements in various cellular functions. Reducing agents, such as N-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-beta, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-beta receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that "redox therapy" is promising for fibrosis-related disease.

Effect of L-Tryptophan and L-Leucine on Gut Hormone Secretion, Appetite Feelings and Gastric Emptying Rates in Lean and Non-Diabetic Obese Participants: A Randomized, Double-Blind, Parallel-Group Trial

PubMed Central

Meyer-Gerspach, Anne Christin; Häfliger, Simon; Meili, Julian; Doody, Alison; Rehfeld, Jens F; Drewe, Jürgen; Beglinger, Christoph; Wölnerhanssen, Bettina

2016-01-01

Background/Objectives Gut hormones such as cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) play a role as satiation factors. Strategies to enhance satiation peptide secretion could provide a therapeutic approach for obesity. Carbohydrates and lipids have been extensively investigated in relation to peptide release. In contrast, the role of proteins or amino acids is less clear. Our aim was to compare the effects of the amino acids L-tryptophan (L-trp) and L-leucine (L-leu) separately on gastric emptying and gut peptide secretion. Participants/Methods The study was conducted as a randomized (balanced), double-blind, parallel-group trial. A total of 10 lean and 10 non-diabetic obese participants were included. Participants received intragastric loads of L-trp (0.52 g and 1.56 g) and L-leu (1.56 g), dissolved in 300 mL tap water; 75 g glucose and 300 mL tap water served as control treatments. Results Results of the study are: i) L-trp at the higher dose stimulates CCK release (p = 0.0018), and induces a significant retardation in gastric emptying (p = 0.0033); ii) L-trp at the higher dose induced a small increase in GLP-1 secretion (p = 0.0257); iii) neither of the amino acids modulated subjective appetite feelings; and iv) the two amino acids did not alter insulin or glucose concentrations. Conclusions L-trp is a luminal regulator of CCK release with effects on gastric emptying, an effect that could be mediated by CCK. L-trp’s effect on GLP-1 secretion is only minor. At the doses given, the two amino acids did not affect subjective appetite feelings. Trial Registration ClinicalTrials.gov NCT02563847 PMID:27875537

The relationship between insulin secretion, the insulin-like growth factor axis and growth in children with cystic fibrosis.

PubMed

Ripa, Paulus; Robertson, Ian; Cowley, David; Harris, Margaret; Masters, I Brent; Cotterill, Andrew M

2002-03-01

Cystic fibrosis-related diabetes mellitus (CFRD) is an increasingly common complication of cystic fibrosis. CFRD is preceded by a progressive decline in insulin secretion but there is no accepted definition of the prediabetic state in CFRD. This prediabetic state appears to have adverse effects on clinical status, nutrition and lung function, but there is no direct evidence that the impaired glucose homeostasis is the cause of these deteriorations. This study examined the prevalence of glucose intolerance and impaired insulin secretion in a population of children with CF without CFRD. Severe CF lung disease is often associated with poor weight gain and slower growth but the mechanism for this is still unclear. The relationships between the current state of glucose homeostasis, insulin secretion and the insulin-like growth factor axis, height velocity, nutrition status and lung function were therefore studied. Eighteen children with cystic fibrosis aged 9.5-15 years had oral glucose tolerance tests and 14 of these also had intravenous glucose tolerance tests (four refused). Blood samples were collected for insulin, C-peptide, glucose, HbA1c, insulin-like growth factor (IGF)-I, IGF-II, IGF-binding protein (IGFBP)-1 and IGFBP-3. Data on height, weight, puberty status, clinical score (Shwachman score) and lung function were recorded. Height velocity, height and weight standard deviation scores (SDS) were calculated using WHO/CDC data. The mean height SDS (-0.52 +/- 0.17) was less than the normal population (P = 0.007) and the mean height velocity was 4.6 +/- 0.5 cm/year, 39% with a height velocity less than the third percentile for age. The weight SDS and body mass index (BMI) were similar to the normal population. Four children had impaired glucose tolerance. The first-phase insulin response (FPIR) was below the first percentile of normal population values in nine (65%). Impaired FPIR or impaired glucose tolerance did not correlate with the Shwachman score, nutritional status or pulmonary function. There was a significant positive correlation between insulin secretion (area under the curve) and height velocity (P = 0.001) and serum IGFBP-3 levels (P = 0.001). Impaired glucose tolerance was present in 20% of children with cystic fibrosis. Impaired insulin secretion was common (65%) even in children with normal glucose tolerance. The mean height SDS for the group was low and the height velocity was abnormally slow in 39%, yet nutritional status as measured by BMI was appropriate for age. Relative insulin deficiency rather than nutritional deprivation or poor clinical status thus appears to be implicated in the poor linear growth of these children with relatively stable lung disease. This was a small study and firm conclusions on this chronic suppurative disease as to the cause of poor growth are not possible. The causes of poor growth are likely to be complex; nevertheless, the apparent decrease in insulin secretion combined with the expected increased demands on insulin production during pubertal growth raises the question as to whether insulin therapy should be considered in children with cystic fibrosis before the onset of cystic fibrosis-related diabetes mellitus.

Loss of autophagy enhances MIF/macrophage migration inhibitory factor release by macrophages.

PubMed

Lee, Jacinta P W; Foote, Andrew; Fan, Huapeng; Peral de Castro, Celia; Lang, Tali; Jones, Sarah A; Gavrilescu, Nichita; Mills, Kingston H G; Leech, Michelle; Morand, Eric F; Harris, James

2016-06-02

MIF (macrophage migration inhibitory factor [glycosylation-inhibiting factor]) is a pro-inflammatory cytokine expressed in multiple cells types, including macrophages. MIF plays a pathogenic role in a number of inflammatory diseases and has been linked to tumor progression in some cancers. Previous work has demonstrated that loss of autophagy in macrophages enhances secretion of IL1 family cytokines. Here, we demonstrate that loss of autophagy, by pharmacological inhibition or siRNA silencing of Atg5, enhances MIF secretion by monocytes and macrophages. We further demonstrate that this is dependent on mitochondrial reactive oxygen species (ROS). Induction of autophagy with MTOR inhibitors had no effect on MIF secretion, but amino acid starvation increased secretion. This was unaffected by Atg5 siRNA but was again dependent on mitochondrial ROS. Our data demonstrate that autophagic regulation of mitochondrial ROS plays a pivotal role in the regulation of inflammatory cytokine secretion in macrophages, with potential implications for the pathogenesis of inflammatory diseases and cancers.

Vasopressin and angiotensin II in reflex regulation of ACTH, glucocorticoids, and renin: effect of water deprivation

NASA Technical Reports Server (NTRS)

Brooks, V. L.; Keil, L. C.

1992-01-01

Angiotensin II (ANG II) and vasopressin participate in baroreflex regulation of adrenocorticotropic hormone (ACTH), glucocorticoid, and renin secretion. The purpose of this study was to determine whether this participation is enhanced in water-deprived dogs, with chronically elevated plasma ANG II and vasopressin levels, compared with water-replete dogs. The baroreflex was assessed by infusing increasing doses of nitroprusside (0.3, 0.6, 1.5, and 3.0 micrograms.kg-1.min-1) in both groups of animals. To quantitate the participation of ANG II and vasopressin, the dogs were untreated or pretreated with the competitive ANG II antagonist saralasin, a V1-vasopressin antagonist, or combined V1/V2-vasopressin antagonist, either alone or in combination. The findings were as follows. 1) Larger reflex increases in ANG II, vasopressin, and glucocorticoids, but not ACTH, were produced in water-deprived dogs compared with water-replete dogs. 2) ANG II blockade blunted the glucocorticoid and ACTH responses to hypotension in water-deprived dogs, but not water-replete dogs. In contrast, vasopressin blockade reduced the ACTH response only in water-replete dogs. 3) Vasopressin or combined vasopressin and ANG II blockade reduced the plasma level of glucocorticoids related either to the fall in arterial pressure or to the increase in plasma ACTH concentration in water-replete dogs, and this effect was enhanced in water-deprived dogs. 4) In both water-deprived and water-replete animals, saralasin and/or a V1-antagonist increased the renin response to hypotension, but a combined V1/V2-antagonist did not. These results reemphasize the importance of endogenous ANG II and vasopressin in the regulation of ACTH, glucocorticoid, and renin secretion.(ABSTRACT TRUNCATED AT 250 WORDS).

Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors.

PubMed

Rehman, Jalees; Li, Jingling; Orschell, Christie M; March, Keith L

2003-03-04

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood and can enhance angiogenesis after infusion into host animals. It is not known whether the proangiogenic effects are a result of such events as endothelial differentiation and subsequent proliferation of EPCs or secondary to secretion of angiogenic growth factors. Human EPCs were isolated as previously described, and their phenotypes were confirmed by uptake of acetylated LDL and binding of ulex-lectin. EPC proliferation and surface marker expression were analyzed by flow cytometry, and conditioned medium was assayed for growth factors. The majority of EPCs expressed monocyte/macrophage markers such as CD14 (95.7+/-0.3%), Mac-1 (57.6+/-13.5%), and CD11c (90.8+/-4.9%). A much lower percentage of cells expressed the specific endothelial marker VE-cadherin (5.2+/-0.7%) or stem/progenitor-cell markers AC133 (0.16+/-0.05%) and c-kit (1.3+/-0.7%). Compared with circulating monocytes, cultured EPCs showed upregulation of monocyte activation and macrophage differentiation markers. EPCs did not demonstrate any significant proliferation but did secrete the angiogenic growth factors vascular endothelial growth factor, hepatocyte growth factor, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Our findings suggest that acetylated LDL(+)ulex-lectin(+) cells, commonly referred to as EPCs, do not proliferate but release potent proangiogenic growth factors. The majority of acetylated LDL(+)ulex-lectin(+) cells are derived from monocyte/macrophages. The findings of low proliferation and endothelial differentiation suggest that their angiogenic effects are most likely mediated by growth factor secretion. These findings may allow for development of novel angiogenic therapies relying on secreted growth factors or on recruitment of endogenous monocytes/macrophages to sites of ischemia.

Immune-endocrine interactions in the mammalian adrenal gland: facts and hypotheses.

PubMed

Nussdorfer, G G; Mazzocchi, G

1998-01-01

Several cytokines, which are the major mediators of the inflammatory responses, are well-known to stimulate the hypothalamopituitary corticotropin-releasing hormone (CRH)/adrenocorticotropic hormone (ACTH) system, thereby evoking secretory responses by the adrenal cortex. Many of these cytokines, including interleukin-1 (IL-1), IL-2, IL-6, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (INF-gamma) are synthesized in the adrenal gland by both parenchymal cells and resident macrophages, and the release of some of them (e.g., IL-6 and TNF-alpha) is regulated by the main agonists of steroid hormone secretion (e.g., ACTH and angiotensin-II) and bacterial endotoxins. Adrenocortical and adrenomedullary cells are provided with specific receptors for IL-1, IL-2, and IL-6. IL-1 and TNF-alpha directly inhibit aldosterone secretion of zona glomerulosa cells, whereas IL-6 enhances it. IL-2, IL-3, IL-6, and INF-alpha are able to directly stimulate glucocorticoid production by zona fasciculata and zona reticularis cells, whereas IL-1 exerts an analogous effect through an indirect mechanism involving the stimulation of catecholamine release by chromaffin cells and/or the activation of the intramedullary CRH/ACTH system; again, TNF-alpha depresses glucocorticoid synthesis. IL-6 raises androgen secretion by inner adrenocortical layers. IL-1 enhances the proliferation of adrenocortical cells, and findings suggest that cytokines may control the apoptotic deletion of senescent zona reticularis cells. The relevance of the intraadrenal cytokine system in the fine-tuning of the secretion and growth of the adrenal cortex under normal conditions remains to be explored. However, indirect proof is available that local immune-endocrine interactions may play an important role in modulating adrenal responses to inflammatory and immune challenges and stresses.

A single amino acid substitution in the variable region of the light chain specifically blocks immunoglobulin secretion.

PubMed Central

Dul, J L; Argon, Y

1990-01-01

Although immunoglobulin light chains are usually secreted in association with heavy chains, free light chains can be secreted by lymphocytes. To identify the structural features of light chains that are essential for their secretion, we mutated a conserved sequence in the variable domain of a lambda I light chain. The effects of the mutations on secretion were assayed by transient expression in COS-1 cells. One mutant (AV60), which replaced Ala-60 with Val, was secreted as efficiently as wild-type lambda I by transfected COS-1 cells. This result was not surprising because secreted lambda II chains contain valine in this position. However, a second lambda I mutant (AV60FS62), which replaced Phe-62 with Ser as well as Ala-60 with Val, was not secreted. This mutant was arrested in the endoplasmic reticulum, as judged by immunofluorescence and by its association with a lumenal endoplasmic reticulum protein, immunoglobulin heavy chain binding protein (BiP). The defect in secretion was not due to gross misfolding of the lambda I chain, since cells cotransfected with AV60FS62 and an immunoglobulin heavy chain gene produced functional antigen-binding antibodies. These assembled IgM molecules were still not secreted. Hence, the replacement of Phe-62 with Ser specifically affects a determinant on the lambda I light chain that is necessary for the intracellular transport of this molecule. Images PMID:2122454

A Study on the Gastrointestinal Hormones and the Gastric Acid Secretion during Physical Stress in Man,

DTIC Science & Technology

1983-12-15

meal and oral glucose during prolonged severe exercise, caloric deficit and sleep deprivation 43 Paper II Secretin - a new stress hormone? 49 8 Page...secretion (gastrin), and that are influenced by the amount of gastric acid produced (secretin, group I pepsinogens). Our subjects were in caloric deficit...response to a liquid meal and oral glucose during prolonged severe exercise, caloric deficit, and sleep deprivation. O Oektedalen, 0 Flaten, P K Opstad

Flavins secreted by roots of iron-deficient Beta vulgaris enable mining of ferric oxide via reductive mechanisms.

PubMed

Sisó-Terraza, Patricia; Rios, Juan J; Abadía, Javier; Abadía, Anunciación; Álvarez-Fernández, Ana

2016-01-01

Iron (Fe) is abundant in soils but generally poorly soluble. Plants, with the exception of Graminaceae, take up Fe using an Fe(III)-chelate reductase coupled to an Fe(II) transporter. Whether or not nongraminaceous species can convert scarcely soluble Fe(III) forms into soluble Fe forms has deserved little attention so far. We have used Beta vulgaris, one among the many species whose roots secrete flavins upon Fe deficiency, to study whether or not flavins are involved in Fe acquisition. Flavins secreted by Fe-deficient plants were removed from the nutrient solution, and plants were compared with Fe-sufficient plants and Fe-deficient plants without flavin removal. Solubilization of a scarcely soluble Fe(III)-oxide was assessed in the presence or absence of flavins, NADH (nicotinamide adenine dinucleotide, reduced form) or plant roots, and an Fe(II) trapping agent. The removal of flavins from the nutrient solution aggravated the Fe deficiency-induced leaf chlorosis. Flavins were able to dissolve an Fe(III)-oxide in the presence of NADH. The addition of extracellular flavins enabled roots of Fe-deficient plants to reductively dissolve an Fe(III)-oxide. We concluded that root-secretion of flavins improves Fe nutrition in B. vulgaris. Flavins allow B. vulgaris roots to mine Fe from Fe(III)-oxides via reductive mechanisms. © 2015 CSIC New Phytologist © 2015 New Phytologist Trust.

Novel antiosteoclastogenic activity of phloretin antagonizing RANKL-induced osteoclast differentiation of murine macrophages.

PubMed

Kim, Jung-Lye; Kang, Min-Kyung; Gong, Ju-Hyun; Park, Sin-Hye; Han, Seon-Young; Kang, Young-Hee

2012-08-01

Bone-remodeling imbalance resulting in more bone resorption than bone formation is known to cause skeletal diseases such as osteoporosis. Phloretin, a natural dihydrochalcone compound largely present in apple peels, possesses antiphotoaging, and antiinflammatory activity. Phloretin inhibited receptor activator of NF-κB ligand (RANKL)-induced formation of multinucleated osteoclasts and diminished bone resorption area produced during the osteoclast differentiation process. It was also found that ≥ 10 μM phloretin reduced RANKL-enhanced tartrate-resistance acid phosphatase activity and matrix metalloproteinase-9 secretion in a dose-dependent manner. The phloretin treatment retarded RANKL-induced expression of carbonic anhydrase II, vacuolar-type H(+) -ATPase D2 and β3 integrin, all involved in the bone resorption. Furthermore, submicromolar phloretin diminished the expression and secretion of cathepsin K elevated by RANKL, being concurrent with inhibition of TRAF6 induction and NF-κB activation. RANKL-induced activation of nuclear factor of activated T cells c1 (NFATc1) and microphthalmia-associated transcription factor was also suppressed by phloretin. These results demonstrate that the inhibition of osteoclast differentiation and bone resorption by phloretin entail a disturbance of TRAF6-NFATc1-NF-κB pathway triggered by RANKL. Therefore, phloretin may be a potential therapeutic agent targeting osteoclast differentiation and bone resorption in skeletal diseases such as osteoporosis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Triiodothyronine stimulates VEGF expression and secretion via steroids and HIF-1α in murine Leydig cells.

PubMed

Dhole, Bodhana; Gupta, Surabhi; Venugopal, Senthil Kumar; Kumar, Anand

2018-06-01

Leydig cells are the principal steroidogenic cells of the testis. Leydig cells also secrete a number of growth factors including vascular endothelial growth factor (VEGF) which has been shown to regulate both testicular steroidogenesis and spermatogenesis. The thyroid hormone, T 3, is known to stimulate steroidogenesis in Leydig cells. T 3 has also been shown to stimulate VEGF production in a variety of cell lines. However, studies regarding the effect of T 3 on VEGF synthesis and secretion by the Leydig cells were lacking. Therefore, we investigated the effect of T 3 on VEGF synthesis and secretion in a mouse Leydig tumour cell line, MLTC-1. The effect of T 3 was compared with that of LH/cAMP and hypoxia, two known stimulators of Leydig cell functions. The cells were treated with T 3 , 8-Br-cAMP (a cAMP analogue), or CoCl 2 (a hypoxia mimetic) and VEGF secreted in the cell supernatant was measured using ELISA. The mRNA levels of VEGF were measured by quantitative RT-PCR. In the MLTC-1 cells, T 3 , 8-Br-cAMP, and CoCl 2 stimulated VEGF mRNA levels and the protein secretion. T 3 also increased steroid secretion as well as HIF-1α protein levels, two well-established upstream regulators of VEGF. Inhibitors of steroidogenesis as well as HIF-1α resulted in inhibition of T 3 -stimulated VEGF secretion by the MLTC-1 cells. This suggested a mediatory role of steroids and HIF-1α protein in T 3 -stimulated VEGF secretion by MLTC-1 cells. The mediation by steroids and HIF-1α were independent of each other. 8-Br-cAMP: 8-bromo - 3', 5' cyclic adenosine monophosphate; CoCl 2 : cobalt chloride; HIF-1α: hypoxia inducible factor -1α; LH: luteinizing hormone; T 3 : 3, 5, 3'-L-triiodothyronine; VEGF: vascular endothelial growth factor.

Anti-dsDNA Antibodies Bind to Mesangial Annexin II in Lupus Nephritis

PubMed Central

Yung, Susan; Cheung, Kwok Fan; Zhang, Qing

2010-01-01

Production of anti-dsDNA antibodies is a hallmark of lupus nephritis, but how these antibodies deposit in organs and elicit inflammatory damage remains unknown. In this study, we sought to identify antigens on the surface of human mesangial cells (HMC) that mediate the binding of human anti-dsDNA antibodies and the subsequent pathogenic processes. We isolated anti-dsDNA antibodies from patients with lupus nephritis by affinity chromatography. We used multiple methods to identify and characterize antigens from the plasma membrane fraction of mesangial cells that crossreacted with the anti-dsDNA antibodies. We found that annexin II mediated the binding of anti-dsDNA antibodies to HMC. After binding to the mesangial cell surface, anti-dsDNA antibodies were internalized into the cytoplasm and nucleus. This also led to induction of IL-6 secretion and annexin II synthesis, mediated through activation of p38 MAPK, JNK, and AKT. Binding of anti-dsDNA antibodies to annexin II correlated with disease activity in human lupus nephritis. Glomerular expression of annexin II correlated with the severity of nephritis, and annexin II colocalized with IgG and C3 deposits in both human and murine lupus nephritis. Gene silencing of annexin II in HMC reduced binding of anti-dsDNA antibody and partially decreased IL-6 secretion. In summary, our data demonstrate that annexin II mediates the binding of anti-dsDNA antibodies to mesangial cells, contributing to the pathogenesis of lupus nephritis. This interaction provides a potential target for therapeutic intervention. PMID:20847146

Airway epithelial cell response to human metapneumovirus infection

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

Bao, X.; Liu, T.; Spetch, L.

2007-11-10

Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and typemore » I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-{kappa}B, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.« less

Antimicrobial peptides secreted by equine mesenchymal stromal cells inhibit the growth of bacteria commonly found in skin wounds.

PubMed

Harman, Rebecca M; Yang, Steven; He, Megan K; Van de Walle, Gerlinde R

2017-07-04

The prevalence of chronic skin wounds in humans is high, and treatment is often complicated by the presence of pathogenic bacteria. Therefore, safe and innovative treatments to reduce the bacterial load in cutaneous wounds are needed. Mesenchymal stromal cells (MSC) are known to provide paracrine signals that act on resident skin cells to promote wound healing, but their potential antibacterial activities are not well described. The present study was designed to examine the antibacterial properties of MSC from horses, as this animal model offers a readily translatable model for MSC therapies in humans. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC on the growth of representative gram-negative and gram-positive bacterial species commonly found in skin wounds and (ii) define the mechanisms by which MSC inhibit bacterial growth. MSC were isolated from the peripheral blood of healthy horses. Gram-negative E. coli and gram-positive S. aureus were cultured in the presence of MSC and MSC conditioned medium (CM), containing all factors secreted by MSC. Bacterial growth was measured by plating bacteria and counting viable colonies or by reading the absorbance of bacterial cultures. Bacterial membrane damage was detected by incorporation of N-phenyl-1-naphthylamine (NPN). Antimicrobial peptide (AMP) gene and protein expression by equine MSC were determined by RT-PCR and Western blot analysis, respectively. Blocking of AMP activity of MSC CM was achieved using AMP-specific antibodies. We found that equine MSC and MSC CM inhibit the growth of E. coli and S. aureus, and that MSC CM depolarizes the cell membranes of these bacteria. In addition, we found that equine MSC CM contains AMPs, and blocking these AMPs with antibodies reduces the effects of MSC CM on bacteria. Our results demonstrate that equine MSC inhibit bacterial growth and secrete factors that compromise the membrane integrity of bacteria commonly found in skin wounds. We also identified four specific AMPs produced by equine MSC. The secretion of AMPs may contribute to the value of MSC as a therapy for cutaneous wounds in both horses and humans.

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women: II. Identification of the molecule as a Fc-receptor-like molecule: a preliminary report.

PubMed

Aranha, C; Bordekar, A; Shahani, S

1998-11-01

Early pregnancy factor (EPF)-like activity from culture supernatants obtained from stimulated lymphocytes of pregnant women was characterized and identified. The enzyme-linked immunosorbent assay depending on the presence of "Fc" receptors on bovine spermatozoa was used to identify the EPF-like molecule purified by gel filtration and reverse-phase high-performance liquid chromatography. The results indicated that the crude lymphocyte culture supernatant, the EPF-positive G IV fraction obtained on gel filtration, and the EPF-positive reverse-phase high-performance liquid chromatography protein readily bound with the different concentrations of aggregated human gamma-globulin in a manner similar to that in which the standard control of aggregated human gamma-globulin binds to the bovine spermatozoa. EPF-like activity synthesized and secreted by lymphocytes during pregnancy may be a Fc-receptor-like molecule.

Cross-Reactivity between Chlamydia trachomatis Heat Shock Protein 10 and Early Pregnancy Factor

PubMed Central

Betsou, Fotini; Borrego, Maria José; Guillaume, Nicolas; Catry, Maria Anjos; Romão, Sandra; Machado-Caetano, J. A.; Sueur, Jean Marie; Mention, Jacques; Faille, Nicole; Orfila, Jeanne

2003-01-01

Chlamydia trachomatis heat shock protein 10 (Chsp10) is associated with chronic genital tract infection with C. trachomatis. Chsp10 is homologous to human chaperonin 10 (Cpn10) and early pregnancy factor (EPF), a form of human Cpn10 that is specifically secreted at the start of pregnancy. We investigated cross-reactions between serum anti-Chsp10 antibodies and anti-EPF antibodies in pregnant and nonpregnant patients. Pregnancy was found to be associated with the presence of anti-EPF antibodies, which are specifically induced in pregnant women with a history of C. trachomatis infection, and with the presence of serum anti-Chsp10 antibodies. We also found that infertility was associated with the presence of anti-Chsp10 and anti-EPF antibodies. The HLA class II haplotype DR8 DQ4 was associated with the presence of anti-Chsp10 antibodies but not of anti-EPF antibodies. PMID:12738647

Expanded adipose-derived stem cells suppress mixed lymphocyte reaction by secretion of prostaglandin E2.

PubMed

Cui, Lei; Yin, Shuo; Liu, Wei; Li, Ningli; Zhang, Wenjie; Cao, Yilin

2007-06-01

Multipotent mesenchymal stem cells (MSCs) in adult tissue are known to be less immunogenic and immunosuppressive. Previous study showed that primary cultures of human adipose-derived stem cells (ADSCs) shared their immunomodulatory properties with other MSCs. However, whether passaged human ADSCs can retain their immunomodulatory effect after in vitro expansion remains unknown. In addition, the mechanism of ADSC-mediated immunomodulatory effect remains to be elucidated. This study aimed to investigate these issues by using passaged human ADSCs as an in vitro study model. Flow cytometry showed that passaged ADSCs expressed human leukocyte antigen (HLA) class I but not class II molecules, which could be induced to express to a high level with interferon-gamma (IFN-gamma) treatment. The study found that passaged ADSCs could not elicit lymphocyte proliferation after co-culturing with them, even after IFN-gamma treatment. In addition, either IFN-gamma-treated or non-treated ADSCs could inhibit phytohemagglutinin (PHA)-stimulated lymphocyte proliferation. Moreover, passaged ADSCs could serve as the third-party cells to inhibited two-way mixed lymphocyte reaction (MLR). Further study using a transwell system also showed that this type of immunosuppressive effect was not cell-cell contact dependent. In defining possible soluble factors, we found that passaged ADSCs significantly increased their secretion of prostaglandin E2 (PGE2), but not transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF), when they were co-cultured with MLR. Furthermore, the result demonstrated that only PGE2 production inhibitor indomethacine, but not TGF-beta- and HGF-neutralizing antibodies, could significantly counteract ADSC-mediated suppression on allogeneic lymphocyte proliferation. These results indicated that in vitro expanded ADSCs retain low immunogenicity and immunosuppressive effect, and PGE2 might be the major soluble factor involved in the in vitro inhibition of allogeneic lymphocyte reaction.

The prediction of a pathogenesis-related secretome of Puccinia helianthi through high-throughput transcriptome analysis.

PubMed

Jing, Lan; Guo, Dandan; Hu, Wenjie; Niu, Xiaofan

2017-03-11

Many plant pathogen secretory proteins are known to be elicitors or pathogenic factors,which play an important role in the host-pathogen interaction process. Bioinformatics approaches make possible the large scale prediction and analysis of secretory proteins from the Puccinia helianthi transcriptome. The internet-based software SignalP v4.1, TargetP v1.01, Big-PI predictor, TMHMM v2.0 and ProtComp v9.0 were utilized to predict the signal peptides and the signal peptide-dependent secreted proteins among the 35,286 ORFs of the P. helianthi transcriptome. 908 ORFs (accounting for 2.6% of the total proteins) were identified as putative secretory proteins containing signal peptides. The length of the majority of proteins ranged from 51 to 300 amino acids (aa), while the signal peptides were from 18 to 20 aa long. Signal peptidase I (SpI) cleavage sites were found in 463 of these putative secretory signal peptides. 55 proteins contained the lipoprotein signal peptide recognition site of signal peptidase II (SpII). Out of 908 secretory proteins, 581 (63.8%) have functions related to signal recognition and transduction, metabolism, transport and catabolism. Additionally, 143 putative secretory proteins were categorized into 27 functional groups based on Gene Ontology terms, including 14 groups in biological process, seven in cellular component, and six in molecular function. Gene ontology analysis of the secretory proteins revealed an enrichment of hydrolase activity. Pathway associations were established for 82 (9.0%) secretory proteins. A number of cell wall degrading enzymes and three homologous proteins specific to Phytophthora sojae effectors were also identified, which may be involved in the pathogenicity of the sunflower rust pathogen. This investigation proposes a new approach for identifying elicitors and pathogenic factors. The eventual identification and characterization of 908 extracellularly secreted proteins will advance our understanding of the molecular mechanisms of interactions between sunflower and rust pathogen and will enhance our ability to intervene in disease states.

Induction of cross-priming of naive CD8+ T lymphocytes by recombinant bacillus Calmette-Guerin that secretes heat shock protein 70-major membrane protein-II fusion protein.

PubMed

Mukai, Tetsu; Maeda, Yumi; Tamura, Toshiki; Matsuoka, Masanori; Tsukamoto, Yumiko; Makino, Masahiko

2009-11-15

Because Mycobacterium bovis bacillus Calmette-Guérin (BCG) unconvincingly activates human naive CD8(+) T cells, a rBCG (BCG-70M) that secretes a fusion protein comprising BCG-derived heat shock protein (HSP)70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed to potentiate the ability of activating naive CD8(+) T cells through dendritic cells (DC). BCG-70M secreted HSP70-MMP-II fusion protein in vitro, which stimulated DC to produce IL-12p70 through TLR2. BCG-70M-infected DC activated not only memory and naive CD8(+) T cells, but also CD4(+) T cells of both types to produce IFN-gamma. The activation of these naive T cells by BCG-70M was dependent on the MHC and CD86 molecules on BCG-70M-infected DC, and was significantly inhibited by pretreatment of DC with chloroquine. Both brefeldin A and lactacystin significantly inhibited the activation of naive CD8(+) T cells by BCG-70M through DC. Thus, the CD8(+) T cell activation may be induced by cross-presentation of Ags through a TAP- and proteosome-dependent cytosolic pathway. When naive CD8(+) T cells were stimulated by BCG-70M-infected DC in the presence of naive CD4(+) T cells, CD62L(low)CD8(+) T cells and perforin-producing CD8(+) T cells were efficiently produced. MMP-II-reactive CD4(+) and CD8(+) memory T cells were efficiently produced in C57BL/6 mice by infection with BCG-70M. These results indicate that BCG-70M activated DC, CD4(+) T cells, and CD8(+) T cells, and the combination of HSP70 and MMP-II may be useful for inducing better T cell activation.

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis

PubMed Central

Clarke, Cassie J.; Berg, Tracy J.; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L.; Vermeulen, Peter B.; Foo, Shane; Kostaras, Eleftherios; Jones, J. Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R.; Norman, Jim C.

2016-01-01

Summary Expression of the initiator methionine tRNA (tRNAiMet) is deregulated in cancer. Despite this fact, it is not currently known how tRNAiMet expression levels influence tumor progression. We have found that tRNAiMet expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAiMet in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAiMet contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAiMet gene (2+tRNAiMet mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAiMet mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAiMet mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAiMet significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAiMet-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAiMet-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAiMet mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAiMet levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. PMID:26948875

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis.

PubMed

Clarke, Cassie J; Berg, Tracy J; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L; Vermeulen, Peter B; Foo, Shane; Kostaras, Eleftherios; Jones, J Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R; Norman, Jim C

2016-03-21

Expression of the initiator methionine tRNA (tRNAi(Met)) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi(Met) expression levels influence tumor progression. We have found that tRNAi(Met) expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi(Met) in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi(Met) contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi(Met) gene (2+tRNAi(Met) mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi(Met) mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi(Met) mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi(Met) significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi(Met)-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi(Met)-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi(Met) mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi(Met) levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Distinct roles of prolactin, epidermal growth factor, and glucocorticoids in β-casein secretion pathway in lactating mammary epithelial cells.

PubMed

Kobayashi, Ken; Oyama, Shoko; Kuki, Chinatsu; Tsugami, Yusaku; Matsunaga, Kota; Suzuki, Takahiro; Nishimura, Takanori

2017-01-15

Beta-casein is a secretory protein contained in milk. Mammary epithelial cells (MECs) synthesize and secrete β-casein during lactation. However, it remains unclear how the β-casein secretion pathway is developed after parturition. In this study, we focused on prolactin (PRL), epidermal growth factor (EGF), and glucocorticoids, which increase in blood plasma and milk around parturition. MECs cultured with PRL, EGF and dexamethasone (DEX: glucocorticoid analog) developed the β-casein secretion pathway. In the absence of PRL, MECs hardly expressed β-casein. EGF enhanced the expression and secretion of β-casein in the presence of PRL and DEX. DEX treatment rapidly increased secreted β-casein concurrent with enhancing β-casein expression. DEX also up-regulated the expression of SNARE proteins, such as SNAP-23, VAMP-8 and Syntaxin-12. Furthermore, PRL and DEX regulated the expression ratio of α s1 -, β- and κ-casein. These results indicate that PRL, EGF and glucocorticoids have distinct roles in the establishment of β-casein secretion pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Theileria parva infection induces autocrine growth of bovine lymphocytes.

PubMed Central

Dobbelaere, D A; Coquerelle, T M; Roditi, I J; Eichhorn, M; Williams, R O

1988-01-01

Bovine lymphocytes infected with the parasite Theileria parva continuously secrete a growth factor that is essential for their proliferation in vitro and also constitutively express interleukin 2 receptors on their surface. Dilution of the secreted growth factor, caused by culturing cells at low density, results in retardation of culture growth. Human recombinant interleukin 2, however, effectively substitutes for the diluted growth factor by restoring normal growth rates and also allows Theileria-infected cells to be grown at low density without the use of feeder layers. Secretion of the growth factor and expression of the interleukin 2 receptor depend on the presence of the parasite in the cytoplasm of the host cell. Elimination of the parasite from the cell cytoplasm by the specific antitheilerial drug BW 720c results in the arrest of growth factor secretion and the disappearance of interleukin 2 receptors from the cell surface. This is accompanied by growth arrest and reversion of the infected cells to the morphology of resting lymphocytes. We propose that the continuous proliferation of infected cells in vitro is mediated by autocrine receptor activation. Images PMID:3133661

Multipotent human stromal cells improve cardiac function after myocardial infarction in mice without long-term engraftment.

PubMed Central

Iso, Yoshitaka; Spees, Jeffrey L.; Serrano, Claudia; Bakondi, Benjamin; Pochampally, Radhika; Song, Yao-Hua; Sobel, Burton E.; Delafontaine, Patrick; Prockop, Darwin J.

2007-01-01

The aim of this study was to determine whether intravenously-administered multipotent stromal cells from human bone marrow (hMSCs) can improve cardiac function after myocardial infarction (MI) without long-term engraftment and therefore whether transitory paracrine effects or secreted factors are responsible for the benefit conferred. hMSCs were injected systemically into immunodeficient mice with acute MI. Cardiac function and fibrosis after MI in the hMSC-treated group was significantly improved compared with that in controls. However, despite the cardiac improvement, there was no evident hMSC engraftment in the heart 3 weeks after MI. Microarray assays and ELISAs demonstrated that multiple protective factors were expressed and secreted from the hMSCs in culture. Factors secreted by hMSCs prevented cell death of cultured cardiomyocytes and endothelial cells under conditions that mimicked tissue ischemia. The favorable effects of hMSCs appear to reflect the impact of secreted factors rather than engraftment, differentiation, or cell fusion. PMID:17257581

In vitro and in vivo assessment of oral autologous artificial connective tissue characteristics that influence its performance as a graft.

PubMed

Fontanilla, Marta Raquel; Espinosa, Lady Giovanna

2012-09-01

Several studies have evaluated proteins secreted by fibroblasts comprising skin substitutes, finding that they are secreted in combinations and concentrations that promote wound healing. However, assessment of proteins secreted by oral fibroblasts forming a part of oral substitutes is scarce. In our previous work, collagen type-I scaffolds (CSs) and autologous artificial connective tissue (AACT) were produced and implanted in rabbit oral lesions, evidencing that AACT outperforms CS. The present work determined the secreted factor profile of AACT in the time of grafting as well as that of the AACT embedded in the clot. It also evaluated the proliferation and viability of AACT fibroblasts to establish the dwell time of these cells in the grafted area. Finally, it assessed whether CS, AACT, and clot-embedded AACT increase fibroblast recruitment induced by a fibrin clot, because the cell migratory response has been associated with the wound-healing outcome. We found that some of the factors secreted by AACT fibroblasts are significantly different from those secreted by clot-embedded AACT fibroblasts. Also, that the profile of proteins secreted by AACT fibroblasts and clot-embedded AACT fibroblasts is different from already reported protein secretion profiles of other engineered tissues used in treating oral mucosa wounds. It was also found that AACT fibroblasts are viable when grafted and remain in the treated area for almost 2 weeks, and that the migratory response of fibroblasts to tissue-substitute stimulus is significantly less than the migratory response induced by the clot alone. Overall, data suggest that AACT secretion of proteins is modulated by three-dimensionality and environment factors. This bioactivity and the fact that AACT does not increase fibroblast migration can be held accountable for AACT's good performance as a graft.

Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence

PubMed Central

Vallaghe, Julie; Gregor, Nathalie; Donthamsetti, Prashant; Harris, Paul E.; Pierre, Nicolas; Freyberg, Robin; Charrier-Savournin, Fabienne; Javitch, Jonathan A.; Freyberg, Zachary

2016-01-01

Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment. PMID:26849707

Phosphate or phosphite addition promotes the proteolytic turnover of phosphate-starvation inducible tomato purple acid phosphatase isozymes.

PubMed

Bozzo, Gale G; Singh, Vinay K; Plaxton, William C

2004-08-27

Within 48 h of the addition of 2.5 mM phosphate (HPO42-, Pi) or phosphite (H2PO3-, Phi) to 8-day-old Pi-starved (-Pi) tomato suspension cells: (i) secreted and intracellular purple acid phosphatase (PAP) activities decreased by about 12- and 6-fold, respectively and (ii) immunoreactive PAP polypeptides either disappeared (secreted PAPs) or were substantially reduced (intracellular PAP). The degradation of both secreted PAP isozymes was correlated with the de novo synthesis of two extracellular serine proteases having M(r)s of 137 and 121 kDa. In vitro proteolysis of purified secreted tomato PAP isozymes occurred following their 24 h incubation with culture filtrate from Pi-resupplied cells. The results indicate that Pi or Phi addition to -Pi tomato cells induces serine proteases that degrade Pi-starvation inducible extracellular proteins.

Differentiation of human umbilical cord mesenchymal stromal cells into low immunogenic hepatocyte-like cells.

PubMed

Zhao, Qinjun; Ren, Hongying; Li, Xiyuan; Chen, Zhong; Zhang, Xiangyu; Gong, Wei; Liu, Yongjun; Pang, Tianxiang; Han, Zhong Chao

2009-01-01

Mesenchymal stromal cells (MSC) isolated from several human tissues have been known to differentiate into the hepatic lineage in vitro, but the immunogenicity of the differentiated hepatocyte-like cells (DHC) has not been reported. Umbilical cord (UC) MSC are thought to be an attractive cell source for cell therapy because of their young age and low infection rate compared with adult tissue MSC. Hepatic differentiation of UC-MSC was induced with a 2-step protocol. The expressions of hepatic markers were detected by RT-PCR and immunofluorescence staining. Albumin production and urea secretion were measured by ELISA and colorimetric assay respectively. The immunosuppressive properties of DHC was detected by mixed lymphocyte culture. After incubation with specific growth factors, including hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), UC MSC exhibited a high hepatic differentiation ability in an adherent culture condition. The differentiated UC MSC showed hepatocyte-like morphology and expressed several liver-specific markers at gene and protein levels. Furthermore, the DHC exhibited hepatocyte-specific functions, including albumin secretion, low-density lipoprotein uptake and urea production. More importantly, DHC did not express major histocompatibility complex (MHC) II antigen and were not able to induce lymphocyte proliferation in mixed lymphocyte culture, as undifferentiated UC MSC did. Our results indicate that UC MSC are able to differentiate into functional hepatocyte-like cells that still retain their low immunogenicity in vitro. More importantly, DHC incorporated into the parenchyma of liver when transplanted into mice with CCl(4)-induced liver injury. Therefore, DHC may be an ideal source for cell therapy of liver diseases.

The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi.

PubMed

Login, Frédéric H; Shevchik, Vladimir E

2006-11-03

Many pathogenic Gram-negative bacteria secrete toxins and lytic enzymes via a multiprotein complex called the type II secretion system. This system, named Out in Erwinia chrysanthemi, consists of 14 proteins integrated or associated with the two bacterial membranes. OutC, a key player in this process, is probably implicated in the recognition of secreted proteins and signal transduction. OutC possesses a short cytoplasmic sequence, a single transmembrane segment (TMS), and a large periplasmic region carrying a putative PDZ domain. A hydrodynamic study revealed that OutC forms stable dimers of an elongated shape, whereas the PDZ domain adopts a globular shape. Bacterial two-hybrid, cross-linking, and pulldown assays revealed that the self-association of OutC is driven by the TMS, whereas the periplasmic region is dispensable for self-association. Site-directed mutagenesis of the TMS revealed that cooperative interactions between three polar residues located at the same helical face provide adequate stability for OutC self-assembly. An interhelical H-bonding mediated by Gln(29) appears to be the main driving force, and two Arg residues located at the TMS boundaries are essential for the stabilization of OutC oligomers. Stepwise mutagenesis of these residues gradually diminished OutC functionality and self-association ability. The triple OutC mutant R15V/Q29L/R36A became monomeric and nonfunctional. Self-association and functionality of the triple mutant were partially restored by the introduction of a polar residue at an alternative position in the interhelical interface. Thus, the OutC TMS is more than just a membrane anchor; it drives the protein self-association that is essential for formation of a functional secretion system.

Fundamentals of neurogastroenterology: basic science.

PubMed

Grundy, David; Al-Chaer, Elie D; Aziz, Qasim; Collins, Stephen M; Ke, Meiyun; Taché, Yvette; Wood, Jackie D

2006-04-01

The focus of neurogastroenterology in Rome II was the enteric nervous system (ENS). To avoid duplication with Rome II, only advances in ENS neurobiology after Rome II are reviewed together with stronger emphasis on interactions of the brain, spinal cord, and the gut in terms of relevance for abdominal pain and disordered gastrointestinal function. A committee with expertise in selective aspects of neurogastroenterology was invited to evaluate the literature and provide a consensus overview of the Fundamentals of Neurogastroenterology textbook as they relate to functional gastrointestinal disorders (FGIDs). This review is an abbreviated version of a fuller account that appears in the forthcoming book, Rome III. This report reviews current basic science understanding of visceral sensation and its modulation by inflammation and stress and advances in the neurophysiology of the ENS. Many of the concepts are derived from animal studies in which the physiologic mechanisms underlying visceral sensitivity and neural control of motility, secretion, and blood flow are examined. Impact of inflammation and stress in experimental models relative to FGIDs is reviewed as is human brain imaging, which provides a means for translating basic science to understanding FGID symptoms. Investigative evidence and emerging concepts implicate dysfunction in the nervous system as a significant factor underlying patient symptoms in FGIDs. Continued focus on neurogastroenterologic factors that underlie the development of symptoms will lead to mechanistic understanding that is expected to directly benefit the large contingent of patients and care-givers who deal with FGIDs.

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

Indagation of serum and salivary reactive oxygen metabolite and cortisol levels in chronic periodontitis and stress-induced chronic periodontitis patients

PubMed Central

Sudhakar, Uma; Thyagarajan, Ramakrishnan; Jeyapal, Bhagyameena; Jagadeesh, Sushuruthi; Jayakumar, Parvathee

2017-01-01

Background: Periodontal disease is not a conventional bacterial infection but is an inflammatory disease initiated by immune response against a group of microorganisms in susceptible hosts. There are many intriguing researches that unfold the secrets of chronic periodontitis. The current researches in chronic periodontitis are directed toward an approach that respects the scientific relationship between the various risk factors, the genetic factors, and the progression of the disease. Aim: This study aims to evaluate the cortisol and reactive oxygen metabolites (ROM) concentration in serum and to find out their association in periodontal health and disease. Materials and Methods: In this study, totally thirty patients have been taken and divided into two groups of chronic periodontitis (Group I) and stress-induced chronic periodontitis (Group II) and evaluated the correlation between the ROM and cortisol levels in them. This is the first study, where both the levels of ROM and cortisol are checked in the serum and saliva. The analysis is done to check the association between them. Statistical Analysis: The data were statistically analyzed using software program (SPSSV 16), Pearson correlation, and paired t-test. Results: Comparison of the mean ROM levels in Group I and Group II showed that mean ROM level in Group II is highly significant than Group I. Conclusion: Our study suggests that stress can have a role in the progression of periodontal disease by increasing the cortisol and ROM levels. PMID:29491582

Inhibiting renin angiotensin system in rate limiting step by aliskiren as a new approach for preventing indomethacin induced gastric ulcers.

PubMed

Halici, Zekai; Polat, Beyzagul; Cadirci, Elif; Topcu, Atilla; Karakus, Emre; Kose, Duygu; Albayrak, Abdulmecit; Bayir, Yasin

2016-10-25

Previously blocking the renin angiotensin system (RAAS) has been effective in the prevention of gastric damage. Therefore, the aim of this study was to investigate the effects of aliskiren, and thus, direct renin blockage, in indomethacin-induced gastric damage model. Effects of aliskiren were evaluated in indomethacin-induced gastric damage model on Albino Wistar rats. Effects of famotidine has been investigated as standard antiulcer agent. Stereological analyses for ulcer area determination, biochemical analyses for oxidative status determination and molecular analyses for tissue cytokine and cyclooxygenase determination were performed on stomach tissues. In addition, to clarify antiulcer effect mechanism of aliskiren pylorus ligation-induced gastric acid secretion model was applied on rats. Aliskiren was able to inhibit indomethacin-induced ulcer formation. It also inhibited renin, and thus, decreased over-produced Angiotensin-II during ulcer formation. Aliskiren improved the oxidative status and cytokine profile of the stomach, which was most probably impaired by increased Angiotensin II concentration. Aliskiren also increased gastroprotective prostaglandin E2 concentration. Finally, aliskiren did not change the gastric acidity in pylorus ligation model. Aliskiren exerted its protective effects on stomach tissue by decreasing inflammatory cytokines and oxidative stress as a result of inhibiting the RAAS, at a rate-limiting step, as well as its end product, angiotensin II. Aliskiren also significantly increased protective factors such as PGE2, but not affect aggressive factors such as gastric acidity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle

USDA-ARS?s Scientific Manuscript database

The locus of enterocyte effacement (LEE) encodes a type III secretion system (T3SS) for secreting factors that enable Escherichia coli O157:H7 to produce attaching and effacing lesions (A/E) on epithelial cells. The importance of LEE-encoded proteins in intestinal colonization of cattle is well-stud...

The regulated secretory pathway and human disease: insights from gene variants and single nucleotide polymorphisms.

PubMed

Lin, Wei-Jye; Salton, Stephen R

2013-01-01

The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs), where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs), with neuropsychiatric, endocrine, and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A) of the human brain-derived neurotrophic factor gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired.

The Regulated Secretory Pathway and Human Disease: Insights from Gene Variants and Single Nucleotide Polymorphisms

PubMed Central

Lin, Wei-Jye; Salton, Stephen R.

2013-01-01

The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs), where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs), with neuropsychiatric, endocrine, and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A) of the human brain-derived neurotrophic factor gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired. PMID:23964269

Individual, household and community level factors associated with keeping tuberculosis status secret in Ghana.

PubMed

Amo-Adjei, Joshua

2016-11-25

In tuberculosis (TB) control, early disclosure is recommended for the purposes of treatment as well as a means of reducing or preventing person-to-person transmission of the bacteria. However, disclosure maybe avoided as a means of escaping stigma, and possible discrimination. This study aimed at providing insights into factors associated with intentions of Ghanaians to keep positive TB diagnosis in their families' a secret. The paper was based on data from the 2014 Ghana Demographic and Health Survey. Descriptive statistics of proportions with Chi-square test and binary logistic regression were used to identify individual, household and community level factors that predicted the outcome variable (keeping TB secret). Women were more inclined (33%) than men (25%) to keep TB in the family a secret. Views about keeping TB secret declined with age for both sexes. For women, higher education had a positive association with whether TB in the family would be kept a secret or not but the same was not observed for men. In a multivariable regression model, the strongest predictor of keeping TB secret was whether the respondent would keep HIV secret, and this was uniform among women (OR = 6.992, p < 0.001) and men (OR = 9.870, p < 0.001). Unwillingness towards disclosing TB status in Ghana is associated with varied socioeconomic and demographic characteristics, which may be driven by fears of stigma and discrimination. Addressing TB-related stigma and discrimination can enhance positive attitudes towards TB disclosure. For an infectious disease such as TB, openness towards status disclosure is important for public health.

Intracellular and extracellular adenosine triphosphate in regulation of insulin secretion from pancreatic β cells (β).

PubMed

Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun

2014-03-01

Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

Effect of Swimming on the Production of Aldosterone in Rats

PubMed Central

Wang, Paulus S.; Jian, Cai-Yun; Yeh, Yung-Hsing; Chen, Yi-An; Wang, Kai-Lee; Lin, Yi-Chun; Chang, Ling-Ling; Wang, Guei-Jane; Wang, Shyi-Wu

2014-01-01

It has been demonstrated that exercise is one of the stresses known to increase the aldosterone secretion. Both potassium and angiotensin II (Ang II) levels are shown to be correlated with aldosterone production during exercise, but the mechanism is still unclear. In an in vivo study, male rats were catheterized via right jugular vein (RJV), and divided into four groups namely water immersion, swimming, lactate infusion (13 mg/kg/min) and pyruvate infusion (13 mg/kg/min) groups. Each group was treated for 10 min. Blood samples were collected at 0, 10, 15, 30, 60 and 120 min from RJV after administration. In an in vitro study, rat zona glomerulosa (ZG) cells were challenged by lactate (1–10 mM) in the presence or absence of Ang II (10−8 M) for 60 min. The levels of aldosterone in plasma and medium were measured by radioimmunoassay. Cell lysates were analyzed by immunoblotting assay. After exercise and lactate infusion, plasma levels of aldosterone and lactate were significantly higher than those in the control group. Swimming for 10 min significantly increased the plasma Ang II levels in male rats. Administration of lactate plus Ang II significantly increased aldosterone production and enhanced protein expression of steroidogenic acute regulatory protein (StAR) in ZG cells. These results demonstrated that acute exercise led to the increase of both aldosterone and Ang II secretion, which is associated with lactate action on ZG cells and might be dependent on the activity of renin-angiotensin system. PMID:25289701

Assessment of Growth Factors Secreted by Human Breastmilk Mesenchymal Stem Cells.

PubMed

Kaingade, Pankaj Mahipatrao; Somasundaram, Indumathi; Nikam, Amar Babaso; Sarang, Shabari Amit; Patel, Jagdish Shantilal

2016-01-01

Human breastmilk is a dynamic, multifaceted biological fluid containing nutrients, bioactive substances, and growth factors. It is effective in supporting growth and development of an infant. As breastmilk has been found to possess mesenchymal stem cells, the importance of the components of breastmilk and their physiological roles is increasing day by day. The present study was intended to identify the secretions of growth factors, mainly vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), from human breastmilk mesenchymal stem cells under basal conditions of in vitro cell culture using synthetic media and human cord serum. The growth factors were analyzed with the enzyme-linked immunosorbent assay technique. The cultured mesenchymal stem cells of breastmilk without serum revealed significant differences in secretions of the VEGF and HGF growth factors (8.55 ± 2.26402 pg/mL and 230.8 ± 45.9861 pg/mL, respectively) compared with mesenchymal stem cells of breastmilk with serum (21.31 ± 4.69 pg/mL and 2,404.42 ± 481.593 pg/mL, respectively). Results obtained from our study demonstrate that both VEGF and HGF are secreted in vitro by human breastmilk mesenchymal stem cells. The roles of VEGF and HGF in surfactant secretion, pulmonary maturation, and neonatal maturity have been well established. Thus, we emphasize that breastmilk-derived MSCs could be a potent therapeutic source in treating neonatal diseases. Besides, due to its immense potency, the study also emphasizes the importance of breastfeeding, which is promoted by organizations like the World Heatlh Organization and UNICEF.

Tumour necrosis factor receptor trafficking dysfunction opens the TRAPS door to pro-inflammatory cytokine secretion

PubMed Central

Turner, Mark D.; Chaudhry, Anupama; Nedjai, Belinda

2011-01-01

Cytokines are secreted from macrophages and other cells of the immune system in response to pathogens. Additionally, in autoinflammatory diseases cytokine secretion occurs in the absence of pathogenic stimuli. In the case of TRAPS [TNFR (tumour necrosis factor receptor)-associated periodic syndrome], inflammatory episodes result from mutations in the TNFRSF1A gene that encodes TNFR1. This work remains controversial, however, with at least three distinct separate mechanisms of receptor dysfunction having been proposed. Central to these hypotheses are the NF-κB (nuclear factor κB) and MAPK (mitogen-activated protein kinase) families of transcriptional activators that are able to up-regulate expression of a number of genes, including pro-inflammatory cytokines. The present review examines each proposed mechanism of TNFR1 dysfunction, and addresses how these processes might ultimately impact upon cytokine secretion and disease pathophysiology. PMID:22115362

[Effect of different nutritional support on pancreatic secretion in acute pancreatitis].

PubMed

Achkasov, E E; Pugaev, A V; Nabiyeva, Zh G; Kalachev, S V

To develop and justify optimal nutritional support in early phase of acute pancreatitis (AP). 140 AP patients were enrolled. They were divided into groups depending on nutritional support: group I (n=70) - early enteral tube feeding (ETF) with balanced mixtures, group II (n=30) - early ETF with oligopeptide mixture, group III (n=40) - total parenteral nutrition (TPN). The subgroups were also isolated depending on medication: A - Octreotide, B - Quamatel, C - Octreotide + Quamatel. Pancreatic secretion was evaluated by using of course of disease, instrumental methods, APUD-system hormone levels (secretin, cholecystokinin, somatostatin, vasointestinal peptide). ETF was followed by pancreas enlargement despite ongoing therapy, while TPN led to gradual reduction of pancreatic size up to normal values. α-amylase level progressively decreased in all groups, however in patients who underwent ETF (I and II) mean values of the enzyme were significantly higher compared with TPN (group III). Secretin, cholecystokinin and vasointestinal peptide were increasing in most cases, while the level of somatostatin was below normal in all groups. Enteral tube feeding (balanced and oligopeptide mixtures) contributes to pancreatic secretion compared with TPN, but this negative impact is eliminated by antisecretory therapy. Dual medication (Octreotide + Quamatel) is more preferable than monotherapy (Octreotide or Quamatel).

An {alpha}1(II) Gly{sup 913} to cys substitution prevents the matrix incorporation of type II collagen which is replaced with type I and III collagens in cartilage from a patient with hypochondrogenesis

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

Mundlos, S.; Chan, D.; Bateman, J.F.

1996-05-03

A heterozygous mutation in the COL2A1 gene was identified in a patient with hypochondrogenesis. The mutation was a single nucleotide transition of G3285T that resulted in an amino acid substitution of Cys for Gly{sup 913} in the {alpha}1(II) chain of type II collagen. This amino acid change disrupted the obligatory Gly-X-Y triplet motif required for the normal formation of a stable collagen triple helix and prevented the deposition of type II collagen into the proposita`s cartilage, which contained predominantly type I and III collagens and minor amounts of type XI collagen. Biosynthetic analysis of collagens produced and secreted by themore » patient`s chondrocytes cultured in alginate beads was consistent with the in vivo matrix composition, demonstrating that the main products were type I and III collagens, along with type XI collagen. The synthesis of the cartilage-specific type XI collagen at similar levels to controls indicated that the isolated cartilage cells had re-differentiated to the chondrocyte phenotype. The chondrocytes also produced small amounts of type II collagen, but this was post-translationally overmodified and not secreted. These data further delineate the biochemical and phenotypic consequences of mutations in the COL2A1 gene and suggest that cartilage formation and bone development can take place in the absence of type II collagen. 23 refs., 5 figs.« less

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.

Angiotensin II Type 1 Receptor Knockdown Impairs Interleukin-1β-Induced Cytokines in Human Periodontal Fibroblasts.

PubMed

Gabriele, Lilian Gobbo; Morandini, Ana Carolina; Dionísio, Thiago José; Santos, Carlos Ferreira

2017-01-01

The renin-angiotensin (Ang) system (RAS) has been reported as an important modulator of inflammatory and immune responses. Evidence suggests an alternative Ang 1-7/Mas receptor axis as counter-regulatory to the classic RAS Ang II/Ang II Type 1 (AT1) receptor axis. It is known that periodontal pathogens elicit host-derived immune response due to release of cytokines such as interleukin (IL)-1β, and fibroblasts are among the most numerous sentinel cells that contribute to this production. The aim of this study is to determine whether AT1 receptor (AT1R) contributes to production of inflammatory cytokines that are important for periodontal pathogenesis using primary human gingival fibroblasts (HGFs) and human periodontal ligament fibroblasts (HPLFs) stimulated with IL-1β. Through RNA interference or pharmacologic inhibition using AT1R antagonist losartan, HGF and HPLF were stimulated by IL-1β for 3 (messenger RNA [mRNA]) or 24 (protein) hours. IL-1β upregulated mRNA expression of AT1R, IL-1β, IL-6, IL-8, tumor necrosis factor-alpha, and osteoprotegerin (OPG) in HGF and HPLF. AT1R knockdown impaired IL-1β-induced IL-6 and IL-8 secretion in cultured HGF and HPLF. AT1R silencing also increased OPG gene expression in HGF only. Pharmacologic inhibition of AT1R through losartan modulated mRNA transcription of IL-6 and IL-8 in HPLF but not in HGF. In contrast, IL-1β-induced secretion of IL-6 and IL-8 was not influenced by losartan in HGF or HPLF. These results suggest that AT1R knockdown and AT1R pharmacologic blockade by losartan may differently control balance of inflammatory cytokines, such as IL-6 and IL-8, in primary human periodontal fibroblasts.

Night work, long working hours, psychosocial work stress and cortisol secretion in mid-life: evidence from a British birth cohort.

PubMed

Thomas, C; Hertzman, C; Power, C

2009-12-01

To examine the relationships between exposure to workplace factors (night work, extended working hours, psychosocial work stress) and cortisol secretion, and to test whether workplace factors interact, resulting in combined effects. Multiple linear and logistic regression was used to test relationships between workplace factors and cortisol secretion in the 1958 British birth cohort at 45 years. Salivary cortisol was measured twice on the same day to capture the post-waking decline, facilitating the analysis of different cortisol patterns: (1) time 1 (T1, 45 minutes post-waking); (2) time 2 (T2, 3 h after T1); (3) average 3 h exposure from T1 to T2 cortisol; and (4) T1 to T2 change. To identify altered diurnal cortisol patterns we calculated: (1) flat T1-T2 change in cortisol; (2) top 5% T1; (3) bottom 5% T1; and (4) T1 hypo-secretion or hyper-secretion. Models were adjusted for socioeconomic position at birth and in adulthood, qualifications, marital status, dependent children, and smoking status. 25% of men and 8% of women were exposed to >1 workplace factor (night work, extended work hours, job strain). Night work was associated with a 4.28% (95% CI 1.21 to 7.45) increase in average 3 h cortisol secretion independently of job strain or work hours. Night workers not exposed to job strain had elevated T1 cortisol (5.81%, 95% CI 1.61 to 10.19), although for T2 cortisol it was night workers exposed to low job control who had elevated levels (11.72%, 95% CI 4.40 to 19.55). Men (but not women) working >48 h/week had lower average 3 h cortisol secretion (4.55%, 95% CI -8.43 to -0.50). There were no main effects for psychosocial work stress. All associations for T2 and average 3 h cortisol secretion weakened slightly after adjustment for confounding factors, but associations for T1 cortisol were unaffected by adjustment. Our study suggests that night work in particular is associated with elevated cortisol secretion and that cortisol dysregulation may exist in subgroups with specific combinations of stressors.

Enhancement of glucose uptake in skeletal muscle L6 cells and insulin secretion in pancreatic hamster-insulinoma-transfected cells by application of non-thermal plasma jet

NASA Astrophysics Data System (ADS)

Kumar, Naresh; Kaushik, Nagendra K.; Park, Gyungsoon; Choi, Eun H.; Uhm, Han S.

2013-11-01

Type-II diabetes Mellitus is characterized by defects in insulin action on peripheral tissues, such as skeletal muscle, adipose tissue, and liver and pancreatic beta cells. Since the skeletal muscle accounts for approximately 75% of insulin-stimulated glucose-uptake in our body, impaired insulin secretion from defected beta cell plays a major role in the afflicted glucose homoeostasis. It was shown that the intracellular reactive oxygen species and nitric oxide level was increased by non-thermal-plasma treatment in ambient air. These increased intracellular reactive species may enhance glucose uptake and insulin secretion through the activation of intracellular calcium (Ca+) and cAMP production.

Profiling of acarviostatin family secondary metabolites secreted by Streptomyces coelicoflavus ZG0656 using ultraperformance liquid chromatography coupled with electrospray ionization mass spectrometry.

PubMed

Geng, Peng; Meng, Xiansheng; Bai, Gang; Luo, Guoan

2008-10-01

Profiling of acarviostatin family secondary metabolites secreted by Streptomyces coelicoflavus ZG0656 was performed by means of a rapid and facile procedure using ultraperformance liquid chromatography coupled with electrospray ionization mass spectrometry (UPLC/ESI-MS). The acarviostatins were separated on a C18 UPLC column with a series of acetonitrile-aqueous ammonia gradients. The target homologues were detected using the multiple reaction monitoring mode, and the chemical structures were confirmed by analyzing the diagnostic fragment ions in their MS/MS spectra. All six known reference acarviostatins (I03, II03, II13, II23, III03, IV03) were thus identified. In addition, at least 74 acarviostatin homologues, including 65 novel compounds, were characterized. Some of the features of the novel structures included having up to five acarviosine moieties, an acarviosine moiety at the reducing terminus, or an incomplete acarviosine moiety at the nonreducing terminus. This type of investigation may be useful for researchers who study secondary metabolomics in microorganisms and plants, especially those who perform metabolic profiling of aminooligosaccharides and other natural products with similar structures.

Structural changes of oviduct of freshwater shrimp, Macrobrachium nipponense (Decapoda, Palaemonidae), during spawning*

PubMed Central

Lu, Jian-ping; Zhang, Xiao-hui; Yu, Xiao-yun

2006-01-01

The structural change of the oviduct of freshwater shrimp (Macrobrachium nipponense) during spawning was examined by electron microscopy. The oviduct wall structural characteristics seem to be influenced significantly by the spawning process. Before the parturition and ovulation, two types of epithelial cells (types I and II) are found in the epithelium. The free surfaces of type I and type II cells have very dense long microvilli. Under the type I and type II cells, are a relatively thick layer of secreting material and a layer of mostly dead cells. After ovulation, two other types of epithelial cells (types III and IV) are found in the oviduct wall epithelium. The free surface of type III cells only has short microvilli scattered on the surface. The thick layer with secreting material and the dead cell layer disappeared at this stage. In some type III cells, the leaking out of cytoplasm from broken cell membrane led to the death of these type III cells. The transformation of all four types of epithelial cells was in the order: IV→I→II→III. PMID:16365928

A century old renin-angiotensin system still grows with endless possibilities: AT1 receptor signaling cascades in cardiovascular physiopathology.

PubMed

Balakumar, Pitchai; Jagadeesh, Gowraganahalli

2014-10-01

Ang II, the primary effector pleiotropic hormone of the renin-angiotensin system (RAS) cascade, mediates physiological control of blood pressure and electrolyte balance through its action on vascular tone, aldosterone secretion, renal sodium absorption, water intake, sympathetic activity and vasopressin release. It affects the function of most of the organs far beyond blood pressure control including heart, blood vessels, kidney and brain, thus, causing both beneficial and deleterious effects. However, the protective axis of the RAS composed of ACE2, Ang (1-7), alamandine, and Mas and MargD receptors might oppose some harmful effects of Ang II and might promote beneficial cardiovascular effects. Newly identified RAS family peptides, Ang A and angioprotectin, further extend the complexities in understanding the cardiovascular physiopathology of RAS. Most of the diverse actions of Ang II are mediated by AT1 receptors, which couple to classical Gq/11 protein and activate multiple downstream signals, including PKC, ERK1/2, Raf, tyrosine kinases, receptor tyrosine kinases (EGFR, PDGF, insulin receptor), nuclear factor κB and reactive oxygen species (ROS). Receptor activation via G12/13 stimulates Rho-kinase, which causes vascular contraction and hypertrophy. The AT1 receptor activation also stimulates G protein-independent signaling pathways such as β-arrestin-mediated MAPK activation and Src-JAK/STAT. AT1 receptor-mediated activation of NADPH oxidase releases ROS, resulting in the activation of pro-inflammatory transcription factors and stimulation of small G proteins such as Ras, Rac and RhoA. The components of the RAS and the major Ang II-induced signaling cascades of AT1 receptors are reviewed. Copyright © 2014 Elsevier Inc. All rights reserved.

Cathepsin B is the driving force of esophageal cell invasion in a fibroblast-dependent manner.

PubMed

Andl, Claudia D; McCowan, Kelsey M; Allison, Gillian L; Rustgi, Anil K

2010-06-01

Esophageal cancer, which frequently exhibits coordinated loss of E-cadherin (Ecad) and transforming growth factor beta (TGFbeta) receptor II (TbetaRII), has a high mortality rate. In a three-dimensional organotypic culture model system, esophageal keratinocytes expressing dominant-negative mutant versions of both Ecad and TbetaRII (ECdnT) invade into the underlying matrix embedded with fibroblasts. We also find that cathepsin B induction is necessary for fibroblast-mediated invasion. Furthermore, the ECdnT cells in this physiological context activate fibroblasts through the secretion of TGFbeta1, which, in turn, is activated by cathepsin B. These results suggest that the interplay between the epithelial compartment and the surrounding microenvironment is crucial to invasion into the extracellular matrix.

Effect of long-term administration of dietary fiber on the exocrine pancreas in the rat.

PubMed

Sommer, H; Kasper, H

1984-08-01

Male Sprague-Dawley rats (50--70g) were fed a standard laboratory diet containing 6% dietary fiber substances (diet I), the same diet supplemented with 5% guar (diet II), 10% wheat bran (diet III), or 5% pectin of high (76%) methylic esterification (diet IV), or a fiber-free diet (diet V). After a 6-week feeding period, the body weight of the animals had increased to 300--350g. The common bile duct was then canulated and the exocrine pancreatic function tested under urethane anesthesia (1.5 g/kg body weight). The tested fiber substances had no effect on the basal pancreatic secretion of volume, bicarbonate, lipase, amylase or protein, or on the wet weight and histological appearance of the organ. However, the fiber substances influenced the pancreatic response to maximal exogenous stimulation with secretin (3.0 CU/100 g X hour) and cholecystokinin (0.6 IDU/100 g X hour) and the enzyme content of the gland significantly. Compared with diet V, diet I increased the DNA content of the pancreas and its secretion of bicarbonate and protein, and decreased the protein concentration in the gland. Diet II reduced the pancreatic content of trypsinogen and protein. Diet III decreased the protein content, but increased the bicarbonate secretion, which was also increased by diet IV. -- We conclude that fiber substances influence stimulated secretion and the enzyme content of the pancrease to a varying degree.

Members of the amylovora group of Erwinia are cellulolytic and possess genes homologous to the type II secretion pathway.

PubMed

Riekki, R; Palomäki, T; Virtaharju, O; Kokko, H; Romantschuk, M; Saarilahti, H T

2000-07-01

A cellulase-producing clone was isolated from a genomic library of the Erwinia rhapontici (Millard) Burkholder strain NCPPB2989. The corresponding gene, named celA, encodes an endoglucanase (EC 3.2.1.4) with the extremely low pH optimum of 3.4 and a temperature optimum between 40 and 50 degrees C. A single ORF of 999 nt was found to be responsible for the Cel activity. The corresponding protein, named CelA, showed 67% identity to the endoglucanase Y of E. chrysanthemi and 51.5% identity to the endoglucanase of Cellulomonas uda, and thus belongs to the glycosyl hydrolase family 8. The celA gene, or its homologue, was found to be present in all E. rhapontici isolates analysed, in E. chrysanthemi, and in E. amylovora. The presence of plant cell wall-degrading enzymes in the amylovora group of Erwinia spp. had not previously been established. Furthermore, the DNA of both E. rhapontici and E. amylovora was found to exhibit homology to genes encoding the type II (GSP) secretion pathway, which is known to be responsible for extracellular targeting of cellulases and pectinases in Erwinia spp. that cause soft rotting, such as E. carotovora and E. chrysanthemi. Secretion of the CelA protein by E. rhapontici could not be verified. However, the CelA protein itself was found to include the information necessary for heterologous secretion by E. chrysanthemi.

DBSecSys: a database of Burkholderia mallei secretion systems.

PubMed

Memišević, Vesna; Kumar, Kamal; Cheng, Li; Zavaljevski, Nela; DeShazer, David; Wallqvist, Anders; Reifman, Jaques

2014-07-16

Bacterial pathogenicity represents a major public health concern worldwide. Secretion systems are a key component of bacterial pathogenicity, as they provide the means for bacterial proteins to penetrate host-cell membranes and insert themselves directly into the host cells' cytosol. Burkholderia mallei is a Gram-negative bacterium that uses multiple secretion systems during its host infection life cycle. To date, the identities of secretion system proteins for B. mallei are not well known, and their pathogenic mechanisms of action and host factors are largely uncharacterized. We present the Database of Burkholderia malleiSecretion Systems (DBSecSys), a compilation of manually curated and computationally predicted bacterial secretion system proteins and their host factors. Currently, DBSecSys contains comprehensive experimentally and computationally derived information about B. mallei strain ATCC 23344. The database includes 143 B. mallei proteins associated with five secretion systems, their 1,635 human and murine interacting targets, and the corresponding 2,400 host-B. mallei interactions. The database also includes information about 10 pathogenic mechanisms of action for B. mallei secretion system proteins inferred from the available literature. Additionally, DBSecSys provides details about 42 virulence attenuation experiments for 27 B. mallei secretion system proteins. Users interact with DBSecSys through a Web interface that allows for data browsing, querying, visualizing, and downloading. DBSecSys provides a comprehensive, systematically organized resource of experimental and computational data associated with B. mallei secretion systems. It provides the unique ability to study secretion systems not only through characterization of their corresponding pathogen proteins, but also through characterization of their host-interacting partners.The database is available at https://applications.bhsai.org/dbsecsys.

Leishmaniosis and generalized demodicosis in three dogs: a clinicopathological and immunohistochemical study.

PubMed

Mozos, E; Pérez, J; Day, M J; Lucena, R; Ginel, P J

1999-04-01

This paper describes the clinicopathological and immunohistochemical aspects of the skin lesions in three dogs with leishmaniosis and generalized demodicosis. Diffuse alopecia, crusts, folliculitis and furunculosis, as commonly seen in generalized demodicosis, were prominent in all the dogs. MicroIscopically, there was a diffuse and perifollicular superficial and deep granulomatous dermatitis and, in two dogs, both Copyright Demodex canis mites and Leishmania spp. amastigotes were observed in the same lesions. Numerous Mac387(+)macrophages were observed in the inflammatory infiltrates, but macrophages loaded with amastigotes were Mac387(-). In all cases, immunoreactive CD3 lymphocytes were sparse, both in the granulomatous and perifollicular infiltrates. There were numerous IgG+, IgG4(+)-secreting plasma cells in areas of folliculitis and furunculosis and fewer IgG2(+), IgG3(+), IgA+and IgM+-secreting plasma cells in the inflammatory infiltrate. In all cases, MHC Class II was expressed by the majority of dermal macrophages and dendritic cells, as well as by lymphocytes and fibroblasts. The paucity of CD3(+)lymphocytes, usually abundant in D. canis lesions, points to leishmania-induced cell-mediated immunosuppression as a predisposing factor for generalized demodicosis. 1999 W.B. Saunders and Company Ltd.

[Alterations in tears aqueous layer during cytostatics treatment].

PubMed

Wojciechowska, Katarzyna; Wieckowska-Szakiel, Marzena; Rózalska, Barbara; Jurowski, Piotr

2013-01-01

The aim of the study was to evaluate tears secretion, pH and lysozyme activity in tears aqueous layer during chemotherapy in lung, breast and bowel cancer. 36 patients were enrolled to the study. Depending on the type of cancer and type of chemotherapy patients were divided into three groups. Group I (12 patients) diagnosed with non-small-cell lung cancer treated with PE schema (cisplatin, etoposide), Group II (12 patients) with breast cancer treated with FAC schema (fluorouracil, doxorubicin, cyclophosphamide), Group III (12 patients) with bowel cancer treated with FU/LV schema (fluorouracil, leucovorin). In all the patients: Schirmer's I test, pH measurements and lysozyme test were performed. Patients were examined before chemotherapy, after 2nd, 4th, 6th cycle. In group I and II lowering of tears secretion (p < 0.001) was revealed. In group III there was higher tears secretion (p < 0.001). PH was lowered after 2nd chemotherapy course in group I and II. In further treatment pH value were in the same lower level as after the second course. In group III there was higher pH--more alkaline (p < 0.001) after 2nd cycle of treatment and it was on the same level to the end of the examination process. Lowering of lysozyme activity in the tears film in all groups (p < 0.001) was established. The higher alterations of the lysozyme activity were observed in group treated with FAC schema. Cytostatic treatment has major influence on tears aqueous layer causing alterations of tears secretions. PH alterations depending on type of chemotherapy was observed. Lowering of lysozyme activity in tears was observed. All the deteriorations aggravate with duration of chemotherapy. Alterations of tears film parameters during chemotherapy may influence upon eye surface homeostasis and infectious complication. tears aqueous layer, Schirmer's test, lysozyme activity, tears pH.

Secretion imbalance between tumour necrosis factor and its inhibitor in inflammatory bowel disease

PubMed Central

Noguchi, M; Hiwatashi, N; Liu, Z; Toyota, T

1998-01-01

Background—Tumour necrosis factor (TNF) α and TNF-β are soluble ligands binding to TNF receptors with similar activities; soluble TNF receptors neutralise TNF activity by acting as inhibitors. Little is known about the cytokine/soluble receptor role in inflammatory bowel disease (IBD). 
Aims—To test the hypothesis that an imbalance in secretion between TNF and TNF inhibitors plays a role in gut inflammation in patients with IBD. 
Methods—The secretion of TNF-α, TNF-β, and soluble TNF receptors was compared in the culture supernatants of colonic biopsy specimens and isolated lamina propria mononuclear cells from patients with active colonic IBD. 
Results—Spontaneous secretion of TNF-α in involved IBD mucosa was higher than in normal control and self limited colitis mucosa. Secretion of TNF-β was higher in patients with Crohn's disease than in those with ulcerative colitis. Soluble TNF receptor in IBD mucosa inhibited TNF activity. Type 2 soluble receptor release from IBD mucosa was increased in active inflammation; release from lamina propria cells was not increased. Mucosal TNF-α production correlated with severity of disease. 
Conclusions—Results showed enhanced secretion of TNF-α but failure to release enhanced amounts of soluble TNF receptor in lamina propria mononuclear cells of patients with IBD. An imbalance in secretion between TNF and TNF inhibitor may be implicated in the pathogenesis of IBD. 

 Keywords: Crohn's disease; inflammation; mucosal immunology; soluble TNF receptor; tumour necrosis factor; ulcerative colitis PMID:10189845

Exogenous bFGF or TGFβ1 accelerates healing of reconstructed dura by CO2 laser soldering in minipigs.

PubMed

Wang, Zhenmin; Zhong, Hongliang; Yang, Zhijun; Zhao, Fu; Wang, Bo; Qu, Peiran; Liu, Pinan

2014-05-01

This study aims to explore the probable mechanism of better result of dural reconstruction by CO2 laser soldering and the effect of exogenous basic fibroblast growth factor (bFGF) or transforming growth factor-beta1(TGFβ1) on wound healing. In part I of the study, ten minipigs were randomized into two equal groups, and the dural defects were reconstructed by conventional fibrin glue (FG) bonding (group I a) or by CO2 laser soldering (group Ib). In part II, 36 minipigs were randomized into three equal groups, and the dural defect was reconstructed by CO2 laser soldering; then exogenous bFGF or TGFβ1 was administered in group IIb and group IIc, respectively, while group IIa served as control group. The dural specimens were harvested at 1st week postoperatively in part I; and at 1st, 2nd, 3rd, and 4th week postoperatively in part II, they were examined for healing condition and subjected to hematoxylin-eosin (HE) staining and immunohistochemical (IHC) staining with antibodies against bFGF and TGFβ1. In part I, group Ib showed higher fibroblast cell density than group Ia (P < 0.05). The optical density (OD) for IHC staining with antibodies against bFGF of group Ib was significantly higher than that of group Ia (P < 0.05), and for IHC staining with antibodies against TGFβ1, group Ib showed positive staining while group Ia was negative. In part II, administering exogenous bFGF or TGFβ1 made a left shift of fibroblast cell number-time curve compared with control group. For specimens' IHC staining with antibodies against bFGF, the OD of group IIb was higher than that of group IIa in the corresponding time. For specimens' IHC staining with antibodies against TGFβ1, the OD of groups IIb and IIc was both higher than that of group IIa (P < 0.05 and P < 0.01, respectively). In conclusion, CO2 laser may trigger fibroblast proliferation through stimulating the secretion of bFGF and TGFβ1. Topically administering exogenous bFGF or TGFβ1 could accelerate the healing of the reconstructed dura by enhancing secretion of bFGF and/or TGFβ1 and promoting the process of fibroblast gathering-degrading.

[Endocrinology 1999-2000].

PubMed

Schreiber, V

2001-02-15

Long-lasting problem on the differentiation of adenohypophyseal cell, which prepares them for their specific tasks (somatotropic, lactotropic ect.), becomes elucidated after recognition of the differentiational effect of transcription factor Pit-1. Expression of that factor in somatotrops results in STH secretion, contrary to lactotrops producing prolactin. Subclinical hypothyreosis (increased TSH with normal T3 and T4) endangers vessel not because of hypercholesterolemia, but because of changes in the dynamics of the blood flow. The idea of cardiotropic effect of thyroidal hormones is supported by the finding that administration of trijodthyronine to children after the surgical correction of heart malformations (cardiopulmonary bypass) improves myocardial function--it elevates cardiac output and decreases requirements on the intensive care. Receptors for hormones in tissues are flexible, they can be "heterooligomers" for dopamine and somatostatin. Mutations of mineralocorticoid receptor may cause hypertension in pregnancy and progesterone receptors have several isoforms. Receptors can be also activated by short exposition to a hormone. Glucocorticoids have probably also membrane receptors. Diabetes mellitus "type I" needn't to be immunogenic and DM type II not only results from down-regulation of receptors and subsequent insulin resistance, but it can be also caused by defects in insulin secretion. Insulin has receptors in the brain and participates in the appetite regulation. The attempt to use "desensibilisation" by peroraly administered insulin in patients with immunogenic DM had no effect. Stress affects memory mechanisms, heavy emotional stress during gravidity can bring congenital malformations. The decrease of mental functions in aged women depends on the level of free estradiol (the fraction, which is not bound to plasma proteins). Activation of dopaminergic neurons can be achieved by neurotropic growth factors. Nesiritide is a recombinant brain natriuretic hormone successfully tested in heart failure. The role of leptin in the appetite regulation in man is still not clear, other signalling molecules may have also an effect, e.g., ghrelin, which primarily stimulates STH secretion and brings about weight gain. Sildenafil influences nitrergic neurons elsewhere than in penis, for example it has positive effects in patients with oesophageal achalasia.

Identification of novel secreted virulence factors from Xylella fastidiosa using a TRV expression system

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa is a bacterium that causes leaf scorch diseases of agriculturally important crops including grapevines and almonds. Little is known about virulence factors that are necessary for X. fastidiosa to grow and cause disease in the xylem vessels of a plant host. Any protein secreted by ...

SNARE-encoding genes VdSec22 and VdSso1 mediate protein secretion required for full virulence in Verticillium dahliae

USDA-ARS?s Scientific Manuscript database

Proteins that mediate cellular and subcellular membrane fusion are key factors in vesicular trafficking in all eukaryotic cells, including the secretion and transport of plant pathogen virulence factors. In this study, we identified vesicle fusion components that included 22 soluble N-ethylmaleimide...

Leukocytosis due to markedly elevated granulocyte-colony stimulating factor levels in a patient with endometrial cancer: Case report and literature review.

PubMed

Clark, Leslie H; Moll, Stephan; Houghton, Damon; O'Connor, Siohban; Soper, John T

2017-05-01

•Granulocyte-colony stimulating factor (GCSF) secretion by gynecologic tumors is rare.•Elevations in serum GCSF can be seen in the absence of tumor GSCF secretion.•Extreme leukocytosis is associated with autocrine tumor growth and poor prognosis.

Reactive Oxygen Species Alter Autocrine and Paracrine Signaling

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

Zangar, Richard C.; Bollinger, Nikki; Weber, Thomas J.

2011-12-01

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endo-plasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a varietymore » of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A customELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-{kappa}B pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.« less

Novel bio-synthetic hybrid materials and coculture systems for musculoskeletal tissue engineering

NASA Astrophysics Data System (ADS)

Lee, Hyeseung Janice

Tissue Engineering is a truly exciting field of this age, trying to regenerate and repair impaired tissues. Unlike the old artificial implants, tissue engineering aims at making a long-term functional biological replacement. One strategy for such tissue engineering requires the following three components: cells, scaffolds, and soluble factors. Cells are cultured in a three-dimensional (3D) scaffold with medium containing various soluble factors. Once a tissue is developed in vitro, then it is implanted in vivo. The overall goal of this thesis was to develop novel bio-synthetic hybrid scaffolds and coculture system for musculoskeletal tissue engineering. The most abundant cartilage extracellular matrix (ECM) components are collagen and glycosaminoglycan (GAG), which are the natural scaffold for chondrocytes. As two different peptides, collagen mimetic peptide (CMP) and hyaluronic acid binding peptide (HABPep) were previously shown to bind to collagen and hyaluronic acid (HA) of GAG, respectively, it was hypothesized that immobilizing CMP and HABP on 3D scaffold would results in an interaction between ECM components and synthetic scaffolds via peptide-ECM bindings. CMP or HABPep-conjugated photopolymerizable poly(ethylene oxide) diacrylate (PEODA) hydrogels were synthesized and shown to retain encapsulated collagen or HA, respectively. This result supported that conjugated CMP and HABPep can interact with collagen and HA, respectively, and can serve as biological linkers in 3D synthetic hydrogels. When chondrocytes or mesenchymal stem cells (MSCs) were seeded, cells in CMP-conjugated scaffolds produced significantly more amount of type II collagen and GAG, compared to those in control scaffolds. Moreover, MSCs cultured in CMP-conjugated scaffolds exhibited lower level of hypertrophic markers, cbfa-1 and type X collagen. These results demonstrated that enhanced interaction between collagen and scaffold via CMP improves chondrogenesis of chondrocytes and MSCs and further reduces hypertrophy of differentiating MSCs. On the other hand, although cells in HABPep-conjugated scaffolds produced less ECM components, they survived and proliferated significantly more than those in control, resulting in overall increase in ECM contents per scaffold. Once implanted in vivo, HABPep-conjugated constructs increased GAG and type II collagen contents further, compared to those of the control hydrogel. These results showed that enhanced interaction between HA and scaffold via HABPep improved the in vitro culture expansion of MSCs and further ECM production in vivo. Effects of cell-secreted bioactive factors via cell-cell communication on stem cell differentiation were also investigated in 3D bilayer system. First, when mesenchymal progenitor cells (MPCs) were cocultured with ES-derived cells (ESDC), morphogenetic factors secreted by ESDCs showed a potential to improve MPC chondrogenesis in both control and chondrogenic medium by increasing not only MPC's chondrogenic gene expression, but also ECM production. Moreover, the effect of ESDC cell-mediated chondrogenesis of MSC could not be mimicked by chondrogenic medium supplemented with TGF-beta1 and dexamethasone. Secondly, coculturing hepatic cells enhanced specific chondrogenic differentiation of ES cells in the 3D bilayer system. These studies demonstrated that cell-secreted soluble factors can be used to guide stem cell differentiation.

A tunable hydrogel system for long-term release of cell-secreted cytokines and bioprinted in situ wound cell delivery.

PubMed

Skardal, Aleksander; Murphy, Sean V; Crowell, Kathryn; Mack, David; Atala, Anthony; Soker, Shay

2017-10-01

For many cellular therapies being evaluated in preclinical and clinical trials, the mechanisms behind their therapeutic effects appear to be the secretion of growth factors and cytokines, also known as paracrine activity. Often, delivered cells are transient, and half-lives of the growth factors that they secrete are short, limiting their long-term effectiveness. The goal of this study was to optimize a hydrogel system capable of in situ cell delivery that could sequester and release growth factors secreted from those cells after the cells were no longer present. Here, we demonstrate the use of a fast photocross-linkable heparin-conjugated hyaluronic acid (HA-HP) hydrogel as a cell delivery vehicle for sustained growth factor release, which extends paracrine activity. The hydrogel could be modulated through cross-linking geometries and heparinization to support sustained release proteins and heparin-binding growth factors. To test the hydrogel in vivo, we used it to deliver amniotic fluid-derived stem (AFS) cells, which are known to secrete cytokines and growth factors, in full thickness skin wounds in a nu/nu murine model. Despite transience of the AFS cells in vivo, the HA-HP hydrogel with AFS cells improved wound closure and reepithelialization and increased vascularization and production of extracellular matrix in vivo. These results suggest that HA-HP hydrogel has the potential to prolong the paracrine activity of cells, thereby increasing their therapeutic effectiveness in wound healing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1986-2000, 2017. © 2016 Wiley Periodicals, Inc.

Discovery of the type VII ESX-1 secretion needle?

PubMed

Ates, Louis S; Brosch, Roland

2017-01-01

Mycobacterium tuberculosis, the etiological agent of human tuberculosis, harbours five ESAT-6/type VII secretion (ESX/T7S) systems. The first esx gene clusters were identified during the genome-sequencing project of M. tuberculosis H37Rv. Follow-up studies revealed additional genes playing important roles in ESX/T7S systems. Among the latter genes, one can find those that encode Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins as well as a gene cluster that is encoded >260 kb upstream of the esx-1 locus and encodes ESX-1 secretion-associated proteins EspA (Rv3616c), EspC (Rv3615c) and EspD (Rv3614c). The espACD cluster has been suggested to have an important function in ESX-1 secretion since EspA-EspC and EsxA-EsxB are mutually co-dependent on each other for secretion. However, the molecular mechanism of this co-dependence and interaction between the substrates remained unknown. In this issue of Molecular Microbiology, Lou and colleagues show that EspC forms high-molecular weight polymerization complexes that resemble selected components of type II, III and/or IV secretion systems of Gram-negative bacteria. Indeed, EspC-multimeric complexes form filamentous structures that could well represent a secretion needle of ESX-1 type VII secretion systems. This exciting observation opens new avenues for research to discover and characterize ESX/T7S components and elucidates the co-dependence of EsxA/B secretion with EspA/C. © 2016 John Wiley & Sons Ltd.

Enhanced angiogenic effect of adipose-derived stromal cell spheroid with low-level light therapy in hind limb ischemia mice.

PubMed

Park, In-Su; Chung, Phil-Sang; Ahn, Jin Chul

2014-11-01

The aim of this study was to investigate the effects of low-level laser therapy (LLLT) on transplanted human adipose-derived mesenchymal stem cells (hASCs) spheroid in a hind limb ischemia animal model. LLLT, hASCs spheroid and hASCs spheroid transplantation with LLLT (spheroid + LLLT) were applied to the ischemic hind limbs in athymic mice. The survival, differentiation and secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) of the spheroid ASCs were evaluated by immunohistochemistry and western blots. Spheroid + LLLT group had enhanced the tissue regeneration, including angiogenesis, compared with the ASC group. The spheroid ASCs contributed to tissue regeneration via differentiation and secretion of growth factors. In the spheroid + LLLT group, the survival of spheroid hASCs increased with a concomitant decrease in apoptosis of spheroid hASCs in the ischemic hind limb. The secretion of growth factors was stimulated in the spheroid + LLLT group compared with the ASCs and spheroid group. These data suggested that LLLT is an effective biostimulator of spheroid hASCs in tissue regeneration that enhanced the survival of ASCs and stimulated the secretion of growth factors in the ischemic hind limb. Copyright © 2014 Elsevier Ltd. All rights reserved.

Glucose in vaginal secretions before and after oral glucose tolerance testing in women with and without recurrent vulvovaginal candidiasis.

PubMed

Ehrström, Sophia; Yu, Anna; Rylander, Eva

2006-12-01

To measure the change of glucose in vaginal secretions during glucose tolerance testing in women with recurrent vulvovaginal candidiasis and in healthy control subjects. Thirty-eight women with recurrent vulvovaginal candidiasis and 45 healthy, age-matched controls completed a health questionnaire regarding general and gynecologic health and food and alcohol habits. They all underwent an oral glucose tolerance test and a vaginal examination. Vaginal secretion was collected from the proximal part of the vagina. Glucose in plasma and in vaginal secretions were measured at fasting and after 2 hours and analyzed with the hexokinase method. A sample size analysis showed that the number of subjects included in the study was sufficient for a beta value of 0.80, at the significance level of alpha=.05, at a difference in glucose in vaginal secretions of 30% after oral glucose tolerance test. In healthy women, the median level of glucose in vaginal secretions was 5.2 mM before and 3.7 mM after oral glucose tolerance test, and plasma glucose was 5.0 mM before and 5.8 mM after oral glucose tolerance test. No significant difference was seen regarding change of glucose level in vaginal secretions and plasma glucose after testing, compared with before oral glucose tolerance testing. There were no differences between women with recurrent vulvovaginal candidiasis and control subjects regarding change in glucose level in vaginal secretions or in plasma during oral glucose tolerance test. II-2.

The relationship between the structures of four beta-lactamases obtained from Bacillus cereus.

PubMed

Cid, H; Carrillo, O; Bunster, M; Martínez, J; Vargas, V

1988-06-01

Bacillus cereus has proved to be one of the most interesting microorganisms in the study of beta-lactamases. It secrets these enzymes very efficiently and, frequently, in multiple forms. Three different forms are produced by strain 569/H; mutant 5/B of the same microorganism is constitutive for the secretion of beta-lactamases I and II. The present study, based on secondary structure prediction by two independent methods, states the relationship among the structures of beta-lactamases I, II and III produced by B. cereus 569/H and beta-lactamase I from the strain 5/B of this microorganism. A strong similarity is also established for the enzyme type III of B. cereus and the enzyme type I produced by B. licheniformis which could have an evolutionary explanation. A structural analysis of the leader peptide regions of these enzymes by the method of Mohana and Argos is also reported.

The extracellular calcium-sensing receptor is required for cholecystokinin secretion in response to l-phenylalanine in acutely isolated intestinal I cells

PubMed Central

Liou, Alice P.; Sei, Yoshitatsu; Zhao, Xilin; Feng, Jianying; Lu, Xinping; Thomas, Craig; Pechhold, Susanne; Raybould, Helen E.

2011-01-01

The extracellular calcium-sensing receptor (CaSR) has recently been recognized as an l-amino acid sensor and has been implicated in mediating cholecystokinin (CCK) secretion in response to aromatic amino acids. We investigated whether direct detection of l-phenylalanine (l-Phe) by CaSR results in CCK secretion in the native I cell. Fluorescence-activated cell sorting of duodenal I cells from CCK-enhanced green fluorescent protein (eGFP) transgenic mice demonstrated CaSR gene expression. Immunostaining of fixed and fresh duodenal tissue sections confirmed CaSR protein expression. Intracellular calcium fluxes were CaSR dependent, stereoselective for l-Phe over d-Phe, and responsive to type II calcimimetic cinacalcet in CCK-eGFP cells. Additionally, CCK secretion by an isolated I cell population was increased by 30 and 62% in response to l-Phe in the presence of physiological (1.26 mM) and superphysiological (2.5 mM) extracellular calcium concentrations, respectively. While the deletion of CaSR from CCK-eGFP cells did not affect basal CCK secretion, the effect of l-Phe or cinacalcet on intracellular calcium flux was lost. In fact, both secretagogues, as well as superphysiological Ca2+, evoked an unexpected 20–30% decrease in CCK secretion compared with basal secretion in CaSR−/− CCK-eGFP cells. CCK secretion in response to KCl or tryptone was unaffected by the absence of CaSR. The present data suggest that CaSR is required for hormone secretion in the specific response to l-Phe by the native I cell, and that a receptor-mediated mechanism may inhibit hormone secretion in the absence of a fully functional CaSR. PMID:21252045

Effects of 3-dimensional culture conditions (collagen-chitosan nano-scaffolds) on maturation of dendritic cells and their capacity to interact with T-lymphocytes.

PubMed

Daneshmandi, Saeed; Dibazar, Shaghayegh Pishkhan; Fateh, Shirin

2016-01-01

In the body, there is a natural three-dimensional (3D) microenvironment in which immune cells, including dendritic cells (DC), play their functions. This study evaluated the impact of using collagen-chitosan 3D nano-scaffolds in comparisons to routine 2D culture plates on DC phenotype and functions. Bone marrow-derived DC were cultured on scaffolds and plates and then stimulated with lipopolysaccharide (LPS) or chitosan-based nanoparticles (NP) for 24 h. Thereafter, DC viability, expression of maturation markers and levels of cytokines secretion were evaluated. In another set of studies, the DC were co-cultured with allogenic T-lymphocytes in both the 2D and 3D systems and effects on DC-induction of T-lymphocyte proliferation and cytokine release were analyzed. The results indicated that CD40, CD86 and MHC II marker expression and interleukin (IL)-12, IL-6 and tumor necrosis factor (TNF)-α secretion by DC were enhanced in 3D cultures in comparison to by cells maintained in the 2D states. The data also showed that DNA/chitosan NP activated DC more than LPS in the 3D system. T-Lymphocyte proliferation was induced to a greater extent by DNA/NP-treated DC when both cell types were maintained on the scaffolds. Interestingly, while DC induction of T-lymphocyte interferon (IFN)-γ and IL-4 release was enhanced in the 3D system (relative to controls), there was a suppression of transforming growth factor (TGF)-β production; effects on IL-10 secretion were variable. The results here suggested that collagen-chitosan scaffolds could provide a pro-inflammatory and activator environment to perform studies to analyze effects of exogenous agents on the induction of DC maturation, NP uptake and/or cytokines release, as well as for the ability of these cells to potentially interact with other immune system cells in vitro.

Mesenchymal stromal cell secretomes are modulated by suspension time, delivery vehicle, passage through catheter, and exposure to adjuvants.

PubMed

Parsha, Kaushik; Mir, Osman; Satani, Nikunj; Yang, Bing; Guerrero, Waldo; Mei, Zhuyong; Cai, Chunyan; Chen, Peng R; Gee, Adrian; Hanley, Patrick J; Aronowski, Jaroslaw; Savitz, Sean I

2017-01-01

Extensive animal data indicate that mesenchymal stromal cells (MSCs) improve outcome in stroke models. Intra-arterial (IA) injection is a promising route of delivery for MSCs. Therapeutic effect of MSCs in stroke is likely based on the broad repertoire of secreted trophic and immunomodulatory cytokines produced by MSCs. We determined the differential effects of exposing MSCs to different types of clinically relevant vehicles, and/or different additives and passage through a catheter relevant to IA injections. MSCs derived from human bone marrow were tested in the following vehicles: 5% albumin (ALB), 6% Hextend (HEX) and 40% dextran (DEX). Each solution was tested (i) alone, (ii) with low-dose heparin, (iii) with 10% Omnipaque, or (iv) a combination of heparin and Omnipaque. Cells in vehicles were collected directly or passed through an IA catheter, and MSC viability and cytokine release profiles were assessed. Cell viability remained above 90% under all tested conditions with albumin being the highest at 97%. Viability was slightly reduced after catheter passage or exposure to heparin or Omnipaque. Catheter passage had little effect on MSC cytokine secretion. ALB led to increased release of angiogenic factors such as vascular endothelial growth factor compared with other vehicles, while HEX and DEX led to suppression of pro-inflammatory cytokines such as interleukin-6. However, when these three vehicles were subjected to catheter passage and/or exposure to additives, the cytokine release profile varied depending on the combination of conditions to which MSCs were exposed. Exposure of MSCs to certain types of vehicles or additives changes the profile of cytokine secretion. The activation phenotype of MSCs may therefore be affected by the vehicles used for these cells or the exposure to the adjuvants used in their administration. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Adipose stem cells can secrete angiogenic factors that inhibit hyaline cartilage regeneration.

PubMed

Lee, Christopher Sd; Burnsed, Olivia A; Raghuram, Vineeth; Kalisvaart, Jonathan; Boyan, Barbara D; Schwartz, Zvi

2012-08-24

Adipose stem cells (ASCs) secrete many trophic factors that can stimulate tissue repair, including angiogenic factors, but little is known about how ASCs and their secreted factors influence cartilage regeneration. Therefore, the aim of this study was to determine the effects ASC-secreted factors have in repairing chondral defects. ASCs isolated from male Sprague Dawley rats were cultured in monolayer or alginate microbeads supplemented with growth (GM) or chondrogenic medium (CM). Subsequent co-culture, conditioned media, and in vivo cartilage defect studies were performed. ASC monolayers and microbeads cultured in CM had decreased FGF-2 gene expression and VEGF-A secretion compared to ASCs cultured in GM. Chondrocytes co-cultured with GM-cultured ASCs for 7 days had decreased mRNAs for col2, comp, and runx2. Chondrocytes treated for 12 or 24 hours with conditioned medium from GM-cultured ASCs had reduced sox9, acan, and col2 mRNAs; reduced proliferation and proteoglycan synthesis; and increased apoptosis. ASC-conditioned medium also increased endothelial cell tube lengthening whereas conditioned medium from CM-cultured ASCs had no effect. Treating ASCs with CM reduced or abolished these deleterious effects while adding a neutralizing antibody for VEGF-A eliminated ASC-conditioned medium induced chondrocyte apoptosis and restored proteoglycan synthesis. FGF-2 also mitigated the deleterious effects VEGF-A had on chondrocyte apoptosis and phenotype. When GM-grown ASC pellets were implanted in 1 mm non-critical hyaline cartilage defects in vivo, cartilage regeneration was inhibited as evaluated by radiographic and equilibrium partitioning of an ionic contrast agent via microCT imaging. Histology revealed that defects with GM-cultured ASCs had no tissue ingrowth from the edges of the defect whereas empty defects and defects with CM-grown ASCs had similar amounts of neocartilage formation. ASCs must be treated to reduce the secretion of VEGF-A and other factors that inhibit cartilage regeneration, which can significantly influence how ASCs are used for repairing hyaline cartilage.

Cancer-associated fibroblasts drive glycolysis in a targetable signaling loop implicated in head and neck squamous cell carcinoma progression.

PubMed

Kumar, Dhruv; New, Jacob; Vishwakarma, Vikalp; Joshi, Radhika; Enders, Jonathan; Lin, Fangchen; Dasari, Sumana; Gutierrez, Wade R; Leef, George; Ponnurangam, Sivapriya; Chavan, Hemantkumar; Ganaden, Lydia; Thornton, Mackenzie M; Dai, Hongying; Tawfik, Ossama; Straub, Jeffrey; Shnayder, Yelizaveta; Kakarala, Kiran; Tsue, Terance Ted; Girod, Douglas A; Van Houten, Bennett; Anant, Shrikant; Krishnamurthy, Partha; Thomas, Sufi Mary

2018-05-16

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC) is associated with a less than 50% 5-year survival rate. Late stage HNSCC frequently consists of up to 80% cancer-associated fibroblasts (CAF). We previously reported that CAF-secreted hepatocyte growth factor (HGF) facilitates HNSCC progression, however very little is known about the role of CAFs in HNSCC metabolism. Here we demonstrate that CAF-secreted HGF increases extracellular lactate levels in HNSCC via upregulation of glycolysis. CAF-secreted HGF induced basic fibroblast growth factor (bFGF) secretion from HNSCC. CAFs were more efficient than HNSCC in using lactate as a carbon source. HNSCC-secreted bFGF increased mitochondrial oxidative phosphorylation (OXPHOS) and HGF secretion from CAFs. Combined inhibition of c-Met and FGFR significantly inhibited CAF-induced HNSCC growth in vitro and in vivo (p<0.001). Our cumulative findings underscore reciprocal signaling between CAF and HNSCC involving bFGF and HGF. This contributes to metabolic symbiosis and a targetable therapeutic axis involving c-Met and FGFR. Copyright ©2018, American Association for Cancer Research.

Trimeric autotransporter adhesins contribute to Actinobacillus pleuropneumoniae pathogenicity in mice and regulate bacterial gene expression during interactions between bacteria and porcine primary alveolar macrophages.

PubMed

Qin, Wanhai; Wang, Lei; Zhai, Ruidong; Ma, Qiuyue; Liu, Jianfang; Bao, Chuntong; Zhang, Hu; Sun, Changjiang; Feng, Xin; Gu, Jingmin; Du, Chongtao; Han, Wenyu; Langford, P R; Lei, Liancheng

2016-01-01

Actinobacillus pleuropneumoniae is an important pathogen that causes respiratory disease in pigs. Trimeric autotransporter adhesin (TAA) is a recently discovered bacterial virulence factor that mediates bacterial adhesion and colonization. Two TAA coding genes have been found in the genome of A. pleuropneumoniae strain 5b L20, but whether they contribute to bacterial pathogenicity is unclear. In this study, we used homologous recombination to construct a double-gene deletion mutant, ΔTAA, in which both TAA coding genes were deleted and used it in in vivo and in vitro studies to confirm that TAAs participate in bacterial auto-aggregation, biofilm formation, cell adhesion and virulence in mice. A microarray analysis was used to determine whether TAAs can regulate other A. pleuropneumoniae genes during interactions with porcine primary alveolar macrophages. The results showed that deletion of both TAA coding genes up-regulated 36 genes, including ene1514, hofB and tbpB2, and simultaneously down-regulated 36 genes, including lgt, murF and ftsY. These data illustrate that TAAs help to maintain full bacterial virulence both directly, through their bioactivity, and indirectly by regulating the bacterial type II and IV secretion systems and regulating the synthesis or secretion of virulence factors. This study not only enhances our understanding of the role of TAAs but also has significance for those studying A. pleuropneumoniae pathogenesis.

In Vivo Efficacy of Fresh vs. Frozen Osteochondral Allografts in the Goat at 6 Months is Associated with PRG4 Secretion

PubMed Central

Pallante-Kichura, Andrea L.; Chen, Albert C.; Temple-Wong, Michele M.; Bugbee, William D.; Sah, Robert L.

2014-01-01

The long-term efficacy of osteochondral allografts is due to the presence of viable chondrocytes within graft cartilage. Chondrocytes in osteochondral allografts, especially those at the articular surface that normally produce the lubricant proteoglycan-4 (PRG4), are susceptible to storage-associated death. The hypothesis of this study was that the loss of chondrocytes within osteochondral grafts leads to decreased PRG4 secretion, after graft storage and subsequent implant. The objectives were to determine the effect of osteochondral allograft treatment (FROZEN vs. FRESH) on secretion of functional PRG4 after (i) storage, and (ii) 6months in vivo in adult goats. FROZEN allograft storage reduced PRG4 secretion from cartilage by ~85% compared to FRESH allograft storage. After 6months in vivo, the PRG4-secreting function of osteochondral allografts was diminished with prior FROZEN storage by ~81% versus FRESH allografts and by ~84% versus non-operated control cartilage. Concomitantly, cellularity at the articular surface in FROZEN allografts was ~96% lower than FRESH allografts and non-operated cartilage. Thus, the PRG4-secreting function of allografts appears to be maintained in vivo based on its state after storage. PRG4 secretion may be not only a useful marker of allograft performance, but also a biological process protecting the articular surface of grafts following cartilage repair. PMID:23362152

Nutritional Ingredients Modulate Adipokine Secretion and Inflammation in Human Primary Adipocytes

PubMed Central

Romacho, Tania; Glosse, Philipp; Richter, Isabel; Elsen, Manuela; Schoemaker, Marieke H.; van Tol, Eric A.; Eckel, Jürgen

2015-01-01

Nutritional factors such as casein hydrolysates and long chain polyunsaturated fatty acids have been proposed to exert beneficial metabolic effects. We aimed to investigate how a casein hydrolysate (eCH) and long chain polyunsaturated fatty acids could affect human primary adipocyte function in vitro. Incubation conditions with the different nutritional factors were validated by assessing cell vitality with lactate dehydrogenase (LDH) release and neutral red incorporation. Intracellular triglyceride content was assessed with Oil Red O staining. The effect of eCH, a non-peptidic amino acid mixture (AA), and long-chain polyunsaturated fatty acids (LC-PUFAs) on adiponectin and leptin secretion was determined by enzyme-linked immunosorbent assay (ELISA). Intracellular adiponectin expression and nuclear factor-κB (NF-κB) activation were analyzed by Western blot, while monocyte chemoattractant protein-1 (MCP-1) release was explored by ELISA. The eCH concentration dependently increased adiponectin secretion in human primary adipocytes through its intrinsic peptide bioactivity, since the non-peptidic mixture, AA, could not mimic eCH’s effects on adiponectin secretion. Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and DHA combined with arachidonic acid (ARA) upregulated adiponectin secretion. However, only DHA and DHA/ARA exerted a potentanti-inflammatory effect reflected by prevention of tumor necrosis factor-α (TNF-α) induced NF-κB activation and MCP-1 secretion in human adipocytes. eCH and DHA alone or in combination with ARA, may hold the key for nutritional programming through their anti-inflammatory action to prevent diseases with low-grade chronic inflammation such as obesity or diabetes. PMID:25629558

Tec-kinase-mediated phosphorylation of fibroblast growth factor 2 is essential for unconventional secretion.

PubMed

Ebert, Antje D; Laussmann, Mareike; Wegehingel, Sabine; Kaderali, Lars; Erfle, Holger; Reichert, Jürgen; Lechner, Johannes; Beer, Hans-Dietmar; Pepperkok, Rainer; Nickel, Walter

2010-06-01

Fibroblast growth factor 2 (FGF2) is a potent mitogen that is exported from cells by an endoplasmic reticulum (ER)/Golgi-independent mechanism. Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes, a process that depends on the phosphoinositide phosphatidylinositol 4,5-biphosphate (PI(4,5)P(2)) at the inner leaflet as well as heparan sulfate proteoglycans at the outer leaflet of plasma membranes; however, additional core and regulatory components of the FGF2 export machinery have remained elusive. Here, using a highly effective RNAi screening approach, we discovered Tec kinase as a novel factor involved in unconventional secretion of FGF2. Tec kinase does not affect FGF2 secretion by an indirect mechanism, but rather forms a heterodimeric complex with FGF2 resulting in phosphorylation of FGF2 at tyrosine 82, a post-translational modification shown to be essential for FGF2 membrane translocation to cell surfaces. Our findings suggest a crucial role for Tec kinase in regulating FGF2 secretion under various physiological conditions and, therefore, provide a new perspective for the development of a novel class of antiangiogenic drugs targeting the formation of the FGF2/Tec complex.

C-type natriuretic peptide is synthesized and secreted from leukemia cell lines, peripheral blood cells, and peritoneal macrophages.

PubMed

Kubo, A; Isumi, Y; Ishizaka, Y; Tomoda, Y; Kangawa, K; Dohi, K; Matsuo, H; Minamino, N

2001-05-01

C-type natriuretic peptide (CNP) is the third member of the natriuretic peptide family. Cultured endothelial cells secrete CNP, and its secretion rate from the endothelial cells is augmented by lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha, which participate in the pathophysiology of inflammation. In this study, we investigated the regulation of CNP secretion from monocytes and macrophages to estimate its contribution to the progression of inflammation. CNP secretion rates from two human leukemia cell lines (THP-1 and HL-60), human peripheral blood lymphocytes, granulocytes, monocytes, monocyte-derived macrophages, and mouse peritoneal macrophages were measured under conditions with or without stimulation. Immunoreactive CNP levels in the culture media of these cells were measured by a specific radioimmunoassay. The secretion rates of CNP from THP-1 and HL-60 cells were augmented according to the degree of their differentiation into macrophage-like cells under the stimulation with phorbol ester. Peripheral blood monocytes also increased the CNP secretion rate after their differentiation into macrophages. Retinoic acid elicited synergistic effects on the CNP secretion rate from HL-60 cells when administered with lipopolysaccharide, interferon-gamma, interleukin-1beta, tumor necrosis factor-alpha, or phorbol ester. In contrast, the phorbol ester-stimulated CNP secretion rate from THP-1 cells was suppressed with dexamethasone, which inhibits monocyte differentiation into macrophage. The secretion rate of CNP from monocytes was shown to be regulated based on the degree of their differentiation. This study provides evidence that the monocyte/macrophage system is one of the sources of CNP, especially under inflammatory conditions.

NADPH Oxidase-Derived H2O2 Contributes to Angiotensin II-Induced Aldosterone Synthesis in Human and Rat Adrenal Cortical Cells

PubMed Central

Rajamohan, Senthilkumar B.; Raghuraman, Gayatri; Prabhakar, Nanduri R.

2012-01-01

Abstract Background The Renin-Angiotensin-Aldosterone-System plays a pivotal role in hypertension. Angiotensin II (Ang II) is a major regulator of aldosterone synthesis and secretion, and it is known to facilitate reactive oxygen species (ROS) generation in many cell types. Aims: Here, we assessed the role of ROS signaling in Ang II-induced aldosterone synthesis by focusing on the regulation of aldosterone synthase (CYP11B2), a cytochrome P450 oxidase that catalyzes the final step in aldosterone biosynthetic pathway. Results: Ang II increased CYP11B2 activity, mRNA and protein with a concomitant elevation of 6-Carboxy- 2′,7′-dichlorodihydrofluorescein diacetate fluorescence, malondialdehyde and protein carbonyl levels (indices of ROS), NADPH oxidase (Nox) activity, and H2O2 levels in human and rat adrenal cortical cells. The expression of nuclear receptor related 1 protein, a transcription factor known to regulate CYP11B2 expression, was also augmented by Ang II. These Ang II-evoked effects were either abolished or attenuated by pretreatment of cells with either Ang II type I receptor (AT1R) antagonist, or antioxidants or Nox inhibitor or siRNA silencing of Nox1, 2 and 4, or inhibitors of phospholipase C and protein kinase C. Exogenous H2O2 mimicked the facilitatory effects of Ang II on CYP11B2 activity, mRNA, and protein expression, and these changes were significantly reduced by PEG-catalase. Innovation: ROS, particularly H2O2, is identified as a key regulator of aldosterone production. Conclusion: Our results suggest that Ang II facilitates CYP11B2 activity and the ensuing aldosterone production via activation of AT1R-Nox-H2O2 signaling pathway. Antioxid. Redox Signal. 17, 445–459. PMID:22214405

Osteogenesis and Trophic Factor Secretion are Influenced by the Composition of Hydroxyapatite/Poly(Lactide-Co-Glycolide) Composite Scaffolds

PubMed Central

He, Jiawei; Genetos, Damian C.

2010-01-01

The use of composite biomaterials for bone repair capitalizes on the beneficial aspects of individual materials while tailoring the mechanical properties of the composite. We hypothesized that substrate composition would modulate the osteogenic response and secretion of potent trophic factors by human mesenchymal stem cells (hMSCs). Composite scaffolds were prepared by combining nanosized hydroxyapatite (HA) and microspheres formed of poly(lactic-co-glycolic acid) (PLG) at varying ratios between 0:1 and 5:1. Scaffolds were seeded with hMSCs for culture in osteogenic conditions or subcutaneous implantation into nude rats. Compressive moduli increased with HA content in a near-linear fashion. The osteogenic differentiation of hMSCs increased in a dose-dependent manner as determined by alkaline phosphatase activity and osteopontin secretion after 4 weeks of culture. Further, endogenous secretion of vascular endothelial growth factor was sustained at significantly higher levels over 28 days for hMSCs seeded in 2.5:1 and 5:1 HA:PLG scaffolds. Eight weeks after implantation, scaffolds with higher HA:PLG ratios exhibited greater vascularization and more mineralized tissue. These data demonstrate that the mechanical properties, osteogenic differentiation, as well as the timing and duration of trophic factor secretion by hMSCs can be tailored through controlling the composition of the polymer–bioceramic composite. PMID:19642853

Cellular and molecular mechanism for secretory autophagy.

PubMed

Kimura, Tomonori; Jia, Jingyue; Claude-Taupin, Aurore; Kumar, Suresh; Choi, Seong Won; Gu, Yuexi; Mudd, Michal; Dupont, Nicolas; Jiang, Shanya; Peters, Ryan; Farzam, Farzin; Jain, Ashish; Lidke, Keith A; Adams, Christopher M; Johansen, Terje; Deretic, Vojo

2017-06-03

Macroautophagy/autophagy plays a role in unconventional secretion of leaderless cytosolic proteins. Whether and how secretory autophagy diverges from conventional degradative autophagy is unclear. We have shown that the prototypical secretory autophagy cargo IL1B/IL-1β (interleukin 1 β) is recognized by TRIM16, and that this first to be identified secretory autophagy receptor interacts with the R-SNARE SEC22B to jointly deliver cargo to the MAP1LC3B-II-positive sequestration membranes. Cargo secretion is unaffected by knockdowns of STX17, a SNARE catalyzing autophagosome-lysosome fusion as a prelude to cargo degradation. Instead, SEC22B in combination with plasma membrane syntaxins completes cargo secretion. Thus, secretory autophagy diverges from degradative autophagy by using specialized receptors and a dedicated SNARE machinery to bypass fusion with lysosomes.

Regulation of adipocytokine secretion and adipocyte hypertrophy by polymethoxyflavonoids, nobiletin and tangeretin.

PubMed

Miyata, Yoshiki; Tanaka, Haruyuki; Shimada, Arata; Sato, Takashi; Ito, Akira; Yamanouchi, Toshikazu; Kosano, Hiroshi

2011-03-28

The polymethoxyflavonoids nobiletin and tangeretin possess several important biological properties such as neuroprotective, antimetastatic, anticancer, and anti-inflammatory properties. The present study was undertaken to examine whether nobiletin and tangeretin could modulate adipocytokine secretion and to evaluate the effects of these flavonoids on the hypertrophy of mature adipocytes. All experiments were performed on the murine preadipocyte cell line 3T3-L1. We studied the formation of intracellular lipid droplets in adipocytes and the apoptosis-inducing activity to evaluate the effects of polymethoxyflavonoids on adipocyte differentiation and hypertrophy, respectively. The secretion of adipocytokines was measured using ELISA. We demonstrated that the combined treatment of differentiation reagents with nobiletin or tangeretin differentiated 3T3-L1 preadipocytes into adipocytes possessing less intracellular triglyceride as compared to vehicle-treated differentiated 3T3-L1 adipocytes. Both flavonoids increased the secretion of an insulin-sensitizing factor, adiponectin, but concomitantly decreased the secretion of an insulin-resistance factor, MCP-1, in 3T3-L1 adipocytes. Furthermore, nobiletin was found to decrease the secretion of resistin, which serves as an insulin-resistance factor. In mature 3T3-L1 adipocytes, nobiletin induced apoptosis; tangeretin, in contrast, did not induce apoptosis, but suppressed further triglyceride accumulation. Our results suggest that nobiletin and tangeretin are promising therapeutic candidates for the prevention and treatment of insulin resistance by modulating the adipocytokine secretion balance. We also demonstrated the different effects of nobiletin and tangeretin on mature adipocytes. Copyright © 2011 Elsevier Inc. All rights reserved.

Insulin secretion and action in North Indian women during pregnancy.

PubMed

Arora, G P; Almgren, P; Thaman, R G; Pal, A; Groop, L; Vaag, A; Prasad, R B; Brøns, C

2017-10-01

The relative roles(s) of impaired insulin secretion vs. insulin resistance in the development of gestational diabetes mellitus depend upon multiple risk factors and diagnostic criteria. Here, we explored their relative contribution to gestational diabetes as defined by the WHO 1999 (GDM1999) and adapted WHO 2013 (GDM2013) criteria, excluding the 1-h glucose value, in a high-risk Indian population from Punjab. Insulin secretion (HOMA2-B) and insulin action (HOMA2-IR) were assessed in 4665 Indian women with or without gestational diabetes defined by the GDM1999 or adapted GDM2013 criteria. Gestational diabetes defined using both criteria was associated with decreased insulin secretion compared with pregnant women with normal glucose tolerance. Women with gestational diabetes defined by the adapted GDM2013, but not GDM1999 criteria, were more insulin resistant than pregnant women with normal glucose tolerance, and furthermore displayed lower insulin secretion than GDM1999 women. Urban habitat, illiteracy, high age and low BMI were independently associated with reduced insulin secretion, whereas Sikh religion, increasing age and BMI, as well as a family history of diabetes were independently associated with increased insulin resistance. Gestational diabetes risk factors influence insulin secretion and action in North Indian women in a differential manner. Gestational diabetes classified using the adapted GDM2013 compared with GDM1999 criteria is associated with more severe impairments of insulin secretion and action. © 2017 Diabetes UK.

MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia.

PubMed

Rowe, P S; de Zoysa, P A; Dong, R; Wang, H R; White, K E; Econs, M J; Oudet, C L

2000-07-01

Oncogenic hypophosphatemic osteomalacia (OHO) is characterized by a renal phosphate leak, hypophosphatemia, low-serum calcitriol (1,25-vitamin-D3), and abnormalities in skeletal mineralization. Resection of OHO tumors results in remission of the symptoms, and there is evidence that a circulating phosphaturic factor plays a role in the bone disease. This paper describes the characterization and cloning of a gene that is a candidate for the tumor-secreted phosphaturic factor. This new gene has been named MEPE (matrix extracellular phosphoglycoprotein) and has major similarities to a group of bone-tooth mineral matrix phospho-glycoproteins (osteopontin (OPN; HGMW-approved symbol SPP1), dentin sialo phosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein II (IBSP), and bone morphogenetic proteins (BMP). All the proteins including MEPE contain RGD sequence motifs that are proposed to be essential for integrin-receptor interactions. Of further interest is the finding that MEPE, OPN, DSPP, DMP1, IBSP, and BMP3 all map to a defined region in chromosome 4q. Refined mapping localizes MEPE to 4q21.1 between ESTs D4S2785 (WI-6336) and D4S2844 (WI-3770). MEPE is 525 residues in length with a short N-terminal signal peptide. High-level expression of MEPE mRNA occurred in all four OHO tumors screened. Three of 11 non-OHO tumors screened contained trace levels of MEPE expression (detected only after RT-PCR and Southern 32P analysis). Normal tissue expression was found in bone marrow and brain with very-low-level expression found in lung, kidney, and human placenta. Evidence is also presented for the tumor secretion of clusterin (HGMW-approved symbol CLU) and its possible role as a cytotoxic factor in one of the OHO patients described.

Integrated Assessment of Diclofenac Biotransformation, Pharmacokinetics, and Omics-Based Toxicity in a Three-Dimensional Human Liver-Immunocompetent Coculture System

PubMed Central

Ravindra, Kodihalli C.; Large, Emma; Young, Carissa L.; Rivera-Burgos, Dinelia; Yu, Jiajie; Cirit, Murat; Hughes, David J.; Wishnok, John S.; Lauffenburger, Douglas A.; Griffith, Linda G.

2017-01-01

In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immunocompetent coculture model and evaluated diclofenac (DCF) metabolic profiles, in vitro–in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography–tandem mass spectrometry. DCF biotransformation was assessed after 48 hours of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this three-dimensional liver microphysiological system. Protein markers were significantly elevated in the culture medium at high micromolar doses of DCF, which were also observed previously for acute drug-induced toxicity in humans. In this immunocompetent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins. Kupffer cell–mediated cytokine release recapitulated an in vivo proinflammatory response exemplified by a cohort of 11 cytokines that were differentially regulated after lipopolysaccharide induction, including interleukin (IL)-1β, IL-1Ra, IL-6, IL-8, IP-10, tumor necrosis factor-α, RANTES (regulated on activation normal T cell expressed and secreted), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, macrophage inflammatory protein-1β, and IL-5. In summary, our findings indicate that three-dimensional liver microphysiological systems may serve as preclinical investigational platforms from the perspective of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins, and cytokines to predict early drug-induced liver toxicity in humans. PMID:28450578

RIC-7 Promotes Neuropeptide Secretion

PubMed Central

Hao, Yingsong; Hu, Zhitao; Sieburth, Derek; Kaplan, Joshua M.

2012-01-01

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV–mediated secretion. PMID:22275875

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

Konishi, Hiroyuki, E-mail: konishi@med.osaka-cu.ac.jp; The 21st Century COE Program 'Base to Overcome Fatigue', Osaka City University Graduate School of Medicine, Osaka; Department of Anatomy and Neuroscience, Nagoya University, Graduate School of Medicine, Nagoya

Research highlights: {yields} We focused on the rat pituitary intermediate lobe (IL) under continuous stress (CS). {yields} CS induced PAP-I and PAP-II expression in melanotrophs of the IL. {yields} This gene induction was triggered by CS-related dopamine dysregulation. {yields} PAP-I and PAP-II may sustain homeostasis of the IL under CS. -- Abstract: Under continuous stress (CS) in rats, melanotrophs, the predominant cell-type in the intermediate lobe (IL) of the pituitary, are hyperactivated to secrete {alpha}-melanocyte-stimulating hormone and thereafter degenerate. Although these phenomena are drastic, the molecular mechanisms underlying the cellular changes are mostly unknown. In this study, we focused onmore » the pancreatitis-associated protein (PAP) family members of the secretory lectins and characterized their expression in the IL of CS model rats because we had identified two members of this family as up-regulated genes in our previous microarray analysis. RT-PCR and histological studies demonstrated that prominent PAP-I and PAP-II expression was induced in melanotrophs in the early stages of CS, while another family member, PAP-III, was not expressed. We further examined the regulatory mechanisms of PAP-I and PAP-II expression and revealed that both were induced by the decreased dopamine levels in the IL under CS. Because the PAP family members are implicated in cell survival and proliferation, PAP-I and PAP-II secreted from melanotrophs may function to sustain homeostasis of the IL under CS conditions in an autocrine or a paracrine manner.« less

An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

PubMed

Wiemels, Richard E; Cech, Stephanie M; Meyer, Nikki M; Burke, Caleb A; Weiss, Andy; Parks, Anastacia R; Shaw, Lindsey N; Carroll, Ronan K

2017-01-01

Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease. Copyright © 2016 American Society for Microbiology.

Alterations in the alveolar epithelium after injury leading to pulmonary fibrosis.

PubMed

Kasper, M; Haroske, G

1996-04-01

This review discusses current knowledge of the involvement of the alveolar epithelium in tissue remodelling during fibrogenesis. The purpose of the present paper is to give an overview, including the authors' own results, of knowledge of ultrastructural alterations, proliferation kinetics and phenotypic changes of pneumocytes in experimental and clinical pathology of pulmonary fibrosis. After lung injury, the alveolar epithelial cells show ultrastructural alterations, hypertrophy and hyperplasia, and a modulation of a series of structural and membrane proteins such as cytoskeletal changes, loss or de novo expression of epithelial adhesion molecules, and altered lectin binding. Furthermore, enhanced secretion of proteases, of cytokines and other soluble factors can be observed in the alveolar epithelium. These findings suggest the contribution of the epithelium in the remodelling process to be greater than expected. Estimations of the cell kinetics show that type II pneumocytes have the proliferative capacity to restore high proportions of damaged type I cells within few hours. In fibrosis this capacity also seems to be affected seriously, resulting in transitional phenotypes between type II and type I cells. Additionally, in the light of the detection of CD44 type of adhesion molecules at the foot processes of type II pneumocytes, some aspects of epithelial-fibroblast interaction are described.

Edaravone suppresses degradation of type II collagen.

PubMed

Huang, Chen; Liao, Guangjun; Han, Jian; Zhang, Guofeng; Zou, Benguo

2016-05-13

Osteoarthritis (OA) is a degenerative joint disease affecting millions of people. The degradation and loss of type II collagen induced by proinflammatory cytokines secreted by chondrocytes, such as factor-α (TNF-α) is an important pathological mechanism to the progression of OA. Edaravone is a potent free radical scavenger, which has been clinically used to treat the neuronal damage following acute ischemic stroke. However, whether Edaravone has a protective effect in articular cartilage hasn't been reported before. In this study, we investigated the chondrocyte protective effects of Edaravone on TNF-α induced degradation of type Ⅱ collagen. And our results indicated that TNF-α treatment resulted in degradation of type Ⅱ collagen, which can be ameliorated by treatment with Edaravone in a dose dependent manner. Notably, it was found that the inhibitory effects of Edaravone on TNF-α-induced reduction of type Ⅱ collagen were mediated by MMP-3 and MMP-13. Mechanistically, we found that Edaravone alleviated TNF-α induced activation of STAT1 and expression of IRF-1. These findings suggest a potential protective effect of Edaravone in OA. Copyright © 2016. Published by Elsevier Inc.

Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells

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

Vaca, Pilar; Berna, Genoveva; Araujo, Raquel

2008-03-10

The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice.more » Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells.« less

Characterisation and expression analysis of B-cell activating factor (BAFF) in spiny dogfish (Squalus acanthias): cartilaginous fish BAFF has a unique extra exon that may impact receptor binding.

PubMed

Li, Ronggai; Dooley, Helen; Wang, Tiehui; Secombes, Christopher J; Bird, Steve

2012-04-01

B-cell activating factor (BAFF), also known as tumour necrosis factor (TNF) ligand superfamily member 13B, is an important immune regulator with critical roles in B-cell survival, proliferation, differentiation and immunoglobulin secretion. A BAFF gene has been cloned from spiny dogfish (Squalus acanthias) and its expression studied. The dogfish BAFF encodes for an anchored type-II transmembrane protein of 288 aa with a putative furin protease cleavage site and TNF family signature as seen in BAFFs from other species. The identity of dogfish BAFF has also been confirmed by conserved cysteine residues, and phylogenetic tree analysis. The dogfish BAFF gene has an extra exon not seen in teleost fish, birds and mammals that encodes for 29 aa and may impact on receptor binding. The dogfish BAFF is highly expressed in immune tissues, such as spleen, and is up-regulated by PWM in peripheral blood leucocytes, suggesting a potentially important role in the immune system. Copyright © 2011 Elsevier Ltd. All rights reserved.

Structure of the Minor Pseudopilin EpsH From the Type 2 Secretion System of Vibrio Cholerae

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

Yanez, M.E.; Korotkov, K.V.; Abendroth, J.

2009-05-28

Many Gram-negative bacteria use the multi-protein type II secretion system (T2SS) to selectively translocate virulence factors from the periplasmic space into the extracellular environment. In Vibrio cholerae the T2SS is called the extracellular protein secretion (Eps) system, which translocates cholera toxin and several enzymes in their folded state across the outer membrane. Five proteins of the T2SS, the pseudopilins, are thought to assemble into a pseudopilus, which may control the outer membrane pore EpsD, and participate in the active export of proteins in a 'piston-like' manner. We report here the 2.0 {angstrom} resolution crystal structure of an N-terminally truncated variantmore » of EpsH, a minor pseudopilin from Vibrio cholerae. While EpsH maintains an N-terminal {alpha}-helix and C-terminal {beta}-sheet consistent with the type 4a pilin fold, structural comparisons reveal major differences between the minor pseudopilin EpsH and the major pseudopilin GspG from Klebsiella oxytoca: EpsH contains a large {beta}-sheet in the variable domain, where GspG contains an {alpha}-helix. Most importantly, EpsH contains at its surface a hydrophobic crevice between its variable and conserved {beta}-sheets, wherein a majority of the conserved residues within the EpsH family are clustered. In a tentative model of a T2SS pseudopilus with EpsH at its tip, the conserved crevice faces away from the helix axis. This conserved surface region may be critical for interacting with other proteins from the T2SS machinery.« less

Transdifferentiation of brain-derived neurotrophic factor (BDNF)-secreting mesenchymal stem cells significantly enhance BDNF secretion and Schwann cell marker proteins.

PubMed

Bierlein De la Rosa, Metzere; Sharma, Anup D; Mallapragada, Surya K; Sakaguchi, Donald S

2017-11-01

The use of genetically modified mesenchymal stem cells (MSCs) is a rapidly growing area of research targeting delivery of therapeutic factors for neuro-repair. Cells can be programmed to hypersecrete various growth/trophic factors such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF) to promote regenerative neurite outgrowth. In addition to genetic modifications, MSCs can be subjected to transdifferentiation protocols to generate neural cell types to physically and biologically support nerve regeneration. In this study, we have taken a novel approach by combining these two unique strategies and evaluated the impact of transdifferentiating genetically modified MSCs into a Schwann cell-like phenotype. After 8 days in transdifferentiation media, approximately 30-50% of transdifferentiated BDNF-secreting cells immunolabeled for Schwann cell markers such as S100β, S100, and p75 NTR . An enhancement was observed 20 days after inducing transdifferentiation with minimal decreases in expression levels. BDNF production was quantified by ELISA, and its biological activity tested via the PC12-TrkB cell assay. Importantly, the bioactivity of secreted BDNF was verified by the increased neurite outgrowth of PC12-TrkB cells. These findings demonstrate that not only is BDNF actively secreted by the transdifferentiated BDNF-MSCs, but also that it has the capacity to promote neurite sprouting and regeneration. Given the fact that BDNF production remained stable for over 20 days, we believe that these cells have the capacity to produce sustainable, effective, BDNF concentrations over prolonged time periods and should be tested within an in vivo system for future experiments. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Angiotensin 1-7 Is a Negative Modulator of Aldosterone Secretion In Vitro and In Vivo.

PubMed

Shefer, Gabi; Marcus, Yonit; Knoll, Esther; Dolkart, Oleg; Foichtwanger, Shulamit; Nevo, Nava; Limor, Rona; Stern, Naftali

2016-08-01

Angiotensin (1-7) [Ang 1-7] is a 7 amino acid peptide generated predominantly from Ang II by the action of Ang-converting enzyme 2. We previously showed that Ang 1-7 reduced plasma aldosterone and plasma renin activity in high fructose-fed rats, and that the reduction in circulating aldosterone seemed to accord a parallel reduction in plasma renin activity. Here, we tested the possibility that Ang 1-7 affects aldosterone secretion acting directly in glomerulosa cells. First, as detected by immunofluorescence, the receptor for Ang 1-7, Mas1 is localized predominantly at the rat adrenal subcapsular region. Second, in isolated rat glomerulosa cells incubates, Ang 1-7 attenuated the aldosterone response to Ang II, with the strongest effect seen on Ang II (10(-9) M) (control 22±2.5 pg/10(5) cells; Ang II [10(-9) M] 189±11 pg/10(5) cells; Ang II [10(-9) M]+Ang 1-7 [10(-6) M] 33±3.6 pg/10(5) cells; P<0.001) and the largest effect on adrenocorticotropic hormone (10(-8) M) (control 30±3.4 pg/10(5) cells; ACTH [10(-8) M] 409±32.5 pg/10(5) cells; ACTH [10(-8) M]+Ang 1-7 [10(-6) M] 280±12.5 pg/10(5) cells; P<0.001). In contrast, Ang 1-7 did not affect the aldosterone response to potassium (K(+)). In rats subjected to a low-salt diet for 7 days, continuous infusion of Ang 1-7 (576 μg/kg per day) resulted in a lesser rise in aldosterone (salt deplete+Ang 1-7, 16.4±4.8 ng/dL) compared with rats receiving vehicle (salt deplete+vehicle, 27.6±5.3 ng/dL; P<0.01) but did not modify plasma renin activity. Taken together, these results indicate that Ang 1-7 can act as a negative modulator of aldosterone secretion in vitro and in vivo. © 2016 American Heart Association, Inc.

Spontaneous hypertension occurs with adipose tissue dysfunction in perilipin-1 null mice.

PubMed

Zou, Liangqiang; Wang, Weiyi; Liu, Shangxin; Zhao, Xiaojing; Lyv, Ying; Du, Congkuo; Su, Xueying; Geng, Bin; Xu, Guoheng

2016-02-01

Perilipin-1 (Plin1) coats lipid droplets exclusively in adipocytes and regulates two principle functions of adipose tissue, triglyceride storage and hydrolysis, which are disrupted upon Plin1 deficiency. In the present study, we investigated the alterations in systemic metabolites and hormones, vascular function and adipose function in spontaneous hypertensive mice lacking perilipin-1 (Plin1-/-). Plin1-/- mice developed spontaneous hypertension without obvious alterations in systemic metabolites and hormones. Plin1 expressed only in adipose cells but not in vascular cells, so its ablation would have no direct effect in situ on blood vessels. Instead, Plin1-/- mice showed dysfunctions of perivascular adipose tissue (PVAT), a fat depot that anatomically surrounds systemic arteries and has an anticontractile effect. In Plin1-/- mice, aortic and mesenteric PVAT were reduced in mass and adipocyte derived relaxing factor secretion, but increased in basal lipolysis, angiotensin II secretion, macrophage infiltration and oxidative stress. Such multiple culprits impaired the anticontractile effect of PVAT to promote vasoconstriction of aortic and mesenteric arteries of Plin1-/- mice. Furthermore, arterial vessels of Plin1-/- mice showed increasing angiotensin II receptor type 1, monocyte chemotactic protein-1 and interlukin-6 expression, structural damage of endothelial and smooth muscle cells, along with impaired endothelium-dependent relaxation. Hypertension in Plin1-/- mice might occur as a deleterious consequence of PVAT dysfunction. This finding provides the direct evidence that links dysfunctional PVAT to vascular dysfunction and hypertension, particularly in pathophysiological states. This hypertensive mouse model might mimic and explain the hypertension occurring in patients with adipose tissue dysfunction, particularly with Plin1 mutations. Copyright © 2015 Elsevier B.V. All rights reserved.

The Pearling Transition Provides Evidence of Force-Driven Endosomal Tubulation during Salmonella Infection.

PubMed

Gao, Yunfeng; Spahn, Christoph; Heilemann, Mike; Kenney, Linda J

2018-06-19

Bacterial pathogens exploit eukaryotic pathways for their own end. Upon ingestion, Salmonella enterica serovar Typhimurium passes through the stomach and then catalyzes its uptake across the intestinal epithelium. It survives and replicates in an acidic vacuole through the action of virulence factors secreted by a type three secretion system located on Salmonella pathogenicity island 2 (SPI-2). Two secreted effectors, SifA and SseJ, are sufficient for endosomal tubule formation, which modifies the vacuole and enables Salmonella to replicate within it. Two-color, superresolution imaging of the secreted virulence factor SseJ and tubulin revealed that SseJ formed clusters of conserved size at regular, periodic intervals in the host cytoplasm. Analysis of SseJ clustering indicated the presence of a pearling effect, which is a force-driven, osmotically sensitive process. The pearling transition is an instability driven by membranes under tension; it is induced by hypotonic or hypertonic buffer exchange and leads to the formation of beadlike structures of similar size and regular spacing. Reducing the osmolality of the fixation conditions using glutaraldehyde enabled visualization of continuous and intact tubules. Correlation analysis revealed that SseJ was colocalized with the motor protein kinesin. Tubulation of the endoplasmic reticulum is driven by microtubule motors, and in the present work, we describe how Salmonella has coopted the microtubule motor kinesin to drive the force-dependent process of endosomal tubulation. Thus, endosomal tubule formation is a force-driven process catalyzed by Salmonella virulence factors secreted into the host cytoplasm during infection. IMPORTANCE This study represents the first example of using two-color, superresolution imaging to analyze the secretion of Salmonella virulence factors as they are secreted from the SPI-2 type three secretion system. Previous studies imaged effectors that were overexpressed in the host cytoplasm. The present work reveals an unusual force-driven process, the pearling transition, which indicates that Salmonella -induced filaments are under force through the interactions of effector molecules with the motor protein kinesin. This work provides a caution by highlighting how fixation conditions can influence the images observed.

Genome-wide analysis of gene expression and protein secretion of Babesia canis during virulent infection identifies potential pathogenicity factors.

PubMed

Eichenberger, Ramon M; Ramakrishnan, Chandra; Russo, Giancarlo; Deplazes, Peter; Hehl, Adrian B

2017-06-13

Infections of dogs with virulent strains of Babesia canis are characterized by rapid onset and high mortality, comparable to complicated human malaria. As in other apicomplexan parasites, most Babesia virulence factors responsible for survival and pathogenicity are secreted to the host cell surface and beyond where they remodel and biochemically modify the infected cell interacting with host proteins in a very specific manner. Here, we investigated factors secreted by B. canis during acute infections in dogs and report on in silico predictions and experimental analysis of the parasite's exportome. As a backdrop, we generated a fully annotated B. canis genome sequence of a virulent Hungarian field isolate (strain BcH-CHIPZ) underpinned by extensive genome-wide RNA-seq analysis. We find evidence for conserved factors in apicomplexan hemoparasites involved in immune-evasion (e.g. VESA-protein family), proteins secreted across the iRBC membrane into the host bloodstream (e.g. SA- and Bc28 protein families), potential moonlighting proteins (e.g. profilin and histones), and uncharacterized antigens present during acute crisis in dogs. The combined data provides a first predicted and partially validated set of potential virulence factors exported during fatal infections, which can be exploited for urgently needed innovative intervention strategies aimed at facilitating diagnosis and management of canine babesiosis.

Unlocking the Secrets of the Brain, Part II: A Continuing Look at Techniques for Exploring the Brain.

ERIC Educational Resources Information Center

Powledge, Tabitha M.

1997-01-01

Describes techniques for delving into the brain including positron emission tomography (PET), single photon emission computed tomography (SPECT), electroencephalogram (EEG), magnetoencephalography (MEG), transcranial magnetic stimulation (TMS), and low-tech indirect studies. (JRH)

sRAGE attenuates angiotensin II-induced cardiomyocyte hypertrophy by inhibiting RAGE-NFκB-NLRP3 activation.

PubMed

Lim, Soyeon; Lee, Myung Eun; Jeong, Jisu; Lee, Jiye; Cho, Soyoung; Seo, Miran; Park, Sungha

2018-05-23

The receptor for advanced glycation endproducts (RAGE) is an innate immunity receptor that has been implicated in the pathogenesis of atherosclerotic cardiovascular disease. However, the possibility that RAGE-mediated signaling is involved in angiotensin II (Ang II)-induced cardiac left ventricular hypertrophy has yet to be investigated. We therefore determined whether RAGE has a role in regulating pathological cardiac hypertrophy. Protein abundance was estimated using Western blotting and intracellular ROS level and phospho-p65 were detected using fluorescence microscopy. Enzyme-linked immunosorbent assay was used to detect HMGB1 and IL-1β. All in vitro experiments were performed using H9C2 cells. To induce cardiomyocyte hypertrophy, 300 nM Ang II was treated for 48 h and 2 µg/ml sRAGE was treated 1 h prior to addition of Ang II. sRAGE attenuated Ang II-induced cardiomyocyte hypertrophy by downregulating RAGE and angiotensin II type 1 receptor expression. Secretion levels of high motility group box 1 and interleukin-1β, estimated from a cell culture medium, were significantly reduced by sRAGE. Activated PKCs and ERK1/2, important signals in left ventricular hypertrophy (LVH) development, were downregulated by sRAGE treatment. Furthermore, we found that nuclear factor-κB and NOD-like receptor protein 3 (NLRP3) were associated with RAGE-mediated cardiomyocyte hypertrophy. In the context of these results, we conclude that RAGE induces cardiac hypertrophy through the activation of the PKCs-ERK1/2 and NF-κB-NLRP3-IL1β signaling pathway, and suggest that RAGE-NLRP3 may be an important mediator of Ang II-induced cardiomyocyte hypertrophy. In addition, we determined that inhibition of RAGE activation with soluble RAGE (sRAGE) has a protective effect on Ang II-induced cardiomyocyte hypertrophy.

Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture

PubMed Central

Takeuchi, Hiroki; Nakatsuji, Norio; Suemori, Hirofumi

2014-01-01

Insulin-producing cells (IPCs) derived from human pluripotent stem cells (hPSCs) may be useful in cell therapy and drug discovery for diabetes. Here, we examined various growth factors and small molecules including those previously reported to develop a robust differentiation method for induction of mature IPCs from hPSCs. We established a protocol that induced PDX1-positive pancreatic progenitor cells at high efficiency, and further induced mature IPCs by treatment with forskolin, dexamethasone, Alk5 inhibitor II and nicotinamide in 3D culture. The cells that differentiated into INSULIN-positive and C-PEPTIDE-positive cells secreted insulin in response to glucose stimulation, indicating a functional IPC phenotype. We also found that this method was applicable to different types of hPSCs. PMID:24671046

Tributyltin (TBT) and Dibutyltin (DBT) Alter Secretion of Tumor Necrosis Factor Alpha (TNFα) from Human Natural Killer (NK) Cells and a Mixture of T cells and NK Cells

PubMed Central

Hurt, Kelsi; Hurd-Brown, Tasia; Whalen, Margaret

2012-01-01

Butyltins (BTs) have been in widespread use. Tributyltin (TBT) has been used as a biocide in a variety of applications and is found in human blood samples. Dibutyltin (DBT) has been used as a stabilizer in polyvinyl chloride plastics and as a de-worming agent in poultry. DBT, like TBT, is found in human blood. Human natural killer (NK) cells are the earliest defense against tumors and viral infections and secrete the cytokine tumor necrosis factor (TNF) alpha (α). TNFα is an important regulator of adaptive and innate immune responses. TNFα promotes inflammation and an association between malignant transformation and inflammation has been established. Previously, we have shown that TBT and DBT were able to interfere with the ability of NK cells to lyse tumor target cells. Here we show that BTs alter cytokine secretion by NK cells as well as a mixture of T and NK lymphocytes (T/NK cells). We examined 24 h, 48 h, and 6 day exposures to TBT (200- 2.5 nM) and DBT (5- 0.05 µM) on TNFα secretion by highly enriched human NK cells and T/NK cells. The results indicate that TBT (200 - 2.5 nM) decreased TNFα secretion from NK cells. In the T/NK cells 200 nM TBT decreased secretion while 100-5 nM TBT increased secretion of TNFα. NK cells or T/NK cells exposed to higher concentrations of DBT showed decreased TNFα secretion while lower concentrations showed increased secretion. The effects of BTs on TNFα secretion are seen at concentrations present in human blood. PMID:23047847

Predictive Factors in Undergraduates' Involvement in Campus Secret Cults in Public Universities in Edo State of Nigeria

ERIC Educational Resources Information Center

Azetta Arhedo, Philip; Aluede, Oyaziwo; Adomeh, Ilu O. C.

2011-01-01

This study examined the predictive factors in undergraduates' involvement in campus secret cults in public universities in Edo State of Nigeria. The study employed the descriptive method, specifically the survey format. A random sample of three hundred and eighty (380) undergraduates was drawn from the two public universities. Data were elicited…

Ovarian stimulation for in vitro fertilization alters the intrauterine cytokine, chemokine, and growth factor milieu encountered by the embryo.

PubMed

Boomsma, Carolien M; Kavelaars, Annemieke; Eijkemans, Marinus J C; Fauser, Bart C J M; Heijnen, Cobi J; Macklon, Nick S

2010-10-01

To elucidate the impact of ovarian stimulation on the intrauterine milieu represented by the cytokine, chemokine, and growth factor profile in endometrial secretions aspirated before embryo transfer. Prospective cohort study. Fertility center in tertiary referral university hospital. Forty-two patients undergoing ovarian stimulation with GnRH analogues were recruited. They participated in both a natural and an ovarian-stimulated cycle for within patient comparisons. Endometrial secretion aspiration was performed immediately before embryo transfer. The concentrations of 17 mediators known to be involved in human embryo implantation were assessed by multiplex immunoassay. After correction for multiple testing, significantly higher concentrations of interleukin (IL)-1β, IL-5, IL-10, IL-12, IL-17, tumor necrosis factor (TNF)-α, heparin-binding epidermal growth factor (HbEGF), eotaxin, and dickkopf homologue-1 were present in endometrial secretions obtained in stimulated compared with natural cycles. Endometrial secretion analysis provides a novel means of investigating the effect of ovarian stimulation on the intrauterine milieu. The in vivo milieu encountered by the embryo after transfer is significantly altered by ovarian stimulation. Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Entropy-driven motility of Sinorhizobium meliloti on a semi-solid surface

PubMed Central

Dilanji, Gabriel E.; Teplitski, Max; Hagen, Stephen J.

2014-01-01

Sinorhizobium meliloti growing on soft agar can exhibit an unusual surface spreading behaviour that differs from other bacterial surface motilities. Bacteria in the colony secrete an exopolysaccharide-rich mucoid fluid that expands outward on the surface, carrying within it a suspension of actively dividing cells. The moving slime disperses the cells in complex and dynamic patterns indicative of simultaneous bacterial growth, swimming and aggregation. We find that while flagellar swimming is required to maintain the cells in suspension, the spreading and the associated pattern formation are primarily driven by the secreted exopolysaccharide EPS II, which creates two entropy-increasing effects: an osmotic flow of water from the agar to the mucoid fluid and a crowding or depletion attraction between the cells. Activation of these physical/chemical phenomena may be a useful function for the high molecular weight EPS II, a galactoglucan whose biosynthesis is tightly regulated by the ExpR/SinI/SinR quorum-sensing system: unlike bacterial colonies that spread via bacterium-generated, physical propulsive forces, S. meliloti under quorum conditions may use EPS II to activate purely entropic forces within its environment, so that it can disperse by passively ‘surfing’ on those forces. PMID:24741008

New factors controlling the balance between osteoblastogenesis and adipogenesis.

PubMed

Abdallah, Basem M; Kassem, Moustapha

2012-02-01

The majority of conditions associated with bone loss, including aging, are accompanied by increased marrow adiposity possibly due to shifting of the balance between osteoblast and adipocyte differentiation in bone marrow stromal (skeletal) stem cells (MSC). In order to study the relationship between osteoblastogenesis and adipogenesis in bone marrow, we have characterized cellular models of multipotent MSC as well as pre-osteoblastic and pre-adipocytic cell populations. Using these models, we identified two secreted factors in the bone marrow microenviroment: secreted frizzled-related protein 1 (sFRP-1) and delta-like1 (preadipocyte factor 1) (Dlk1/Pref-1). Both exert regulatory effects on osteoblastogenesis and adipogenesis. Our studies suggest a model for lineage fate determination of MSC that is regulated through secreted factors in the bone marrow microenvironment that mediate a cross-talk between lineage committed cell populations in addition to controlling differentiation choices of multipotent MSC. Copyright © 2011 Elsevier Inc. All rights reserved.

Relationship between nitric oxide- and calcium-dependent signal transduction pathways in growth hormone release from dispersed goldfish pituitary cells.

PubMed

Chang, John P; Sawisky, Grant R; Davis, Philip J; Pemberton, Joshua G; Rieger, Aja M; Barreda, Daniel R

2014-09-15

Nitric oxide (NO) and Ca(2+) are two of the many intracellular signal transduction pathways mediating the control of growth hormone (GH) secretion from somatotropes by neuroendocrine factors. We have previously shown that the NO donor sodium nitroprusside (SNP) elicits Ca(2+) signals in identified goldfish somatotropes. In this study, we examined the relationships between NO- and Ca(2+)-dependent signal transduction mechanisms in GH secretion from primary cultures of dispersed goldfish pituitary cells. Morphologically identified goldfish somatotropes stained positively for an NO-sensitive dye indicating they may be a source of NO production. In 2h static incubation experiments, GH release responses to the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) were attenuated by CoCl2, nifedipine, verapamil, TMB-8, BHQ, and KN62. In column perifusion experiments, the ability of SNP to induce GH release was impaired in the presence of TMB-8, BHQ, caffeine, and thapsigargin, but not ryanodine. Caffeine-elicited GH secretion was not affected by the NO scavenger PTIO. These results suggest that NO-stimulated GH release is dependent on extracellular Ca(2+) availability and voltage-sensitive Ca(2+) channels, as well as intracellular Ca(2+) store(s) that possess BHQ- and/or thapsigargin-inhibited sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases, as well as TMB-8- and/or caffeine-sensitive, but not ryanodine-sensitive, Ca(2+)-release channels. Calmodulin kinase-II also likely participates in NO-elicited GH secretion but caffeine-induced GH release is not upstream of NO production. These findings provide insights into how NO actions many integrate with Ca(2+)-dependent signalling mechanisms in goldfish somatotropes and how such interactions may participate in the GH-releasing actions of regulators that utilize both NO- and Ca(2+)-dependent transduction pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

Hypothesis: kisspeptin mediates male hypogonadism in obesity and type 2 diabetes.

PubMed

George, Jyothis T; Millar, Robert P; Anderson, Richard A

2010-01-01

Hypogonadism occurs commonly in men with type 2 diabetes (T2DM) and severe obesity. Current evidence points to a decreased secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus and thereby decreased secretion of gonadotropins from the pituitary gland as a central feature of the pathophysiology in these men. Hyperglycaemia, inflammation, leptin and oestrogen-related feedback have been proposed to make aetiological contributions to the hypogonadotropic hypogonadism of T2DM. However, the neuroendocrine signals that link these factors with modulation of GnRH neurons have yet to be identified. Kisspeptins play a central role in the modulation of GnRH secretion and, thus, downstream regulation of gonadotropins and testosterone secretion in men. Inactivating mutations of the kisspeptin receptor have been shown to cause hypogonadotropic hypogonadism in man, whilst an activating mutation is associated with precocious puberty. Data from studies in experimental animals link kisspeptin expression with individual factors known to regulate GnRH secretion, including hyperglycaemia, inflammation, leptin and oestrogen. We therefore hypothesise that decreased endogenous kisspeptin secretion is the common central pathway that links metabolic and endocrine factors in the pathology of testosterone deficiency seen in men with obesity and T2DM. We propose that the kisspeptin system plays a central role in integrating a range of metabolic inputs, thus constituting the link between energy status with the hypothalamic-pituitary-gonadal axis, and put forward potential clinical studies to test the hypothesis. Copyright 2010 S. Karger AG, Basel.

Signal Factors Secreted by 2D and Spheroid Mesenchymal Stem Cells and by Cocultures of Mesenchymal Stem Cells Derived Microvesicles and Retinal Photoreceptor Neurons

PubMed Central

Mao, Mao; Zhou, Liang

2017-01-01

We aim to identify levels of signal factors secreted by MSCs cultured in 2D monolayers (2D-MSCs), spheroids (spheroids MSCs), and cocultures of microvesicles (MVs) derived from 2D-MSCs or spheroid MSCs and retinal photoreceptor neurons. We seeded 2D-MSCs, spheroid MSCs, and cells derived from spheroids MSCs at equal numbers. MVs isolated from all 3 culture conditions were incubated with 661W cells. Levels of 51 signal factors in conditioned medium from those cultured conditions were quantified with bead-based assay. We found that IL-8, IL-6, and GROα were the top three most abundant signal factors. Moreover, compared to 2D-MSCs, levels of 11 cytokines and IL-2Rα were significantly increased in conditioned medium from spheroid MSCs. Finally, to test if enhanced expression of these factors reflects altered immunomodulating activities, we assessed the effect of 2D-MSC-MVs and 3D-MSC-MVs on CD14+ cell chemoattraction. Compared to 2D-MSC-MVs, 3D-MSC-MVs significantly decreased the chemotactic index of CD14+ cells. Our results suggest that spheroid culture conditions improve the ability of MSCs to selectively secrete signal factors. Moreover, 3D-MSC-MVs also possessed an enhanced capability to promote signal factors secretion compared to 2D-MSC-MVs and may possess enhanced immunomodulating activities and might be a better regenerative therapy for retinal degenerative diseases. PMID:28194184

Regulation of SFRP-1 expression in the rat dental follicle.

PubMed

Liu, Dawen; Yao, Shaomian; Wise, Gary E

2012-01-01

Tooth eruption requires osteoclastogenesis and subsequent bone resorption. Secreted frizzled-related protein-1 (SFRP-1) negatively regulates osteoclastogenesis. Our previous studies indicated that SFRP-1 is expressed in the rat dental follicle (DF), with reduced expression at days 3 and 9 close to the times for the major and minor bursts of osteoclastogenesis, respectively; but it remains unclear as to what molecules contribute to its reduced expression at these critical times. Thus, it was the aim of this study to determine which molecules regulate the expression of SFRP-1 in the DF. To that end, the DF cells were treated with cytokines that are maximally expressed at days 3 or 9, and SFRP-1 expression was determined. Our study indicated that colony-stimulating factor-1 (CSF-1), a molecule maximally expressed in the DF at day 3, down-regulated SFRP-1 expression. As to endothelial monocyte-activating polypeptide II (EMAP-II), a highly expressed molecule in the DF at day 3, it had no effect on the expression of SFRP-1. However, when EMAP-II was knocked down by siRNA, the expression of SFRP-1 was elevated, and this elevated SFRP-1 expression could be reduced by adding recombinant EMAP-II protein. This suggests that EMAP-II maintained a lower level of SFRP-1 in the DF. TNF-α is a molecule maximally expressed at day 9, and this study indicated that it also down-regulated the expression of SFRP-1 in the DF cells. In conclusion, CSF-1 and EMAP-II may contribute to the reduced SFRP-1 expression seen on day 3, while TNF-α may contribute to the reduced SFRP-1 expression at day 9.

Klebsiella pneumoniae Siderophores Induce Inflammation, Bacterial Dissemination, and HIF-1α Stabilization during Pneumonia.

PubMed

Holden, Victoria I; Breen, Paul; Houle, Sébastien; Dozois, Charles M; Bachman, Michael A

2016-09-13

Klebsiella pneumoniae is a Gram-negative pathogen responsible for a wide range of infections, including pneumonia and bacteremia, and is rapidly acquiring antibiotic resistance. K. pneumoniae requires secretion of siderophores, low-molecular-weight, high-affinity iron chelators, for bacterial replication and full virulence. The specific combination of siderophores secreted by K. pneumoniae during infection can impact tissue localization, systemic dissemination, and host survival. However, the effect of these potent iron chelators on the host during infection is unknown. In vitro, siderophores deplete epithelial cell iron, induce cytokine secretion, and activate the master transcription factor hypoxia inducible factor-1α (HIF-1α) protein that controls vascular permeability and inflammatory gene expression. Therefore, we hypothesized that siderophore secretion by K. pneumoniae directly contributes to inflammation and bacterial dissemination during pneumonia. To examine the effects of siderophore secretion independently of bacterial growth, we performed infections with tonB mutants that persist in vivo but are deficient in siderophore import. Using a murine model of pneumonia, we found that siderophore secretion by K. pneumoniae induces the secretion of interleukin-6 (IL-6), CXCL1, and CXCL2, as well as bacterial dissemination to the spleen, compared to siderophore-negative mutants at an equivalent bacterial number. Furthermore, we determined that siderophore-secreting K. pneumoniae stabilized HIF-1α in vivo and that bacterial dissemination to the spleen required alveolar epithelial HIF-1α. Our results indicate that siderophores act directly on the host to induce inflammatory cytokines and bacterial dissemination and that HIF-1α is a susceptibility factor for bacterial invasion during pneumonia. Klebsiella pneumoniae causes a wide range of bacterial diseases, including pneumonia, urinary tract infections, and sepsis. To cause infection, K. pneumoniae steals iron from its host by secreting siderophores, small iron-chelating molecules. Classically, siderophores are thought to worsen infections by promoting bacterial growth. In this study, we determined that siderophore-secreting K. pneumoniae causes lung inflammation and bacterial dissemination to the bloodstream independently of bacterial growth. Furthermore, we determined that siderophore-secreting K. pneumoniae activates a host protein, hypoxia inducible factor (HIF)-1α, and requires it for siderophore-dependent bacterial dissemination. Although HIF-1α can protect against some infections, it appears to worsen infection with K. pneumoniae Together, these results indicate that bacterial siderophores directly alter the host response to pneumonia in addition to providing iron for bacterial growth. Therapies that disrupt production of siderophores could provide a two-pronged attack against K. pneumoniae infection by preventing bacterial growth and preventing bacterial dissemination to the blood. Copyright © 2016 Holden et al.

The Fas/Fap-1/Cav-1 complex regulates IL-1RA secretion in mesenchymal stem cells to accelerate wound healing.

PubMed

Kou, Xiaoxing; Xu, Xingtian; Chen, Chider; Sanmillan, Maria Laura; Cai, Tao; Zhou, Yanheng; Giraudo, Claudio; Le, Anh; Shi, Songtao

2018-03-14

Mesenchymal stem cells (MSCs) are capable of secreting exosomes, extracellular vesicles, and cytokines to regulate cell and tissue homeostasis. However, it is unknown whether MSCs use a specific exocytotic fusion mechanism to secrete exosomes and cytokines. We show that Fas binds with Fas-associated phosphatase-1 (Fap-1) and caveolin-1 (Cav-1) to activate a common soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-mediated membrane fusion mechanism to release small extracellular vesicles (sEVs) in MSCs. Moreover, we reveal that MSCs produce and secrete interleukin-1 receptor antagonist (IL-1RA) associated with sEVs to maintain rapid wound healing in the gingiva via the Fas/Fap-1/Cav-1 cascade. Tumor necrosis factor-α (TNF-α) serves as an activator to up-regulate Fas and Fap-1 expression via the nuclear factor κB pathway to promote IL-1RA release. This study identifies a previously unknown Fas/Fap-1/Cav-1 axis that regulates SNARE-mediated sEV and IL-1RA secretion in stem cells, which contributes to accelerated wound healing. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Insulin-like growth factor and fibroblast growth factor expression profiles in growth-restricted fetal sheep pancreas.

PubMed

Chen, Xiaochuan; Rozance, Paul J; Hay, William W; Limesand, Sean W

2012-05-01

Placental insufficiency results in intrauterine growth restriction (IUGR), impaired fetal insulin secretion and less fetal pancreatic β-cell mass, partly due to lower β-cell proliferation rates. Insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs) regulate fetal β-cell proliferation and pancreas development, along with transcription factors, such as pancreatic and duodenal homeobox 1 (PDX-1). We determined expression levels for these growth factors, their receptors and IGF binding proteins in ovine fetal pancreas and isolated islets. In the IUGR pancreas, relative mRNA expression levels of IGF-I, PDX-1, FGF7 and FGFR2IIIb were 64% (P < 0.01), 76% (P < 0.05), 76% (P < 0.05) and 52% (P < 0.01) lower, respectively, compared with control fetuses. Conversely, insulin-like growth factor binding protein 2 (IGFBP-2) mRNA and protein concentrations were 2.25- and 1.2-fold greater (P < 0.05) in the IUGR pancreas compared with controls. In isolated islets from IUGR fetuses, IGF-II and IGFBP-2 mRNA concentrations were 1.5- and 3.7-fold greater (P < 0.05), and insulin mRNA was 56% less (P < 0.05) than control islets. The growth factor expression profiles for IGF and FGF signaling pathways indicate that declines in β-cell mass are due to decreased growth factor signals for both pancreatic progenitor epithelial cell and mature β-cell replication.

Dedifferentiated Schwann Cell Precursors Secreting Paracrine Factors Are Required for Regeneration of the Mammalian Digit Tip.

PubMed

Johnston, Adam P W; Yuzwa, Scott A; Carr, Matthew J; Mahmud, Neemat; Storer, Mekayla A; Krause, Matthew P; Jones, Karen; Paul, Smitha; Kaplan, David R; Miller, Freda D

2016-10-06

Adult mammals have lost multi-tissue regenerative capacity, except for the distal digit, which is able to regenerate via mechanisms that remain largely unknown. Here, we show that, after adult mouse distal digit removal, nerve-associated Schwann cell precursors (SCPs) dedifferentiate and secrete growth factors that promote expansion of the blastema and digit regeneration. When SCPs were dysregulated or ablated, mesenchymal precursor proliferation in the blastema was decreased and nail and bone regeneration were impaired. Transplantation of exogenous SCPs rescued these regeneration defects. We found that SCPs secrete factors that promote self-renewal of mesenchymal precursors, and we used transcriptomic and proteomic analysis to define candidate factors. Two of these, oncostatin M (OSM) and platelet-derived growth factor AA (PDGF-AA), are made by SCPs in the regenerating digit and rescued the deficits in regeneration caused by loss of SCPs. As all peripheral tissues contain nerves, these results could have broad implications for mammalian tissue repair and regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

Regulation of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of MMP, and progesterone secretion in luteinized granulosa cells from normally ovulating women with polycystic ovary disease.

PubMed

Ben-Shlomo, Izhar; Goldman, Shlomit; Shalev, Eliezer

2003-03-01

To investigate the regulation of MMP-9, TIMP-1, and progesterone via three signal transduction pathways in luteinized granulosa cells from normal ovulatory and PCOD women. In vitro study. Laboratory for Research in Reproductive Sciences, Department of Obstetrics and Gynecology, Ha'Emek Hospital, Afula, Israel. Ten normal ovulatory and 10 women with polycystic ovary disease (PCOD) treated in an assisted reproduction program. Cultured cells were exposed to phorbol 12-myristate 13-acetate (TPA), acting via protein kinase C (PKC), to epidermal growth factor (EGF), acting via protein tyrosine kinase (PTK), and to forskolin, acting via protein kinase A (PKA). Secretion of MMP-9, TIMP-1, and progesterone. Phorbol 12-myristate 13-acetate elicited an increase in MMP-9 and TIMP-1 secretion in both groups and apparently did not affect progesterone secretion. Epidermal growth factor did not change significantly neither MMP-9 nor TIMP-1 secretion but dose dependently decreased MMP-9-TIMP-1 ratio and increased progesterone secretion in the PCOD group. Forskolin inhibited MMP-9 activity and increased TIMP-1 and progesterone secretion in both groups. Progesterone production was inversely related to the ratio of MMP-9-TIMP-1 regardless of cell origin. In this preliminary study, similar and divergent patterns have emerged in the regulation of MMP-9 and TIMP-1 in human luteinized granulosa cells. Repressing MMP-9-TIMP-1 ratio may have an important modulatory effect on progesterone secretion.

Secretion of acid phosphatase by axenic Entamoeba histolytica NIH-200 and properties of the extracellular enzyme.

PubMed

Agrawal, A; Pandey, V C; Kumar, S; Sagar, P

1989-01-01

Entamoeba histolytica (NIH-200) secreted large amounts of acid phosphatase in its external environment when grown axenically in modified TPS-II medium. Fractionation by DEAE-cellulose chromatography of the precipitate obtained from the cell-free medium at 60% ammonium sulfate saturation yielded 3 distinct peaks of enzyme activity. The enzyme in all the peaks showed resistance to tartrate but was inhibited by fluoride, cupric chloride, ethylene diamine-tetra acetic acid, ammonium molybdate and cysteine; however, enzyme associated with different peaks differed in its polyacrylamide gel electrophoretic profiles and behavior towards concanavalin A.

Use of plant roots for phytoremediation and molecular farming

PubMed Central

Gleba, Doloressa; Borisjuk, Nikolai V.; Borisjuk, Ludmyla G.; Kneer, Ralf; Poulev, Alexander; Skarzhinskaya, Marina; Dushenkov, Slavik; Logendra, Sithes; Gleba, Yuri Y.; Raskin, Ilya

1999-01-01

Alternative agriculture, which expands the uses of plants well beyond food and fiber, is beginning to change plant biology. Two plant-based biotechnologies were recently developed that take advantage of the ability of plant roots to absorb or secrete various substances. They are (i) phytoextraction, the use of plants to remove pollutants from the environment and (ii) rhizosecretion, a subset of molecular farming, designed to produce and secrete valuable natural products and recombinant proteins from roots. Here we discuss recent advances in these technologies and assess their potential in soil remediation, drug discovery, and molecular farming. PMID:10339526

Intellectual property and information controversy (II)

NASA Astrophysics Data System (ADS)

Aoyama, Hirokazu

As advanced information has been proceeded rapidly, intellectual property has become more important than ever as business resources of enterprises. Based on the former report by the author "present status of and trend in intellectual property" this paper describes "information" related intellectual property controversy which have been occurred, that is, 1) affairs related to computer hardwares and softwares (the case of compatible machines and OS, the case of application softwares, computer crimes) and 2) affairs on trade secret (the case of revealing enterprises'secret, the case of industrial espionage). It also discusses how intellectual property should be protected and utilized from now on.

The tad locus: postcards from the widespread colonization island.

PubMed

Tomich, Mladen; Planet, Paul J; Figurski, David H

2007-05-01

The Tad (tight adherence) macromolecular transport system, which is present in many bacterial and archaeal species, represents an ancient and major new subtype of type II secretion. The tad genes are present on a genomic island named the widespread colonization island (WCI), and encode the machinery that is required for the assembly of adhesive Flp (fimbrial low-molecular-weight protein) pili. The tad genes are essential for biofilm formation, colonization and pathogenesis in the genera Aggregatibacter (Actinobacillus), Haemophilus, Pasteurella, Pseudomonas, Yersinia, Caulobacter and perhaps others. Here we review the structure, function and evolution of the Tad secretion system.

Targeting GH-1 splicing as a novel pharmacological strategy for growth hormone deficiency type II.

PubMed

Miletta, Maria Consolata; Flück, Christa E; Mullis, Primus-E

2017-01-15

Isolated growth hormone deficiency type II (IGHD II) is a rare genetic splicing disorder characterized by reduced growth hormone (GH) secretion and short stature. It is mainly caused by autosomal dominant-negative mutations within the growth hormone gene (GH-1) which results in missplicing at the mRNA level and the subsequent loss of exon 3, producing the 17.5-kDa GH isoform: a mutant and inactive GH protein that reduces the stability and the secretion of the 22-kDa GH isoform, the main biologically active GH form. At present, patients suffering from IGHD II are treated with daily injections of recombinant human GH (rhGH) in order to reach normal height. However, this type of replacement therapy, although effective in terms of growth, does not prevent the toxic effects of the 17.5-kDa mutant on the pituitary gland, which may eventually lead to other hormonal deficiencies. As the severity of the disease inversely correlates with the 17.5-kDa/22-kDa ratio, increasing the inclusion of exon 3 is expected to ameliorate disease symptoms. This review focuses on the recent advances in experimental and therapeutic strategies applicable to treat IGHD II in clinical and preclinical contexts. Several avenues for alternative IGHD II therapy will be discussed including the use of small interfering RNA (siRNA) and short hairpin RNA (shRNA) constructs that specifically target the exon 3-deleted transcripts as well as the application of histone deacetylase inhibitors (HDACi) and antisense oligonucleotides (AONs) to enhance full-length GH-1 transcription, correct GH-1 exon 3 splicing and manipulate GH pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Immunopathological and Modulatory Effects of Cag A+ Genotype on Gastric Mucosa, Inflammatory Response, Pepsinogens, and Gastrin-17 Secretion in Iraqi Patients infected with H. pylori.

PubMed

Al-Ezzy, Ali Ibrahim Ali

2018-05-20

To determine the immunopathological correlation between Cag A+ H. pylori -specific IgG; pepsinogen I&II (PI&PII); gastrin-17 (G-17); status of gastric and duodenal mucosa and inflammatory activities on different gastroduodenal disorders. Eighty gastroduodenal biopsies were taken from patients with gastroduodenal disorders for histopathological evaluation and H. pylori diagnosis. Serum samples were used for evaluation of gastric hormones and detection of H. pylori -specific IgG antibodies. The tissue expression of H. pylori Cag A gene was detected by in situ hybridisation. H. pylori IgG antibodies were detected in (88.8%) of enrolled patients. According to Cag A gene expression, Significant difference (P value ˂ 0.05) was detected in levels of PG I; PGII, PG I/PG II among patients with gastric disorders. Serum G-17 level was negatively correlated with Cag A gene expression (P-value = 0.04). There was a significant correlation between H. pylori IgG and PG I; PG II; G-17. The current study revealed that corpus atrophic gastritis was diagnosed histologically with (5%) gastric ulcer cases; (3.75%) of duodenal ulcer cases; (3.75%) of duodenitis cases; (1.25%) of gastropathy cases and (8.75%) of gastritis cases. At the same time H. pylori gastritis diagnosed concurrently with (8.75%) of gastric ulcer cases; (11.25%) of duodenal ulcer cases; (17.5%) of gastropathy cases; (3.75%) of duodenitis cases and (2.5%) of prepyloric ulcer cases. A significant correlation was reported between the Immunopathological status of gastric mucosa and endoscopic mucosal finding among duodenal ulcer cases and gastritis cases only. A positive correlation was reported between serum levels of PGI; PGII; PGI/PGII; G-17; PMNs grade and Immunopathological status of the gastroduodenal mucosa of H. pylori Infected patients. A significant difference was reported in lymphocyte grades among gastric disorders without correlation with immunohistopathological changes in the mucosa (P-value = 0.002). A significant difference was reported in lymphocyte grades among different disorders according to H. pylori IgG. A significant difference was reported in serum level of PG I; PG II; PG I/PG II; G-17 according to PMN and lymphocyte grades (P-value ˂ 0.01). PMNs grades positively correlated with gastric Cag A expression; H. pylori IgG; PG II; G-17 levels. PG I; PG I/ PG II correlated with lymphocyte grades (P-value ˂ 0.05); while PGII has a negative correlation (P-value = 0.039). Endoscopic mucosal finding does not reflect exactly the actual immunopathological changes of gastric mucosa during H. pylori infection. Secretion of gastrin was not affected by the presence of Cag A in gastric tissue. Instead, the fluctuation in the hormone level appears to be due to the presence of H. pylori infection in gastric tissue. Gastric tissue infiltration with PMNs & lymphocytes inflammatory infiltrates has a direct effect on PGs and gastrin levels in serum of infected patients. The level of PG I; PG II; G-17 secretion correlated with the development of immune response against H. pylori and production of specific H. pylori IgG. Finally, H. pylori can modulate gastric secretions through Cag A dependent and independent pathways.

Hypokalemia decreases testosterone production in male mice by altering luteinizing hormone secretion.

PubMed

Sánchez-Capelo, A; Castells, M T; Cremades, A; Peñafiel, R

1996-09-01

Potassium deficiency produced by feeding mice a low potassium diet caused a marked decrease in plasma and testicular testosterone concentrations and a concomitant fall in the weight of seminal vesicles and in renal ornithine decarboxylase activity. All of these parameters were rapidly restored when potassium supply was normalized. Immunocytochemical analysis of gonadotropes and plasma LH values suggested that the pulsatile liberation of LH by the pituitary was impaired in the potassium-deficient male mice. Because the synthesis of testosterone in the potassium-deficient mice was stimulated by exogenous LH, hCG, or GnRH, one can conclude that alteration of the transcellular potassium gradient could affect the regulation of the hypothalamo-hypophyseal-testicular axis by affecting the pulsatile release of GnRH. Our results showing that the stimulation of LH secretion after castration was similar in control and potassium-deficient male mice suggest that a testicular factor(s) different from testosterone could be implicated in the abnormal regulation of LH secretion in potassium-deficient mice. We conclude that plasma potassium concentration is an important factor in the regulation of gonadotropin secretion and testicular functions.

Natural Killer Cells Promote Fetal Development through the Secretion of Growth-Promoting Factors.

PubMed

Fu, Binqing; Zhou, Yonggang; Ni, Xiang; Tong, Xianhong; Xu, Xiuxiu; Dong, Zhongjun; Sun, Rui; Tian, Zhigang; Wei, Haiming

2017-12-19

Natural killer (NK) cells are present in large populations at the maternal-fetal interface during early pregnancy. However, the role of NK cells in fetal growth is unclear. Here, we have identified a CD49a + Eomes + subset of NK cells that secreted growth-promoting factors (GPFs), including pleiotrophin and osteoglycin, in both humans and mice. The crosstalk between HLA-G and ILT2 served as a stimulus for GPF-secreting function of this NK cell subset. Decreases in this GPF-secreting NK cell subset impaired fetal development, resulting in fetal growth restriction. The transcription factor Nfil3, but not T-bet, affected the function and the number of this decidual NK cell subset. Adoptive transfer of induced CD49a + Eomes + NK cells reversed impaired fetal growth and rebuilt an appropriate local microenvironment. These findings reveal properties of NK cells in promoting fetal growth. In addition, this research proposes approaches for therapeutic administration of NK cells in order to reverse restricted nourishments within the uterine microenvironment during early pregnancy. Copyright © 2017 Elsevier Inc. All rights reserved.

Bromelain Treatment Decreases Secretion of Pro-Inflammatory Cytokines and Chemokines by Colon Biopsies In Vitro

PubMed Central

Onken, Jane E.; Greer, Paula K.; Calingaert, Brian; Hale, Laura P.

2008-01-01

Oral bromelain has been anecdotally reported to decrease inflammation in ulcerative colitis (UC). Proteolytically active bromelain is known to decrease expression of mRNAs encoding pro-inflammatory cytokines by human leukocytes in vitro. To assess the effect of bromelain on mucosal secretion of cytokines in inflammatory bowel disease (IBD), endoscopic colon biopsies from patients with UC, Crohn’s disease (CD), and non-IBD controls were treated in vitro with bromelain or media, then cultured. Secretion of pro-inflammatory cytokines and chemokines was measured. Significant increases in granulocyte colony stimulating factor (G-CSF), interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF) were detected in the media from actively inflamed areas in UC and CD as compared with non-inflamed IBD tissue and non-IBD controls. In vitro bromelain treatment decreased secretion of G-CSF, granulocyte-macrophage colony stimulating factor (GM-CSF), IFN-γ, CCL4/macrophage inhibitory protein (MIP)-1β, and TNF by inflamed tissue in IBD. Bromelain may be a novel therapy for IBD. PMID:18160345

Phospholipase PlaB is a new virulence factor of Legionella pneumophila.

PubMed

Schunder, Eva; Adam, Patrick; Higa, Futoshi; Remer, Katharina A; Lorenz, Udo; Bender, Jennifer; Schulz, Tino; Flieger, Antje; Steinert, Michael; Heuner, Klaus

2010-06-01

We previously identified Legionella pneumophila PlaB as the major cell-associated phospholipase A/lysophospholipase A with contact-dependent hemolytic activity. In this study, we further characterized this protein and found it to be involved in the virulence of L. pneumophila. PlaB was mainly expressed and active during exponential growth. Active PlaB was outer membrane-associated and at least in parts surface-exposed. Transport to the outer membrane was not dependent on the type I (T1SS), II (T2SS), IVB (T4BSS) or Tat secretion pathways. Furthermore, PlaB activity was not dependent on the presence of the macrophage infectivity potentiator (Mip) or the major secreted zinc metalloproteinase A (MspA). Despite the fact that PlaB is not essential for replication in protozoa or macrophage cell lines, we found that plaB mutants were impaired for replication in the lungs and dissemination to the spleen in the guinea pig infection model. Histological sections monitored less inflammation and destruction of the lung tissue after infection with the plaB mutants compared to L. pneumophila wild type. Taken together, PlaB is the first phospholipase A/lysophospholipase A with a confirmed role in the establishment of Legionnaires' disease. Copyright 2010 Elsevier GmbH. All rights reserved.

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

PubMed Central

Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping

2013-01-01

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174

Modulation of hepatocyte growth factor secretion in human female reproductive tract stromal fibroblasts by poly (I:C) and estradiol.

PubMed

Coleman, Kimberly D; Ghosh, Mimi; Crist, Sarah G; Wright, Jacqueline A; Rossoll, Richard M; Wira, Charles R; Fahey, John V

2012-01-01

Hepatocyte Growth Factor (HGF) secretion facilitates epithelial cell growth and development in the female reproductive tract (FRT) and may contribute to pathological conditions such as cancer and endometriosis. We hypothesized that estradiol and poly (I:C), a synthetic RNA mimic, may have a regulatory effect on HGF secretion by stromal fibroblasts from FRT tissues. Following hysterectomies, normal tissue from the uterus, endocervix, and ectocervix were dispersed into stromal cell fractions by enzymatic digestion and differential filtering. Stromal fibroblasts were cultured and treated with estradiol and/or poly (I:C), and conditioned media were analyzed for HGF via enzyme-linked immunosorbent assay. Treating uterine fibroblasts with estradiol or poly (I:C) significantly increased HGF secretion. When uterine fibroblasts were co-treated with estradiol and poly (I:C), the effect on HGF secretion was additive. In contrast, stromal fibroblasts from endo- and ecto-cervix were unresponsive to estradiol, but were stimulated to secrete HGF by poly (I:C). HGF secretion is uniquely regulated in the uterus, but not in ecto- and endo-cervix, by estradiol. Moreover, potential viral pathogens further induce HGF. These findings have potential applications in understanding both hormonal regulation of normal tissue as well as the role of HGF in tumorogenesis, endometriosis, and human immunodeficiency virus infection. © 2011 John Wiley & Sons A/S.

Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

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

Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku

2013-06-14

Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs)more » and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.« less

Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition.

PubMed

de Groot, A; Koster, M; Gérard-Vincent, M; Gerritse, G; Lazdunski, A; Tommassen, J; Filloux, A

2001-02-01

Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).

Industrial production of clotting factors: Challenges of expression, and choice of host cells.

PubMed

Kumar, Sampath R

2015-07-01

The development of recombinant forms of blood coagulation factors as safer alternatives to plasma derived factors marked a major advance in the treatment of common coagulation disorders. These are complex proteins, mostly enzymes or co-enzymes, involving multiple post-translational modifications, and therefore are difficult to express. This article reviews the nature of the expression challenges for the industrial production of these factors, vis-à-vis the translational and post-translational bottlenecks, as well as the choice of host cell lines for high-fidelity production. For achieving high productivities of vitamin K dependent proteins, which include factors II (prothrombin), VII, IX and X, and protein C, host cell limitation of γ-glutamyl carboxylation is a major bottleneck. Despite progress in addressing this, involvement of yet unidentified protein(s) impedes a complete cell engineering solution. Human factor VIII expresses at very low levels due to limitations at several steps in the protein secretion pathway. Protein and cell engineering, vector improvement and alternate host cells promise improvement in the productivity. Production of Von Willebrand factor is constrained by its large size, complex structure, and the need for extensive glycosylation and disulfide-bonded oligomerization. All the licensed therapeutic factors are produced in CHO, BHK or HEK293 cells. While HEK293 is a recent adoption, BHK cells appear to be disfavored. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Treatment with specific soluble factors promotes the functional maturation of transcription factor-mediated, pancreatic transdifferentiated cells.

PubMed

Motoyama, Hiroaki; Kobayashi, Akira; Yokoyama, Takahide; Shimizu, Akira; Sakai, Hiroshi; Notake, Tsuyoshi; Fukushima, Kentaro; Miyagawa, Shin-Ichi

2018-01-01

Pancreatic lineage-specific transcription factors (TFs) display instructive roles in converting adult cells to endocrine pancreatic cells through a process known as transdifferentiation. However, little is known about potential factors capable of accelerating transdifferentiation following transduction to achieve the functional maturation of transdifferentiated cells. In this study, we demonstrated, using adult liver-derived progenitor cells, that soluble factors utilized in pancreatic differentiation protocols of pluripotent stem cells promote functional maturation of TFs-mediated transdifferentiated cells. Treatment with an N2 supplement in combination with three soluble factors (glucagon-like peptide-1 [GLP-1] receptor agonist, notch inhibitor, and transforming growth factor-β [TGF-β] inhibitor) enhanced liver-to-pancreas transdifferentiation based on the following findings: i) the incidence of c-peptide-positive cells increased by approximately 1.2-fold after the aforementioned treatment; ii) the c-peptide expression level in the treated cells increased by approximately 12-fold as compared with the level in the untreated cells; iii) the treated cells secreted insulin in a glucose-dependent manner, whereas the untreated cells did not; and iv) transplantation of treated-transdifferentiated cells into streptozotocin-induced immunodeficient diabetic mice led to the amelioration of hyperglycemia. These results suggest that treatment with specific soluble factors promotes the functional maturation of transdifferentiated cells. Our findings could facilitate the development of new modalities for cell-replacement therapy for patients with diabetes.

The cytoprotective role of DJ-1 and p45 NFE2 against human primary alveolar type II cell injury and emphysema.

PubMed

Tan, Li Hui; Bahmed, Karim; Lin, Chih-Ru; Marchetti, Nathaniel; Bolla, Sudhir; Criner, Gerard J; Kelsen, Steven; Madesh, Muniswamy; Kosmider, Beata

2018-02-23

Emphysema is characterized by irreversibly enlarged airspaces and destruction of alveolar walls. One of the factors contributing to this disease pathogenesis is an elevation in extracellular matrix (ECM) degradation in the lung. Alveolar type II (ATII) cells produce and secrete pulmonary surfactants and proliferate to restore the epithelium after damage. We isolated ATII cells from control non-smokers, smokers and patients with emphysema to determine the role of NFE2 (nuclear factor, erythroid-derived 2). NFE2 is a heterodimer composed of two subunits, a 45 kDa (p45 NFE2) and 18 kDa (p18 NFE2) polypeptides. Low expression of p45 NFE2 in patients with emphysema correlated with a high ECM degradation. Moreover, we found that NFE2 knockdown increased cell death induced by cigarette smoke extract. We also studied the cross talk between p45 NFE2 and DJ-1. DJ-1 protein is a redox-sensitive chaperone that protects cells from oxidative stress. We detected that cigarette smoke significantly increased p45 NFE2 levels in DJ-1 KO mice compared to wild-type mice. Our results indicate that p45 NFE2 expression is induced by exposure to cigarette smoke, has a cytoprotective activity against cell injury, and its downregulation in human primary ATII cells may contribute to emphysema pathogenesis.

Disruption of Sorting Nexin 5 Causes Respiratory Failure Associated with Undifferentiated Alveolar Epithelial Type I Cells in Mice

PubMed Central

Im, Sun-Kyoung; Jeong, HyoBin; Jeong, Hyun-Woo; Kim, Kyong-Tai; Hwang, Daehee; Ikegami, Machiko; Kong, Young-Yun

2013-01-01

Sorting nexin 5 (Snx5) has been posited to regulate the degradation of epidermal growth factor receptor and the retrograde trafficking of cation-independent mannose 6-phosphate receptor/insulin-like growth factor II receptor. Snx5 has also been suggested to interact with Mind bomb-1, an E3 ubiquitin ligase that regulates the activation of Notch signaling. However, the in vivo functions of Snx5 are largely unknown. Here, we report that disruption of the Snx5 gene in mice (Snx5-/- mice) resulted in partial perinatal lethality; 40% of Snx5-/- mice died shortly after birth due to cyanosis, reduced air space in the lungs, and respiratory failure. Histological analysis revealed that Snx5-/- mice exhibited thickened alveolar walls associated with undifferentiated alveolar epithelial type I cells. In contrast, alveolar epithelial type II cells were intact, exhibiting normal surfactant synthesis and secretion. Although the expression levels of surfactant proteins and saturated phosphatidylcholine in the lungs of Snx5-/- mice were comparable to those of Snx5+/+ mice, the expression levels of T1α, Aqp5, and Rage, markers for distal alveolar epithelial type I cells, were significantly decreased in Snx5 -/- mice. These results demonstrate that Snx5 is necessary for the differentiation of alveolar epithelial type I cells, which may underlie the adaptation to air breathing at birth. PMID:23526992

The Role of Salivary Gland Scintigraphy in the Evaluation of Salivary Gland Dysfunction in Uncontrolled Type II Diabetic Patients.

PubMed

Senthilkumar, B; Sathasivasubramanian, S

2013-09-01

The aim of the present study was to evaluate the salivary gland dysfunction in patients with uncontrolled type II diabetes using salivary gland scintigraphy and then to compare these ratios with quantitative whole salivary secretion rates. Using a gamma camera (siemens-diacam) equipped with a low energy all-purpose collimator, 32 uncontrolled type II diabetic patients and 30 normal healthy patients were studied by injecting a radio isotope (technetium 99m pertechnetate) about 5 mCi was injected intravenously in to anticubital vein and the activity was measured for the 1(st), 20(th) and 40(th) min. At 20 min after injection, vitamin C chewable tablet was given to stimulate the secretion and continued until the end of the study period (40 min). Before scintigraphy, salivary sampling was carried out in both diabetic and normal individuals in a quiet room, saliva was allowed to accumulate and was expectorated into the collecting vessel approximately once a minute for 15 min and the volume was recorded as Unstimulated salivary flow rate and after 5 min break vitamin C chewable tablet was given to stimulate the secretion and the patient was asked to expectorate the saliva in the collecting vessel for 5 min. The expectorated volume was recorded as stimulated salivary flow rate. The mean of the measurements of scintigraphic ratio and salivary secretion rates were compared using the paired Student's t-test. The scintigraphic mean uptake and excretory ratio (ER) and the salivary flow rates were correlated. The result shows that there was a significant correlation between salivary flow rate and scintigraphic uptake and ER. However, statistically significant result could not be derived as it may be due to smaller sample size and marginal difference in the scintigraphic values between the groups. Salivary gland scintigraphy plays a significant role in the evaluation of salivary gland dysfunction. However, its role as an independent investigative procedure in the evaluation of salivary gland dysfunction requires a study with a larger sample size, may yield a statistical significant result and it can also act as an adjunct along with salivary flow rate procedure.

Crystal structure of the Yersinia type III secretion protein YscE

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

Phan, Jason; Austin, Brian P.; Waugh, David S.

2010-12-06

The plague-causing bacterium Yersinia pestis utilizes a contact-dependent (type III) secretion system (T3SS) to transport virulence factors from the bacterial cytosol directly into the interior of mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. The type III secretion apparatus is composed of 20-25 different Yersinia secretion (Ysc) proteins. We report here the structure of YscE, the smallest Ysc protein, which is a dimer in solution. The probable mode of oligomerization is discussed.

Triiodothyronine regulates angiogenic growth factor and cytokine secretion by isolated human decidual cells in a cell-type specific and gestational age-dependent manner.

PubMed

Vasilopoulou, E; Loubière, L S; Lash, G E; Ohizua, O; McCabe, C J; Franklyn, J A; Kilby, M D; Chan, S Y

2014-06-01

Does triiodothyronine (T3) regulate the secretion of angiogenic growth factors and cytokines by human decidual cells isolated from early pregnancy? T3 modulates the secretion of specific angiogenic growth factors and cytokines, with different regulatory patterns observed amongst various isolated subpopulations of human decidual cells and with a distinct change between the first and second trimesters of pregnancy. Maternal thyroid dysfunction during early pregnancy is associated with complications of malplacentation including miscarriage and pre-eclampsia. T3 regulates the proliferation and apoptosis of fetal-derived trophoblasts, as well as promotes the invasive capability of extravillous trophoblasts (EVT). We hypothesize that T3 may also have a direct impact on human maternal-derived decidual cells, which are known to exert paracrine regulation upon trophoblast behaviour and vascular development at the uteroplacental interface. This laboratory-based study used human decidua from first (8-11 weeks; n = 18) and second (12-16 weeks; n = 12) trimester surgical terminations of apparently uncomplicated pregnancies. Primary cultures of total decidual cells, and immunomagnetic bead-isolated populations of stromal-enriched (CD10+) and stromal-depleted (CD10-) cells, uterine natural killer cells (uNK cells; CD56+) and macrophages (CD14+) were assessed for thyroid hormone receptors and transporters by immunocytochemistry. Each cell population was treated with T3 (0, 1, 10, 100 nM) and assessments were made of cell viability (MTT assay) and angiogenic growth factor and cytokine secretion (immunomediated assay). The effect of decidual cell-conditioned media on EVT invasion through Matrigel(®) was evaluated. Immunocytochemistry showed the expression of thyroid hormone transporters (MCT8, MCT10) and receptors (TRα1, TRβ1) required for thyroid hormone-responsiveness in uNK cells and macrophages from the first trimester. The viability of total decidual cells and the different cell isolates were unaffected by T3 so changes in cell numbers could not account for any observed effects. In the first trimester, T3 decreased VEGF-A secretion by total decidual cells (P < 0.05) and increased angiopoietin-2 secretion by stromal-depleted cells (P < 0.05) but in the second trimester total decidual cells showed only increased angiogenin secretion (P < 0.05). In the first trimester, T3 reduced IL-10 secretion by total decidual cells (P < 0.05), and reduced granulocyte macrophage colony stimulating factor (P < 0.01), IL-8 (P < 0.05), IL-10 (P < 0.01), IL-1β (P < 0.05) and monocyte chemotactic protein -1 (P < 0.001) secretion by macrophages, but increased tumour necrosis factor-α secretion by stromal-depleted cells (P < 0.05) and increased IL-6 by uNK cells (P < 0.05). In contrast, in the second trimester T3 increased IL-10 secretion by total decidual cells (P < 0.01) but did not affect cytokine secretion by uNK cells and macrophages. Conditioned media from first trimester T3-treated total decidual cells and macrophages did not alter EVT invasion compared with untreated controls. Thus, treatment of decidual cells with T3 resulted in changes in both angiogenic growth factor and cytokine secretion in a cell type-specific and gestational age-dependent manner, with first trimester decidual macrophages being the most responsive to T3 treatment, but these changes in decidual cell secretome did not affect EVT invasion in vitro. Our results are based on in vitro findings and we cannot be certain if a similar response occurs in human pregnancy in vivo. Optimal maternal thyroid hormone concentrations could play a critical role in maintaining a balanced inflammatory response in early pregnancy to prevent fetal immune rejection and promote normal placental development through the regulation of the secretion of critical cytokines and angiogenic growth factors by human decidual cells. Our data suggest that there is an ontogenically determined regulatory 'switch' in T3 responsiveness between the first and second trimesters, and support the notion that the timely and early correction of maternal thyroid dysfunction is critical in influencing pregnancy outcomes. This study is funded by Wellbeing of Women (RG/1082/09 to S.Y.C., M.D.K., J.A.F., L.S.L., G.E.L.) and Action Medical Research - Henry Smith Charity (SP4335 to M.D.K., S.Y.C., L.S.L., J.A.F.). The authors have no conflicts of interest to disclose.

Triiodothyronine regulates angiogenic growth factor and cytokine secretion by isolated human decidual cells in a cell-type specific and gestational age-dependent manner

PubMed Central

Vasilopoulou, E.; Loubière, L.S.; Lash, G.E.; Ohizua, O.; McCabe, C.J.; Franklyn, J.A.; Kilby, M.D.; Chan, S.Y.

2014-01-01

STUDY QUESTION Does triiodothyronine (T3) regulate the secretion of angiogenic growth factors and cytokines by human decidual cells isolated from early pregnancy? SUMMARY ANSWER T3 modulates the secretion of specific angiogenic growth factors and cytokines, with different regulatory patterns observed amongst various isolated subpopulations of human decidual cells and with a distinct change between the first and second trimesters of pregnancy. WHAT IS KNOWN ALREADY Maternal thyroid dysfunction during early pregnancy is associated with complications of malplacentation including miscarriage and pre-eclampsia. T3 regulates the proliferation and apoptosis of fetal-derived trophoblasts, as well as promotes the invasive capability of extravillous trophoblasts (EVT). We hypothesize that T3 may also have a direct impact on human maternal-derived decidual cells, which are known to exert paracrine regulation upon trophoblast behaviour and vascular development at the uteroplacental interface. STUDY DESIGN, SIZE, DURATION This laboratory-based study used human decidua from first (8–11 weeks; n = 18) and second (12–16 weeks; n = 12) trimester surgical terminations of apparently uncomplicated pregnancies. PARTICIPANTS/MATERIALS, SETTING, METHODS Primary cultures of total decidual cells, and immunomagnetic bead-isolated populations of stromal-enriched (CD10+) and stromal-depleted (CD10−) cells, uterine natural killer cells (uNK cells; CD56+) and macrophages (CD14+) were assessed for thyroid hormone receptors and transporters by immunocytochemistry. Each cell population was treated with T3 (0, 1, 10, 100 nM) and assessments were made of cell viability (MTT assay) and angiogenic growth factor and cytokine secretion (immunomediated assay). The effect of decidual cell-conditioned media on EVT invasion through Matrigel® was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE Immunocytochemistry showed the expression of thyroid hormone transporters (MCT8, MCT10) and receptors (TRα1, TRβ1) required for thyroid hormone-responsiveness in uNK cells and macrophages from the first trimester. The viability of total decidual cells and the different cell isolates were unaffected by T3 so changes in cell numbers could not account for any observed effects. In the first trimester, T3 decreased VEGF-A secretion by total decidual cells (P < 0.05) and increased angiopoietin-2 secretion by stromal-depleted cells (P < 0.05) but in the second trimester total decidual cells showed only increased angiogenin secretion (P < 0.05). In the first trimester, T3 reduced IL-10 secretion by total decidual cells (P < 0.05), and reduced granulocyte macrophage colony stimulating factor (P < 0.01), IL-8 (P < 0.05), IL-10 (P < 0.01), IL-1β (P < 0.05) and monocyte chemotactic protein -1 (P < 0.001) secretion by macrophages, but increased tumour necrosis factor-α secretion by stromal-depleted cells (P < 0.05) and increased IL-6 by uNK cells (P < 0.05). In contrast, in the second trimester T3 increased IL-10 secretion by total decidual cells (P < 0.01) but did not affect cytokine secretion by uNK cells and macrophages. Conditioned media from first trimester T3-treated total decidual cells and macrophages did not alter EVT invasion compared with untreated controls. Thus, treatment of decidual cells with T3 resulted in changes in both angiogenic growth factor and cytokine secretion in a cell type-specific and gestational age-dependent manner, with first trimester decidual macrophages being the most responsive to T3 treatment, but these changes in decidual cell secretome did not affect EVT invasion in vitro. LIMITATIONS, REASONS FOR CAUTION Our results are based on in vitro findings and we cannot be certain if a similar response occurs in human pregnancy in vivo. WIDER IMPLICATIONS OF THE FINDINGS Optimal maternal thyroid hormone concentrations could play a critical role in maintaining a balanced inflammatory response in early pregnancy to prevent fetal immune rejection and promote normal placental development through the regulation of the secretion of critical cytokines and angiogenic growth factors by human decidual cells. Our data suggest that there is an ontogenically determined regulatory ‘switch’ in T3 responsiveness between the first and second trimesters, and support the notion that the timely and early correction of maternal thyroid dysfunction is critical in influencing pregnancy outcomes. STUDY FUNDING/COMPETING INTEREST(S) This study is funded by Wellbeing of Women (RG/1082/09 to S.Y.C., M.D.K., J.A.F., L.S.L., G.E.L.) and Action Medical Research – Henry Smith Charity (SP4335 to M.D.K., S.Y.C., L.S.L., J.A.F.). The authors have no conflicts of interest to disclose. PMID:24626803

Measurement of gastric meal and secretion volumes using magnetic resonance imaging

PubMed Central

Hoad, C.L.; Parker, H.; Hudders, N.; Costigan, C.; Cox, E.F.; Perkins, A.C.; Blackshaw, P.E.; Marciani, L.; Spiller, R.C.; Fox, M.R.; Gowland, P.A.

2015-01-01

MRI can assess multiple gastric functions without ionizing radiation. However, time consuming image acquisition and analysis of gastric volume data, plus confounding of gastric emptying measurements by gastric secretions mixed with the test meal have limited its use to research centres. This study presents an MRI acquisition protocol and analysis algorithm suitable for the clinical measurement of gastric volume and secretion volume. Reproducibility of gastric volume measurements was assessed using data from 10 healthy volunteers following a liquid test meal with rapid MRI acquisition within one breath-hold and semi-automated analysis. Dilution of the ingested meal with gastric secretion was estimated using a respiratory-triggered T1 mapping protocol. Accuracy of the secretion volume measurements was assessed using data from 24 healthy volunteers following a mixed (liquid/solid) test meal with MRI meal volumes compared to data acquired using gamma scintigraphy (GS) on the same subjects studied on a separate study day. The mean (SD) coefficient of variance between 3 observers for both total gastric contents (including meal, secretions and air) and just the gastric contents (meal and secretion only) was 3 (2) % at large gastric volumes (> 200 ml). Mean (SD) secretion volumes post meal ingestion were 64 (51) ml and 110 (40) ml at 15 and 75 minutes respectively. Comparison with GS meal volumes, showed that MRI meal only volume (after correction for secretion volume) were similar to GS, with a linear regression gradient (std err) of 1.06 (0.10) and intercept −11 (24) ml. In conclusion, (i) rapid acquisition removed the requirement to image during prolonged breath-hold (ii) semi-automatic analysis greatly reduced time required to derive measurements and (iii) correction for secretion volumes provides accurate assessment of gastric meal volumes and emptying. Together these features provide the scientific basis of a protocol which would be suitable in clinical practice. PMID:25592405

Collagen and Stretch Modulate Autocrine Secretion of Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Proteins from Differentiated Skeletal Muscle Cells

NASA Technical Reports Server (NTRS)

Perrone, Carmen E.; Fenwick-Smith, Daniela; Vandenburgh, Herman H.

1995-01-01

Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micro-g of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.

Celecoxib enhances the anti-inflammatory effects of farnesylthiosalicylic acid on T cells independent of prostaglandin E(2) production.

PubMed

Mor, Adam; Aizman, Elizabeta; Kloog, Yoel

2012-10-01

Celecoxib (Celebrex(®)), a non-steroidal anti-inflammatory drug and selective cyclooxygenase-2 inhibitor, is widely used to treat arthritis and other inflammatory disorders. Awareness of its anti-proliferative properties has prompted another indication for its use, in preventing colon polyps in high-risk populations. Farnesylthiosalicylic acid (FTS; Salirasib(®)), designed to inhibit oncogenic Ras and currently under evaluation in phase I/II and II clinical trials, was recently shown by our group to exert anti-inflammatory effects on both lymphocytes and mast cells. Here we examined whether celecoxib combined with FTS would enhance this anti-inflammatory activity. While each drug separately inhibited Ras activation in these cells, their combination yielded more marked inhibition as well as further inhibition of ERK phosphorylation, lymphocyte adhesion, and interleukin-2 secretion. The inhibitory effects, moreover, were independent of prostaglandin E(2) secretion. These data point to the promising potential of combined treatment with celecoxib and FTS for inflammatory disorders involving lymphocytes.

Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay.

PubMed

Amano, Satoshi; Ogura, Yuki; Akutsu, Nobuko; Nishiyama, Toshio

2007-02-01

Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme-linked immunoassay with two kinds of specific monoclonal antibodies against the non-collagenous domain-1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor-beta1 (TGF-beta1). The synthesis of type VII collagen was elevated by TGF-beta1, platelet-derived growth factor, tumor necrosis factor-alpha, and interleukin-1beta, but not by TGF-alpha. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.

Prevention of skin flap necrosis by use of adipose-derived stromal cells with light-emitting diode phototherapy.

PubMed

Park, In-Su; Mondal, Arindam; Chung, Phil-Sang; Ahn, Jin Chul

2015-03-01

The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted human adipose-derived mesenchymal stromal cells (ASCs) in the skin flap of mice. LLLT, ASC transplantation and ASC transplantation with LLLT (ASC + LLLT) were applied to the skin flap. Immunostaining and Western blot analysis were performed to evaluate cell survival and differentiation and secretion of vascular endothelial growth factor and basic fibroblast growth factor by the ASCs. Vascular regeneration was assessed by means of immunostaining in addition to hematoxylin and eosin staining. In the ASC + LLLT group, the survival of ASCs was increased as the result of the decreased apoptosis of ASCs. The secretion of growth factors was higher in this group as compared with ASCs alone. ASCs contributed to tissue regeneration through vascular cell differentiation and secretion of angiogenic growth factors. The ASC + LLLT group displayed improved treatment efficacy including neovascularization and tissue regeneration compared with ASCs alone. Transplanting ASCs to ischemic skin flaps improved therapeutic efficacy for ischemia treatment as the result of enhanced cell survival and paracrine effects. These data suggest that LLLT is an effective biostimulator of ASCs in vascular regeneration, which enhances the survival of ASCs and stimulates the secretion of growth factors in skin flaps. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Incidence and prognostic value of serotonin secretion in pancreatic neuroendocrine tumours.

PubMed

Zandee, Wouter T; van Adrichem, Roxanne C; Kamp, Kimberly; Feelders, Richard A; van Velthuysen, Marie-Louise F; de Herder, Wouter W

2017-08-01

Serotonin secretion occurs in approximately 1%-4% of patients with a pancreatic neuroendocrine tumour (PNET), but the incidence is not well defined. The aim of this study was to determine the incidence of serotonin secretion with and without carcinoid syndrome and the prognostic value for overall survival (OS). Data were collected from 255 patients with a PNET if 24-hours urinary 5-hydroxyindoleacetic acid excretion (5-HIAA) was assessed. Patients were diagnosed with serotonin secretion if 24-hours urinary 5-HIAA excretion was more than 3× the upper limit of normal (ULN) of 50 μmol/24 hours during follow-up. The effect of serotonin secretion on OS was estimated with uni- and multivariate analyses using a Cox regression. Two (0.8%) patients were diagnosed with carcinoid syndrome, and another 20 (7.8%) had a serotonin-secreting PNET without symptoms. These patients mostly had ENETS stage IV disease with high chromogranin A (CgA). Serotonin secretion was a negative prognostic factor in univariate analysis (HR 2.2, 95% CI: 1.27-3.81), but in multivariate analysis, only CgA>10× ULN (HR: 1.81, 95% CI: 1.10-2.98) and neuron-specific enolase (NSE) >ULN (HR: 3.51, 95% CI: 2.26-5.46) were predictors for OS. Immunohistochemical staining for serotonin was positive in 28.6% of serotonin-secreting PNETs (one with carcinoid syndrome) and negative in all controls. Carcinoid syndrome is rare in patients with a PNET, but serotonin secretion occurs often. This is a negative prognostic factor for OS, but after correction for CgA and NSE, it is no longer a predictor and probably only a "not-so innocent bystander" in patients with high tumour burden. © 2017 John Wiley & Sons Ltd.

Regulation of BDNF Release by ARMS/Kidins220 through Modulation of Synaptotagmin-IV Levels.

PubMed

López-Benito, Saray; Sánchez-Sánchez, Julia; Brito, Verónica; Calvo, Laura; Lisa, Silvia; Torres-Valle, María; Palko, Mary E; Vicente-García, Cristina; Fernández-Fernández, Seila; Bolaños, Juan P; Ginés, Silvia; Tessarollo, Lino; Arévalo, Juan C

2018-06-06

BDNF is a growth factor with important roles in the nervous system in both physiological and pathological conditions, but the mechanisms controlling its secretion are not completely understood. Here, we show that ARMS/Kidins220 negatively regulates BDNF secretion in neurons from the CNS and PNS. Downregulation of the ARMS/Kidins220 protein in the adult mouse brain increases regulated BDNF secretion, leading to its accumulation in the striatum. Interestingly, two mouse models of Huntington's disease (HD) showed increased levels of ARMS/Kidins220 in the hippocampus and regulated BDNF secretion deficits. Importantly, reduction of ARMS/Kidins220 in hippocampal slices from HD mice reversed the impaired regulated BDNF release. Moreover, there are increased levels of ARMS/Kidins220 in the hippocampus and PFC of patients with HD. ARMS/Kidins220 regulates Synaptotagmin-IV levels, which has been previously observed to modulate BDNF secretion. These data indicate that ARMS/Kidins220 controls the regulated secretion of BDNF and might play a crucial role in the pathogenesis of HD. SIGNIFICANCE STATEMENT BDNF is an important growth factor that plays a fundamental role in the correct functioning of the CNS. The secretion of BDNF must be properly controlled to exert its functions, but the proteins regulating its release are not completely known. Using neuronal cultures and a new conditional mouse to modulate ARMS/Kidins220 protein, we report that ARMS/Kidins220 negatively regulates BDNF secretion. Moreover, ARMS/Kidins220 is overexpressed in two mouse models of Huntington's disease (HD), causing an impaired regulation of BDNF secretion. Furthermore, ARMS/Kidins220 levels are increased in brain samples from HD patients. Future studies should address whether ARMS/Kidins220 has any function on the pathophysiology of HD. Copyright © 2018 the authors 0270-6474/18/385415-14$15.00/0.

Fibrin Glue Improves the Therapeutic Effect of MSCs by Sustaining Survival and Paracrine Function

PubMed Central

Kim, Inok; Lee, Sung Koo; Yoon, Jung In; Kim, Da Eun

2013-01-01

Fibrin glue has been widely investigated as a cell delivery vehicle for improving the therapeutic effects of mesenchymal stem cells (MSCs). Implanted MSCs produce their therapeutic effects by secreting paracrine factors and by replacing damaged tissues after differentiation. While the influence of fibrin glue on the differentiation potential of MSCs has been well documented, its effect on paracrine function of MSCs is largely unknown. Herein we investigated the influence of fibrin glue on the paracrine effects of MSCs. MSCs were isolated from human adipose tissue. The effects of fibrin glue on survival, migration, secretion of growth factors, and immune suppression of MSCs were investigated in vitro. MSCs in fibrin glue survived and secreted growth factors such as the vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) over 14 days. VEGF and immune modulators, including the transforming growth factor (TGF)-β1 and prostaglandin E2, secreted from MSCs in fibrin glue significantly increased under inflammatory conditions. Thus, MSCs in fibrin glue effectively suppressed immune reactions. In addition, fibrin glue protected the MSCs from oxidative stress and prevented human dermal fibroblast death induced by exposure to extreme stress. In contrast, MSCs within fibrin glue hardly migrated. These results suggest that fibrin glue may sustain survival of implanted MSCs and their paracrine function. Our results provide a mechanistic data to allow further development of MSCs with fibrin glue as a clinical treatment. PMID:23701237

Adiponectin promotes VEGF-C-dependent lymphangiogenesis by inhibiting miR-27b through a CaMKII/AMPK/p38 signaling pathway in human chondrosarcoma cells.

PubMed

Huang, Chun-Yin; Chang, An-Chen; Chen, Hsien-Te; Wang, Shih-Wei; Lo, Yuan-Shun; Tang, Chih-Hsin

2016-09-01

Chondrosarcoma is the second most frequently occurring type of bone malignancy characterized by distant metastatic propensity. Vascular endothelial growth factor-C (VEGF-C) is the major lymphangiogenic factor, and makes crucial contributions to tumour lymphangiogenesis and lymphatic metastasis. Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes. In recent years, adiponectin has also been indicated as facilitating tumorigenesis, angiogenesis and metastasis. However, the effect of adiponectin on VEGF-C regulation and lymphangiogenesis in chondrosarcoma has remained largely a mystery. In the present study, we have shown a clinical correlation between adiponectin and VEGF-C, as well as tumour stage, in human chondrosarcoma tissues. We further demonstrated that adiponectin promoted VEGF-C expression and secretion in human chondrosarcoma cells. The conditioned medium from adiponectin-treated cells significantly induced tube formation and migration of human lymphatic endothelial cells. In addition, adiponectin knock down inhibited lymphangiogenesis in vitro and in vivo We also found that adiponectin-induced VEGF-C is mediated by the calmodulin-dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK) and p38 signaling pathway. Furthermore, the expression of miR-27b was negatively regulated by adiponectin via the CaMKII, AMPK and p38 cascade. The present study is the first to describe the mechanism of adiponectin-promoted lymphangiogenesis by up-regulating VEGF-C expression in chondrosarcomas. Thus, adiponectin could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Stem cell factor supports migration in canine mesenchymal stem cells.

PubMed

Enciso, Nathaly; Ostronoff, Luciana L K; Mejías, Guillermo; León, Leticia G; Fermín, María Luisa; Merino, Elena; Fragio, Cristina; Avedillo, Luis; Tejero, Concepción

2018-03-01

Adult Mesenchymal Stem Cells (MSC) are cells that can be defined as multipotent cells able to differentiate into diverse lineages, under appropriate conditions. These cells have been widely used in regenerative medicine, both in preclinical and clinical settings. Initially discovered in bone marrow, MSC can now be isolated from a wide spectrum of adult and foetal tissues. Studies to evaluate the therapeutic potential of these cells are based on their ability to arrive to damaged tissues. In this paper we have done a comparative study analyzing proliferation, surface markers and OCT4, SOX9, RUNX2, PPARG genes expression in MSC cells from Bone marrow (BMMSC) and Adipose tissue (ASC). We also analyzed the role of Stem Cell Factor (SCF) on MSC proliferation and on ASCs metalloproteinases MMP-2, MMP-9 secretion. Healthy dogs were used as BMMSC donors, and ASC were collected from omentum during elective ovariohysterectomy surgery. Both cell types were cultured in IMDM medium with or without SCF, 10% Dog Serum (DS), and incubated at 38 °C with 5% CO2. Growth of BMMSCs and ASCs was exponential until 25-30 days. Flow citometry of MSCs revealed positive results for CD90 and negative for CD34, CD45 and MCH-II. Genes were evaluated by RT-PCR and metalloproteinases by zymografy. Our findings indicate morphological and immunological similarities as well as expression of genes from both origins on analyzed cells. Furthermore, SCF did not affect proliferation of MSCs, however it up-regulated MMP-2 and MMP-9 secretion in ASCs. These results suggest that metalloproteinases are possibly essential molecules pivoting migration.

The subcommissural organ of the rat secretes Reissner's fiber glycoproteins and CSF-soluble proteins reaching the internal and external CSF compartments

PubMed Central

Vio, Karin; Rodríguez, Sara; Yulis, Carlos R; Oliver, Cristian; Rodríguez, Esteban M

2008-01-01

Background The subcommissural organ (SCO) is a highly conserved brain gland present throughout the vertebrate phylum; it secretes glycoproteins into the cerebrospinal fluid (CSF), where they aggregate to form Reissner's fiber (RF). SCO-spondin is the major constituent protein of RF. Evidence exists that the SCO also secretes proteins that remain soluble in the CSF. The aims of the present investigation were: (i) to identify and partially characterize the SCO-secretory compounds present in the SCO gland itself and in the RF of the Sprague-Dawley rat and non-hydrocephalic hyh mouse, and in the CSF of rat; (ii) to make a comparative analysis of the proteins present in these three compartments; (iii) to identify the proteins secreted by the SCO into the CSF at different developmental periods. Methods The proteins of the SCO secreted into the CSF were studied (i) by injecting specific antibodies into ventricular CSF in vivo; (ii) by immunoblots of SCO, RF and CSF samples, using specific antibodies against the SCO secretory proteins (AFRU and anti-P15). In addition, the glycosylated nature of SCO-compounds was analysed by concanavalin A and wheat germ agglutinin binding. To analyse RF-glycoproteins, RF was extracted from the central canal of juvenile rats and mice; to investigate the CSF-soluble proteins secreted by the SCO, CSF samples were collected from the cisterna magna of rats at different stages of development (from E18 to PN30). Results Five glycoproteins were identified in the rat SCO with apparent molecular weights of 630, 450, 390, 320 and 200 kDa. With the exception of the 200-kDa compound, all other compounds present in the rat SCO were also present in the mouse SCO. The 630 and 390 kDa compounds of the rat SCO have affinity for concanavalin A but not for wheat germ agglutinin, suggesting that they correspond to precursor forms. Four of the AFRU-immunoreactive compounds present in the SCO (630, 450, 390, 320 kDa) were absent from the RF and CSF. These may be precursor and/or partially processed forms. Two other compounds (200, 63 kDa) were present in SCO, RF and CSF and may be processed forms. The presence of these proteins in both, RF and CSF suggests a steady-state RF/CSF equilibrium for these compounds. Eight AFRU-immunoreactive bands were consistently found in CSF samples from rats at E18, E20 and PN1. Only four of these compounds were detected in the cisternal CSF of PN30 rats. The 200 kDa compound appears to be a key compound in rats since it was consistently found in all samples of SCO, RF and embryonic and juvenile CSF. Conclusion It is concluded that (i) during the late embryonic life, the rat SCO secretes compounds that remain soluble in the CSF and reach the subarachnoid space; (ii) during postnatal life, there is a reduction in the number and concentration of CSF-soluble proteins secreted by the SCO. The molecular structure and functional significance of these proteins remain to be elucidated. The possibility they are involved in brain development has been discussed. PMID:18218138

Green tea polyphenol epigallocatechin-3-gallate and cranberry proanthocyanidins act in synergy with cathelicidin (LL-37) to reduce the LPS-induced inflammatory response in a three-dimensional co-culture model of gingival epithelial cells and fibroblasts.

PubMed

Lombardo Bedran, Telma Blanca; Palomari Spolidorio, Denise; Grenier, Daniel

2015-06-01

The human antimicrobial peptide cathelicidin (LL-37) possesses anti-inflammatory properties that may contribute to attenuating the inflammatory process associated with chronic periodontitis. Plant polyphenols, including those from cranberry and green tea, have been reported to reduce inflammatory cytokine secretion by host cells. In the present study, we hypothesized that A-type cranberry proanthocyanidins (AC-PACs) and green tea epigallocatechin-3-gallate (EGCG) act in synergy with LL-37 to reduce the secretion of inflammatory mediators by oral mucosal cells. A three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts treated with non-cytotoxic concentrations of AC-PACs (25 and 50 μg/ml), EGCG (1 and 5 μg/ml), and LL-37 (0.1 and 0.2 μM) individually and in combination (AC-PACs+LL-37 and EGCG+LL-37) were stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). Multiplex ELISA assays were used to quantify the secretion of 54 host factors, including chemokines, cytokines, growth factors, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). LL-37, AC-PACs, and EGCG, individually or in combination, had no effect on the regulation of MMP and TIMP secretion but inhibited the secretion of several cytokines. AC-PACs and LL-37 acted in synergy to reduce the secretion of CXC-chemokine ligand 1 (GRO-α), granulocyte colony-stimulating factor (G-CSF), and interleukin-6 (IL-6), and had an additive effect on reducing the secretion of interleukin-8 (IL-8), interferon-γ inducible protein 10 (IP-10), and monocyte chemoattractant protein-1 (MCP-1) in response to LPS stimulation. EGCG and LL-37 acted in synergy to reduce the secretion of GRO-α, G-CSF, IL-6, IL-8, and IP-10, and had an additive effect on MCP-1 secretion. The combination of LL-37 and natural polyphenols from cranberry and green tea acted in synergy to reduce the secretion of several cytokines by an LPS-stimulated 3D co-culture model of oral mucosal cells. Such combinations show promising results as potential adjunctive therapies for treating inflammatory periodontitis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Encephalitozoon intestinalis Inhibits Dendritic Cell Differentiation through an IL-6-Dependent Mechanism

PubMed Central

Bernal, Carmen E.; Zorro, Maria M.; Sierra, Jelver; Gilchrist, Katherine; Botero, Jorge H.; Baena, Andres; Ramirez-Pineda, Jose R.

2016-01-01

Microsporidia are a group of intracellular pathogens causing self-limited and severe diseases in immunocompetent and immunocompromised individuals, respectively. A cellular type 1 adaptive response, mediated by IL-12, IFNγ, CD4+, and CD8+ T cells has been shown to be essential for host resistance, and dendritic cells (DC) play a key role at eliciting anti-microsporidial immunity. We investigated the in vitro response of DC and DC precursors/progenitors to infection with Encephalitozoon intestinalis (Ei), a common agent of human microsporidosis. Ei-exposed DC cultures up-regulated the surface expression of MHC class II and the costimulatory molecules CD86 and CD40, only when high loads of spores were used. A vigorous secretion of IL-6 but not of IL-1β or IL-12p70 was also observed in these cultures. Ei-exposed DC cultures consisted of immature infected and mature bystander DC, as assessed by MHC class II and costimulatory molecules expression, suggesting that intracellular Ei spores deliver inhibitory signals in DC. Moreover, Ei selectively inhibited the secretion of IL-12p70 in LPS-stimulated DC. Whereas Ei-exposed DC promoted allogeneic naïve T cell proliferation and IL-2 and IFNγ secretion in DC-CD4+ T cell co-cultures, separated co-cultures with bystander or infected DCs showed stimulation or inhibition of IFNγ secretion, respectively. When DC precursors/progenitors were exposed to Ei spores, a significant inhibition of DC differentiation was observed without shifting the development toward cells phenotypically or functionally compatible with myeloid-derived suppressor cells. Neutralization experiments demonstrated that this inhibitory effect is IL-6-dependent. Altogether this investigation reveals a novel potential mechanism of immune escape of microsporidian parasites through the modulation of DC differentiation and maturation. PMID:26870700

The novel Legionella pneumophila type II secretion substrate NttC contributes to infection of amoebae Hartmannella vermiformis and Willaertia magna.

PubMed

Tyson, Jessica Y; Vargas, Paloma; Cianciotto, Nicholas P

2014-12-01

The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires' disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts. © 2014 The Authors.

The surfactant of Legionella pneumophila Is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species.

PubMed

Stewart, Catherine R; Burnside, Denise M; Cianciotto, Nicholas P

2011-11-01

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment.

The Surfactant of Legionella pneumophila Is Secreted in a TolC-Dependent Manner and Is Antagonistic toward Other Legionella Species ▿†

PubMed Central

Stewart, Catherine R.; Burnside, Denise M.; Cianciotto, Nicholas P.

2011-01-01

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent “sliding” motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment. PMID:21890700

Exosome secretion affects social motility in Trypanosoma brucei

PubMed Central

Shaked, Hadassa; Arvatz, Gil; Tkacz, Itai Dov; Binder, Lior; Waldman Ben-Asher, Hiba; Okalang, Uthman; Chikne, Vaibhav; Cohen-Chalamish, Smadar; Michaeli, Shulamit

2017-01-01

Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite Trypanosoma brucei, exosome secretion is induced by stress that affects trans-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites. PMID:28257521

Secretion of Pseudomonas aeruginosa type III cytotoxins is dependent on pseudomonas quinolone signal concentration.

PubMed

Singh, G; Wu, B; Baek, M S; Camargo, A; Nguyen, A; Slusher, N A; Srinivasan, R; Wiener-Kronish, J P; Lynch, S V

2010-10-01

Pseudomonas aeruginosa is an opportunistic pathogen that can, like other bacterial species, exist in antimicrobial resistant sessile biofilms and as free-swimming, planktonic cells. Specific virulence factors are typically associated with each lifestyle and several two component response regulators have been shown to reciprocally regulate transition between biofilm-associated chronic, and free-swimming acute infections. Quorum sensing (QS) signal molecules belonging to the las and rhl systems are known to regulate virulence gene expression by P. aeruginosa. However the impact of a recently described family of novel quorum sensing signals produced by the Pseudomonas Quinolone Signal (PQS) biosynthetic pathway, on the transition between these modes of infection is less clear. Using clonal isolates from a patient developing ventilator-associated pneumonia, we demonstrated that clinical observations were mirrored by an in vitro temporal shift in isolate phenotype from a non-secreting, to a Type III cytotoxin secreting (TTSS) phenotype and further, that this phenotypic change was PQS-dependent. While intracellular type III cytotoxin levels were unaffected by PQS concentration, cytotoxin secretion was dependent on this signal molecule. Elevated PQS concentrations were associated with inhibition of cytotoxin secretion coincident with expression of virulence factors such as elastase and pyoverdin. In contrast, low concentrations or the inability to biosynthesize PQS resulted in a reversal of this phenotype. These data suggest that expression of specific P. aeruginosa virulence factors appears to be reciprocally regulated and that an additional level of PQS-dependent post-translational control, specifically governing type III cytotoxin secretion, exists in this species. Copyright 2010 Elsevier Ltd. All rights reserved.

Two Sulfur Glycoside Compounds Isolated from Lepidium apetalum Willd Protect NRK52e Cells against Hypertonic-Induced Adhesion and Inflammation by Suppressing the MAPK Signaling Pathway and RAAS.

PubMed

Yuan, Peipei; Zheng, Xiaoke; Li, Meng; Ke, Yingying; Fu, Yang; Zhang, Qi; Wang, Xiaolan; Feng, Weisheng

2017-11-12

Lepidium apetalum Willd has been used to reduce edema and promote urination. Cis -desulfoglucotropaeolin ( cis -DG) and trans -desulfoglucotropaeolin ( trans -DG) were isolated from Lepidium apetalum Willd, and caused a significant increase in cell viability in a hypertonic model in NRK52e cells. In the hypertonic model, cis -DG and trans -DG significantly promoted the cell viability of NRK52e cells and inhibited the elevation of Na⁺ in the supernatant, inhibited the renin-angiotensin-aldosterone (RAAS) system, significantly reduced the levels of angiotensin II (Ang II) and aldosterone (ALD), and lowered aquaporin-2 (AQP2) and Na⁺-K⁺ ATP content in renal medulla. After treatment with cis -DG and trans -DG, expression of calcineurin (CAN) and Ca/calmodulin-dependent protein kinase II (CaMK II) was decreased in renal tissue and Ca 2+ influx was inhibited, thereby reducing the secretion of transforming growth factor-β (TGFβ), reversing the increase in adhesion and inflammatory factor E-selectin and monocyte chemotactic protein 1 (MCP-1) induced by high NaCl, while reducing oxidative stress status and decreasing the expression of cyclooxygenase-2 (COX2). Furthermore, inhibition of protein kinase C (PKC) expression also contributed to these improvements. The cis -DG and trans -DG reduced the expression of p-p44/42 MAPK, p-JNK and p-p38, inhibited the phosphorylation of the MAPK signaling pathway in NRN52e cells induced by high salt, decreased the overexpression of p-p38 and p-HSP27, and inhibited the overactivation of the p38-MAPK signaling pathway, suggesting that the p38-MAPK pathway may play a vital role in the hypertonic-induced adhesion and inflammatory response. From the results of this study, it can be concluded that the mechanism of cis -DG and trans -DG may mainly be through inhibiting the p38-MAPK signaling pathway, inhibiting the excessive activation of the RAAS system, and thereby reducing adhesion and inflammatory factors.

Restoring conjunctival tolerance by topical nuclear factor-κB inhibitors reduces preservative-facilitated allergic conjunctivitis in mice.

PubMed

Guzmán, Mauricio; Sabbione, Florencia; Gabelloni, María Laura; Vanzulli, Silvia; Trevani, Analía Silvina; Giordano, Mirta Nilda; Galletti, Jeremías Gastón

2014-09-04

To evaluate the role of nuclear factor-κB (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis. Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T-cell responses and antigen-induced ocular inflammation were evaluated. Nuclear factor-κB activation and associated inflammatory changes also were assessed in murine eyes and in an epithelial cell line after BAK exposure. Benzalkonium chloride promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of IL-6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB-dependent fashion after interaction with T cells. Benzalkonium chloride triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Isolation and Identification of Proteins Secreted by Cells Cultured within Synthetic Hydrogel-Based Matrices

PubMed Central

2018-01-01

Cells interact with and remodel their microenvironment, degrading large extracellular matrix (ECM) proteins (e.g., fibronectin, collagens) and secreting new ECM proteins and small soluble factors (e.g., growth factors, cytokines). Synthetic mimics of the ECM have been developed as controlled cell culture platforms for use in both fundamental and applied studies. However, how cells broadly remodel these initially well-defined matrices remains poorly understood and difficult to probe. In this work, we have established methods for widely examining both large and small proteins that are secreted by cells within synthetic matrices. Specifically, human mesenchymal stem cells (hMSCs), a model primary cell type, were cultured within well-defined poly(ethylene glycol) (PEG)-peptide hydrogels, and these cell-matrix constructs were decellularized and degraded for subsequent isolation and analysis of deposited proteins. Shotgun proteomics using liquid chromatography and mass spectrometry identified a variety of proteins, including the large ECM proteins fibronectin and collagen VI. Immunostaining and confocal imaging confirmed these results and provided visualization of protein organization within the synthetic matrices. Additionally, culture medium was collected from the encapsulated hMSCs, and a Luminex assay was performed to identify secreted soluble factors, including vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), basic fibroblast growth factor (FGF-2), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α). Together, these methods provide a unique approach for studying dynamic reciprocity between cells and synthetic microenvironments and have the potential to provide new biological insights into cell responses during three-dimensional (3D) controlled cell culture. PMID:29552635

Isolation and Identification of Proteins Secreted by Cells Cultured within Synthetic Hydrogel-Based Matrices.

PubMed

Sawicki, Lisa A; Choe, Leila H; Wiley, Katherine L; Lee, Kelvin H; Kloxin, April M

2018-03-12

Cells interact with and remodel their microenvironment, degrading large extracellular matrix (ECM) proteins (e.g., fibronectin, collagens) and secreting new ECM proteins and small soluble factors (e.g., growth factors, cytokines). Synthetic mimics of the ECM have been developed as controlled cell culture platforms for use in both fundamental and applied studies. However, how cells broadly remodel these initially well-defined matrices remains poorly understood and difficult to probe. In this work, we have established methods for widely examining both large and small proteins that are secreted by cells within synthetic matrices. Specifically, human mesenchymal stem cells (hMSCs), a model primary cell type, were cultured within well-defined poly(ethylene glycol) (PEG)-peptide hydrogels, and these cell-matrix constructs were decellularized and degraded for subsequent isolation and analysis of deposited proteins. Shotgun proteomics using liquid chromatography and mass spectrometry identified a variety of proteins, including the large ECM proteins fibronectin and collagen VI. Immunostaining and confocal imaging confirmed these results and provided visualization of protein organization within the synthetic matrices. Additionally, culture medium was collected from the encapsulated hMSCs, and a Luminex assay was performed to identify secreted soluble factors, including vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), basic fibroblast growth factor (FGF-2), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α). Together, these methods provide a unique approach for studying dynamic reciprocity between cells and synthetic microenvironments and have the potential to provide new biological insights into cell responses during three-dimensional (3D) controlled cell culture.

Altered cytokine profiles of human retinal pigment epithelium: Oxidant injury and replicative senescence

PubMed Central

Cao, Sijia; Walker, Gregory B.; Wang, Xuefeng; Cui, Jing Z.

2013-01-01

Purpose Age-related macular degeneration (AMD) is a local, chronic inflammatory disease of the eye that is influenced by oxidative stress and dysregulation of the retinal pigment epithelium (RPE) associated with aging. The purpose of this study is to characterize the effects of oxidative stress and replicative senescence on the secreted cytokine profiles of RPE in vitro. Methods We used multiple, serial passages of human RPE cells from primary culture as an in vitro model of aging. Responses of early passage 5 (P5) and late passage 21 (P21) RPE cells were compared. Oxidative stress was induced in RPE cells (P5) by exposure to 75 μM hydroquinone (HQ) for 24 h. The secretome profiles of the RPE cells were measured with a multiplex suspension assay that assayed human cytokine, chemokine, and growth factors. Immunohistochemistry on younger (≤55 years old) and older (≥70 years old) human post-mortem donor eyes was used to verify selected cytokines. Results Supernatant of HQ-treated RPE cultures exhibited increased secreted levels of vascular endothelial growth factor (VEGF), interleukin (IL)-12, and IL-10 that reached statistical significance (p<0.05). Supernatant of late passage P21 RPE cultures exhibited decreased secreted levels of stromal cell-derived factor (SDF)-1α, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-8, IL-15, IL-6, and an increased level of IL-1ra compared to early passage P5 RPE cultures that reached statistical significance (p<0.05). Immunohistochemical analysis demonstrated increased expression of IL-1ra in RPE cells from older post-mortem donor eyes (≥70 years old) versus younger eyes (≤55 years old). Conclusions Our data demonstrate a unique cytokine secretion profile of primary culture RPE cells at early and late passage. Our in vitro data suggest an age-specific modulation of cytokine secretion in RPE and is consistent with immunohistochemical analysis on post-mortem eyes. The secretion profile associated with RPE under conditions that mimic oxidative stress, another factor associated with the pathogenesis of AMD, emphasizes upregulation of the angiogenic growth factor, vascular endothelial growth factor. Together, these data support the role of advanced age and oxidative stress in inflammatory cytokine modulation in RPE cells. PMID:23559866

Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis.

PubMed

East, Alexandra; Mechaly, Ariel E; Huysmans, Gerard H M; Bernarde, Cédric; Tello-Manigne, Diana; Nadeau, Nathalie; Pugsley, Anthony P; Buschiazzo, Alejandro; Alzari, Pedro M; Bond, Peter J; Francetic, Olivera

2016-01-05

The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural Basis of Chemokine Sequestration by CrmD, a Poxvirus-Encoded Tumor Necrosis Factor Receptor

PubMed Central

Wang, Dongli; Chen, Dongwei; He, Guangjun; Huang, Li; Wang, Hanzhong; Wang, Xinquan

2011-01-01

Pathogens have evolved sophisticated mechanisms to evade detection and destruction by the host immune system. Large DNA viruses encode homologues of chemokines and their receptors, as well as chemokine-binding proteins (CKBPs) to modulate the chemokine network in host response. The SECRET domain (smallpox virus-encoded chemokine receptor) represents a new family of viral CKBPs that binds a subset of chemokines from different classes to inhibit their activities, either independently or fused with viral tumor necrosis factor receptors (vTNFRs). Here we present the crystal structures of the SECRET domain of vTNFR CrmD encoded by ectromelia virus and its complex with chemokine CX3CL1. The SECRET domain adopts a β-sandwich fold and utilizes its β-sheet I surface to interact with CX3CL1, representing a new chemokine-binding manner of viral CKBPs. Structure-based mutagenesis and biochemical analysis identified important basic residues in the 40s loop of CX3CL1 for the interaction. Mutation of corresponding acidic residues in the SECRET domain also affected the binding for other chemokines, indicating that the SECRET domain binds different chemokines in a similar manner. We further showed that heparin inhibited the binding of CX3CL1 by the SECRET domain and the SECRET domain inhibited RAW264.7 cell migration induced by CX3CL1. These results together shed light on the structural basis for the SECRET domain to inhibit chemokine activities by interfering with both chemokine-GAG and chemokine-receptor interactions. PMID:21829356

Activation and desensitization of platelets by platelet-activating factor (PAF) derived from IgE-sensitized basophils. I. Characteristics of the secretory response

PubMed Central

1976-01-01

The secretion of vasoactive amines from rabbit platelets induced by the platelet-activating factor (PAF) derived from IgE-sensitized rabbit basophils, was examined. The secretion required calcium has previously been shown to be noncytotoxic and was optimal in both rate and extent at 37 degrees C and pH 7.2. Different temperature-sensitive steps were rate limiting for secretion above or below 20 degrees C. The rate of secretion was dependent upon the concentration of PAF and also of platelets. Maximal rates were observed with relatively low concentrations of platelets (2.5 X 10(8)/ml), sharply contrasting with other platelet stimuli such as C3 or thrombin. The extent of secretion was dependent upon PAF concentration until a maximum of 50 or 60% of the serotonin was released and then declined with increasing amounts of PAF. This was interpreted to result from the platelets becoming desensitized to the PAF, a process that shuts off the secretion. Such a desensitization was demonstrated and was shown to be stimulus specific, i.e., other stimuli could still induce secretion from PAF-desensitized platelets. PAF extracted with ethanol from the albumin to which it is usually bound during preparation, exhibited similar characteristics, except that secretion of up to 90% of the serotonin was induced. The extracted PAF thus seemed less able to induce the desensitization. Its use did provide important evidence that populations of rabbit platelets are relatively homogenous in their ability to respond to PAF. PMID:3618

Discovery of Novel Secreted Virulence Factors from Salmonella enterica Serovar Typhimurium by Proteomic Analysis of Culture Supernatants

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

Niemann, George; Brown, Roslyn N.; Gustin, Jean K.

The intracellular pathogen Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis in the world. This pathogen has two type-III secretion systems (TTSS) necessary for virulence that are encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) and are expressed during extracellular or intracellular infectious states, respectively, to deliver virulence factors (effectors) to the host cell cytoplasm. While many have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this mass spectrometry-based proteomics study, we identified effector proteins secreted under minimal acidic medium growth conditions that induced themore » SPI-2 TTSS and its effectors, and compared the secretome from the parent strain to the secretome from strains missing either essential (SsaK) or regulatory components (SsaL) of the SPI-2 secretion apparatus. We identified 75% of the known TTSS effector repertoire. Excluding translocon components, 95% of the known effectors were biased for identification in the ssaL mutant background, which demonstrated that SsaL regulates SPI-2 type III secretion. To confirm secretion to animal cells, we made translational fusions of several of the best candidates to the calmodulin-dependent adenylate cyclase of Bordetella pertussis and assayed cAMP levels of infected J774 macrophage-like cells. From these infected cells we identified six new TTSS effectors and two others that are secreted independent of TTSS. Our results substantiate reports of additional secretion systems encoded by Salmonella other than TTSS.« less

Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.

PubMed

Lallemand, Mathilde; Login, Frédéric H; Guschinskaya, Natalia; Pineau, Camille; Effantin, Géraldine; Robert, Xavier; Shevchik, Vladimir E

2013-01-01

The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

Dynamic Interplay between the Periplasmic and Transmembrane Domains of GspL and GspM in the Type II Secretion System

PubMed Central

Guschinskaya, Natalia; Pineau, Camille; Effantin, Géraldine; Robert, Xavier; Shevchik, Vladimir E.

2013-01-01

The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine. PMID:24223969

Profiling of proteins secreted in the bovine oviduct reveals diverse functions of this luminal microenvironment.

PubMed

Pillai, Viju Vijayan; Weber, Darren M; Phinney, Brett S; Selvaraj, Vimal

2017-01-01

The oviductal microenvironment is a site for key events that involve gamete maturation, fertilization and early embryo development. Secretions into the oviductal lumen by either the lining epithelium or by transudation of plasma constituents are known to contain elements conducive for reproductive success. Although previous studies have identified some of these factors involved in reproduction, knowledge of secreted proteins in the oviductal fluid remains rudimentary with limited definition of function even in extensively studied species like cattle. In this study, we used a shotgun proteomics approach followed by bioinformatics sequence prediction to identify secreted proteins present in the bovine oviductal fluid (ex vivo) and secretions from the bovine oviductal epithelial cells (in vitro). From a total of 2087 proteins identified, 266 proteins could be classified as secreted, 109 (41%) of which were common for both in vivo and in vitro conditions. Pathway analysis indicated different classes of proteins that included growth factors, metabolic regulators, immune modulators, enzymes, and extracellular matrix components. Functional analysis revealed mechanisms in the oviductal lumen linked to immune homeostasis, gamete maturation, fertilization and early embryo development. These results point to several novel components that work together with known elements mediating functional homeostasis, and highlight the diversity of machinery associated with oviductal physiology and early events in cattle fertility.

Ovarian and endocrine characteristics during an estrous cycle in Angus, Brahman, and Senepol cows in a subtropical environment.

PubMed

Alvarez, P; Spicer, L J; Chase, C C; Payton, M E; Hamilton, T D; Stewart, R E; Hammond, A C; Olson, T A; Wettemann, R P

2000-05-01

To determine breed differences in ovarian function and endocrine secretion, daily rectal ultrasonography was conducted on multiparous lactating Angus (temperate Bos taurus; n = 12), Brahman (tropical Bos indicus; n = 12), and Senepol (tropical Bos taurus; n = 12) cows during an estrous cycle in summer. Blood was collected daily to quantify plasma concentrations of FSH, LH, progesterone, estradiol, GH, insulin-like growth factor (IGF)-I, IGF-II, IGF binding proteins (IGFBP), insulin, glucose, and plasma urea nitrogen (PUN). Numbers of small (2 to 5 mm), medium (6 to 8 mm), and large follicles (> or = 9 mm) were greater (P < .05) in Brahman than in Angus and(or) Senepol cows. Length of the estrous cycle (SEM = .6 d) was similar (P > .10) among Senepol (20.4 d), Angus (19.5 d), and Brahman (19.7 d) cows. Senepol cows had greater (P < .05) diameters of the corpus luteum (CL) and a delayed regression of the CL as compared with Angus cows. The secondary surge of FSH (between d 1 and 2; d 0 = estrus) was greater in Angus than Brahman or Senepol cows (breed x day, P < .05). Between d 2 and 14 of the estrous cycle, concentrations of progesterone, LH, IGF-II, and binding activities of IGFBP-3, IGFBP-2, and the 27- to 29-kDa IGFBP in plasma did not differ (P > .10) among breeds. Concentrations of GH, IGF-I, insulin, and PUN were greater (P < .001) and binding activities of the 22-kDa and 20-kDa IGFBP tended (P < .10) to be greater in plasma of Brahman than in Angus or Senepol cows. Plasma glucose concentrations were greater (P < .05) in Senepol than in Brahman or Angus cows. In conclusion, Brahman (Bos indicus) and Senepol cows (tropical Bos taurus) had greater numbers of follicles in all size categories and greater diameter of CL than Angus (temperate Bos taurus) cows. These ovarian differences may be due to changes in the pattern of secretion of FSH, insulin, IGF-I, and GH but not LH, IGF-II, or IGFBP-2 or -3.

Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets

PubMed Central

Möhle, Robert; Green, David; Moore, Malcolm A. S.; Nachman, Ralph L.; Rafii, Shahin

1997-01-01

We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial growth factor (VEGF). Megakaryocytes (CD41a+) were generated by ex vivo expansion of hematopoietic progenitor cells with kit-ligand and thrombopoietin for 10 days and further purified with immunomagnetic microbeads. Using reverse transcription–PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (121, 165, and 189 amino acids). Large quantities of VEGF (>1 ng/106 cells/3 days) were detected in the supernatant of Dami cells, ex vivo-generated megakaryocytes, and CD41a+ cells isolated from bone marrow. The constitutive secretion of VEGF by CD41a+ cells was stimulated by growth factors of the megakaryocytic lineage (interleukin 3, thrombopoietin). Western blotting of heparin–Sepharose-enriched supernatant mainly detected the isoform VEGF165. In addition, immunohistochemistry showed intracytoplasmic VEGF in polyploid megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner. Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells. VEGF delivered to sites of vascular injury by activated platelets may initiate angiogenesis. PMID:9012841

Astrocyte-Secreted Factors Selectively Alter Neural Stem and Progenitor Cell Proliferation in the Fragile X Mouse

PubMed Central

Sourial, Mary; Doering, Laurie C.

2016-01-01

An increasing body of evidence indicates that astrocytes contribute to the governance and fine tuning of stem and progenitor cell production during brain development. The effect of astrocyte function in cell production in neurodevelopmental disorders is unknown. We used the Neural Colony Forming Cell assay to determine the effect of astrocyte conditioned media (ACM) on the generation of neurospheres originating from either progenitor cells or functional stem cells in the knock out (KO) Fragile X mouse model. ACM from both normal and Fmr1-KO mice generated higher percentages of smaller neurospheres indicative of restricted proliferation of the progenitor cell population in Fmr1-KO brains. Wild type (WT) neurospheres, but not KO neurospheres, showed enhanced responses to ACM from the Fmr1-KO mice. In particular, Fmr1-KO ACM increased the percentage of large neurospheres generated, representative of spheres produced from neural stem cells. We also used 2D DIGE to initiate identification of the astrocyte-secreted proteins with differential expression between Fmr1-KO and WT cortices and hippocampi. The results further support the critical role of astrocytes in governing neural cell production in brain development and point to significant alterations in neural cell proliferation due to astrocyte secreted factors from the Fragile X brain. Highlights: • We studied the proliferation of neural stem and progenitor cells in Fragile X. • We examined the role of astrocyte-secreted factors in neural precursor cell biology. • Astrocyte-secreted factors with differential expression in Fragile X identified. PMID:27242437

Secreted Proteins Defy the Expression Level–Evolutionary Rate Anticorrelation

PubMed Central

Feyertag, Felix; Berninsone, Patricia M.; Alvarez-Ponce, David

2017-01-01

The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E–R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein–protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E–R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E–R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E–R anticorrelation. PMID:28007979

Effect of erythropoietin on mesenchymal stem cell differentiation and secretion in vitro in an acute kidney injury microenvironment.

PubMed

Liu, N M; Tian, J; Wang, W W; Han, G F; Cheng, J; Huang, J; Zhang, J Y

2013-02-28

We investigated the effect of erythropoietin (EPO) on differentiation and secretion of bone marrow-derived mesenchymal stem cells in an acute kidney injury microenvironment. Acute kidney injury mouse models were prepared. Both renal cortices were then immediately collected to produce the ischemia/reperfusion kidney homogenate supernatant. The morphological and ultrastructural changes in the cells were observed using an inverted microscope and a transmission electron microscope. Cytokeratin-18 was detected using flow cytometry. Bone morphogenetic protein-7 levels, hepatocyte growth factor, and vascular endothelial growth factor in the culture medium were detected using an enzyme-linked immunosorbent assay. The cells had high CD29 and CD44 expression, as well as low CD34 and CD45 expression. More round and oval cells with cobble-like appearances were observed after EPO treatment. In addition, an increase in the number of rough endoplasmic reticula, lysosomes, and mitochondria was observed in the cytoplasm; the intercellular junction peculiar to epithelial cells was also seen on the cell surface. After treatment with ischemia/reperfusion kidney homogenate supernatant, cytokeratin-18 expression increased significantly and EPO could magnify its expression. Bone morphogenetic protein-7 levels, hepatocyte growth factor, and vascular endothelial growth factor levels after treatment with ischemia/reperfusion kidney homogenate supernatant significantly decreased, whereas EPO increased the cytokine secretion. The acute kidney injury microenvironment can induce the bone marrow-derived mesenchymal stem cells to partially differentiate into renal tubular epithelium-shaped cells, but weaken their secretion function. EPO intervention can boost up their differentiation function and reverse their low secretion effect.

SIMPL Systems, or: Can We Design Cryptographic Hardware without Secret Key Information?

NASA Astrophysics Data System (ADS)

Rührmair, Ulrich

This paper discusses a new cryptographic primitive termed SIMPL system. Roughly speaking, a SIMPL system is a special type of Physical Unclonable Function (PUF) which possesses a binary description that allows its (slow) public simulation and prediction. Besides this public key like functionality, SIMPL systems have another advantage: No secret information is, or needs to be, contained in SIMPL systems in order to enable cryptographic protocols - neither in the form of a standard binary key, nor as secret information hidden in random, analog features, as it is the case for PUFs. The cryptographic security of SIMPLs instead rests on (i) a physical assumption on their unclonability, and (ii) a computational assumption regarding the complexity of simulating their output. This novel property makes SIMPL systems potentially immune against many known hardware and software attacks, including malware, side channel, invasive, or modeling attacks.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2014-05-27

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2014-10-28

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2015-04-14

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2013-10-01

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

An immunohistochemical study of the endocrine pancreas in raptors.

PubMed

Palmieri, C; Shivaprasad, H L

2014-12-01

The cytoarchitecture of the endocrine pancreas of 10 raptors (golden eagles, peregrine falcons, Saker falcon, turkey vultures, red-tailed hawk and unspecified falcon) was examined by immunohistochemistry. Three islet types were identified: type A mixed islets composed mainly by glucagon (A)-secreting cells, type B mixed islets with predominantly insulin (B)-secreting cell component and type M mixed islets (type M) consisting of variable number of glucagon-, insulin- and somatostatin (D)-secreting cells. The latter were further characterized into Type I, II or III according to the cell distribution of the three cell types. A and D cells were also randomly scattered within the exocrine pancreas. The results of this study suggest that the classical concept in birds of a segregation of A and B cells in well-defined and distinct islets is not applicable in raptors, reflecting an evolutionary adaptation to different dietary habits and variation in developmental mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Model of bicarbonate secretion by resting frog stomach fundus mucosa. II. Role of the oxyntopeptic cells.

PubMed

Debellis, L; Iacovelli, C; Frömter, E; Curci, S

1994-10-01

In the present publication we report mainly electrophysiological studies on oxyntopeptic cells of frog gastric mucosa which aim at clarifying a possible involvement of these cells in the process of resting gastric alkali (HCO3-) secretion, described in the preceding publication. The experiments were performed on intact gastric fundus mucosa of Rana esculenta mounted in Ussing chambers. After removal of the muscle and connective tissue layer oxyntopeptic cells were punctured from the serosal surface with conventional or pH-sensitive microelectrodes to measure, besides transepithelial voltage and resistance, the basolateral cell membrane potential, the voltage divider ratio, and the cell pH in response to secretagogues and/or changes in serosal ion concentration. Carbachol (10(-4) mol/l), which transiently stimulated HCO3- secretion by 0.22 mumol.cm-2.h-1, transiently acidified the cells by 0.09 +/- SEM 0.03 pH units (n = 6) and transiently induced an apical cell membrane anion conductance. According to the model of gastric HCO3- secretion presented in the preceding publication, this anion conductance could be involved in gastric HCO3- secretion, mediating, besides Cl- efflux, also apical HCO3- efflux. In addition carbachol stimulated basolateral Na+(HCO3-)n-cotransport, which according to the results from the preceding publication mediates basolateral HCO3- uptake for secretion. By contrast, cAMP-mediated secretagogues, such as histamine or others, which stimulate HCl secretion and transiently alkalinize the oxyntopeptic cells, were found to down-regulate the basolateral Na+(HCO3-)n-cotransporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of cell survival, growth, migration, and mesenchymal transdifferentiation effects of fibroblast secretome on tumor cells of head and neck squamous cell carcinoma.

PubMed

Metzler, Veronika Maria; Pritz, Christian; Riml, Anna; Romani, Angela; Tuertscher, Raphaela; Steinbichler, Teresa; Dejaco, Daniel; Riechelmann, Herbert; Dudás, József

2017-11-01

Fibroblasts play a central role in tumor invasion, recurrence, and metastasis in head and neck squamous cell carcinoma. The aim of this study was to investigate the influence of tumor cell self-produced factors and paracrine fibroblast-secreted factors in comparison to indirect co-culture on cancer cell survival, growth, migration, and epithelial-mesenchymal transition using the cell lines SCC-25 and human gingival fibroblasts. Thereby, we particularly focused on the participation of the fibroblast-secreted transforming growth factor beta-1.Tumor cell self-produced factors were sufficient to ensure tumor cell survival and basic cell growth, but fibroblast-secreted paracrine factors significantly increased cell proliferation, migration, and epithelial-mesenchymal transition-related phenotype changes in tumor cells. Transforming growth factor beta-1 generated individually migrating disseminating tumor cell groups or single cells separated from the tumor cell nest, which were characterized by reduced E-cadherin expression. At the same time, transforming growth factor beta-1 inhibited tumor cell proliferation under serum-starved conditions. Neutralizing transforming growth factor beta antibody reduced the cell migration support of fibroblast-conditioned medium. Transforming growth factor beta-1 as a single factor was sufficient for generation of disseminating tumor cells from epithelial tumor cell nests, while other fibroblast paracrine factors supported tumor nest outgrowth. Different fibroblast-released factors might support tumor cell proliferation and invasion, as two separate effects.

29 CFR 95.36 - Intangible property.

Code of Federal Regulations, 2011 CFR

2011-07-01

... excludes physical objects (e.g., laboratory samples). Research data also do not include: (A) Trade secrets... privacy, such as information that could be used to identify a particular person in a research study. (ii... Freedom of Information Act (FOIA) request for research data relating to published research findings...

29 CFR 95.36 - Intangible property.

Code of Federal Regulations, 2012 CFR

2012-07-01

... excludes physical objects (e.g., laboratory samples). Research data also do not include: (A) Trade secrets... privacy, such as information that could be used to identify a particular person in a research study. (ii... Freedom of Information Act (FOIA) request for research data relating to published research findings...

29 CFR 95.36 - Intangible property.

Code of Federal Regulations, 2013 CFR

2013-07-01

... excludes physical objects (e.g., laboratory samples). Research data also do not include: (A) Trade secrets... privacy, such as information that could be used to identify a particular person in a research study. (ii... Freedom of Information Act (FOIA) request for research data relating to published research findings...

29 CFR 95.36 - Intangible property.

Code of Federal Regulations, 2010 CFR

2010-07-01

... excludes physical objects (e.g., laboratory samples). Research data also do not include: (A) Trade secrets... privacy, such as information that could be used to identify a particular person in a research study. (ii... Freedom of Information Act (FOIA) request for research data relating to published research findings...

29 CFR 95.36 - Intangible property.

Code of Federal Regulations, 2014 CFR

2014-07-01

... excludes physical objects (e.g., laboratory samples). Research data also do not include: (A) Trade secrets... privacy, such as information that could be used to identify a particular person in a research study. (ii... Freedom of Information Act (FOIA) request for research data relating to published research findings...

Virulence from vesicles: Novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain.

PubMed

Kunsmann, Lisa; Rüter, Christian; Bauwens, Andreas; Greune, Lilo; Glüder, Malte; Kemper, Björn; Fruth, Angelika; Wai, Sun Nyunt; He, Xiaohua; Lloubes, Roland; Schmidt, M Alexander; Dobrindt, Ulrich; Mellmann, Alexander; Karch, Helge; Bielaszewska, Martina

2015-08-18

The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and enteroaggregative E. coli, but their secretion pathways are unknown. We demonstrate that the outbreak strain releases a cocktail of virulence factors via outer membrane vesicles (OMVs) shed during growth. The OMVs contain Shiga toxin (Stx) 2a, the major virulence factor of the strain, Shigella enterotoxin 1, H4 flagellin, and O104 lipopolysaccharide. The OMVs bind to and are internalised by human intestinal epithelial cells via dynamin-dependent and Stx2a-independent endocytosis, deliver the OMV-associated virulence factors intracellularly and induce caspase-9-mediated apoptosis and interleukin-8 secretion. Stx2a is the key OMV component responsible for the cytotoxicity, whereas flagellin and lipopolysaccharide are the major interleukin-8 inducers. The OMVs represent novel ways for the E. coli O104:H4 outbreak strain to deliver pathogenic cargoes and injure host cells.

Induction of virulence factors in Giardia duodenalis independent of host attachment

PubMed Central

Emery, Samantha J.; Mirzaei, Mehdi; Vuong, Daniel; Pascovici, Dana; Chick, Joel M.; Lacey, Ernest; Haynes, Paul A.

2016-01-01

Giardia duodenalis is responsible for the majority of parasitic gastroenteritis in humans worldwide. Host-parasite interaction models in vitro provide insights into disease and virulence and help us to understand pathogenesis. Using HT-29 intestinal epithelial cells (IEC) as a model we have demonstrated that initial sensitisation by host secretions reduces proclivity for trophozoite attachment, while inducing virulence factors. Host soluble factors triggered up-regulation of membrane and secreted proteins, including Tenascins, Cathepsin-B precursor, cystatin, and numerous Variant-specific Surface Proteins (VSPs). By comparison, host-cell attached trophozoites up-regulated intracellular pathways for ubiquitination, reactive oxygen species (ROS) detoxification and production of pyridoxal phosphate (PLP). We reason that these results demonstrate early pathogenesis in Giardia involves two independent host-parasite interactions. Motile trophozoites respond to soluble secreted signals, which deter attachment and induce expression of virulence factors. Trophozoites attached to host cells, in contrast, respond by up-regulating intracellular pathways involved in clearance of ROS, thus anticipating the host defence response. PMID:26867958

Uterine flushing proteome of the tammar wallaby after reactivation from diapause.

PubMed

Martin, Florine C; Ang, Ching-Seng; Gardner, David K; Renfree, Marilyn B; Shaw, Geoff

2016-11-01

The marsupial tammar wallaby has the longest period of embryonic diapause of any mammal, up to 11 months, during which there is no cell division or blastocyst growth. Since the blastocyst in diapause is surrounded by acellular coats, the signals that maintain or terminate diapause involve factors that reside in uterine secretions. The nature of such factors remains to be resolved. In this study, uterine flushings (UFs) were used to assess changes in uterine secretions of tammars using liquid chromatography-mass spectrometry (LC-MS/MS) during diapause (day 0 and 3) and reactivation days (d) 4, 5, 6, 8, 9, 11 and 24 after removal of pouch young (RPY), which initiates embryonic development. This study supports earlier suggestions that the presence of specific factors stimulate reactivation, early embryonic growth and cell proliferation. A mitogen, hepatoma-derived growth factor and soluble epidermal growth factor receptors were observed from d3 until at least d11 RPY when these secreted proteins constituted 21% of the UF proteome. Binding of these factors to specific cellular receptors or growth factors may directly stimulate DNA synthesis and division in endometrial gland cells. Proteins involved in the p53/CDKN1A (p21) cell cycle inhibition pathway were also observed in the diapause samples. Progesterone and most of the oestrogen-regulated proteins were present in the UF after d3, which is concomitant with the start of blastocyst mitoses at d4. We propose that once the p21 inhibition of the cell cycle is lost, growth factors including HDGF and EGFR are responsible for reactivation of the diapausing blastocyst via the uterine secretions. © 2016 Society for Reproduction and Fertility.

Mucinous (colloid) adenocarcinomas secrete distinct O-acylated forms of sialomucins: a histochemical study of gastric, colorectal and breast adenocarcinomas.

PubMed

Sáez, C; Japón, M A; Poveda, M A; Segura, D I

2001-12-01

Mucinous (colloid) adenocarcinomas represent a distinct group of tumours defined by the presence of large amounts of extracellular mucins. By using histochemical methods, we analysed mucins secreted by mucinous versus non-mucinous adenocarcinomas and looked for differential secretion profiles. Sixty-four adenocarcinomas were studied (23 colorectal, 17 gastric, and 24 breast tumours). Thirty-two tumours were of the colloid type. The following methods were applied to paraffin tissue sections: (i) Alcian blue (pH 2.5) and periodic acid-Schiff (PAS); (ii) high iron diamine and Alcian blue (pH 2.5); (iii) periodic acid borohydride, potassium hydroxide, and PAS; (iv) periodic acid-thionine Schiff, potassium hydroxide, and PAS; and (v) periodic acid-borohydride and PAS. Most adenocarcinomas secreted acidic mucins, with sialomucins predominating over sulfomucins, except for non-mucinous adenocarcinomas of the breast which showed predominant neutral mucins. All mucinous adenocarcinomas contained C9-O-acyl sialic acid as mono, di(C8,C9)-, or tri(C7,C8,C9)-O-acyl forms. Acidic mucins secreted by the majority of non-colloid adenocarcinomas consisted of non-O-acylated sialomucins. C9-O-acylation of sialic acid is a characteristic feature of mucinous adenocarcinomas and can be readily detected by histochemical methods.

Saponins from the traditional medicinal plant Momordica charantia stimulate insulin secretion in vitro

PubMed Central

Keller, Amy C.; Ma, Jun; Kavalier, Adam; He, Kan; Brillantes, Anne-Marie B.; Kennelly, Edward J.

2012-01-01

The antidiabetic activity of Momordica charantia (L.), Cucurbitaceae, a widely-used treatment for diabetes in a number of traditional medicine systems, was investigated in vitro. Antidiabetic activity has been reported for certain saponins isolated from M. charantia. In this study insulin secretion was measured in MIN6 β-cells incubated with an ethanol extract, saponin-rich fraction, and five purified saponins and cucurbitane triterpenoids from M. charantia, 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al (1), momordicine I (2), momordicine II (3), 3-hydroxycucurbita-5,24-dien-19-al-7,23-di-O-β-glucopyranoside (4), and kuguaglycoside G (5). Treatments were compared to incubation with high glucose (27 mM) and the insulin secretagogue, glipizide (50 μM). At 125 μg/ml, an LC-ToF-MS characterized saponin-rich fraction stimulated insulin secretion significantly more than the DMSO vehicle, p=0.02. At concentrations 10 and 25 μg/ml, compounds 3 and 5 also significantly stimulated insulin secretion as compared to the vehicle, p≤0.007, and p= 0.002, respectively. This is the first report of a saponin-rich fraction, and isolated compounds from M. charantia, stimulating insulin secretion in an in vitro, static incubation assay. PMID:22133295

Photoacoustic analysis of the solubilization kinetics of pulmonary secretions from cystic fibrosis patients - secretor and non-secretor phenotypes

NASA Astrophysics Data System (ADS)

Barja, P. R.; Coelho, C. C.; Paiva, R. F.; Barboza, M. A.; Matos, L. C.; Matos, C. C. B.; Oliveira, L. V. F.

2010-03-01

Cystic fibrosis (CF) is an autosomal recessive inherited disease that increases viscoelasticity of pulmonary secretions. Affected patients are required to use therapeutic aerosols continuously. The expression of ABH glycoconjugates in exocrine secretions determines the nature of part of the carbohydrates present in these secretions, allowing the classification of individuals into the so-called "secretor" and "non secretor" phenotypes. The aim of this work was to employ photoacoustic (PA) measurements to monitor the solubilization kinetics of pulmonary secretions from CF patients, analyzing the influence of the secretor status in the solubilization kinetics of samples nebulized with different therapeutic aerosols. Sputum samples were obtained by spontaneous expectoration from positive and negative secretor CF patients. Each sample was nebulized with i) tobramycin, ii) alpha dornase, and iii) N-acetylcysteine in a PA cell; fitting of the data with the Boltzmann equation led to the determination of t0 (typical interaction time) and Δt (solubilization interval) for each curve. Differences between the secretor and non-secretor phenotypes were statistically significant in the groups for tobramycin and alpha dornase, but not for N-acetylcysteine. Results show that the secretor status influences the solubilization of pulmonary mucus of CF patients nebulized with tobramycin and alpha dornase.

Endocrinological control of growth.

PubMed

Sizonenko, P C

1978-01-01

Many endocrinological factors control cellular growth of different tissues (cell multiplication and cell volume) and skeletal growth. The role of neuro-transmitters and of hypothalamic releasing and inhibiting factors of growth hormone secretion will be reviewed. The importance of the somatomedins on cartilage growth will be stressed. Thyroid hormones, androgens, and oestrogens have important stimulating actions on skeletal growth and maturation. Conversely, glucocorticoids have an important inhibitory effect on growth. The precise roles of these hormone factors in the regulation of growth hormone secretion, somatomedin production and tissue growth, particularly the cartilage, remain to be completely elucidated.

The Crystal Structure of a Binary Complex of Two Pseudopilins: EpsI And EpsJ From the Type 2 Secretion System of Vibrio Vulnificus

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

Yanez, M.E.; Korotkov, K.V.; Abendroth, J.

2009-05-28

Type II secretion systems (T2SS) translocate virulence factors from the periplasmic space of many pathogenic bacteria into the extracellular environment. The T2SS of Vibrio cholerae and related species is called the extracellular protein secretion (Eps) system that consists of a core of multiple copies of 11 different proteins. The pseudopilins, EpsG, EpsH, EpsI, EpsJ and EpsK, are five T2SS proteins that are thought to assemble into a pseudopilus, which is assumed to interact with the outer membrane pore, and may actively participate in the export of proteins. We report here biochemical evidence that the minor pseudopilins EpsI and EpsJ frommore » Vibrio species interact directly with one another. Moreover, the 2.3 {angstrom} resolution crystal structure of a complex of EspI and EpsJ from Vibrio vulnificus represents the first atomic resolution structure of a complex of two different pseudopilin components from the T2SS. Both EpsI and EpsJ appear to be structural extremes within the family of type 4a pilin structures solved to date, with EpsI having the smallest, and EpsJ the largest, 'variable pilin segment' seen thus far. A high degree of sequence conservation in the EpsI:EpsJ interface indicates that this heterodimer occurs in the T2SS of a large number of bacteria. The arrangement of EpsI and EpsJ in the heterodimer would correspond to a right-handed helical character of proteins assembled into a pseudopilus.« less

Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB

PubMed Central

Lieberman, Joshua A.; Frost, Nicholas A.; Hoppert, Michael; Fernandes, Paula J.; Vogt, Stefanie L.; Raivio, Tracy L.; Blanpied, Thomas A.

2012-01-01

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis. PMID:22247509

The multi-faceted outcomes of conjunct diabetes and cardiovascular familial history in type 2 diabetes.

PubMed

Hermans, Michel P; Ahn, Sylvie A; Rousseau, Michel F

2012-01-01

Familial history of early-onset CHD (EOCHD) is a major risk factor for CHD. Familial diabetes history (FDH) impacts β-cell function. Some transmissible, accretional gradient of CHD risk may exist when diabetes and EOCHD familial histories combine. We investigated whether the impact of such combination is neutral, additive, or potentiating in T2DM descendants, as regards cardiometabolic phenotype, glucose homeostasis and micro-/macroangiopathies. Cross-sectional retrospective cohort study of 796 T2DM divided according to presence (Diab[+]) or absence (Diab[-]) of 1st-degree diabetes familial history and/or EOCHD (CVD(+) and (-)). Four subgroups: (i) [Diab(-)CVD(-)] (n=355); (ii) [Diab(+)CVD(-)] (n=338); (iii) [Diab(-)CVD(+)] (n=47); and (iv) [Diab(+)CVD(+)] (n=56). No interaction on subgroup distribution between presence of both familial histories, the combination of which translated into additive detrimental outcomes and higher rates of fat mass, sarcopenia, (hs)CRP and retinopathy. FDH(+) had lower insulinemia, insulin secretion, hyperbolic product, and accelerated hyperbolic product loss. An EOCHD family history affected neither insulin secretion nor sensitivity. There were significant differences regarding macroangiopathy/CAD, more prevalent in [Diab(-)CVD(+)] and [Diab(+)CVD(+)]. Among CVD(+), the highest macroangiopathy prevalence was observed in [Diab(-)CVD(+)], who had 66% macroangiopathy, and 57% CAD, rates higher (absolute-relative) by 23%-53% (overall) and 21%-58% (CAD) than [Diab(+)CVD(+)], who inherited the direst cardiometabolic familial history (p 0.0288 and 0.0310). A parental history for diabetes markedly affects residual insulin secretion and secretory loss rate in T2DM offspring without worsening insulin resistance. It paradoxically translated into lower macroangiopathy with concurrent familial EOCHD. Conjunct diabetes and CV familial histories generate multi-faceted vascular outcomes in offspring, including lesser macroangiopathy/CAD. Copyright © 2012 Elsevier Inc. All rights reserved.

Rab3a Is Critical for Trapping Alpha-MSH Granules in the High Ca2+-Affinity Pool by Preventing Constitutive Exocytosis

PubMed Central

Sedej, Simon; Klemen, Maša Skelin; Schlüter, Oliver M.; Rupnik, Marjan Slak

2013-01-01

Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca2+-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca2+-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca2+] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α-MSH) and β-endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca2+-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca2+-sensitive release-ready vesicles as determined by slow photo-release of caged Ca2+. Radioimmunoassay revealed that α-MSH, but not β-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca2+-dependent release of α-MSH. PMID:24205339

Virulence factors encoded by Legionella longbeachae identified on the basis of the genome sequence analysis of clinical isolate D-4968.

PubMed

Kozak, Natalia A; Buss, Meghan; Lucas, Claressa E; Frace, Michael; Govil, Dhwani; Travis, Tatiana; Olsen-Rasmussen, Melissa; Benson, Robert F; Fields, Barry S

2010-02-01

Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.

Pleiotropic function of DLX3 in amelogenesis: from regulating pH and keratin expression to controlling enamel rod decussation.

PubMed

Duverger, Olivier; Morasso, Maria I

2018-12-01

DLX3 is essential for tooth enamel development and is so far the only transcription factor known to be mutated in a syndromic form of amelogenesis imperfecta. Through conditional deletion of Dlx3 in the dental epithelium in mouse, we have previously established the involvement of DLX3 in enamel pH regulation, as well as in controlling the expression of sets of keratins that contribute to enamel rod sheath formation. Here, we show that the decussation pattern of enamel rods was lost in conditional knockout animals, suggesting that DLX3 controls the coordinated migration of ameloblasts during enamel secretion. We further demonstrate that DLX3 regulates the expression of some components of myosin II complexes potentially involved in driving the movement of ameloblasts that leads to enamel rod decussation.

Structural studies of a bifunctional inhibitor of neprilysin and DPP-IV.

PubMed

Oefner, Christian; Pierau, Sabine; Schulz, Henk; Dale, Glenn E

2007-09-01

Neutral endopeptidase (NEP) is the major enzyme involved in the metabolic inactivation of a number of bioactive peptides including the enkephalins, substance P, endothelin, bradykinin and atrial natriuretic factor, as well as the incretin hormone glucagon-like peptide 1 (GLP-1), which is a potent stimulator of insulin secretion. The activity of GLP-1 is also rapidly abolished by the serine protease dipeptidyl peptidase IV (DPP-IV), which led to an elevated interest in inhibitors of this enzyme for the treatment of type II diabetes. A dual NEP/DPP-IV inhibitor concept is proposed, offering an alternative strategy for the treatment of type 2 diabetes. Here, the synthesis and crystal structures of the soluble extracellular domain of human NEP (residues 52-749) complexed with the NEP, competitive and potent dual NEP/DPP-IV inhibitor MCB3937 are described.

Stretch-Induced Hypertrophy Activates NFkB-Mediated VEGF Secretion in Adult Cardiomyocytes

PubMed Central

Leychenko, Anna; Konorev, Eugene; Jijiwa, Mayumi; Matter, Michelle L.

2011-01-01

Hypertension and myocardial infarction are associated with the onset of hypertrophy. Hypertrophy is a compensatory response mechanism to increases in mechanical load due to pressure or volume overload. It is characterized by extracellular matrix remodeling and hypertrophic growth of adult cardiomyocytes. Production of Vascular Endothelial Growth Factor (VEGF), which acts as an angiogenic factor and a modulator of cardiomyocyte function, is regulated by mechanical stretch. Mechanical stretch promotes VEGF secretion in neonatal cardiomyocytes. Whether this effect is retained in adult cells and the molecular mechanism mediating stretch-induced VEGF secretion has not been elucidated. Our objective was to investigate whether cyclic mechanical stretch induces VEGF secretion in adult cardiomyocytes and to identify the molecular mechanism mediating VEGF secretion in these cells. Isolated primary adult rat cardiomyocytes (ARCMs) were subjected to cyclic mechanical stretch at an extension level of 10% at 30 cycles/min that induces hypertrophic responses. Cyclic mechanical stretch induced a 3-fold increase in VEGF secretion in ARCMs compared to non-stretch controls. This increase in stretch-induced VEGF secretion correlated with NFkB activation. Cyclic mechanical stretch-mediated VEGF secretion was blocked by an NFkB peptide inhibitor and expression of a dominant negative mutant IkBα, but not by inhibitors of the MAPK/ERK1/2 or PI3K pathways. Chromatin immunoprecipitation assays demonstrated an interaction of NFkB with the VEGF promoter in stretched primary cardiomyocytes. Moreover, VEGF secretion is increased in the stretched myocardium during pressure overload-induced hypertrophy. These findings are the first to demonstrate that NFkB activation plays a role in mediating VEGF secretion upon cyclic mechanical stretch in adult cardiomyocytes. Signaling by NFkB initiated in response to cyclic mechanical stretch may therefore coordinate the hypertrophic response in adult cardiomyocytes. Elucidation of this novel mechanism may provide a target for developing future pharmacotherapy to treat hypertension and heart disease. PMID:22174951

Effect of thyrotropin-releasing factor on serum thyroid-stimulating hormone

PubMed Central

Costom, Bruce H.; Grumbach, Melvin M.; Kaplan, Selna L.

1971-01-01

To test the hypothesis that the primary defect in some patients with idiopathic hypopituitary dwarfism is failure to secrete hypothalamic hypophysiotropic-releasing factors, synthetic thyrotropin-releasing factor (TRF), 500 μg, wa given intravenously, and timed venous samples obtained for determination of the concentration of plasma TSH by radioimmunoassay in three groups of subjects: (a) 11 patients without evidence of endocrine or systemic disease, (group I) (b) 8 with isolated growth hormone deficiency and normal thyroid function, (group II) and (c) 9 patients with idiopathic hypopituitary dwarfism and thyroid-stimulating hormone (TSH) deficiency (group III). The mean fasting plasma TSH value was 4.1 μU/ml in group I, and 3.9 μU/ml in group II; in both groups there was a brisk rise in plasma TSH to peak levels of 12-45 μU/ml at 30-45 min, and a fall toward base line levels at 120 min. All children in group III had basal TSH levels of < 1.5 μU/ml; one failed to respond to TRF; eight exhibited a rise in plasma TSH with peak values comparable with those in groups I and II. In four of eight children in group III who responded to TRF, the TSH response was delayed and the initial rise in plasma TSH was not detectable until 10-60 min. In these four patients, plasma TSH levels continued to rise at 120 min. The mean fasting concentration of plasma thyroxine iodide (T4) in subjects with normal thyroid function (groups I and II) was 5.6 μg/100 ml, and the mean plasma T4 level at 120 min was 6.6 μg/100 ml. This difference between fasting and postTRF plasma T4 was significant (P < 0.001) by paired analysis. Mean fasting plasma T4 concentration in group III patients was 1.3 μg/100 ml; after TRF a significant rise in T4 concentration was not detected in this group. The results indicate that TRF test is useful in distinguishing between primary hypothalamic and pituitary forms of TSH deficiency. In light of the evidence of TRF deficiency in eight of nine patients with idiopathic hypopituitary dwarfism, it seems likely that in these patients, other pituitary hormone deficiencies may be attributable to deficiency of their respective releasing factors. Images PMID:4330007

Biosurfactant mannosyl-erythritol lipid inhibits secretion of inflammatory mediators from RBL-2H3 cells.

PubMed

Morita, Yosuke; Tadokoro, Satoshi; Sasai, Masao; Kitamoto, Dai; Hirashima, Naohide

2011-12-01

Biosurfactant mannosyl-erythritol lipids (MELs) are glycolipids produced by microbes that have various biological activities. It has been reported that MELs exhibit excellent surface-activity and also various bioactivities, such as induction of cell differentiation and apoptosis. However, little is known about their action related to drug discovery or drug seeds. We investigated the effects of MELs on the secretion of inflammatory mediators from mast cells that play a central role in allergic responses. Mast cells secrete three kinds of inflammatory mediators and we quantified these secreted mediators by photometer or ELISA. The action mechanisms of MELs were studied by Ca(2+)-sensitive fluorescence dye and Western blotting of phosphorylated proteins. MELs inhibited exocytotic release by antigen stimulation in a dose-dependent manner. We also found that MELs inhibited antigen-induced secretion of leukotriene C(4) and cytokine TNF-α (tumor necrosis factor-α). The inhibitory action of MELs on mediator secretion was mediated by inhibition of Ca(2+) increase, phosphorylation of MAP kinases and SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) that serve as a molecular machinery for exocytotic membrane fusion. MELs have anti-inflammatory action inhibiting the secretion of inflammatory mediators from mast cells. MELs affects two of major intracellular signaling pathways including Ca(2+) increase and MAP kinases. MELs also inhibited the phosphorylation of SNARE proteins that is crucial for not only exocytosis but also intracellular vesicular trafficking. 2011 Elsevier B.V. All rights reserved.

Control of very low-density lipoprotein secretion by N-ethylmaleimide-sensitive factor and miR-33.

PubMed

Allen, Ryan M; Marquart, Tyler J; Jesse, Jordan J; Baldán, Angel

2014-06-20

Several reports suggest that antisense oligonucleotides against miR-33 might reduce cardiovascular risk in patients by accelerating the reverse cholesterol transport pathway. However, conflicting reports exist about the impact of anti-miR-33 therapy on the levels of very low-density lipoprotein-triglycerides (VLDL-TAG). We test the hypothesis that miR-33 controls hepatic VLDL-TAG secretion. Using therapeutic silencing of miR-33 and adenoviral overexpression of miR-33, we show that miR-33 limits hepatic secretion of VLDL-TAG by targeting N-ethylmaleimide-sensitive factor (NSF), both in vivo and in primary hepatocytes. We identify conserved sequences in the 3'UTR of NSF as miR-33 responsive elements and show that Nsf is specifically recruited to the RNA-induced silencing complex following induction of miR-33. In pulse-chase experiments, either miR-33 overexpression or knock-down of Nsf lead to decreased secretion of apolipoproteins and TAG in primary hepatocytes, compared with control cells. Importantly, Nsf rescues miR-33-dependent reduced secretion. Finally, we show that overexpression of Nsf in vivo increases global hepatic secretion and raises plasma VLDL-TAG. Together, our data reveal key roles for the miR-33-NSF axis during hepatic secretion and suggest that caution should be taken with anti-miR-33-based therapies because they might raise proatherogenic VLDL-TAG levels. © 2014 American Heart Association, Inc.

The production of coagulation factor VII by adipocytes is enhanced by tumor necrosis factor-α or isoproterenol.

PubMed

Takahashi, N; Yoshizaki, T; Hiranaka, N; Kumano, O; Suzuki, T; Akanuma, M; Yui, T; Kanazawa, K; Yoshida, M; Naito, S; Fujiya, M; Kohgo, Y; Ieko, M

2015-05-01

A relationship has been reported between blood concentrations of coagulation factor VII (FVII) and obesity. In addition to its role in coagulation, FVII has been shown to inhibit insulin signals in adipocytes. However, the production of FVII by adipocytes remains unclear. We herein investigated the production and secretion of FVII by adipocytes, especially in relation to obesity-related conditions including adipose inflammation and sympathetic nerve activation. C57Bl/6J mice were fed a low- or high-fat diet and the expression of FVII messenger RNA (mRNA) was then examined in adipose tissue. 3T3-L1 cells were used as an adipocyte model for in vitro experiments in which these cells were treated with tumor necrosis factor-α (TNF-α) or isoproterenol. The expression and secretion of FVII were assessed by quantitative real-time PCR, Western blotting and enzyme-linked immunosorbent assays. The expression of FVII mRNA in the adipose tissue of mice fed with high-fat diet was significantly higher than that in mice fed with low-fat diet. Expression of the FVII gene and protein was induced during adipogenesis and maintained in mature adipocytes. The expression and secretion of FVII mRNA were increased in the culture medium of 3T3-L1 adipocytes treated with TNF-α, and these effects were blocked when these cells were exposed to inhibitors of mitogen-activated kinases or NF-κB activation. The β-adrenoceptor agonist isoproterenol stimulated the secretion of FVII from mature adipocytes via the cyclic AMP/protein kinase A pathway. Blockade of secreted FVII with the anti-FVII antibody did not affect the phosphorylation of Akt in the isoproterenol-stimulated adipocytes. Obese adipose tissue produced FVII. The production and secretion of FVII by adipocytes was enhanced by TNF-α or isoproterenol via different mechanisms. These results indicate that FVII is an adipokine that plays an important role in the pathogenesis of obesity.

Irx1 regulates dental outer enamel epithelial and lung alveolar type II epithelial differentiation

PubMed Central

Yu, Wenjie; Li, Xiao; Eliason, Steven; Romero-Bustillos, Miguel; Ries, Ryan J.; Cao, Huojun; Amendt, Brad A.

2017-01-01

The Iroquois genes (Irx) appear to regulate fundamental processes that lead to cell proliferation, differentiation, and maturation during development. In this report, the Iroquois homeobox 1 (Irx1) transcription factor was functionally disrupted using a LacZ insert and LacZ expression demonstrated stage-specific expression during embryogenesis. Irx1 is highly expressed in the brain, lung, digits, kidney, testis and developing teeth. Irx1 null mice are neonatal lethal and this lethality it due to pulmonary immaturity. Irx1−/− mice show delayed lung maturation characterized by defective surfactant protein secretion and Irx1 marks a population of SP-C expressing alveolar type II cells. Irx1 is specifically expressed in the outer enamel epithelium (OEE), stellate reticulum (SR) and stratum intermedium (SI) layers of the developing tooth. Irx1 mediates dental epithelial cell differentiation in the lower incisors resulting in delayed growth of the lower incisors. Irx1 is specifically and temporally expressed during developmental stages and we have focused on lung and dental development in this report. Irx1+ cells are unique to the development of the incisor outer enamel epithelium, patterning of Lef-1+ and Sox2+ cells as well as a new marker for lung alveolar type II cells. Mechanistically, Irx1 regulates Foxj1 and Sox9 to control cell differentiation during development. PMID:28746823

Irx1 regulates dental outer enamel epithelial and lung alveolar type II epithelial differentiation.

PubMed

Yu, Wenjie; Li, Xiao; Eliason, Steven; Romero-Bustillos, Miguel; Ries, Ryan J; Cao, Huojun; Amendt, Brad A

2017-09-01

The Iroquois genes (Irx) appear to regulate fundamental processes that lead to cell proliferation, differentiation, and maturation during development. In this report, the Iroquois homeobox 1 (Irx1) transcription factor was functionally disrupted using a LacZ insert and LacZ expression demonstrated stage-specific expression during embryogenesis. Irx1 is highly expressed in the brain, lung, digits, kidney, testis and developing teeth. Irx1 null mice are neonatal lethal and this lethality it due to pulmonary immaturity. Irx1 -/- mice show delayed lung maturation characterized by defective surfactant protein secretion and Irx1 marks a population of SP-C expressing alveolar type II cells. Irx1 is specifically expressed in the outer enamel epithelium (OEE), stellate reticulum (SR) and stratum intermedium (SI) layers of the developing tooth. Irx1 mediates dental epithelial cell differentiation in the lower incisors resulting in delayed growth of the lower incisors. Irx1 is specifically and temporally expressed during developmental stages and we have focused on lung and dental development in this report. Irx1+ cells are unique to the development of the incisor outer enamel epithelium, patterning of Lef-1+ and Sox2+ cells as well as a new marker for lung alveolar type II cells. Mechanistically, Irx1 regulates Foxj1 and Sox9 to control cell differentiation during development. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Placental lactogen secretion during prolonged-pregnancy in the rat: the ovary plays a pivotal role in the control of placental function.

PubMed

Shiota, K; Furuyama, N; Takahashi, M

1991-10-01

The serum of rats at mid-pregnancy contains at least 2 distinct placental lactogen (PL)-like substances tentatively termed placental lactogen-alpha (PL-alpha) and placental lactogen-beta (PL-beta) (Endocrinol Japon 38: 533-540, 1991). We have investigated the secretory patterns of three placental lactogens (PL-alpha, PL-beta and placental lactogen-II) during normal pregnancy and in two prolonged-pregnancy models. Pregnancy was prolonged by the introduction of new corpora lutea by inducing ovulation on day 15 of pregnancy by successive treatments with PMSG (30 IU/rat, sc on day 12) and hCG (10 IU/rat, iv on day 14), and in the second model by progesterone implants on day 15 of pregnancy. During normal pregnancy, each of the 3 PLs exhibited only one secretory peak in the serum; PL-alpha and PL-beta on day 12 and placental lactogen II (PL-II) on day 20. Interestingly, in the rats with new sets of corpora lutea, serum PL-alpha and PL-beta levels began to increase again on day 18 and showed peaks on day 20 for PL-alpha and on day 22 for PL-beta. In this model, the initiation of PL-II secretion was not affected, but high levels were maintained until day 26, when parturition occurred. In rats receiving either PMSG or hCG, the secretory patterns of the PLs were similar to as those during normal pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble Factors from Biofilms of Wound Pathogens Modulate Human Bone Marrow-derived Stromal Cell Differentiation, Migration, Angiogenesis, and Cytokine Secretion

DTIC Science & Technology

2015-03-28

Becerra, Christopher R Rathbone and Joseph C Wenke Abstract Background: Chronic, non- healing wounds are often characterized by the persistence of bacteria...within biofilms - aggregations of cells encased within a self -produced polysaccharide matrix. Biofilm bacteria exhibit unique characteristics from...modulation of host-immune responses by secreting factors that promote wound healing . While these characteristics make MSCs an attractive therapeutic

Presynaptic elements involved in the maintenance of the neuromuscular junction

NASA Technical Reports Server (NTRS)

Burrows, G. H.

1984-01-01

Alterations in the neuromuscular junction were observed in rats preceding loss of muscle mass. In view of the possibility that these alterations involve changes in the secretion of myotrophic agents by presynaptic motor neurons, an investigation was undertaken to characterize a neuronall factor which is thought to be involved in the initiation and maintenance of cholinergic synapses. This factor, which is secreted into the incubation medium by NG108-15 neuroblastoma x glioma hybrid cells, induces the aggregation of nicotinic acetylcholine receptors on primary cultures of rat hindlimb myotubes. Previous attempts to purify this factor failed. Extensive washing of the NG108-15 cells with hepes-buffered salt solution followed by short (4 hour) collection times resulted in the collection of incubation medium containing maximal aggregation activity with as little as 5 ug secreted protein per ml of fresh medium. A three-fold increase in specific activity was obtained after anion exchange chromatography.

Vaccination with Irradiated Autologous Melanoma Cells Engineered to Secrete Human Granulocyte--Macrophage Colony-Stimulating Factor Generates Potent Antitumor Immunity in Patients with Metastatic Melanoma

NASA Astrophysics Data System (ADS)

Soiffer, Robert; Lynch, Thomas; Mihm, Martin; Jung, Ken; Rhuda, Catherine; Schmollinger, Jan C.; Hodi, F. Stephen; Liebster, Laura; Lam, Prudence; Mentzer, Steven; Singer, Samuel; Tanabe, Kenneth K.; Benedict Cosimi, A.; Duda, Rosemary; Sober, Arthur; Bhan, Atul; Daley, John; Neuberg, Donna; Parry, Gordon; Rokovich, Joseph; Richards, Laurie; Drayer, Jan; Berns, Anton; Clift, Shirley; Cohen, Lawrence K.; Mulligan, Richard C.; Dranoff, Glenn

1998-10-01

We conducted a Phase I clinical trial investigating the biologic activity of vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte--macrophage colony-stimulating factor in patients with metastatic melanoma. Immunization sites were intensely infiltrated with T lymphocytes, dendritic cells, macrophages, and eosinophils in all 21 evaluable patients. Although metastatic lesions resected before vaccination were minimally infiltrated with cells of the immune system in all patients, metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction (at least 80%), fibrosis, and edema in 11 of 16 patients examined. Antimelanoma cytotoxic T cell and antibody responses were associated with tumor destruction. These results demonstrate that vaccination with irradiated autologous melanoma cells engineered to secrete granulocyte--macrophage colony-stimulating factor stimulates potent antitumor immunity in humans with metastatic melanoma.

Pre-Treatment of Human Mesenchymal Stem Cells With Inflammatory Factors or Hypoxia Does Not Influence Migration to Osteoarthritic Cartilage and Synovium.

PubMed

Leijs, Maarten J C; van Buul, Gerben M; Verhaar, Jan A N; Hoogduijn, Martin J; Bos, Pieter K; van Osch, Gerjo J V M

2017-04-01

Mesenchymal stem cells (MSCs) are promising candidates as a cell-based therapy for osteoarthritis (OA), although current results are modest. Pre-treatment of MSCs before application might improve their therapeutic efficacy. Pre-treatment of MSCs with inflammatory factors or hypoxia will improve their migration and adhesion capacities toward OA-affected tissues. Controlled laboratory study. We used real-time polymerase chain reaction to determine the effects of different fetal calf serum (FCS) batches, platelet lysate (PL), hypoxia, inflammatory factors, factors secreted by OA tissues, and OA synovial fluid (SF) on the expression of 12 genes encoding chemokine or adhesion receptors. Migration of MSCs toward factors secreted by OA tissues was studied in vitro, and attachment of injected MSCs was evaluated in vivo in healthy and OA knees of male Wistar rats. Different FCS batches, PL, or hypoxia did not influence the expression of the migration and adhesion receptor genes. Exposure to inflammatory factors altered the expression of CCR1, CCR4, CD44, PDGFRα, and PDGFRβ. MSCs migrated toward factors secreted by OA tissues in vitro. Neither pre-treatment with inflammatory factors nor the presence of OA influenced MSC migration in vitro or adhesion in vivo. Factors secreted by OA tissues increase MSC migration in vitro. In vivo, no difference in MSC adhesion was found between OA and healthy knees. Pre-treatment with inflammatory factors influenced the expression of migration and adhesion receptors of MSCs but not their migration in vitro or adhesion in vivo. To improve the therapeutic capacity of intra-articular injection of MSCs, they need to remain intra-articular for a longer period of time. Pre-treatment of MSCs with hypoxia or inflammatory factors did not increase the migration or adhesion capacity of MSCs and will therefore not likely prolong their intra-articular longevity. Alternative approaches to prolong the intra-articular presence of MSCs should be developed to increase the therapeutic effect of MSCs in OA.

No association of coffee consumption with gastric ulcer, duodenal ulcer, reflux esophagitis, and non-erosive reflux disease: a cross-sectional study of 8,013 healthy subjects in Japan.

PubMed

Shimamoto, Takeshi; Yamamichi, Nobutake; Kodashima, Shinya; Takahashi, Yu; Fujishiro, Mitsuhiro; Oka, Masashi; Mitsushima, Toru; Koike, Kazuhiko

2013-01-01

Probably due to caffeine-induced gastric acid secretion, negative effects of coffee upon various upper-gastrointestinal diseases have been precariously accepted, despite the inadequate epidemiological evidence. Our aim is to evaluate the effect of coffee consumption on four major acid-related diseases: gastric ulcer (GU), duodenal ulcer (DU), reflux esophagitis (RE), and non-erosive reflux disease (NERD) based on the large-scale multivariate analysis. Of the 9,517 healthy adults, GU, DU, and RE were diagnosed by endoscopy, and NERD was diagnosed by the symptoms of heartburn and regurgitation without esophageal erosion. Associations between coffee consumption and the four disorders were evaluated, together with age, gender, body mass index (BMI), Helicobacter pylori (HP) infection status, pepsinogen I/II ratio, smoking, and alcohol. We further performed meta-analysis using the random effects model to redefine the relationship between coffee intake and peptic ulcer disease. The eligible 8,013 study subjects comprised of 5,451 coffee drinkers and 2,562 non-coffee drinkers. By univariate analysis, age, BMI, pepsinogen I/II ratio, smoking, and alcohol showed significant associations with coffee consumption. By multiple logistic regression analysis, positively correlated factors with significance were HP infection, current smoking, BMI, and pepsinogen I/II ratio for GU; HP infection, pepsinogen I/II ratio, and current smoking for DU; HP non-infection, male, BMI, pepsinogen I/II ratio, smoking, age, and alcohol for RE; younger age, smoking, and female for NERD. The meta-analyses could detect any association of coffee consumption with neither GU nor DU. In conclusion, there are no significant relationship between coffee consumption and the four major acid-related upper gastrointestinal disorders.

22 CFR 226.36 - Intangible property.

Code of Federal Regulations, 2013 CFR

2013-04-01

... excludes physical objects (e.g., laboratory samples). Research data also do not include: (A) Trade secrets... privacy, such as information that could be used to identify a particular person in a research study. (ii...) request for research data relating to published research findings produced under an award that were used...

22 CFR 226.36 - Intangible property.

Code of Federal Regulations, 2011 CFR

2011-04-01

... excludes physical objects (e.g., laboratory samples). Research data also do not include: (A) Trade secrets... privacy, such as information that could be used to identify a particular person in a research study. (ii...) request for research data relating to published research findings produced under an award that were used...

22 CFR 226.36 - Intangible property.

Code of Federal Regulations, 2014 CFR

2014-04-01

... excludes physical objects (e.g., laboratory samples). Research data also do not include: (A) Trade secrets... privacy, such as information that could be used to identify a particular person in a research study. (ii...) request for research data relating to published research findings produced under an award that were used...

22 CFR 226.36 - Intangible property.

Code of Federal Regulations, 2012 CFR

2012-04-01

... excludes physical objects (e.g., laboratory samples). Research data also do not include: (A) Trade secrets... privacy, such as information that could be used to identify a particular person in a research study. (ii...) request for research data relating to published research findings produced under an award that were used...

21 CFR 170.35 - Affirmation of generally recognized as safe (GRAS) status.

Code of Federal Regulations, 2010 CFR

2010-04-01

... as provided in § 170.30(e), except those subject to the NAS/NRC GRAS list survey (36 FR 20546...) Quantitative compositions. (h) Manufacturing process (excluding any trade secrets). (ii) Use of the substance... the substance in food, including: (a) References to qualitative and quantitative methods for...

21 CFR 170.35 - Affirmation of generally recognized as safe (GRAS) status.

Code of Federal Regulations, 2012 CFR

2012-04-01

... as provided in § 170.30(e), except those subject to the NAS/NRC GRAS list survey (36 FR 20546...) Quantitative compositions. (h) Manufacturing process (excluding any trade secrets). (ii) Use of the substance... the substance in food, including: (a) References to qualitative and quantitative methods for...

21 CFR 170.35 - Affirmation of generally recognized as safe (GRAS) status.

Code of Federal Regulations, 2011 CFR

2011-04-01

... as provided in § 170.30(e), except those subject to the NAS/NRC GRAS list survey (36 FR 20546...) Quantitative compositions. (h) Manufacturing process (excluding any trade secrets). (ii) Use of the substance... the substance in food, including: (a) References to qualitative and quantitative methods for...

21 CFR 170.35 - Affirmation of generally recognized as safe (GRAS) status.

Code of Federal Regulations, 2013 CFR

2013-04-01

... as provided in § 170.30(e), except those subject to the NAS/NRC GRAS list survey (36 FR 20546...) Quantitative compositions. (h) Manufacturing process (excluding any trade secrets). (ii) Use of the substance... the substance in food, including: (a) References to qualitative and quantitative methods for...

Effect of pH values on the extracellular polysaccharide secreted by Acidithiobacillus ferrooxidans during chalcopyrite bioleaching

NASA Astrophysics Data System (ADS)

Yu, Run-lan; Liu, Jing; Tan, Jian-xi; Zeng, Wei-min; Shi, Li-juan; Gu, Guo-hua; Qin, Wen-qing; Qiu, Guan-zhou

2014-04-01

The pH value plays an important role in the bioleaching of sulphide minerals. The effect of pH values on the extracellular polysaccharide secreted by Acidithiobacillus ferrooxidans was investigated in different phases of bacterial growth during chalcopyrite bioleaching. It is found that extracellular polysaccharide secretion from the cells attached to chalcopyrite is more efficiently than that of the free cells in the bioleaching solution. Three factors, pH values, the concentration of soluble metal ions, and the bacterial growth and metabolism, affect extracellular polysaccharide secretion in the free cells, and are related to the bacterial growth phase. Extracellular polysaccharide secretion from the attached cells is mainly dependent on the pH value of the bacterial culture.

Substance P as a putative efferent transmitter mediates GABAergic inhibition in mouse taste buds.

PubMed

Huang, Anthony Y; Wu, Sandy Y

2018-04-01

Capsaicin-mediated modulation of taste nerve responses is thought to be produced indirectly by the actions of neuropeptides, for example, CGRP and substance P (SP), on taste cells implying they play a role in taste sensitivity. During the processing of gustatory information in taste buds, CGRP shapes peripheral taste signals via serotonergic signalling. The underlying assumption has been that SP exerts its effects on taste transmitter secretion in taste buds of mice. To test this assumption, we investigated the net effect of SP on taste-evoked ATP secretion from mouse taste buds, using functional calcium imaging with CHO cells expressing high-affinity transmitter receptors as cellular biosensors. Our results showed that SP elicited PLC activation-dependent intracellular Ca 2+ transients in taste cells via neurokinin 1 receptors, most likely on glutamate-aspartate transporter-expressing Type I cells. Furthermore, SP caused Type I cells to secrete GABA. Combined with the recent findings that GABA depresses taste-evoked ATP secretion, the current results indicate that SP elicited secretion of GABA, which provided negative feedback onto Type II (receptor) cells to reduce taste-evoked ATP secretion. These findings are consistent with a role for SP as an inhibitory transmitter that shapes the peripheral taste signals, via GABAergic signalling, during the processing of gustatory information in taste buds. Notably, the results suggest that SP is intimately associated with GABA in mammalian taste signal processing and demonstrate an unanticipated route for sensory information flow within the taste bud. © 2018 The British Pharmacological Society.

Effect of mitomycin C on IL-1R expression, IL-1-related hepatocyte growth factor secretion and corneal epithelial cell migration.

PubMed

Chen, Tsan-Chi; Chang, Shu-Wen

2010-03-01

To investigate how mitomycin C (MMC) modulates hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) secretions in human corneal fibroblasts and regulates human corneal epithelial (HCE) cell migration. Primary human corneal fibroblasts were treated with MMC (0.05, 0.1, or 0.2 mg/mL for 5 minutes) and were cultivated with or without interleukin (IL)-1beta. Transcript and secretion of HGF and KGF were determined by quantitative real-time RT-PCR and Western blot analysis, respectively. The effect of MMC-treated fibroblasts on HCE cell migration was evaluated using a transwell migration assay. The influence of MMC on HGF expression/secretion and HCE cell migration was further confirmed by RNA interference. The number of IL-1 receptors (IL-1R) on the fibroblast surface was analyzed by flow cytometry. MMC alone did not affect endogenous HGF expression, whereas IL-1beta alone significantly upregulated HGF transcripts and secretion. By modifying IL-1R numbers, MMC further upregulated IL-1beta-related HGF expression at a concentration of 0.05 mg/mL but to a lesser extent at 0.1 and 0.2 mg/mL. KGF transcripts and intracellular expression were suppressed by MMC dose dependently in the presence or absence of IL-1beta, whereas KGF secretion was not affected. Conditioned medium from MMC-treated fibroblasts exerted a similar concentration-dependent effect on HCE cell migration, enhancing migration most significantly at 0.05 mg/mL MMC in the presence of IL-1beta. The MMC dose-dependent modulation of HCE cell migration was abolished in HGF-silenced fibroblasts. MMC differentially modulated IL-1R expression at various concentrations and regulated HGF and KGF differently. MMC alone did not alter HGF expression. In the presence of IL-1beta, MMC-treated corneal fibroblasts modified HCE cell migration through IL-1beta-induced HGF secretion.

Medicinal mushroom Lingzhi or Reishi, Ganoderma lucidum (W.Curt.:Fr.) P. Karst., beta-glucan induces Toll-like receptors and fails to induce inflammatory cytokines in NF-kappaB inhibitor-treated macrophages.

PubMed

Batbayar, Sainkhuu; Kim, Mi Jeong; Kim, Ha Won

2011-01-01

Beta-Glucan of medicinal Lingzhi or Reishi mushroom, Ganoderma lucidum (BGG), possesses immunostimulatory and anti-tumor activities. Innate immune cells are activated by the binding of beta-glucan to the dectin-1 receptor. The present study investigated the immunostimulating activities of BGG, including binding to dectin-1, secretion of cytokines and reactive oxygen species, and induction of Toll-like receptors (TLRs) in RAW264.7 mouse macrophages. Reverse transcription-polymerase chain reaction and flow cytometry were used for the cytokine and TLR analyses. A mouse inflammation antibody array was used for protein-level cytokine analysis. BGG bound to dectin-1 and induced RAW264.7 cell secretion of several cytokines, including granulocyte colony-stimulating factor, interleukin (IL)-6, regulated upon activation normal T cell expressed and secreted (RANTES), tissue inhibitor of metalloproteinase-1, and tumor necrosis factor-alpha. The secretion of these cytokines was further increased by the addition of lipopolysaccharide (LPS). BGG also induced both nitric oxide and inducible nitric oxide synthase (iNOS). Treatment with an inhibitor of nuclear factor-kappa B (NF-kappaB) reduced the induction of IL-1, IL-6, and iNOS in a concentration-dependent manner. Expressions of TLR2, TLR4, and TLR6 were increased by BGG treatment, and addition of LPS induced further induction of TLR4 and TLR6. Our result indicates that BGG induces macrophage secretion of inflammatory cytokines, which can be potentiated by the presence of LPS, likely by binding to dectin-1 and TLR-2/6 receptors, which activate NF-kappaB and prompt the secretion of cytokines.

Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures.

PubMed

Becerra-Arteaga, Alejandro; Mason, Hugh S; Shuler, Michael L

2006-01-01

Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.

Secretomes reveal several novel proteins as well as TGF-β1 as the top upstream regulator of metastatic process in breast cancer.

PubMed

Erin, Nuray; Ogan, Nur; Yerlikaya, Azmi

2018-03-20

Metastatic breast cancer is resistant to many conventional treatments and novel therapeutic targets are needed. We previously isolated subsets of 4T1 murine breast cancer cells which metastasized to liver (4TLM), brain (4TBM), and heart (4THM). Among these cells, 4TLM is the most aggressive one, demonstrating mesenchymal phenotype. Here we compared secreted proteins from 4TLM, 4TBM, and 4THM cells and compared with that of hardly metastatic 67NR cells to detect differentially secreted factors involved in organ-specific metastasis. Label-free LC-MS/MS proteomic technique was used to detect the differentially secreted proteins. Eighty-five of over 500 secreted proteins were significantly altered in metastatic breast cancer cells. Differential expression of several proteins such as fibulin-4, Bone Morphogenetic Protein 1, TGF-β1 MMP-3, MMP-9, and Thymic Stromal Lymphopoietin were further verified using ELISA or Western blotting. Many of these identified proteins were also present in human metastatic breast carcinomas. Annexin A1 and A5, laminin beta 1, Neutral alpha-glucosidase AB were commonly found at least in three out of six studies examined here. Ingenuity Pathway Analysis showed that proteins differentially secreted from metastatic cells are involved primarily in carcinogenesis and TGF-β1 is the top upstream regulator in all metastatic cells. Cells metastasized to different organs displayed significant differences in several of secreted proteins. Proteins differentially altered were fibronectin, insulin-like growth factor-binding protein 7, and Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1. On the other hand, many exosomal proteins were also common to all metastatic cells, demonstrating involvement of key universal factors in distant metastatic process.

Differential bioaccumulation and translocation patterns in three mangrove plants experimentally exposed to iron. Consequences for environmental sensing.

PubMed

Arrivabene, Hiulana Pereira; Campos, Caroline Quenupe; Souza, Iara da Costa; Wunderlin, Daniel Alberto; Milanez, Camilla Rozindo Dias; Machado, Silvia Rodrigues

2016-08-01

Avicennia schaueriana, Laguncularia racemosa and Rhizophora mangle were experimentally exposed to increasing levels of iron (0, 10, 20 and 100 mg L(-1) added Fe(II) in Hoagland's nutritive medium). The uptake and translocation of iron from roots to stems and leaves, Fe-secretion through salt glands (Avicennia schaueriana and Laguncularia racemosa) as well as anatomical and histochemical changes in plant tissues were evaluated. The main goal of this work was to assess the diverse capacity of these plants to detect mangroves at risk in an area affected by iron pollution (Vitoria, Espírito Santo, Brazil). Results show that plants have differential patterns with respect to bioaccumulation, translocation and secretion of iron through salt glands. L. racemosa showed the best environmental sensing capacity since the bioaccumulation of iron in both Fe-plaque and roots was higher and increased as the amount of added-iron rose. Fewer changes in translocation factors throughout increasing added-iron were observed in this species. Furthermore, the amount of iron secreted through salt glands of L. racemosa was strongly inhibited when exposed to added-iron. Among three studied species, A. schaueriana showed the highest levels of iron in stems and leaves. On the other hand, Rhizophora mangle presented low values of iron in these compartments. Even so, there was a significant drop in the translocation factor between aerial parts with respect to roots, since the bioaccumulation in plaque and roots of R. mangle increased as iron concentration rose. Moreover, rhizophores of R. mangle did not show changes in bioaccumulation throughout the studied concentrations. So far, we propose L. racemosa as the best species for monitoring iron pollution in affected mangroves areas. To our knowledge, this is the first detailed report on the response of these plants to increasing iron concentration under controlled conditions, complementing existing data on the behavior of the same plants under field exposure. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interleukin-13 associates with life-threatening rhinovirus infections in infants and young children.

PubMed

Caballero, Mauricio T; Hijano, Diego R; Acosta, Patricio L; Mateu, Cecilia G; Marcone, Débora N; Linder, Jodell E; Talarico, Laura B; Elder, John M; Echavarria, Marcela; Miller, Eva Kathryn; Polack, Fernando P

2018-04-17

Delineate risk factors associated with severe hypoxemia (O 2 sat ≤87%) in infants and children younger than 2 years hospitalized with single pathogen HRV infection. Prospective study in a yearly catchment population of 56 560 children <2 years old between 2011 and 2013 in Argentina. All children with respiratory signs and O 2 sat <93% on admission were included. HRV infections were identified by reverse transcriptase-polymerase chain reaction. Epidemiologic, clinical, viral, and immunological risk factors were assessed. Among 5012 hospitalized patients, HRV was detected as a single pathogen in 347 (6.92%) subjects. Thirty-two (9.2%) had life-threatening disease. Traditional risk factors for severe bronchiolitis did not affect severity of illness. HRV viral load, HRV groups, and type II and III interferons did not associate with severe hypoxemia. Interleukin-13 Levels in respiratory secretions at the time of admission (OR = 7.43 (3-18.4); P < 0.001 for IL-13 >10 pg/mL) predisposed to life-threatening disease. Targeted interventions against IL-13 should be evaluated to decrease severity of HRV illness in infancy and early childhood. © 2018 Wiley Periodicals, Inc.

HrpN of Erwinia amylovora functions in the translocation of DspA/E into plant cells.

PubMed

Bocsanczy, Ana M; Nissinen, Riitta M; Oh, Chang-Sik; Beer, Steven V

2008-07-01

The type III secretion system (T3SS) is required by plant pathogenic bacteria for the translocation of certain bacterial proteins to the cytoplasm of plant cells or secretion of some proteins to the apoplast. The T3SS of Erwinia amylovora, which causes fire blight of pear, apple and other rosaceous plants, secretes DspA/E, which is an indispensable pathogenicity factor. Several other proteins, including HrpN, a critical virulence factor, are also secreted by the T3SS. Using a CyaA reporter system, we demonstrated that DspA/E is translocated into the cells of Nicotiana tabacum'Xanthi'. To determine if other T3-secreted proteins are needed for translocation of DspA/E, we examined its translocation in several mutants of E. amylovora strain Ea321. DspA/E was translocated by both hrpW and hrpK mutants, although with some delay, indicating that these two proteins are dispensable in the translocation of DspA/E. Remarkably, translocation of DspA/E was essentially abolished in both hrpN and hrpJ mutants; however, secretion of DspA/E into medium was not affected in any of the mentioned mutants. In contrast to the more virulent strain Ea273, secretion of HrpN was abolished in a hrpJ mutant of strain Ea321. In addition, HrpN was weakly translocated into plant cytoplasm. These results suggest that HrpN plays a significant role in the translocation of DspA/E, and HrpJ affects the translocation of DspA/E by affecting secretion or stability of HrpN. Taken together, these results explain the critical importance of HrpN and HrpJ to the development of fire blight.

Mechanisms of estradiol-induced insulin secretion by the G protein-coupled estrogen receptor GPR30/GPER in pancreatic beta-cells.

PubMed

Sharma, Geetanjali; Prossnitz, Eric R

2011-08-01

Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes.

Differential profiles of immune mediators and in vitro HIV infectivity between endocervical and vaginal secretions from women with Chlamydia trachomatis infection: a pilot study.

PubMed

Sperling, Rhoda; Kraus, Thomas A; Ding, Jian; Veretennikova, Alina; Lorde-Rollins, Elizabeth; Singh, Tricia; Lo, Yungtai; Quayle, Alison J; Chang, Theresa L

2013-09-01

Chlamydia trachomatis infection is one of the most prevalent bacterial STIs in the USA and worldwide, and women with C. trachomatis infection are at increased risk of acquiring HIV. Because immune activation at the genital mucosa facilitates HIV/SIV infection, C. trachomatis-mediated cytokine induction may contribute to increased HIV transmission in asymptomatic women. To begin to elucidate the mechanisms, we longitudinally analyzed profiles of innate immune factors and HIV infectivity in genital secretions from anatomically specific sites in asymptomatic women during C. trachomatis infection and post-antibiotic treatment. We found higher levels of cytokines and chemokines in endocervical secretions than vaginal secretions. Compared with the convalescent state, G-CSF, IL-1α, and RANTES were elevated in endocervical secretions, IFN-γ and TNF-α were elevated in vaginal secretions, and IFNγ, IL-1β, and MIP1-α were elevated in cervicolavage fluid (CVL), before adjustment of multiple comparisons. Elevated endocervical levels of IP-10 and MCP-1 were associated with the use of hormonal contraception in infected women after successful treatment, suggesting the role of hormonal contraception in inflammation independent of STIs. Importantly, soluble factors found in endocervical secretions during infection enhanced HIV infectivity while no difference in HIV infectivity was found with vaginal secretions or CVL during infection or at convalescence. Taken together, the profiles of immune mediators and in vitro HIV infectivity indicate that the endocervical and vaginal mucosa are immunologically distinct. Our results underscore the importance of considering anatomical site and local sampling methodology when measuring mucosal responses, particularly in the presence of C. trachomatis infection. Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Lipopolysaccharide-induced IL-18 secretion from murine Kupffer cells independently of myeloid differentiation factor 88 that is critically involved in induction of production of IL-12 and IL-1beta.

PubMed

Seki, E; Tsutsui, H; Nakano, H; Tsuji, N; Hoshino, K; Adachi, O; Adachi, K; Futatsugi, S; Kuida, K; Takeuchi, O; Okamura, H; Fujimoto, J; Akira, S; Nakanishi, K

2001-02-15

IL-18, produced as biologically inactive precursor, is secreted from LPS-stimulated macrophages after cleavage by caspase-1. In this study, we investigated the mechanism underlying caspase-1-mediated IL-18 secretion. Kupffer cells constantly stored IL-18 and constitutively expressed caspase-1. Inhibition of new protein synthesis only slightly reduced IL-18 secretion, while it decreased and abrogated their IL-1beta and IL-12 secretion, respectively. Kupffer cells deficient in Toll-like receptor (TLR) 4, an LPS-signaling receptor, did not secrete IL-18, IL-1beta, and IL-12 upon LPS stimulation. In contrast, Kupffer cells lacking myeloid differentiation factor 88 (MyD88), an adaptor molecule for TLR-mediated-signaling, secreted IL-18 without IL-1beta and IL-12 production in a caspase-1-dependent and de novo synthesis-independent manner. These results indicate that MyD88 is essential for IL-12 and IL-1beta production from Kupffer cells while their IL-18 secretion is mediated via activation of endogenous caspase-1 without de novo protein synthesis in a MyD88-independent fashion after stimulation with LPS. In addition, infection with Listeria monocytogenes, products of which have the capacity to activate TLR, increased serum levels of IL-18 in wild-type and MyD88-deficient mice but not in caspase-1-deficient mice, whereas it induced elevation of serum levels of IL-12 in both wild-type and caspase-1-deficient mice but not in MyD88-deficient mice. Taken together, these results suggested caspase-1-dependent, MyD88-independent IL-18 release in bacterial infection.

Renin-angiotensin-aldosterone (RAAS): The ubiquitous system for homeostasis and pathologies.

PubMed

Patel, Seema; Rauf, Abdur; Khan, Haroon; Abu-Izneid, Tareq

2017-10-01

Renin-angiotensin-aldosterone system (RAAS) is a vital system of human body, as it maintains plasma sodium concentration, arterial blood pressure and extracellular volume. Kidney-secreted renin enzyme acts on its substrate to form angiotensin II, a versatile effector peptide hormone. Every organ is affected by RAAS activation and the resultant hypertension, cell proliferation, inflammation, and fibrosis. The imbalance of renin and angiotensin II can result in an overwhelming number of chronic and acute diseases. RAAS is influenced by other enzymes, hormones, pumps and signaling pathways, hence, this review discusses important facets of this system, its crosstalk with other crucial factors like estrogen, thyroid, cortisol, kallikrein-kinin system, Wnt/β-catenin signaling, and sodium-potassium pump. The nexus of RAAS with the above-discussed systems was scantily explored before. So, this review furnishes a new perspective in comprehension of inflammation diseases. It is followed by the formulation of hypotheses, which can contribute to better management of an array of pathologies plaguing mankind. Manipulation of RAAS, by bending it towards ACE2 expression can regulate endocrine functions, which can be critical for a number of pathological management. Dietary intervention can restore RAAS to normalcy. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Type-IVC Secretion System: A Novel Subclass of Type IV Secretion System (T4SS) Common Existing in Gram-Positive Genus Streptococcus

PubMed Central

Chen, Chen; Gao, George F.

2012-01-01

A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. Type IV secretion system (T4SS) is one of versatile secretion systems essential for the virulence and even survival of some bacteria species, and they enable the secretion of protein and DNA substrates across the cell envelope. T4SS was once believed to be present only in Gram-negative bacteria. In this study, we present evidence of a new subclass of T4SS, Type-IVC secretion system and indicate its common existence in the Gram-positive bacterial genus Streptococcus. We further identified that VirB1, VirB4, VirB6 and VirD4 are the minimal key components of this system. Using genome comparisons and evolutionary relationship analysis, we proposed that Type-IVC secretion system is movable via transposon factors and mediates the conjugative transfer of DNA, enhances bacterial pathogenicity, and could cause large-scale outbreaks of infections in humans. PMID:23056296

Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells

PubMed Central

Zavašnik-Bergant, Tina; Bergant Marušič, Martina

2016-01-01

Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii. PMID:26960148

Cloning and functional characterization of a fatty acid transport protein (FATP) from the pheromone gland of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone.

PubMed

Qian, Shuguang; Fujii, Takeshi; Ito, Katsuhiko; Nakano, Ryo; Ishikawa, Yukio

2011-01-01

Sex pheromones of moths are largely classified into two types based on the presence (Type I) or absence (Type II) of a terminal functional group. While Type-I sex pheromones are synthesized from common fatty acids in the pheromone gland (PG), Type-II sex pheromones are derived from hydrocarbons produced presumably in the oenocytes and transported to the PG via the hemolymph. Recently, a fatty acid transport protein (BmFATP) was identified from the PG of the silkworm Bombyx mori, which produces a Type-I sex pheromone (bombykol). BmFATP was shown to facilitate the uptake of extracellular fatty acids into PG cells for the synthesis of bombykol. To elucidate the presence and function of FATP in the PG of moths that produce Type-II sex pheromones, we explored fatp homologues expressed in the PG of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone (Type II). A fatp homologue cloned from E. japonica (Ejfatp) was predominantly expressed in the PG, and its expression is upregulated shortly after eclosion. Functional expression of EjFATP in Escherichia coli enhanced the uptake of long chain fatty acids (C₁₈ and C₂₀), but not pheromone precursor hydrocarbons. To the best of our knowledge, this is the first report of the cloning and functional characterization of a FATP in the PG of a moth producing a Type-II sex pheromone. Although EjFATP is not likely to be involved in the uptake of pheromone precursors in E. japonica, the expression pattern of Ejfatp suggests a role for EjFATP in the PG not directly linked to pheromone biosynthesis. Copyright © 2010 Elsevier Ltd. All rights reserved.

Cell surface engineering of Bacillus subtilis improves production yields of heterologously expressed α-amylases.

PubMed

Cao, Haojie; van Heel, Auke J; Ahmed, Hifza; Mols, Maarten; Kuipers, Oscar P

2017-04-04

Bacillus subtilis is widely used as a cell factory for numerous heterologous proteins of commercial value and medical interest. To explore the possibility of further enhancing the secretion potential of this model bacterium, a library of engineered strains with modified cell surface components was constructed, and the corresponding influences on protein secretion were investigated by analyzing the secretion of α-amylase variants with either low-, neutral- or high- isoelectric points (pI). Relative to the wild-type strain, the presence of overall anionic membrane phospholipids (phosphatidylglycerol and cardiolipin) increased dramatically in the PssA-, ClsA- and double KO mutants, which resulted in an up to 47% higher secretion of α-amylase. Additionally, we demonstrated that the appropriate net charge of secreted targets (AmyTS-23, AmyBs and AmyBm) was beneficial for secretion efficiency as well. In B. subtilis, the characteristics of cell membrane phospholipid bilayer and the pIs of heterologous α-amylases appear to be important for their secretion efficiency. These two factors can be engineered to reduce the electrostatic interaction between each other during the secretion process, which finally leads to a better secretion yield of α-amylases.

Production of Human Monoclonal Rheumatoid Factor Secreting Hybridomas Derived from Rheumatoid Synovial Cells

DTIC Science & Technology

1989-01-01

lymphocytes obtained from patients with other We were then interested to see whether AD7 RF had types of arthritis who were seronegative were also...seropositive rheumatoid anhrius patients and from synowal cells in arthritis patients seroneganve for rheumatoidfactor. 1.0 Patients No. of hybridso 1gM RF...Production of human monoclonal rheumatoid factor secreting hybridomas derived from rheumatoid s’inovial cells 12. PEFTONAL AUTVOR(S) Robbins DL, Kenny

Absence of CD9 reduces endometrial VEGF secretion and impairs uterine repair after parturition.

PubMed

Kawano, Natsuko; Miyado, Kenji; Yoshii, Noriko; Kanai, Seiya; Saito, Hidekazu; Miyado, Mami; Inagaki, Noboru; Odawara, Yasushi; Hamatani, Toshio; Umezawa, Akihiro

2014-04-16

In mammals, uterine epithelium is remodeled cyclically throughout adult life for pregnancy. Despite the expression of CD9 in the uterine epithelium, its role in maternal reproduction is unclear. Here, we addressed this issue by examining uterine secretions collected from patients undergoing fertility treatment and fertilization-competent Cd9(-/-) mice expressing CD9-GFP in their eggs (Cd9(-/-)TG). CD9 in uterine secretions was observed as extracellular matrix-like feature, and its amount of the secretions associated with repeated pregnancy failures. We also found that the litter size of Cd9(-/-)TG female mice was significantly reduced after their first birth. Severely delayed re-epithelialization of the endometrium was then occurred. Concomitantly, vascular endothelial growth factor (VEGF) was remarkably reduced in the uterine secretions of Cd9(-/-)TG female mice. These results provide the first evidence that CD9-mediated VEGF secretion plays a role in re-epithelialization of the uterus.

[The effect of isoflurane on the secretion of TNF-alpha and IL-1 beta from LPS-stimulated human peripheral blood monocytes].

PubMed

Sato, W; Enzan, K; Masaki, Y; Kayaba, M; Suzuki, M

1995-07-01

The cytokines such as tumor necrosis factor and interleukin-1 secreted from macrophages/monocytes proved to play important roles in the pathogenesis of endotoxemia, severe pancreatitis and other surgical injuries. However, it is still unclear how inhalational anesthetic agents influence the secretion of these cytokines from macrophages/monocytes. We investigated the effects of isoflurane on TNF-alpha and IL-1 beta secretions from human peripheral blood monocytes stimulated by lipopolysaccharide. TNF-alpha and IL-1 beta secretions increased after LPS stimulation and this increase was inhibited by isoflurane in dose-dependent fashion. The inhibitory action of isoflurane disappeared between 1 and 3 hours after stopping isoflurane inhalation. We concluded that isoflurane could inhibit TNF-alpha and IL-1 beta secretions from peripheral blood monocytes stimulated by LPS in a dose-dependent fashion and that the inhibitory action of isoflurane was reversible.

Activation of innate immunity by prostate specific antigen (PSA).

PubMed

Kodak, James A; Mann, Dean L; Klyushnenkova, Elena N; Alexander, Richard B

2006-11-01

Prostate specific antigen (PSA) is a serine protease secreted by the prostatic epithelium. The only known function of the protein is to cleave seminogelin. We wished to determine if PSA activated peripheral blood mononuclear cells (PBMC). PBMC and selected sub-populations were cultured with purified PSA. Secretion of IFNgamma was measured by cytokine capture flow cytometry and enzyme-linked immunosorbent assay. We observed secretion of IFNgamma and a proliferative response in PBMC cultured with PSA. We found that NK cells were the source of the IFNgamma but NK cells were not directly stimulated by PSA. Rather, a soluble factor secreted primarily by CD14 monocytes in response to PSA stimulated NK cells to secrete IFNgamma. PSA induces a pro-inflammatory response that results in the secretion of INFgamma by NK cells. The presence of large amounts of PSA could contribute to the common finding of inflammatory infiltrates in the prostate.

Natural Selection of Human Embryos: Decidualizing Endometrial Stromal Cells Serve as Sensors of Embryo Quality upon Implantation

PubMed Central

Teklenburg, Gijs; Salker, Madhuri; Molokhia, Mariam; Lavery, Stuart; Trew, Geoffrey; Aojanepong, Tepchongchit; Mardon, Helen J.; Lokugamage, Amali U.; Rai, Raj; Landles, Christian; Roelen, Bernard A. J.; Quenby, Siobhan; Kuijk, Ewart W.; Kavelaars, Annemieke; Heijnen, Cobi J.; Regan, Lesley; Brosens, Jan J.; Macklon, Nick S.

2010-01-01

Background Pregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs) into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown. Methodology/Principal Findings We employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1β, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo. Conclusions Human ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits maternal investment in developmentally impaired pregnancies. PMID:20422011

Secreted Proteins Defy the Expression Level-Evolutionary Rate Anticorrelation.

PubMed

Feyertag, Felix; Berninsone, Patricia M; Alvarez-Ponce, David

2017-03-01

The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E-R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein-protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E-R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E-R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E-R anticorrelation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Alcohol alters hypothalamic glial-neuronal communications involved in the neuroendocrine control of puberty: In vivo and in vitro assessments.

PubMed

Dees, W L; Hiney, J K; Srivastava, V K

2015-11-01

The onset of puberty is the result of the increased secretion of hypothalamic luteinizing hormone-releasing hormone (LHRH). The pubertal process can be altered by substances that can affect the prepubertal secretion of this peptide. Alcohol is one such substance known to diminish LHRH secretion and delay the initiation of puberty. The increased secretion of LHRH that normally occurs at the time of puberty is due to a decrease of inhibitory tone that prevails prior to the onset of puberty, as well as an enhanced development of excitatory inputs to the LHRH secretory system. Additionally, it has become increasingly clear that glial-neuronal communications are important for pubertal development because they play an integral role in facilitating the pubertal rise in LHRH secretion. Thus, in recent years attempts have been made to identify specific glial-derived components that contribute to the development of coordinated communication networks between glia and LHRH cell bodies, as well as their nerve terminals. Transforming growth factor-α and transforming growth factor-β1 are two such glial substances that have received attention in this regard. This review summarizes the use of multiple neuroendocrine research techniques employed to assess these glial-neuronal communication pathways involved in regulating prepubertal LHRH secretion and the effects that alcohol can have on their respective functions. Copyright © 2015 Elsevier Inc. All rights reserved.

MHC class II molecules control murine B cell responsiveness to lipopolysaccharide stimulation.

PubMed

Rodo, Joana; Gonçalves, Lígia A; Demengeot, Jocelyne; Coutinho, António; Penha-Gonçalves, Carlos

2006-10-01

LPS is a strong stimulator of the innate immune system and inducer of B lymphocyte activation. Two TLRs, TLR4 and RP105 (CD180), have been identified as mediators of LPS signaling in murine B cells, but little is known about genetic factors that are able to control LPS-induced cell activation. We performed a mouse genome-wide screen that aside from identifying a controlling locus mapping in the TLR4 region (logarithm of odds score, 2.77), also revealed that a locus closely linked to the MHC region (logarithm of odds score, 3.4) governed B cell responsiveness to LPS stimulation. Using purified B cells obtained from MHC congenic strains, we demonstrated that the MHC(b) haplotype is accountable for higher cell activation, cell proliferation, and IgM secretion, after LPS stimulation, when compared with the MHC(d) haplotype. Furthermore, B cells from MHC class II(-/-) mice displayed enhanced activation and proliferation in response to LPS. In addition, we showed that the MHC haplotype partially controls expression of RP105 (a LPS receptor molecule), following a pattern that resembles the LPS responsiveness phenotype. Together, our results strongly suggest that murine MHC class II molecules play a role in constraining the B cell response to LPS and that genetic variation at the MHC locus is an important component in controlling B cell responsiveness to LPS stimulation. This work raises the possibility that constraining of B cell responsiveness by MHC class II molecules may represent a functional interaction between adaptive and innate immune systems.

Development and evaluation of bevacizumab-modified pegylated cationic liposomes using cellular and in vivo models of human pancreatic cancer

NASA Astrophysics Data System (ADS)

Kuesters, Geoffrey M.

Targeting the tumor vascular supply in a homogenous manner is a difficult task to achieve with the use of pegylated cationic liposomes (PCLs) alone. Our formulation consisting of bevacizumab conjugated to the distal end of PEG on PCLs was thus developed in an effort to eliminate some of this heterogeneity as well as to increase tumor targeting overall. This study focuses on pancreatic cancer, which has the poorest five-year survival rate of all cancers because of its late diagnosis. The addition of bevacizumab will target tumor areas because it binds to VEGF which is secreted by tumors in high levels. In vitro, we showed that pancreatic cancer cells (Capan-1, HPAF-II and PANC-1) all secrete VEGF into media at different levels, with Capan-1 producing the most and HPAF-II producing the least. A murine endothelial cell line, MS1-VEGF, produces and secretes the most VEGF. A human microvascular endothelial cell line (HMEC-1) was grown in two different conditions, with and without VEGF in the media. Modifying PCLs with bevacizumab enhanced the binding and uptake of PCLs by some pancreatic and endothelial cells in vitro, particularly the cells that had or secreted the most significant amount of VEGF in the media. This translated into enhanced tumor targeting in a biodistribution study using a Capan-1 subcutaneous pancreatic tumor model. This also showed enhanced blood retention compared to the unmodified PCLs while it diminished uptake by the spleen and increased uptake by the kidney. To test the therapeutic benefit of this enhanced uptake and targeting, an anti-angiogenic agent, 2-methoxyestradiol was incorporated into the formulation with 20% incorporation efficiency. Both the unmodified and modified drug-loaded PCLs were the least efficacious against Capan-1, moderately effective against HPAF-II, PANC-1, MS1-VEGF and HMEC-1 grown without VEGF in the media and most efficacious against HMEC-1 grown with VEGF which had the most VEGF present in the media. Multiple in vivo experiments were performed using two different pancreatic cancer models, one with HPAF-II and the other with Capan-1. The HPAF-II study showed that bevacizumab-modified PCLs significantly enhanced the therapeutic effect over unmodified PCLs, which were ineffective when compared to the untreated control. However, bevacizumab alone was just as efficacious as the bevacizumab-modified PCLs. In the Capan-1 study, both the modified and unmodified PCLs were efficacious but the bevacizumab-modified PCLs were the most efficacious. The addition of bevacizumab not only increased the tumor targeting but also the therapeutic efficacy of 2-methoxyestradiol. Attaching bevacizumab to the distal end of PEG on 2-methoxyestradiol-loaded PCLs was effective at limiting tumor growth. The potential for this formulation is not limited to therapy but also for imaging tumors as well as monitoring the therapeutic response.

The regulatory mechanism of Hsp90{alpha} secretion from endothelial cells and its role in angiogenesis during wound healing

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

Song, Xiaomin; Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084; Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084

2010-07-16

Research highlights: {yields} Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90{alpha} secretion from endothelial cells. {yields} Secreted Hsp90{alpha} localizes on the leading edge of activated endothelial cells. {yields} Secreted Hsp90{alpha} promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90{alpha} (Hsp90{alpha}) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90{alpha} can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation andmore » migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90{alpha} from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90{alpha} in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90{alpha} but not Hsp90{beta} is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90{alpha} localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90{alpha} neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90{alpha} localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90{alpha} can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90{alpha} as a stimulator for wound repair.« less

VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

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

Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica

2010-10-15

Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conducemore » to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.« less

Measurement of gastric meal and secretion volumes using magnetic resonance imaging

NASA Astrophysics Data System (ADS)

Hoad, C. L.; Parker, H.; Hudders, N.; Costigan, C.; Cox, E. F.; Perkins, A. C.; Blackshaw, P. E.; Marciani, L.; Spiller, R. C.; Fox, M. R.; Gowland, P. A.

2015-02-01

MRI can assess multiple gastric functions without ionizing radiation. However, time consuming image acquisition and analysis of gastric volume data, plus confounding of gastric emptying measurements by gastric secretions mixed with the test meal have limited its use to research centres. This study presents an MRI acquisition protocol and analysis algorithm suitable for the clinical measurement of gastric volume and secretion volume. Reproducibility of gastric volume measurements was assessed using data from 10 healthy volunteers following a liquid test meal with rapid MRI acquisition within one breath-hold and semi-automated analysis. Dilution of the ingested meal with gastric secretion was estimated using a respiratory-triggered T1 mapping protocol. Accuracy of the secretion volume measurements was assessed using data from 24 healthy volunteers following a mixed (liquid/solid) test meal with MRI meal volumes compared to data acquired using gamma scintigraphy (GS) on the same subjects studied on a separate study day. The mean ± SD coefficient of variance between 3 observers for both total gastric contents (including meal, secretions and air) and just the gastric contents (meal and secretion only) was 3  ±  2% at large gastric volumes (>200 ml). Mean ± SD secretion volumes post meal ingestion were 64  ±  51 ml and 110  ±  40 ml at 15 and 75 min, respectively. Comparison with GS meal volumes, showed that MRI meal only volume (after correction for secretion volume) were similar to GS, with a linear regression gradient ± std err of 1.06  ±  0.10 and intercept -11  ±  24 ml. In conclusion, (i) rapid volume acquisition and respiratory triggered T1 mapping removed the requirement to image during prolonged breath-holds (ii) semi-automatic analysis greatly reduced the time required to derive measurements and (iii) correction for secretion volumes provided accurate assessment of gastric meal volumes and emptying. Together these features provide the scientific basis of a protocol which would be suitable in clinical practice.

Flavonoid metabolites reduce tumor necrosis factor-α secretion to a greater extent than their precursor compounds in human THP-1 monocytes.

PubMed

di Gesso, Jessica L; Kerr, Jason S; Zhang, Qingzhi; Raheem, Saki; Yalamanchili, Sai Krishna; O'Hagan, David; Kay, Colin D; O'Connell, Maria A

2015-06-01

Flavonoids are generally studied in vitro, in isolation, and as unmetabolized precursor structures. However, in the habitual diet, multiple flavonoids are consumed together and found present in the circulation as complex mixtures of metabolites. Using a unique study design, we investigated the potential for singular or additive anti-inflammatory effects of flavonoid metabolites relative to their precursor structures. Six flavonoids, 14 flavonoid metabolites, and 29 combinations of flavonoids and their metabolites (0.1-10 μM) were screened for their ability to reduce LPS-induced tumor necrosis factor-α (TNF-α) secretion in THP-1 monocytes. One micromolar peonidin-3-glucoside, cyanidin-3-glucoside, and the metabolites isovanillic acid (IVA), IVA-glucuronide, vanillic acid-glucuronide, protocatechuic acid-3-sulfate, and benzoic acid-sulfate significantly reduced TNF-α secretion when in isolation, while there was no effect on TNF-α mRNA expression. Four combinations of metabolites that included 4-hydroxybenzoic acid (4HBA) and/or protocatechuic acid also significantly reduced TNF-α secretion to a greater extent than the precursors or metabolites alone. The effects on LPS-induced IL-1β and IL-10 secretion and mRNA expression were also examined. 4HBA significantly reduced IL-1β secretion but none of the flavonoids or metabolites significantly modified IL-10 secretion. This study provides novel evidence suggesting flavonoid bioactivity results from cumulative or additive effects of circulating metabolites. © 2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single-Cell Detection of Secreted Aβ and sAPPα from Human IPSC-Derived Neurons and Astrocytes.

PubMed

Liao, Mei-Chen; Muratore, Christina R; Gierahn, Todd M; Sullivan, Sarah E; Srikanth, Priya; De Jager, Philip L; Love, J Christopher; Young-Pearse, Tracy L

2016-02-03

Secreted factors play a central role in normal and pathological processes in every tissue in the body. The brain is composed of a highly complex milieu of different cell types and few methods exist that can identify which individual cells in a complex mixture are secreting specific analytes. By identifying which cells are responsible, we can better understand neural physiology and pathophysiology, more readily identify the underlying pathways responsible for analyte production, and ultimately use this information to guide the development of novel therapeutic strategies that target the cell types of relevance. We present here a method for detecting analytes secreted from single human induced pluripotent stem cell (iPSC)-derived neural cells and have applied the method to measure amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα), analytes central to Alzheimer's disease pathogenesis. Through these studies, we have uncovered the dynamic range of secretion profiles of these analytes from single iPSC-derived neuronal and glial cells and have molecularly characterized subpopulations of these cells through immunostaining and gene expression analyses. In examining Aβ and sAPPα secretion from single cells, we were able to identify previously unappreciated complexities in the biology of APP cleavage that could not otherwise have been found by studying averaged responses over pools of cells. This technique can be readily adapted to the detection of other analytes secreted by neural cells, which would have the potential to open new perspectives into human CNS development and dysfunction. We have established a technology that, for the first time, detects secreted analytes from single human neurons and astrocytes. We examine secretion of the Alzheimer's disease-relevant factors amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα) and present novel findings that could not have been observed without a single-cell analytical platform. First, we identify a previously unappreciated subpopulation that secretes high levels of Aβ in the absence of detectable sAPPα. Further, we show that multiple cell types secrete high levels of Aβ and sAPPα, but cells expressing GABAergic neuronal markers are overrepresented. Finally, we show that astrocytes are competent to secrete high levels of Aβ and therefore may be a significant contributor to Aβ accumulation in the brain. Copyright © 2016 the authors 0270-6474/16/361730-17$15.00/0.

The cardiokine story unfolds: ischemic stress-induced protein secretion in the heart.

PubMed

Doroudgar, Shirin; Glembotski, Christopher C

2011-04-01

Intercellular communication depends on many factors, including proteins released via the classical or non-classical secretory pathways, many of which must be properly folded to be functional. Owing to their adverse effects on the secretion machinery, stresses such as ischemia can impair the folding of secreted proteins. Paradoxically, cells rely on secreted proteins to mount a response designed to resist stress-induced damage. This review examines this paradox using proteins secreted from the heart, cardiokines, as examples, and focuses on how the ischemic heart maintains or even increases the release of select cardiokines that regulate important cellular processes in the heart, including excitation-contraction coupling, hypertrophic growth, myocardial remodeling and stem cell function, in ways that moderate ischemic damage and enhance cardiac repair. Copyright © 2010 Elsevier Ltd. All rights reserved.

Protective effect of C-peptide on experimentally induced diabetic nephropathy and the possible link between C-peptide and nitric oxide.

PubMed

Elbassuoni, Eman A; Aziz, Neven M; El-Tahawy, Nashwa F

2018-06-01

Diabetic nephropathy one of the major microvascular diabetic complications. Besides hyperglycemia, other factors contribute to the development of diabetic complications as the proinsulin connecting peptide, C-peptide. We described the role of C-peptide replacement therapy on experimentally induced diabetic nephropathy, and its potential mechanisms of action by studying the role of nitric oxide (NO) as a mediator of C-peptide effects by in vivo modulating its production by N G -nitro-l-arginine methyl ester (L-NAME). Renal injury markers measured were serum urea, creatinine, tumor necrosis factor alpha, and angiotensin II, and malondialdehyde, total antioxidant, Bcl-2, and NO in renal tissue. In conclusion, diabetic induction resulted in islet degenerations and decreased insulin secretion with its metabolic consequences and subsequent renal complications. C-Peptide deficiencies in diabetes might have contributed to the metabolic and renal error, since C-peptide treatment to the diabetic rats completely corrected these errors. The beneficial effects of C-peptide are partially antagonized by L-NAME coadministration, indicating that NO partially mediates C-peptide effects.

Sonic hedgehog promotes neurite outgrowth of cortical neurons under oxidative stress: Involving of mitochondria and energy metabolism.

PubMed

He, Weiliang; Cui, Lili; Zhang, Cong; Zhang, Xiangjian; He, Junna; Xie, Yanzhao; Chen, Yanxia

2017-01-01

Oxidative stress has been demonstrated to be involved in the etiology of several neurobiological disorders. Sonic hedgehog (Shh), a secreted glycoprotein factor, has been implicated in promoting several aspects of brain remodeling process. Mitochondria may play an important role in controlling fundamental processes in neuroplasticity. However, little evidence is available about the effect and the potential mechanism of Shh on neurite outgrowth in primary cortical neurons under oxidative stress. Here, we revealed that Shh treatment significantly increased the viability of cortical neurons in a dose-dependent manner, which was damaged by hydrogen peroxide (H 2 O 2 ). Shh alleviated the apoptosis rate of H 2 O 2 -induced neurons. Shh also increased neuritogenesis injuried by H 2 O 2 in primary cortical neurons. Moreover, Shh reduced the generation of reactive oxygen species (ROS), increased the activities of SOD and and decreased the productions of MDA. In addition, Shh protected mitochondrial functions, elevated the cellular ATP levels and amelioratesd the impairment of mitochondrial complex II activities of cortical neurons induced by H 2 O 2 . In conclusion, all these results suggest that Shh acts as a prosurvival factor playing an essential role to neurite outgrowth of cortical neuron under H 2 O 2 -induced oxidative stress, possibly through counteracting ROS release and preventing mitochondrial dysfunction and ATP as well as mitochondrial complex II activities against oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

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

Watanabe, H.; Grubb, J.H.; Sly, W.S.

1990-10-01

The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functionalmore » receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.« less

Crystallization and preliminary crystallographic studies of LipA, a secretory lipase/esterase from Xanthomonas oryzae pv. oryzae

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

Aparna, Gudlur; Chatterjee, Avradip; Jha, Gopaljee

2007-08-01

The crystallization and preliminary crystallographic studies of LipA, a lipase/esterase secreted by X. oryzae pv. oryzae during its infection of rice plants, are reported. Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. Several enzymes that are secreted through the type II secretion system of this bacterium play an important role in the plant–microbe interaction, being important for virulence and also being able to induce potent host defence responses. One of these enzymes is a secretory lipase/esterase, LipA, which shows a very weak homology to other bacterial lipases and gives a positivemore » tributyrin plate assay. In this study, LipA was purified from the culture supernatant of an overexpressing clone of X. oryzae pv. oryzae and two types of crystals belonging to space group C2 but with two different unit-cell parameters were obtained using the hanging-drop vapour-diffusion method. Type I crystals diffract to a maximum resolution of 1.89 Å and have unit-cell parameters a = 93.1, b = 62.3, c = 66.1 Å, β = 90.8°. Type II crystals have unit-cell parameters a = 103.6, b = 54.6, c = 66.3 Å, β = 92.6° and diffract to 1.86 Å. Solvent-content analysis shows one monomer in the asymmetric unit in both the crystal forms.« less

Changes in expression and secretion patterns of fibroblast growth factor 8 and Sonic Hedgehog signaling pathway molecules during murine neural stem/progenitor cell differentiation in vitro☆

PubMed Central

Lu, Jiang; Lu, Kehuan; Li, Dongsheng

2012-01-01

In the present study, we investigated the dynamic expression of fibroblast growth factor 8 and Sonic Hedgehog signaling pathway related factors in the process of in vitro hippocampal neural stem/progenitor cell differentiation from embryonic Sprague-Dawley rats or embryonic Kunming species mice, using fluorescent quantitative reverse transcription-PCR and western blot analyses. Results demonstrated that the dynamic expression of fibroblast growth factor 8 was similar to fibroblast growth factor receptor 1 expression but not to other fibroblast growth factor receptors. Enzyme-linked immunosorbent assay demonstrated that fibroblast growth factor 8 and Sonic Hedgehog signaling pathway protein factors were secreted by neural cells into the intercellular niche. Our experimental findings indicate that fibroblast growth factor 8 and Sonic Hedgehog expression may be related to the differentiation of neural stem/progenitor cells. PMID:25624789

Pernicious anemia: What are the actual diagnosis criteria?

PubMed Central

Cattan, Daniel

2011-01-01

A gastric intrinsic factor output under 200 U/h after pentagastrin stimulation (N > 2000 U/h) is specific for pernicious anemia. The other findings are either variable or non specific. Serum intrinsic factor antibodies, considered as specific in general practice, are present only in half of the patients with pernicious anemia. In their absence, since the disappearance of the Schilling tests, the gastric tubage currently used for the study of gastric acid secretion, is obligatory for the simultaneous study of intrinsic factor output. This study is important to eliminate another disease much more frequent than pernicious anemia, the protein bound to cobalamin malabsorption was observed in achlorhydric simple atrophic gastritis in the presence of intrinsic factor secretion. PMID:21274387

Pernicious anemia: what are the actual diagnosis criteria?

PubMed

Cattan, Daniel

2011-01-28

A gastric intrinsic factor output under 200 U/h after pentagastrin stimulation (N > 2000 U/h) is specific for pernicious anemia. The other findings are either variable or non specific. Serum intrinsic factor antibodies, considered as specific in general practice, are present only in half of the patients with pernicious anemia. In their absence, since the disappearance of the Schilling tests, the gastric tubage currently used for the study of gastric acid secretion, is obligatory for the simultaneous study of intrinsic factor output. This study is important to eliminate another disease much more frequent than pernicious anemia, the protein bound to cobalamin malabsorption was observed in achlorhydric simple atrophic gastritis in the presence of intrinsic factor secretion.

Inhibition of COX-2 reduces the age-dependent increase of hippocampal inflammatory markers, corticosterone secretion, and behavioral impairments in the rat.

PubMed

Casolini, Paola; Catalani, Assia; Zuena, Anna R; Angelucci, Luciano

2002-05-01

Brain aging as well as brain degenerative processes with accompanying cognitive impairments are generally associated with hyperactivity of the hypothalamus-pituitary-adrenal axis, the end product of which, the glucocorticoid hormone, has been warranted the role of cell damage primum movens ("cascade hypothesis"). However, chronic inflammatory activity occurs in the hippocampus of aged rats as well as in the brain of Alzheimer's disease patients. The concomitant increase in the secretion of the glucocorticoid hormone, the endogenous anti-inflammatory and pro-inflammatory markers, has prompted us to investigate the two phenomena in the aging rat, and to work out its meaning. This study shows that: (I) interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and prostaglandin E(2) (PGE(2)) increase with age in the rats hippocampus, and (II) chronic oral treatment with celecoxib, a selective cycloxygenase-2 (COX-2) inhibitor, is able to contrast the age-dependent increase in hippocampal levels of pro-inflammatory markers and circulating anti-inflammatory corticosterone, provided that it is started at an early stage of aging. Under these conditions, age-related impairments in cognitive ability may be ameliorated. Taken together, these results indicate that there is a natural tendency to offset the age-dependent increase in brain inflammatory processes via the homeostatic increase of the circulating glucocorticoid hormone. Copyright 2002 Wiley-Liss, Inc.

Mechanisms of disease: inflammasome activation and the development of type 2 diabetes

PubMed Central

Grant, Ryan W.; Dixit, Vishwa D.

2013-01-01

Over the recent past, the importance of aberrant immune cell activation as one of the contributing mechanisms to the development of insulin-resistance and type 2 diabetes (T2D) has been recognized. Among the panoply of pro-inflammatory cytokines that are linked to chronic metabolic diseases, new data suggests that interleukin-1β (IL-1β) may play an important role in initiating and sustaining inflammation-induced organ dysfunction in T2D. Therefore, factors that control secretion of bioactive IL-1β have therapeutic implications. In this regard, the identification of multiprotein scaffolding complexes, “inflammasomes,” has been a great advance in our understanding of this process. The secretion of bioactive IL-1β is predominantly controlled by activation of caspase-1 through assembly of a multiprotein scaffold, “inflammasome” that is composed of NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) ASC (apoptosis associated speck-like protein containing a CARD) and procaspase-1. The NLRP3 inflammasome appears to be an important sensor of metabolic dysregulation and controls obesity-associated insulin resistance and pancreatic beta cell dysfunction. Initial clinical “proof of concept” studies suggest that blocking IL-1β may favorably modulate factors related to development and treatment of T2D. However, this potential therapeutic approach remains to be fully substantiated through phase-II clinical studies. Here, we outline the new immunological mechanisms that link metabolic dysfunction to the emergence of chronic inflammation and discuss the opportunities and challenges of future therapeutic approaches to dampen NLRP3 inflammasome activation or IL-1β signaling for controlling type 2 diabetes. PMID:23483669

Optimization of dendritic cell loading with tumor cell lysates for cancer immunotherapy.

PubMed

Hatfield, Paul; Merrick, Alison E; West, Emma; O'Donnell, Dearbhaile; Selby, Peter; Vile, Richard; Melcher, Alan A

2008-09-01

The immune response to cancer is critically determined by the way in which tumor cells die. As necrotic, stress-associated death can be associated with activation of antitumor immunity, whole tumor cell antigen loading strategies for dendritic cell (DC)-based vaccination have commonly used freeze-thaw "necrotic" lysates as an immunogenic source of tumor-associated antigens. In this study, the effect of such lysates on the ability of DCs to mature in response to well-established maturation stimuli was examined, and methods to enhance lysate-induced DC activation explored. Freeze-thaw lysates were prepared from murine tumor cell lines and their effects on bone marrow-derived DC maturation and function examined. Unmodified freeze-thaw tumor cell lysates inhibited the toll-like receptor-induced maturation and function of bone marrow-derived DCs, preventing up-regulation of CD40, CD86, and major histocompatibility complex class II, and reducing secretion of inflammatory cytokines [interleukin (IL)-12 p70, tumor necrosis factor-alpha, and IL-6]. Although IL-10 secretion was increased by lysate-pulsed DCs, this was not responsible for the observed suppression of IL-12. Although activation of the nuclear factor-kappaB pathway remained intact, the kinase activity of phosphorylated p38 mitogen-activated protein kinase was inhibited in lysate-pulsed DCs. Lysate-induced DC suppression was partially reversed in vitro by induction of tumor cell stress before lysis, and only DCs loaded with stressed lysates afforded protection against tumor challenge in vivo. These data suggest that ex vivo freeze-thaw of tumor cells does not effectively mimic in vivo immunogenic necrosis, and advocates careful characterization and optimization of tumor cell-derived vaccine sources for cancer immunotherapy.

Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration.

PubMed

Inoue, Yuji; Iriyama, Aya; Ueno, Shuji; Takahashi, Hidenori; Kondo, Mineo; Tamaki, Yasuhiro; Araie, Makoto; Yanagi, Yasuo

2007-08-01

Because there is no effective treatment for this retinal degeneration, potential application of cell-based therapy has attracted considerable attention. Several investigations support that bone marrow mesenchymal stem cells (MSCs) can be used for a broad spectrum of indications. Bone marrow MSCs exert their therapeutic effect in part by secreting trophic factors to promote cell survival. The current study investigates whether bone marrow MSCs secrete factor(s) to promote photoreceptor cell survival and whether subretinal transplantation of bone marrow MSCs promotes photoreceptor survival in a retinal degeneration model using Royal College of Surgeons (RCS) rats. In vitro, using mouse retinal cell culture, it was demonstrated that the conditioned medium of the MSCs delays photoreceptor cell apoptosis, suggesting that the secreted factor(s) from the MSCs promote photoreceptor cell survival. In vivo, the MSCs were injected into the subretinal space of the RCS rats and histological analysis, real-time RT-PCR and electrophysiological analysis demonstrated that the subretinal transplantation of MSCs delays retinal degeneration and preserves retinal function in the RCS rats. These results suggest that MSC is a useful cell source for cell-replacement therapy for some forms of retinal degeneration.

Game Plan for Learning

ERIC Educational Resources Information Center

Toppo, Greg

2016-01-01

IN 1959, six years before he authored the study that would remake America's segregated public schools, James S. Coleman found himself face to face with a very different foe: the inscrutable desires, evolving tastes, and secret motivations of the post--World War II American teenager. At the time, Coleman was head of Johns Hopkins University's…

[Association between obesity and ovarian cancer].

PubMed

Valladares, Macarena; Corsini, Gino; Romero, Carmen

2014-05-01

Obesity is a risk factor for cancer. Epidemiological evidences associate ovarian cancer with obesity. Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer and accounts for a high rate of mortality. The association between ovarian cancer and obesity could be explained by molecular factors secreted by adipose tissue such as leptin. In EOC, leptin increases cell proliferation and inhibits apoptosis. Additionally, adipose tissue synthesizes endogenous estrogens, which increase cell proliferation of epithelial ovarian cells. Also, obesity associated hyperinsulinism could increase ovarian estrogen secretion.

Fusion-activated cation entry (FACE) via P2X₄ couples surfactant secretion and alveolar fluid transport.

PubMed

Thompson, Kristin E; Korbmacher, Jonas P; Hecht, Elena; Hobi, Nina; Wittekindt, Oliver H; Dietl, Paul; Kranz, Christine; Frick, Manfred

2013-04-01

Two fundamental mechanisms within alveoli are essential for lung function: regulated fluid transport and secretion of surfactant. Surfactant is secreted via exocytosis of lamellar bodies (LBs) in alveolar type II (ATII) cells. We recently reported that LB exocytosis results in fusion-activated cation entry (FACE) via P2X₄ receptors on LBs. We propose that FACE, in addition to facilitating surfactant secretion, modulates alveolar fluid transport. Correlative fluorescence and atomic force microscopy revealed that FACE-dependent water influx correlated with individual fusion events in rat primary ATII cells. Moreover, ATII cell monolayers grown at air-liquid interface exhibited increases in short-circuit current (Isc) on stimulation with ATP or UTP. Both are potent agonists for LB exocytosis, but only ATP activates FACE. ATP, not UTP, elicited additional fusion-dependent increases in Isc. Overexpressing dominant-negative P2X₄ abrogated this effect by ∼50%, whereas potentiating P2X4 lead to ∼80% increase in Isc. Finally, we monitored changes in alveolar surface liquid (ASL) on ATII monolayers by confocal microscopy. Only stimulation with ATP, not UTP, led to a significant, fusion-dependent, 20% decrease in ASL, indicating apical-to-basolateral fluid transport across ATII monolayers. Our data support the first direct link between LB exocytosis, regulation of surfactant secretion, and transalveolar fluid resorption via FACE.

Differential Receptor for Advanced Glycation End Products Expression in Preeclamptic, Intrauterine Growth Restricted, and Gestational Diabetic Placentas.

PubMed

Alexander, Kristen L; Mejia, Camilo A; Jordan, Clinton; Nelson, Michael B; Howell, Brian M; Jones, Cameron M; Reynolds, Paul R; Arroyo, Juan A

2016-02-01

Receptor for advanced glycation end products (RAGE) is a receptor implicated in the modulation of inflammation. Inflammation has been associated with pregnancy pathologies including preeclampsia (PE), intrauterine growth restriction (IUGR), and gestational diabetes mellitus (GDM). Our objective was to examine placental RAGE expression in PE, IUGR, and GDM complications. Human placental tissues were obtained for RAGE determination using Q-PCR, immunohistochemistry, and Western blot. Invasive trophoblast cells were cultured and treated with AGES for RAGE activation studies. Compared to control placenta, we observed: (i) decreased RAGE gene expression during GDM, (ii) increased RAGE protein in the PE placenta, and (iii) decreased RAGE protein in the IUGR placenta. In trophoblast cells exposed AGEs, we observed: (i) decreased trophoblast invasion, (ii) increased c-Jun N-terminal kinases (JNK) and Extracellular signal-regulated kinases (ERK), and (iii) increased TNF-α and IL-1β secretion. We conclude that placental RAGE is activated during PE and that RAGE-mediated inflammation in the trophoblast involves increased pro-inflammatory cytokine secretion. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Pulmonary haptoglobin (pHp) is part of the surfactant system in the human lung.

PubMed

Abdullah, Mahdi; Goldmann, Torsten

2012-11-20

Since the existence of pHp was demonstrated, it has been shown that this molecule and its receptor CD163 are regulated by different stimuli. Furthermore, a comparably fast secretion of pHp was described as well as the immuno-stimulatory effects. The intention of this study was to elucidate the role of pHp in the human lungs further. Here we show, by means of confocal microscopy and immune-electron-microscopy, a clear co-localization of pHp with surfactant protein-B in lamellar bodies of alveolar epithelial cells type II. These results are underlined by immunohistochemical stainings in differently fixed human lung tissues, which show pHp in vesicular and released form. The images of the released form resemble the intended position of surfactant in the human alveolus. pHp is secreted by Alveolar epithelial cells type II as previously shown. Moreover, pHp is co-localized with Surfactant protein-B. We conclude that the presented data shows that pHp is a native part of the surfactant system in the human lung. http://www.diagnosticpathology.diagnomx.eu/vs/2563584738239912.

Calcitonin Gene-Related Peptide Reduces Taste-Evoked ATP Secretion from Mouse Taste Buds.

PubMed

Huang, Anthony Y; Wu, Sandy Y

2015-09-16

Immunoelectron microscopy revealed that peripheral afferent nerve fibers innervating taste buds contain calcitonin gene-related peptide (CGRP), which may be as an efferent transmitter released from peripheral axon terminals. In this report, we determined the targets of CGRP within taste buds and studied what effect CGRP exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura-2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings showed that a subset of Presynaptic (Type III) taste cells (53%) responded to 0.1 μm CGRP with an increase in intracellular Ca(2+). In contrast, Receptor (Type II) taste cells rarely (4%) responded to 0.1 μm CGRP. Using pharmacological tools, the actions of CGRP were probed and elucidated by the CGRP receptor antagonist CGRP(8-37). We demonstrated that this effect of CGRP was dependent on phospholipase C activation and was prevented by the inhibitor U73122. Moreover, applying CGRP caused taste buds to secrete serotonin (5-HT), a Presynaptic (Type III) cell transmitter, but not ATP, a Receptor (Type II) cell transmitter. Further, our previous studies showed that 5-HT released from Presynaptic (Type III) cells provides negative paracrine feedback onto Receptor (Type II) cells by activating 5-HT1A receptors, and reducing ATP secretion. Our data showed that CGRP-evoked 5-HT release reduced taste-evoked ATP secretion. The findings are consistent with a role for CGRP as an inhibitory transmitter that shapes peripheral taste signals via serotonergic signaling during processing gustatory information in taste buds. The taste sensation is initiated with a highly complex set of interactions between a variety of cells located within the taste buds before signal propagation to the brain. Afferent signals from the oral cavity are carried to the brain in chemosensory fibers that contribute to chemesthesis, the general chemical sensitivity of the mucus membranes in the oronasal cavities and being perceived as pungency, irritation, or heat. This is a study of a fundamental question in neurobiology: how are signals processed in sensory end organs, taste buds? More specifically, taste-modifying interactions, via transmitters, between gustatory and chemosensory afferents inside taste buds will help explain how a coherent output is formed before being transmitted to the brain. Copyright © 2015 the authors 0270-6474/15/3512714-11$15.00/0.

[Relationship between sleep disordered breathing and body weight loss in patients with chronic obstructive pulmonary disease].

PubMed

Ito, Eiki; Murata, Akira; Yamamoto, Kazuo; Kudo, Shoji

2003-04-01

We evaluated body weight loss and growth hormone secretion in patients with sleep-disordered breathing associated with chronic obstructive pulmonary disease. Of 11 patients hospitalized for pulmonary rehabilitation, five (WL group) had a history of body weight loss within two years before their interviews, while the other 6 patients (NWL group) had no changes in body weight. All patients underwent body index measurements, pulmonary function tests, blood gas analyses, assessments of nutritional status, and full night polysomnography for two consecutive days. Growth hormone levels were measured in the first 3-hour period following falling asleep. There were no significant inter-group differences between the results of pulmonary function tests, blood gas analyses, or nutritional status assessment. The WL group had a significantly higher percentage loss of body weight than the NWL group (mean +/- S.D. 11.5 +/- 4.7% in the WL group versus 2.7 +/- 1.8% in the NWL group, p < 0.01). The WL group had a significantly higher sleep apnea/hypopnea index than the NWL group (42.4 +/- 9.5/hr in the WL group versus 7.8 +/- 2.9/hr in the NWL group, p < 0.01). The WL group showed a higher rate of stage I + II sleep than the NWL group (84.9 +/- 7.0% versus 64.5 +/- 8.7%), with lower rates of slow wave sleep (2.2 +/- 2.1% versus 15.0 +/- 8.7%) and rapid eye movement sleep (12.9 +/- 6.3% versus 20.6 +/- 1.0%). The WL group showed a low level of growth hormone secretion with no peak in the sequential curve, but had a higher level of insulin growth factor-1 than the NWL group (148 +/- 36 ng/ml versus 90 +/- 22 ng/ml, p < 0.01). These results suggest that chronic obstructive pulmonary disease patients undergoing weight loss are likely to have an increase of growth hormone secretions in the daytime, possibly induced by underlying psychiatric disorders such as depression. Patients with chronic obstructive pulmonary disease may lose weight regardless of nutritional status because of a disturbance of growth hormone secretion resulting of sleep-disordered breathing.

MicroRNAs and toxicology: A love marriage.

PubMed

Schraml, Elisabeth; Hackl, Matthias; Grillari, Johannes

2017-01-01

With the dawn of personalized medicine, secreted microRNAs (miRNAs) have come into the very focus of biomarker development for various diseases. MiRNAs fulfil key requirements of diagnostic tools such as i) non or minimally invasive accessibility, ii) robust, standardized and non-expensive quantitative analysis, iii) rapid turnaround of the test result and iv) most importantly because they provide a comprehensive snapshot of the ongoing physiologic processes in cells and tissues that package and release miRNAs into cell-free space. These characteristics have also established circulating miRNAs as promising biomarker candidates for toxicological studies, where they are used as biomarkers of drug-, or chemical-induced tissue injury for safety assessment. The tissue-specificity and early release of circulating miRNAs upon tissue injury, when damage is still reversible, are main factors for their clinical utility in toxicology. Here we summarize in brief, current knowledge of this field.

The Extended Granin Family: Structure, Function, and Biomedical Implications

PubMed Central

Bartolomucci, Alessandro; Possenti, Roberta; Mahata, Sushil K.; Fischer-Colbrie, Reiner; Loh, Y. Peng

2011-01-01

The chromogranins (chromogranin A and chromogranin B), secretogranins (secretogranin II and secretogranin III), and additional related proteins (7B2, NESP55, proSAAS, and VGF) that together comprise the granin family subserve essential roles in the regulated secretory pathway that is responsible for controlled delivery of peptides, hormones, neurotransmitters, and growth factors. Here we review the structure and function of granins and granin-derived peptides and expansive new genetic evidence, including recent single-nucleotide polymorphism mapping, genomic sequence comparisons, and analysis of transgenic and knockout mice, which together support an important and evolutionarily conserved role for these proteins in large dense-core vesicle biogenesis and regulated secretion. Recent data further indicate that their processed peptides function prominently in metabolic and glucose homeostasis, emotional behavior, pain pathways, and blood pressure modulation, suggesting future utility of granins and granin-derived peptides as novel disease biomarkers. PMID:21862681

The extended granin family: structure, function, and biomedical implications.

PubMed

Bartolomucci, Alessandro; Possenti, Roberta; Mahata, Sushil K; Fischer-Colbrie, Reiner; Loh, Y Peng; Salton, Stephen R J

2011-12-01

The chromogranins (chromogranin A and chromogranin B), secretogranins (secretogranin II and secretogranin III), and additional related proteins (7B2, NESP55, proSAAS, and VGF) that together comprise the granin family subserve essential roles in the regulated secretory pathway that is responsible for controlled delivery of peptides, hormones, neurotransmitters, and growth factors. Here we review the structure and function of granins and granin-derived peptides and expansive new genetic evidence, including recent single-nucleotide polymorphism mapping, genomic sequence comparisons, and analysis of transgenic and knockout mice, which together support an important and evolutionarily conserved role for these proteins in large dense-core vesicle biogenesis and regulated secretion. Recent data further indicate that their processed peptides function prominently in metabolic and glucose homeostasis, emotional behavior, pain pathways, and blood pressure modulation, suggesting future utility of granins and granin-derived peptides as novel disease biomarkers. Copyright © 2011 by The Endocrine Society

miR-Let7A Modulates Autophagy Induction in LPS-Activated Microglia

PubMed Central

Song, Juhyun; Oh, Yumi

2015-01-01

Microglia regulate the secretion of various immunomediators in central nervous system diseases. Microglial autophagy is the crucial process for cell's survival and cytokine productions. Recent studies have reported that several microRNAs are involved in the autophagy system. miR-Let7A is such a microRNA that plays a role in various inflammation responses, and is magnified as a key modulator particularly in the autophagy system. In present study, we investigated whether miR-Let7A is involved in autophagy in activating microglia. Overexpression of miR-Let7A in LPS-stimulated BV2 microglial cells promoted the induction of the autophagy related factors such as LC3II, Beclin1, and ATG3. Our results suggest a potential role of miR-Let7A in the autophagy process of microglia during CNS inflammation. PMID:26113790

DBSecSys 2.0: a database of Burkholderia mallei and Burkholderia pseudomallei secretion systems.

PubMed

Memišević, Vesna; Kumar, Kamal; Zavaljevski, Nela; DeShazer, David; Wallqvist, Anders; Reifman, Jaques

2016-09-20

Burkholderia mallei and B. pseudomallei are the causative agents of glanders and melioidosis, respectively, diseases with high morbidity and mortality rates. B. mallei and B. pseudomallei are closely related genetically; B. mallei evolved from an ancestral strain of B. pseudomallei by genome reduction and adaptation to an obligate intracellular lifestyle. Although these two bacteria cause different diseases, they share multiple virulence factors, including bacterial secretion systems, which represent key components of bacterial pathogenicity. Despite recent progress, the secretion system proteins for B. mallei and B. pseudomallei, their pathogenic mechanisms of action, and host factors are not well characterized. We previously developed a manually curated database, DBSecSys, of bacterial secretion system proteins for B. mallei. Here, we report an expansion of the database with corresponding information about B. pseudomallei. DBSecSys 2.0 contains comprehensive literature-based and computationally derived information about B. mallei ATCC 23344 and literature-based and computationally derived information about B. pseudomallei K96243. The database contains updated information for 163 B. mallei proteins from the previous database and 61 additional B. mallei proteins, and new information for 281 B. pseudomallei proteins associated with 5 secretion systems, their 1,633 human- and murine-interacting targets, and 2,400 host-B. mallei interactions and 2,286 host-B. pseudomallei interactions. The database also includes information about 13 pathogenic mechanisms of action for B. mallei and B. pseudomallei secretion system proteins inferred from the available literature or computationally. Additionally, DBSecSys 2.0 provides details about 82 virulence attenuation experiments for 52 B. mallei secretion system proteins and 98 virulence attenuation experiments for 61 B. pseudomallei secretion system proteins. We updated the Web interface and data access layer to speed-up users' search of detailed information for orthologous proteins related to secretion systems of the two pathogens. The updates of DBSecSys 2.0 provide unique capabilities to access comprehensive information about secretion systems of B. mallei and B. pseudomallei. They enable studies and comparisons of corresponding proteins of these two closely related pathogens and their host-interacting partners. The database is available at http://dbsecsys.bhsai.org .

Experiment K-7-22: Growth Hormone Regulation Synthesis and Secretion in Microgravity. Part 2; Hypothalamic Growth Hormone-Releasing Factor, Somatostatin Immunoreactivity, and Messenger RNA Levels in Microgravity

NASA Technical Reports Server (NTRS)

Sawchenko, P. E.; Arias, C.; Krasnov, I.; Grindeland, R. E.; Vale, W.

1994-01-01

Immunohistochemical analyses of hypothalamic hormones carried out on tissue from rats flown on an earlier flight (Cosmos 1887) suggested preferential effects on hypophysiotropic principles involved in the regulation of growth hormone secretion and synthesis. We found that staining in the median eminence for peptides that provide both stimulatory (growth hormone-releasing factor, or GRF) and inhibitory (somatostatin, SS) influences on growth hormone secretion were depressed in flight animals relative to synchronous controls, while staining for other neuroendocrine peptides, cortocotropin-releasing factor and arginine vasopressin, were similar in these two groups. While this suggests some selective impact of weightlessness on the two principal central nervous system regulators of growth hormone dynamics, the fact that both GRF- and SS-immunoreactivity (IR) appeared affected in the same direction is somewhat problematic, and makes tentative any intimation that effects on CNS control mechanisms may be etiologically significant contributors to the sequelae of reduced growth hormone secretion seen in prolonged space flight. To provide an additional, and more penetrating, analysis we attempted in hypothalamic material harvested from animals flown on Cosmos 2044 to complement immunohistochemical analyses of GRF and SS staining with quantitative, in situ assessments of messenger RNAs encoding the precursors for both these hormones.

Enhanced angiogenic effect of adipose-derived stromal cell spheroid with low-level light therapy in hindlimb ischemia mice

NASA Astrophysics Data System (ADS)

Park, In-Su; Ahn, Jin-Chul; Chung, Phil-Sang

2014-02-01

Adipose-derived stromal cells (ASCs) are attractive cell source for tissue engineering. However, one obstacle to this approach is that the transplanted ASC population can decline rapidly in the recipient tissue. The aim of this study was to investigate the effects of low-level laser therapy (LLLT) on transplanted human ASCs (hASCs) spheroid in a hindlimb ischemia animal model. LLLT, hASCs spheroid and hASCs spheroid transplantation with LLLT (spheroid + LLLT) were applied to the ischemic hindlimbs in athymic mice. The survival, differentiation and secretion of vascular endothelial growth (VEGF) of spheroid ASCs were evaluated by immunohistochemistry. The spheroid + LLLT group enhanced the tissue regeneration, including angiogenesis, compared with other groups. The spheroid contributed tissue regeneration via differentiation and secretion of growth factors. In the spheroid + LLLT group, the survival of spheroid hASCs was increased by the decreased apoptosis of spheroid hASCs in the ischemic hindlimb. The secretion of growth factors was stimulated in the spheroid + LLLT group compared with the ASCs group and spheroid group. These data suggest that LLLT is an effective biostimulator of spheroid hASCs in tissue regeneration that enhances the survival of ASCs and stimulates the secretion of growth factors in the ischemic hindlimb.