Comparative genomic organization and tissue-specific transcription of the duplicated fabp7 and fabp10 genes in teleost fishes.

PubMed

Parmar, Manoj B; Wright, Jonathan M

2013-11-01

A whole-genome duplication (WGD) early in the teleost fish lineage makes fish ideal organisms to study the fate of duplicated genes and underlying evolutionary trajectories that have led to the retention of ohnologous gene duplicates in fish genomes. Here, we compare the genomic organization and tissue-specific transcription of the ohnologous fabp7 and fabp10 genes in medaka, three-spined stickleback, and spotted green pufferfish to the well-studied duplicated fabp7 and fabp10 genes of zebrafish. Teleost fabp7 and fabp10 genes contain four exons interrupted by three introns. Polypeptide sequences of Fabp7 and Fabp10 show the highest sequence identity and similarity with their orthologs from vertebrates. Orthology was evident as the ohnologous Fabp7 and Fabp10 polypeptides of teleost fishes each formed distinct clades and clustered together with their orthologs from other vertebrates in a phylogenetic tree. Furthermore, ohnologous teleost fabp7 and fabp10 genes exhibit conserved gene synteny with human FABP7 and chicken FABP10, respectively, which provides compelling evidence that the duplicated fabp7 and fabp10 genes of teleost fishes most likely arose from the well-documented WGD. The tissue-specific distribution of fabp7a, fabp7b, fabp10a, and fabp10b transcripts provides evidence of diverged spatial transcriptional regulation between ohnologous gene duplicates of fabp7 and fabp10 in teleost fishes.

Developmental and Cell-Specific Expression of Thyroid Hormone Transporters in the Mouse Cochlea

PubMed Central

Sharlin, David S.; Visser, Theo J.

2011-01-01

Thyroid hormone is essential for the development of the cochlea and auditory function. Cochlear response tissues, which express thyroid hormone receptor β (encoded by Thrb), include the greater epithelial ridge and sensory epithelium residing inside the bony labyrinth. However, these response tissues lack direct blood flow, implying that mechanisms exist to shuttle hormone from the circulation to target tissues. Therefore, we investigated expression of candidate thyroid hormone transporters L-type amino acid transporter 1 (Lat1), monocarboxylate transporter (Mct)8, Mct10, and organic anion transporting polypeptide 1c1 (Oatp1c1) in mouse cochlear development by in situ hybridization and immunofluorescence analysis. L-type amino acid transporter 1 localized to cochlear blood vessels and transiently to sensory hair cells. Mct8 localized to the greater epithelial ridge, tympanic border cells underlying the sensory epithelium, spiral ligament fibrocytes, and spiral ganglion neurons, partly overlapping with the Thrb expression pattern. Mct10 was detected in a highly restricted pattern in the outer sulcus epithelium and weakly in tympanic border cells and hair cells. Organic anion transporting polypeptide 1c1 localized primarily to fibrocytes in vascularized tissues of the spiral limbus and spiral ligament and to tympanic border cells. Investigation of hypothyroid Tshr−/− mice showed that transporter expression was delayed consistent with retardation of cochlear tissue maturation but not with compensatory responses to hypothyroidism. The results demonstrate specific expression of thyroid hormone transporters in the cochlea and suggest that a network of thyroid hormone transport underlies cochlear development. PMID:21878515

Adhesion inhibition of Mycoplasma iowae to chicken lymphoma DT40 cells by monoclonal antibodies reacting with a 65-kD polypeptide.

PubMed

Fiorentin, L; Panangala, V S; Zhang, Y; Toivio-Kinnucan, M

1998-01-01

Tissue- and cell-specific attachment of mycoplasmas is a key aspect of the host-parasite relationship. In this study, monoclonal antibodies (MAbs) recognizing surface membrane polypeptides with molecular masses of 46 kD (p46) and 65 kD (p65), respectively, were examined in a microtiter cell attachment (agglutination) inhibition assay. MAbs MI3, MI6, and MI12 reacting with p65 polypeptide of Mycoplasma iowae inhibited attachment of the organisms to chicken lymphoma (DT 40) cells. One MAb (MI2) that reacted with p65 in immunoblots did not inhibit cell attachment, possibly because of the intrinsic native conformation of the epitope(s) in intact mycoplasmas as opposed to the linear state (sodium dodecyl sulfate denatured) in immunoblots. More pronounced M. iowae adherence inhibition was demonstrated by polyclonal turkey and mouse anti-M. iowae antisera compared with MAbs. Immunogold labelling followed by electron microscopy allowed us to localize the MAb-recognized epitopes on the membrane surface of M. iowae. On the basis of the cell attachment inhibition of M. iowae by specific MAbs (MI3, MI6, and MI12), we propose that the p65 polypeptide plays a role in cytadherence. The ability of polyclonal antisera to inhibit attachment of M. iowae more efficiently than the MAbs suggests that additional epitopes within p65 and/or other proteins are involved in cell attachment.

Identification of a major polypeptide of the nuclear pore complex

PubMed Central

1982-01-01

The nuclear pore complex is a prominent structural component of the nuclear envelope that appears to regulate nucleoplasmic molecular movement. Up to now, none of its polypeptides have been defined. To identify possible pore complex proteins, we fractionated rat liver nuclear envelopes and microsomal membranes with strong protein perturbants into peripheral and intrinsic membrane proteins, and compared these fractions on SDS gels. From this analysis, we identified a prominent 190-kilodalton intrinsic membrane polypeptide that occurs specifically in nuclear envelopes. Lectin binding studies indicate that this polypeptide (gp 190) is the major nuclear envelope glycoprotein. Upon treatment of nuclear envelopes with Triton X-100, gp 190 remains associated with a protein substructure of the nuclear envelope consisting of pore complexes and nuclear lamina. We prepared monospecific antibodies to gp 190 for immunocytochemical localization. Immunofluorescence staining of tissue culture cells suggests that gp 190 occurs exclusively in the nucleus during interphase. This polypeptide becomes dispersed throughout the cell in mitotic prophase when the nuclear envelope is disassembled, and subsequently returns to the nuclear surfaces during telophase when the nuclear envelope is reconstructed. Immunoferritin labeling of Triton-treated rat liver nuclei demonstrates that gp 190 occurs exclusively in the nuclear pore complex, in the regions of the cytoplasmic (and possibly nucleoplasmic) pore complex annuli. A polypeptide that cross-reacts with gp 190 is present in diverse vertebrate species, as shown by antibody labeling of nitrocellulose SDS gel transfers. On the basis of its biochemical characteristics, we suggest that gp 190 may be involved in anchoring the pore complex to nuclear envelope membranes. PMID:7153248

Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early

DTIC Science & Technology

2013-07-01

biology, nanotechnology, and imaging technology, molecular imaging utilizes specific probes as contrast agents to visualize cellular processes at the...This reagent was covalently coupled to the oligosaccharides attached to polypeptide side-chains of extracellular membrane proteins on living cells...website. The normal tissue gene expression profile dataset was modified and processed as described by Fang (8) and mean intensities and standard

Tissue-specific changes of glutamine synthetase activity in oats after rhizosphere infestation by Pseudomonas syringae pv. tabaci. Final report

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

Knight, T.J.; Temple, S.; Sengupta-Gopalan, C.

1996-05-15

Oats (Avena sativa L. lodi) tolerant of rhizosphere infestation by Pseudomonas syringae pv. tabaci when challenged by the pathogen experience tissue-specific alterations of ammonia assimilatory capabilities. Altered ammonia assimilatory potentials between root and leaf tissue result from selective inactivation of glutamine synthetase (GS) by the toxin Tabtoxinine-B-lactam (TBL). Root GS is sensitive and leaf GSs are resistant to TBL inactivation. With prolonged challenge by the pathogen root GS activity decreases but leaf GS specific activity increase. Higher leaf GS activity is due to decreased rates of degradation rather than increased GS synthesis. Higher leaf GS activity and elevated levels ofmore » GS polypeptide appear to result from a limited interaction between GS and TBL leading to the accumulation of a less active but more stable GS holoenzyme. Tolerant challenged oats besides surviving rhizosphere infestation, experience enhanced growth. A strong correlation exists between leaf GS activity and whole plant fresh weight, suggesting that tissue-specific changes in ammonia assimilatory capability provides the plant a more efficient mechanism for uptake and utilization of nitrogen.« less

Neuropeptides in Lower Urinary Tract (LUT) Function

PubMed Central

Arms, Lauren; Vizzard, Margaret A.

2014-01-01

Numerous neuropeptide/receptor systems including vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating polypeptide, calcitonin gene-related peptide, substance P, neurokinin A, bradykinin, and endothelin-1 are expressed in the lower urinary tract (LUT) in both neural and non-neural (e.g., urothelium) components. LUT neuropeptide immunoreactivity is present in afferent and autonomic efferent neurons innervating the bladder and urethra and in the urothelium of the urinary bladder. Neuropeptides have tissue-specific distributions and functions in the LUT and exhibit neuroplastic changes in expression and function with LUT dysfunction following neural injury, inflammation and disease. LUT dysfunction with abnormal voiding including urinary urgency, increased voiding frequency, nocturia, urinary incontinence and pain may reflect a change in the balance of neuropeptides in bladder reflex pathways. LUT neuropeptide/receptor systems may represent potential targets for therapeutic intervention. PMID:21290237

The Role of TPS and TPA in the Diagnostics of Distant Metastases.

PubMed

Kucera, Radek; Topolcan, Ondrej; Fiala, Ondrej; Kinkorova, Judita; Treska, Vladislav; Zedníková, Ilona; Slouka, David; Simanek, Vaclav; Safanda, Martin; Babuska, Vaclav

2016-02-01

The aim of the study was to assess the degree to which tissue polypeptide antigen (TPA) and tissue polypeptide-specific antigen (TPS), as well as carcinoembryonic antigen (CEA), can assist in the detection of distant metastases. We assessed 157 patients with colorectal and breast cancer divided into two groups. The first was a group of patients with cancer at stages 1, 2 and 3; the second was a group of patients with cancer at stage 4 with metastasis. We found significantly higher levels of all biomarkers in the metastatic group compared to the group with cancer at stages 1-3 (p<0.0001). The calculated area under the receiver operating characteristic (ROC) curve was 0.9929 for TPS, 0.9337 for TPA and 0.7234 for CEA. The cut-off was calculated for each biomarker at 95% specificity, TPS cut-off=255 IU/l (sensitivity 95%), TPA cut-off=200 IU/l (sensitivity 70%) and CEA cut-off=18 μg/l (sensitivity 37%). We suggest combining CEA with TPS or TPA in the detection of distant metastases or using only cytokeratins. This approach can significantly increase the quality of detection of the metastatic process. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Combinatorial discovery of enzymes with utility in biomass transformation

DOEpatents

Fox, Brian G; Elsen, Nathaniel L

2015-02-03

Methods for the cell-free identification of polypeptide and polypeptide combinations with utility in biomass transformation, as well as specific novel polypeptides and cell-free systems containing polypeptide combinations discovered by such methods are disclosed.

Molecular Cloning and Characterization of Two Pig Vasoactive Intestinal Polypeptide Receptors (VPAC1-R and VPAC2-R)

PubMed Central

He, Xiaping; Meng, Fengyan; Wang, Yajun

2014-01-01

We here report the cloning, tissue expression, and functional analyses of the two pig vasoactive intestinal polypeptide (VIP) receptors (pVPAC1-R and pVPAC2-R). The cloned full-length pVPAC1-R and pVPAC2-R share high structural similarity with their mammalian counterparts. Functional assay revealed that the full-length pVPAC1-R and pVPAC2-R-expressed Chinese hamster ovary (CHO) cells could be activated by pVIP and pPACAP38 potently, indicating that pVPAC1-R and pVPAC2-R are capable of binding VIP and pituitary adenylate cyclase-activating polypeptide (PACAP). In addition to the identification of the transcripts encoding the two full-length receptors, multiple splice transcript variants were isolated. Comparison with the pig genome database revealed that pVPAC1-R and pVPAC2-R share a unique gene structure with 14 exons different from other vertebrates. Reverse transcription and polymerase chain reaction (RT-PCR) assays further showed that the transcript encoding the full-length pVPAC2-R is widely expressed in all adult tissues whereas the splice variants of pVPAC1-R are predominantly expressed in all tissues instead of the transcript encoding the full-length receptor, hinting that pVPAC2-R may play more important roles than pVPAC1-R in mediating VIP and PACAP actions. Our present findings help to elucidate the important role of VIP and PACAP and promote to rethink of their species-specific physiological roles including their actions in regulation of phenotypic traits in pigs. PMID:24520933

Identification of the neurofibromatosis type 1 gene product

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

Gutmann, D.H.; Wood, D.L.; Collins, F.S.

The gene for neurofibromatosis type 1 (NF1) was recently identified by positional cloning. The complete cDNA encodes a polypeptide of 2818 amino acids. To study the NF1 gene product, antibodies were raised against both fusion proteins and synthetic peptides. Initial characterization of two anti-peptide antibodies and one fusion-protein antibody demonstrated a specific protein of {approx}250 kDa by both immunoprecipitation and immunoblotting. This protein was found in all tissues and cell lines examined and is detected in human, rat, and mouse tissues. To demonstrate that these antibodies specifically recognize the NF1 protein, additional fusion proteins containing the sequence specific to themore » synthetic peptide were generated. Both peptide antisera recognize the proper specific fusion proteins so generated. Immunoprecipitates using the peptide antisera were shown to recognize the same protein detected by immunoblotting with either the other peptide antiserum or the fusion-protein antiserum. Immunoblotting using antiserum specific to spatially distinct epitopes conducted on tissue homogenates demonstrated the NF1 protein in all adult tissues. Based on the homology between the NF1 gene product and members of the GTPase-activating protein (GAP) superfamily, the name NF1-GAP-related protein (NF1GRP) is suggested.« less

Common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands

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

Cameron, R.S.; Cameron, P.L.; Castle, J.D.

1986-10-01

A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues - pancreatic, lacrimal, and submandibular - from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pImore » and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion.« less

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

PubMed Central

1986-01-01

A collection of 17 monoclonal antibodies elicited against the light- harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC- II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b. PMID:3528171

The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes.

PubMed Central

German, M S; Moss, L G; Wang, J; Rutter, W J

1992-01-01

The pancreatic beta cell makes several unique gene products, including insulin, islet amyloid polypeptide (IAPP), and beta-cell-specific glucokinase (beta GK). The functions of isolated portions of the insulin, IAPP, and beta GK promoters were studied by using transient expression and DNA binding assays. A short portion (-247 to -197 bp) of the rat insulin I gene, the FF minienhancer, contains three interacting transcriptional regulatory elements. The FF minienhancer binds at least two nuclear complexes with limited tissue distribution. Sequences similar to that of the FF minienhancer are present in the 5' flanking DNA of the human IAPP and rat beta GK genes and also the rat insulin II and mouse insulin I and II genes. Similar minienhancer constructs from the insulin and IAPP genes function as cell-specific transcriptional regulatory elements and compete for binding of the same nuclear factors, while the beta GK construct competes for protein binding but functions poorly as a minienhancer. These observations suggest that the patterns of expression of the beta-cell-specific genes result in part from sharing the same transcriptional regulators. Images PMID:1549125

Polypeptide having an amino acid replaced with N-benzylglycine

DOEpatents

Mitchell, Alexander R.; Young, Janis D.

1996-01-01

The present invention relates to one or more polypeptides having useful biological activity in a mammal, which comprise: a polypeptide related to bradykinin of four to ten amino acid residues wherein one or more specific amino acids in the polypeptide chain are replaced with achiral N-benzylglycine. These polypeptide analogues have useful potent agonist or antagonist pharmacological properties depending upon the structure. A preferred polypeptide is (N-benzylglycine.sup.7)-bradykinin.

A composite model including visfatin, tissue polypeptide-specific antigen, hyaluronic acid, and hematological variables for the diagnosis of moderate-to-severe fibrosis in nonalcoholic fatty liver disease: a preliminary study.

PubMed

Chwist, Alina; Hartleb, Marek; Lekstan, Andrzej; Kukla, Michał; Gutkowski, Krzysztof; Kajor, Maciej

2014-01-01

Histopathological risk factors for end-stage liver failure in patients with nonalcoholic fatty liver disease (NAFLD) include nonalcoholic steatohepatitis (NASH) and advanced liver fibrosis. There is a need for noninvasive diagnostic methods for these 2 conditions. The aim of this study was to investigate new laboratory variables with a predictive potential to detect advanced fibrosis (stages 2 and 3) in NAFLD. The study involved 70 patients with histologically proven NAFLD of varied severity. Additional laboratory variables included zonulin, haptoglobin, visfatin, adiponectin, leptin, tissue polypeptide-specific antigen (TPSA), hyaluronic acid, and interleukin 6. Patients with NASH (NAFLD activity score of ≥5) had significantly higher HOMA-IR values and serum levels of visfatin, haptoglobin, and zonulin as compared with those without NASH on histological examination. Advanced fibrosis was found in 16 patients (22.9%) and the risk factors associated with its prevalence were age, the ratio of erythrocyte count to red blood cell distribution width, platelet count, and serum levels of visfatin and TPSA. Based on these variables, we constructed a scoring system that differentiated between NAFLD patients with and without advanced fibrosis with a sensitivity of 75% and specificity of 100% (area under the receiver operating characteristic curve, 0.93). The scoring system based on the above variables allows to predict advanced fibrosis with high sensitivity and specificity. However, its clinical utility should be verified in further studies involving a larger number of patients.

Molecular Characterization of Tomato 3-Dehydroquinate Dehydratase-Shikimate:NADP Oxidoreductase1

PubMed Central

Bischoff, Markus; Schaller, Andreas; Bieri, Fabian; Kessler, Felix; Amrhein, Nikolaus; Schmid, Jürg

2001-01-01

Analysis of cDNAs encoding the bifunctional 3-dehydroquinate dehydratase-shikimate:NADP oxidoreductase (DHQase-SORase) from tomato (Lycopersicon esculentum) revealed two classes of cDNAs that differed by 57 bp within the coding regions, but were otherwise identical. Comparison of these cDNA sequences with the sequence of the corresponding single gene unequivocally proved that the primary transcript is differentially spliced, potentially giving rise to two polypeptides that differ by 19 amino acids. Quantitative real-time polymerase chain reaction revealed that the longer transcript constitutes at most 1% to 2% of DHQase-SORase transcripts. Expression of the respective polypeptides in Escherichia coli mutants lacking the DHQase or the SORase activity gave functional complementation only in case of the shorter polypeptide, indicating that skipping of a potential exon is a prerequisite for the production of an enzymatically active protein. The deduced amino acid sequence revealed that the DHQase-SORase is most likely synthesized as a precursor with a very short (13-amino acid) plastid-specific transit peptide. Like other genes encoding enzymes of the prechorismate pathway in tomato, this gene is elicitor-inducible. Tissue-specific expression resembles the patterns obtained for 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase 2 and dehydroquinate synthase genes. This work completes our studies of the prechorismate pathway in that cDNAs for all seven enzymes (including isozymes) of the prechorismate pathway from tomato have now been characterized. PMID:11299368

Comparison of CYFRA 21-1 and tissue polypeptide specific antigen (TPS) for detecting nasopharyngeal carcinoma.

PubMed

Tai, Chih-Jaan; Liu, Feng-Yuan; Liang, Ji-An; Yang, Shih-Neng; Tsai, Ming-Hsui; Kao, Cbia-Hung

2002-01-01

CYFRA 21-1 is a tumor marker that is useful for detecting squamous cell carcinoma (SCC) of the lung. Tissue polypeptide specific antigen (TPS) is a tumor marker that indicates tumor proliferative rate rather than tumor burden. Our aim in this study was to compare the clinical value of CYFRA 21-1 and TPS in the detection of nasopharyngeal carcinoma (NPC). Serum levels of CYFRA 21-1 and TPS were measured in 60 patients with untreated NPC (including 36 differentiated SCC and 24 undifferentiated carcinomas) for comparison with each other. The cut-off values of CYFRA 21-1 and TPS, determined at the 95th percentile of the 43 healthy controls, were 2.50 ng/ml and 115.2 U/l, respectively. The results revealed that the mean serum values of CYFRA 21-1 (6.20 +/- 5.21 ng/ml) and TPS (153.9 +/- 17.3 U/l) in all 60 NPC patients were significantly higher than that in the 43 healthy controls (CYFRA 21-1 = 1.32 + 0.50 ng/ml, TPS = 85.0 +/- 16.0 U/l) (p value < 0.05). In addition, the detecting sensitivities of CYFRA 21-1 (60.0%) and TPS (58.3%) for all 60 NPC were not significantly different (p value > 0.05). In conclusion, our results suggest that both CYFRA 21-1 and TPS are potential tumor markers for the detection of NPC.

Control of storage-protein synthesis during seed development in pea (Pisum sativum L.).

PubMed Central

Gatehouse, J A; Evans, I M; Bown, D; Croy, R R; Boulter, D

1982-01-01

The tissue-specific syntheses of seed storage proteins in the cotyledons of developing pea (Pisum sativum L.) seeds have been demonstrated by estimates of their qualitative and quantitative accumulation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and rocket immunoelectrophoresis respectively. Vicilin-fraction proteins initially accumulated faster than legumin, but whereas legumin was accumulated throughout development, different components of the vicilin fraction had their predominant periods of synthesis at different stages of development. The translation products in vitro of polysomes isolated from cotyledons at different stages of development reflected the synthesis in vivo of storage-protein polypeptides at corresponding times. The levels of storage-protein mRNA species during development were estimated by 'Northern' hybridization using cloned complementary-DNA probes. This technique showed that the levels of legumin and vicilin (47000-Mr precursors) mRNA species increased and decreased in agreement with estimated rates of synthesis of the respective polypeptides. The relative amounts of these messages, estimated by kinetic hybridization were also consistent. Legumin mRNA was present in leaf poly(A)+ RNA at less than one-thousandth of the level in cotyledon poly(A)+ (polyadenylated) RNA, demonstrating tissue-specific expression. Evidence is presented that storage-protein mRNA species are relatively long-lived, and it is suggested that storage-protein synthesis is regulated primarily at the transcriptional level. Images Fig. 2. Fig. 3. PMID:6897609

Evaluation of a biomimetic 3D substrate based on the Human Elastin-like Polypeptides (HELPs) model system for elastolytic activity detection.

PubMed

Corich, Lucia; Busetti, Marina; Petix, Vincenzo; Passamonti, Sabina; Bandiera, Antonella

2017-08-10

Elastin is a fibrous protein that confers elasticity to tissues such as skin, arteries and lung. It is extensively cross-linked, highly hydrophobic and insoluble. Nevertheless, elastin can be hydrolysed by bacterial proteases in infectious diseases, resulting in more or less severe tissue damage. Thus, development of substrates able to reliably and specifically detect pathogen-secreted elastolytic activity is needed to improve the in vitro evaluation of the injury that bacterial proteases may provoke. In this work, two human biomimetic elastin polypeptides, HELP and HELP1, as well as the matrices derived from HELP, have been probed as substrates for elastolytic activity detection. Thirty strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients were analyzed in parallel with standard substrates, to detect proteolytic and elastolytic activity. Results point to the HELP-based 3D matrix as an interesting biomimetic model of elastin to assess bacterial elastolytic activity in vitro. Moreover, this model substrate enables to further elucidate the mechanism underlying elastin degradation at molecular level, as well as to develop biomimetic material-based devices responsive to external stimuli. Copyright © 2017 Elsevier B.V. All rights reserved.

Catalytic and reactive polypeptides and methods for their preparation and use

DOEpatents

Schultz, Peter

1993-01-01

Catalytic and reactive polypeptides include a binding site specific for a reactant or reactive intermediate involved in a chemical reaction of interest. The polypeptides further include at least one active functionality proximate the bi.

Distribution of Pancreatic Polypeptide-secreting Endocrine Cells in Nondiabetic and Diabetic Cases.

PubMed

Śliwińska-Mossoń, Mariola; Milnerowicz, Halina

2017-07-01

The aim of the study was to demonstrate the effects of cigarette smoking and ongoing inflammation in chronic pancreatitis on the functioning of pancreatic polypeptide (PP)-secreting cells and to determine the relationship between the occurrence of an increased number of PP cells in the pancreas, the change in their location, and the intensity of their inflammatory changes in the course of pancreatitis and diabetes. Samples of tissues from healthy persons and from patients were verified histopathologically, and then PP was localized by immunohistochemical staining using the monoclonal anti-human PP antibody. The histopathologic evaluation of the hormone expression intensity in tissue sections was carried out using the semiquantitative method and was calculated with digital image analysis. The present study showed a very strong PP expression in the pancreatic tissue (especially in the head of the pancreas) derived from smoking patients with diabetes. The increase in the percentage of cells in the PP islets, between the acinar cells in smoking patients with diabetes and a statistically significant increase in the expression of PP, indicates a pancreatic endocrine dysfunction and suggests that cigarette smoking has a negative impact on the organ's efficiency. Because of its properties, the PP appears to be a useful marker of the endocrine insufficiency of the pancreas and a specific prognostic parameter of developing diabetes due to chronic pancreatitis.

Pituitary adenylate cyclase activating polypeptide reduces A-type K+ currents and caspase activity in cultured adult mouse olfactory neurons.

PubMed

Han, P; Lucero, M T

2005-01-01

Pituitary adenylate cyclase activating polypeptide has been shown to reduce apoptosis in neonatal cerebellar and olfactory receptor neurons, however the underlying mechanisms have not been elucidated. In addition, the neuroprotective effects of pituitary adenylate cyclase activating polypeptide have not been examined in adult tissues. To study the effects of pituitary adenylate cyclase activating polypeptide on neurons in apoptosis, we measured caspase activation in adult olfactory receptor neurons in vitro. Interestingly, we found that the protective effects of pituitary adenylate cyclase activating polypeptide were related to the absence of a 4-aminopyridine (IC50=144 microM) sensitive rapidly inactivating potassium current often referred to as A-type current. In the presence of 40 nM pituitary adenylate cyclase activating polypeptide 38, both A-type current and activated caspases were significantly reduced. A-type current reduction by pituitary adenylate cyclase activating polypeptide was blocked by inhibiting the phospholipase C pathway, but not the adenylyl cyclase pathway. Our observation that 5 mM 4-aminopyridine mimicked the caspase inhibiting effects of pituitary adenylate cyclase activating polypeptide indicates that A-type current is involved in apoptosis. This work contributes to our growing understanding that potassium currents are involved with the activation of caspases to affect the balance between cell life and death.

Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.

PubMed

Mossabeb, Roschanak; Seiberler, Susanne; Mittermann, Irene; Reininger, Renate; Spitzauer, Susanne; Natter, Susanne; Verdino, Petra; Keller, Walter; Kraft, Dietrich; Valenta, Rudolf

2002-10-01

The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells. It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids. Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients. By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library. Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family. Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography. By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity. Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies. Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein. Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient. As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations in atopic dermatitis.

Multifunctional cellulase and hemicellulase

DOEpatents

Fox, Brian G.; Takasuka, Taichi; Bianchetti, Christopher M.

2015-09-29

A multifunctional polypeptide capable of hydrolyzing cellulosic materials, xylan, and mannan is disclosed. The polypeptide includes the catalytic core (cc) of Clostridium thermocellum Cthe_0797 (CelE), the cellulose-specific carbohydrate-binding module CBM3 of the cellulosome anchoring protein cohesion region (CipA) of Clostridium thermocellum (CBM3a), and a linker region interposed between the catalytic core and the cellulose-specific carbohydrate binding module. Methods of using the multifunctional polypeptide are also disclosed.

Component effect of stem cell-loaded thermosensitive polypeptide hydrogels on cartilage repair.

PubMed

Liu, He; Cheng, Yilong; Chen, Jinjin; Chang, Fei; Wang, Jincheng; Ding, Jianxun; Chen, Xuesi

2018-06-01

Biophysical properties of the desired biomimetic scaffolds, such as porosity and elasticity, have been proven associated with the efficacy of cartilage regeneration. In this work, the copolymers of poly(l-alanine)-block-poly(ethylene glycol)-block-poly(l-alanine) (PA-PEG-PA) and poly(l-alanine-co-l-phenylalanine)-block-poly(ethylene glycol)-block-poly(l-alanine-co-l-phenylalanine) (PAF-PEG-PAF) with different ratios of alanine to phenylalanine were synthesized. The introduction of a hydrophobic amino acid, i.e., phenylalanine, into polyalanine-based thermosensitive hydrogel led to the enhanced gelation behaviors and upregulated mechanical properties. Moreover, the increase of phenylalanine content resulted in the enlarged pore size and enhanced mechanical strength of PAF-PEG-PAF thermogel, followed by the regeneration of hyaline-like cartilage with reduced fibrous tissue formation in vivo. The findings indicated the great potential of thermosensitive polypeptide hydrogels in cartilage tissue engineering. Articular cartilage defect has limited self-repair ability due to the lack of blood supply and innervation, which may lead to knee osteoarthritis afterwards. Injectable hydrogels are demonstrated possessing outstanding properties as biomimetic scaffolds in cartilage tissue engineering, while the effect of biophysical properties on the efficacy of cartilage regeneration has not been revealed. Herein, the poly(ethylene glycol)-polypeptide triblock copolymers with different ratios of alanine to phenylalanine were synthesized. The sol-to-gel transition temperature and the critical gelation concentration decreased as the increased amount of phenylalanine unit, resulting in the enlarged pore size and enhanced mechanical strength. These features lead to better regeneration of hyaline-like cartilage with reduced fibrous tissue formation, indicating great potential of thermosensitive polypeptide hydrogels for efficient cartilage repair. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Localization of the human 64kD autoantigen D1 to myofibrils in a subset of extraocular muscle fibers

NASA Technical Reports Server (NTRS)

Conley, C. A.; Fowler, V. M.

1999-01-01

PURPOSE. To evaluate the tissue-specific expression pattern of the 64kD human autoantigen D1, a tropomodulin-related protein that may be involved in thyroid-associated ophthalmopathy. METHODS. Recombinant 64kD human autoantigen D1 was generated in a bacterial expression system and used to immunize rabbits. Specific antibodies were affinity-purified and used for Western blots on normal and hyperthyroid rat and rabbit tissue, and immunofluorescence localization on cryosections of rat tissue. RESULTS. Anti-64kD human autoantigen D1 antibodies recognize specifically a approximately 70kD polypeptide in western blots of extraocular muscle, sternothyroid muscle, and smooth muscle. Immunofluorescence staining demonstrates that the 64kD human autoantigen D1 localizes to myofibrils in slow fibers from rat extraocular and sternothyroid muscle. The level of this protein is not altered in extraocular muscles from hyperthyroid rabbits. CONCLUSIONS. The 64kD human autoantigen D1 is expressed in slow fibers of extraocular and sternothyroid muscles as a component of myofibrils, and is not upregulated in conditions of hyperthyroidism.

Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

NASA Technical Reports Server (NTRS)

Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

1992-01-01

The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

Ectopic expression of the gastric inhibitory polypeptide receptor gene is a sufficient genetic event to induce benign adrenocortical tumor in a xenotransplantation model.

PubMed

Mazzuco, Tania L; Chabre, Olivier; Sturm, Nathalie; Feige, Jean-Jacques; Thomas, Michaël

2006-02-01

Aberrant expression of ectopic G protein-coupled receptors (GPCRs) in adrenal cortex tissue has been observed in several cases of ACTH-independent macronodular adrenal hyperplasias and adenomas associated with Cushing's syndrome. Although there is clear clinical evidence for the implication of these ectopic receptors in abnormal regulation of cortisol production, whether this aberrant GPCR expression is the cause or the consequence of the development of an adrenal hyperplasia is still an open question. To answer it, we genetically engineered primary bovine adrenocortical cells to have them express the gastric inhibitory polypeptide receptor. After transplantation of these modified cells under the renal capsule of adrenalectomized immunodeficient mice, tissues formed had their functional and histological characteristics analyzed. We observed the formation of an enlarged and hyperproliferative adenomatous adrenocortical tissue that secreted cortisol in a gastric inhibitory polypeptide-dependent manner and induced a mild Cushing's syndrome with hyperglycemia. Moreover, we show that tumor development was ACTH independent. Thus, a single genetic event, inappropriate expression of a nonmutated GPCR gene, is sufficient to initiate the complete phenotypic alterations that ultimately lead to the formation of a benign adrenocortical tumor.

Serum tumor markers in breast cancer: are they of clinical value?

PubMed

Duffy, Michael J

2006-03-01

Although multiple serum-based tumor markers have been described for breast cancer, such as CA 15-3, BR 27.29 (CA27.29), carcinoembryonic antigen (CEA), tissue polypeptide antigen, tissue polypeptide specific antigen, and HER-2 (the extracellular domain), the most widely used are CA 15-3 and CEA. The literature relevant to serum tumor markers in breast cancer was reviewed. Particular attention was given to systematic reviews, prospective randomized trials, and guidelines issued by expert panels. Because of a lack of sensitivity for early disease and lack of specificity, none of the available markers is of value for the detection of early breast cancer. High preoperative concentrations of CA 15-3 are, however, associated with adverse patient outcome. Although serial determinations of tumor markers after primary treatment for breast cancer can preclinically detect recurrent/metastatic disease with lead times of approximately 2-9 months, the clinical value of this lead time remains to be determined. Serum markers, however, are the only validated approach for monitoring treatment in patients with advanced disease that cannot be evaluated by use of conventional criteria. CA 15-3 is one of the first circulating prognostic factors for breast cancer. Preoperative concentrations thus might be combined with existing prognostic factors for predicting outcome in patients with newly diagnosed breast cancer. At present, the most important clinical application of CA 15-3 is in monitoring therapy in patients with advanced breast cancer that is not assessable by existing clinical or radiologic procedures.

Biochemical, molecular, and phylogenetic analysis of pyruvate carboxylase in the yellow fever mosquito, Aedes aegypti.

PubMed

Tu, Z; Hagedorn, H H

1997-02-01

Pyruvate carboxylase (PC, pyruvate: carbon dioxide ligase [ADP-forming], EC 6.4.1.1) was purified from the yellow fever mosquito, Aedes aegypti. The purified PC showed two polypeptides of similar M(r) (133 and 128 k). The N-terminal sequences of both polypeptides were shown to be very similar, if not identical. A polyclonal antiserum against the 133 kDa polypeptide cross-reacted strongly with the 128 kDa polypeptide. PC was found in all tissues examined. Using a semi-quantitative Western blot assay, PC was shown to be concentrated in the indirect flight muscles and fat body preparations. The ratios of the 133 to 128 kDa polypeptides were shown to differ in various tissues and an Aedes albopictus cell line. The indirect flight muscle was the only tissue in which the 128 kDa polypeptide was more abundant, while both the midgut and the cell line showed almost exclusively the 133 kDa polypeptide. Both peptides were present in varying amounts in brain, malpighian tubule, ovary and fat body preparation. The two isoforms of PC could play different roles in the flight muscle and other tissues. Clones covering a complete cDNA of PC of A. aegypti were obtained using a directional approach. The 3952 bp nucleotide sequence, including a 3585 bp coding region, was determined from these cDNA clones. The deduced 1195 amino acid sequence has a calculated M(r) of 132,200. A putative mitochondrial targeting sequence was determined by comparing the deduced amino acid sequence to the N-terminal sequences of the mature protein. The presence of a mitochondrial targeting sequence indicates that the mosquito PC encoded by the cloned cDNA may be localized in the mitochondria. After the targeting sequence, three functional domains were identified in the following order; biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP). The mosquito PC showed very high similarity to PCs from other sources (55.1-75.2% identity). Genomic Southern analysis indicated that there could be two similar PC genes or a single PC gene with allelic polymorphism in the A. aegypti genome. The evolutionary relationship of PCs among different organisms was consistent with the accepted evolutionary relationship of their host organisms. The evolution of the domain structures of the biotin-dependent carboxylases including PC was also investigated. This analysis indicates that biotin-dependent carboxylases evolved from a common origin. The analysis also provides evidence for early gene duplication events that shaped the family of biotin-dependent carboxylases. Clear evidence for the coevolution of BC and BCCP domains is presented, although they are associated with very different CT domains and the relative position of the three functional domains varies between members of the biotin-dependent carboxylases.

Measurements of free and total PSA, tissue polypeptide-specific antigen (TPS), and CYFRA 21-1 in prostate cancer patients under intermittent androgen suppression therapy.

PubMed

Theyer, G; Dürer, A; Theyer, U; Haberl, I; Ulsperger, E; Baumgartner, G; Hamilton, G

1999-10-01

The present study evaluated monthly measurements of free and total prostate-specific antigen (PSA), and the tumor proliferation markers tissue polypeptide-specific antigen (TPS) and cytokeratin fragment 21-1 (CYFRA 21-1) in patients with advanced prostate cancer receiving intermittent androgen suppression therapy (IAS). Thirty-four men received alternating cycles of 8 month androgen suppression and treatment cessation (mean duration, 10.3 months) until PSA increased to >20 microg/l. Measurements of testosterone, percentage of free PSA, TPS, and CYFRA 21-1 were performed using ELISA and RIA assays. Periods of androgen suppression resulted in reversible reductions of testosterone (from 6 +/- 0.8 to <0.58 ng/ml), PSA (from 31.2 +/- 4.5 to <1.7 microg/l), and prostatic volume (mean reduction, 22.2 +/- 4.6%), indicating apoptotic regression of the tumors. Upon treatment cessation, testosterone increased to 6.1 +/- 0.56 ng/ml within 2 months, followed by an increase of PSA to 5.8 +/- 0.8 microg/l. The mean percentage of free PSA (15.1 +/- 2.6%) exhibited no significant change during the whole IAS cycle. TPS showed a decrease of 50% after 3 months, and CYFRA 21-1 a 25% decrease after 7 months of androgen suppression treatment. During treatment cessation, TPS exceeded the normal cutoff value of 90 U/l late in tumor regrowth (9-11 months), whereas CYFRA 21-1 remained below the normal cutoff value of 3.3 ng/ml. PSA is the best and most sensitive marker of prostate cancer regression and regrowth during IAS cycles of the markers tested in this study. Free PSA constitutes approximately 15% of total PSA (range, 5-32%), and its percentage showed no significant change during IAS cycles. The TPS and CYFRA 21-1 proliferation marker changes in IAS seem to be related mainly to effects on normal androgen-dependent tissues. Copyright 1999 Wiley-Liss, Inc.

Chemical determination of polypeptide hormones.

PubMed Central

Tatemoto, K; Mutt, V

1978-01-01

The presence or absence of peptide hormones in tissue extracts may in certain cases be demonstrated by exposing the extracts to conditions under which characteristic fragments of the polypeptide molecule in question are formed and then analyzing for such fragments. An approximate quantitation of the hormones may also be achieved thereby. In the present work the COOH-terminal fragments of polypeptides containing characteristic alpha-amide groups were released enzymatically and then converted into the fluorescent dansyl derivatives, which were identified by thin-layer chromatography. In this way the presence of secretin, cholecystokinin, and the vasoactive intestinal peptide in concentrates of porcine intestinal extracts were demonstrated by their COOH-terminal amide fragments: valine (or leucylvaline) amide, phenylalanine amide, and asparagine (or leucylasparagine) amide, respectively. The analytical methodology used in the present study may also be useful in devising simple and reliable chemical assay methods for the isolation of already known polypeptides and in the isolation of previously uncharacterized polypeptides from natural sources. Images PMID:279902

The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis.

PubMed

Sato, T; Oeller, P W; Theologis, A

1991-02-25

The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14). We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl. The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each. The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product. DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6621-6625). Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da. The data suggest that the enzyme is post-translationally processed during protein purification.

Evidence for a hepatocellular lineage in a combined hepatocellular-cholangiocarcinoma of transitional type.

PubMed

Fisher, H P; Doppl, W; Osborn, M; Altmannsberger, M

1988-01-01

A combined hepatocellular-cholangiocarcinoma (CHC) of transitional subtype and the surrounding cirrhotic liver tissue were investigated immunocytochemically by monoclonal antibodies specific for each of the keratin polypeptides 7, 8, 18 and 19. Different keratin subsets were found in different parts of the tumour. The hepatocellular component reveals keratins 8 and 18, with the bordering cells of trabecular formations additionally expressing keratins 7 and 19. The same keratins i.e. 7, 8, 18, 19 were found in normal bile duct epithelium as well as in cholangiocarcinomatous and transitional areas of hepatocellular and cholangiocellular differentiation. Normal hepatocytes express only keratin 8 and 18. In cirrhotic liver some modified hepatocytes additionally express keratin 7. When ductal transformation is observed in the marginal parts of portal tracts and fibrous septa the keratin polypeptide pattern mimics that of bile duct epithelium. The cholangiocellular metaplasia of hepatocytes observed here correlates well with findings in hepato-organogenesis and hepatocarcinogenesis and suggests that the transitional subtype of combined hepatocellular-cholangiocarcinoma is a variant of hepatocellular carcinoma.

Identification of substance P precursor forms in human brain tissue.

PubMed Central

Nyberg, F; le Grevés, P; Terenius, L

1985-01-01

Substance P prohormones were identified in the caudate nucleus, hypothalamus, and substantia nigra of human brain. A polypeptide fraction of acidic brain extracts was fractionated on Sephadex G-50. The lyophilized fractions were sequentially treated with trypsin and a substance P-degrading enzyme with strong preference toward the Phe7-Phe8 and Phe8-Gly9 bonds. The released substance P(1-7) fragment was isolated by ion-exchange chromatography and quantitated by a specific radioimmunoassay. Confirmation of the structure of the isolated radioimmunoassay-active fragment was achieved by electrophoresis and HPLC. By using this enzymatic/radioimmunoassay procedure, two polypeptide fractions of apparent Mr 5000 and 15,000, respectively, were identified. The latter component was the major one of the two but was estimated to account for only about 5% of total substance P radioimmunoassay activity. Because it is of the size predicted from the nucleotide sequences of cDNA for substance P prohormones in bovine brain, the Mr 15,000 component may represent the full-length prohormone. PMID:2408270

Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate.

PubMed

Chang, J Y

1985-09-02

alpha-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative amino-terminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of alpha-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by alpha-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit alpha-thrombin in 20 microliters of 50 mM ammonium bicarbonate at 25 degrees C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for alpha-thrombin has the structures of (a) P4-P3-Pro-Arg-P1'-P2', where P3 and P4 are hydrophobic amino acid and P1', P2' are nonacidic amino acids and (b) P2-Arg-P1', where P2 or P1' are Gly. The requirement for hydrophobic P3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P1' and P2' residues. These findings confirm the importance of hydrophobic P2-P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by alpha-thrombin can be reasonably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage.

Plants with elevated levels of glucan

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

Pauly, Markus; Kraemer, Florian J.; Hake, Sarah

The present disclosure relates to mutations in licheninase genes encoding polypeptides with decreased licheninase activity, which when expressed in plants results in elevated levels of glucan in the plants. In particular, the disclosure relates to licheninase nucleic acids and polypeptides related to glucan accumulation in plants, plants with reduced expression of a licheninase nucleic acid, and methods related to the generation of plants with increased glucan content in the cell walls of leaf tissue.

Transgenic rescue of adipocyte glucose-dependent insulinotropic polypeptide receptor expression restores high fat diet-induced body weight gain.

PubMed

Ugleholdt, Randi; Pedersen, Jens; Bassi, Maria Rosaria; Füchtbauer, Ernst-Martin; Jørgensen, Signe Marie; Kissow, Hanne-Louise; Nytofte, Nikolaj; Poulsen, Steen Seier; Rosenkilde, Mette Marie; Seino, Yutaka; Thams, Peter; Holst, Peter Johannes; Holst, Jens Juul

2011-12-30

The glucose-dependent insulinotropic polypeptide receptor (GIPr) has been implicated in high fat diet-induced obesity and is proposed as an anti-obesity target despite an uncertainty regarding the mechanism of action. To independently investigate the contribution of the insulinotropic effects and the direct effects on adipose tissue, we generated transgenic mice with targeted expression of the human GIPr to white adipose tissue or beta-cells, respectively. These mice were then cross-bred with the GIPr knock-out strain. The central findings of the study are that mice with GIPr expression targeted to adipose tissue have a similar high fat diet -induced body weight gain as control mice, significantly greater than the weight gain in mice with a general ablation of the receptor. Surprisingly, this difference was due to an increase in total lean body mass rather than a gain in total fat mass that was similar between the groups. In contrast, glucose-dependent insulinotropic polypeptide-mediated insulin secretion does not seem to be important for regulation of body weight after high fat feeding. The study supports a role of the adipocyte GIPr in nutrient-dependent regulation of body weight and lean mass, but it does not support a direct and independent role for the adipocyte or beta-cell GIPr in promoting adipogenesis.

Peptidergic innervation of the human male genital tract.

PubMed

Gu, J; Polak, J M; Probert, L; Islam, K N; Marangos, P J; Mina, S; Adrian, T E; McGregor, G P; O'Shaughnessy, D J; Bloom, S R

1983-08-01

Four peptides--vasoactive intestinal polypeptide, substance P, somatostatin and a peptide-like avian pancreatic polypeptide--have been found in nerves of the human male genitalia using highly sensitive and specific methods of immunocytochemistry and radioimmunoassay. Five other peptides (met-enkephalin, leu-enkephalin, neurotensin, bombesin and cholecystokinin-8) were absent. Vasoactive intestinal polypeptide was the most abundant peptide, its highest concentration being in the proximal corpus cavernosum. Immunoelectron microscopy localized this peptide to large (97 +/- 20 nm), round, electron-dense granules of p-type nerve terminals. Vasoactive intestinal polypeptide-immunoreactive neuronal cell bodies were found in the prostate gland and the root of the corpus cavernosum. Substance P immunoreactive material was present in smaller concentration and was mainly localized in nerves around the corpuscular receptors of the glans penis. Somatostatin immunoreactive nerves were associated mainly with the smooth muscle of the seminal vesicle and the vas deferens. When antiserum to avian pancreatic polypeptide was applied, certain nerves were stained, particularly in the vas deferens, the prostate gland and the seminal vesicle. However, chromatography detected no pure avian pancreatic polypeptide suggesting the presence of a structurally related substance, possibly neuropeptide Y, which cross-reacts with the avian pancreatic polypeptide antiserum. Similar distributions between vasoactive intestinal polypeptide-immunoreactive and acetylcholinesterase-positive nerves and between avian pancreatic polypeptide-immunoreactive and adrenergic nerves were observed. A general neuronal marker, neuron-specific enolase, was used to investigate the general pattern of the organ's innervation. The abundance and distribution patterns of these peptide-immunoreactive nerves indicate that they may play important roles in the male sexual physiology.

Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting.

PubMed Central

Tamura, A; Ohashi, N; Urakami, H; Takahashi, K; Oyanagi, M

1985-01-01

Polyacrylamide gel electrophoresis of lysates of purified Rickettsia tsutsugamushi revealed as many as 30 polypeptide bands, including major bands corresponding to molecular sizes of 70, 60, 54 to 56, and 46 to 47 kilodaltons. Compared with the polypeptide composition of the rickettsiae of Gilliam, Karp, and Kato strains and a newly isolated Shimokoshi strain, the major polypeptide in the Kato strain (54-56K) and in the Karp strain (46-47K) migrated a little faster and slower, respectively, than the corresponding polypeptides in the other strains. The largest major polypeptide (54-56K) was digestible by the treatment of intact rickettsiae with trypsin and variable in content in separate preparations, suggesting that the polypeptide exists on the rickettsial surface and is easily degraded during the handling of these microorganisms. Several surface polypeptides of rickettsiae, including the 54-56K and 46-47K polypeptides, were detected by radioiodination of intact rickettsiae followed by polyacrylamide gel electrophoresis of the lysate; however, the 70K and 60K polypeptides were not labeled. Immunoblotting experiments with hyperimmune sera prepared in guinea pigs against each strain demonstrated that the 70K, 54-56K, and 46-47K polypeptides showed antigenic activities. The 54-56K polypeptide appeared to be strain specific, whereas the 70K and 46-47K polypeptides cross-reacted with the heterologous antisera. Images PMID:3922893

Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

NASA Astrophysics Data System (ADS)

Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

2017-02-01

The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring.

Hormonal control of implantation.

PubMed

Sandra, Olivier

2016-06-01

In mammals, implantation represents a key step of pregnancy and its progression conditions not only the success of pregnancy but health of the offspring. Implantation requires a complex and specific uterine tissue, the endometrium, whose biological functions are tightly regulated by numerous signals, including steroids and polypeptide hormones. Endometrial tissue is endowed with dynamic properties that associate its ability to control the developmental trajectory of the embryo (driver property) and its ability to react to embryos displaying distinct capacities to develop to term (sensor property). Since dynamical properties of the endometrium can be affected by pre- and post-conceptional environment, determining how maternal hormonal signals and their biological actions are affected by environmental factors (e.g. nutrition, stress, infections) is mandatory to reduce or even to prevent their detrimental effects on endometrial physiology in order to preserve the optimal functionality of this tissue. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells.

PubMed

Ghandour, M S; Parkkila, A K; Parkkila, S; Waheed, A; Sly, W S

2000-11-01

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.

Toughening of Thermoresponsive Arrested Networks of Elastin-Like Polypeptides To Engineer Cytocompatible Tissue Scaffolds.

PubMed

Glassman, Matthew J; Avery, Reginald K; Khademhosseini, Ali; Olsen, Bradley D

2016-02-08

Formulation of tissue engineering or regenerative scaffolds from simple bioactive polymers with tunable structure and mechanics is crucial for the regeneration of complex tissues, and hydrogels from recombinant proteins, such as elastin-like polypeptides (ELPs), are promising platforms to support these applications. The arrested phase separation of ELPs has been shown to yield remarkably stiff, biocontinuous, nanostructured networks, but these gels are limited in applications by their relatively brittle nature. Here, a gel-forming ELP is chain-extended by telechelic oxidative coupling, forming extensible, tough hydrogels. Small angle scattering indicates that the chain-extended polypeptides form a fractal network of nanoscale aggregates over a broad concentration range, accessing moduli ranging from 5 kPa to over 1 MPa over a concentration range of 5-30 wt %. These networks exhibited excellent erosion resistance and allowed for the diffusion and release of encapsulated particles consistent with a bicontinuous, porous structure with a broad distribution of pore sizes. Biofunctionalized, toughened networks were found to maintain the viability of human mesenchymal stem cells (hMSCs) in 2D, demonstrating signs of osteogenesis even in cell media without osteogenic molecules. Furthermore, chondrocytes could be readily mixed into these gels via thermoresponsive assembly and remained viable in extended culture. These studies demonstrate the ability to engineer ELP-based arrested physical networks on the molecular level to form reinforced, cytocompatible hydrogel matrices, supporting the promise of these new materials as candidates for the engineering and regeneration of stiff tissues.

Identification of polypeptides necessary for chemotaxis in Escherichia coli.

PubMed Central

Silverman, M; Simon, M

1977-01-01

Molecular cloning techniques were used to construct Escherichia coli-lambda hybrids that contained many of the genes necessary for flagellar rotation and chemotaxis. The properties of specific hybrids that carried the classical "cheA" and "cheB" loci were examined by genetic complementation and by measuring the capacity of the hybrids to direct the synthesis of specific polypeptides. The results of these tests with lambda hybrids and with a series of deletion mutations derived from the hybrids redefined the "cheA" and "cheB" regions. Six genes were resolved: cheA, cheW, cheX, cheB, cheY, and cheZ. They directed the synthesis of specific polypeptides with the following apparent molecular weights: cheA, 76,000 and 66,000; cheW, 12,000; cheX, 28,000; cheB, 38,000; cheY, 8,000; and cheZ, 24,000. The presence of another gene, cheM, was inferred from the protein synthesis experiments. The cheM gene directed the synthesis of polypeptides with apparent molecular weights of 63,000, 61,000, and 60,000. The synthesis of all of these polypeptides is regulated by the same mechanisms that regulate the synthesis of flagellar-related structural components. Images PMID:324984

Advanced biorefinery in lower termite-effect of combined pretreatment during the chewing process

PubMed Central

2012-01-01

Background Currently the major barrier in biomass utilization is the lack of an effective pretreatment of plant cell wall so that the carbohydrates can subsequently be hydrolyzed into sugars for fermentation into fuel or chemical molecules. Termites are highly effective in degrading lignocellulosics and thus can be used as model biological systems for studying plant cell wall degradation. Results We discovered a combination of specific structural and compositional modification of the lignin framework and partial degradation of carbohydrates that occurs in softwood with physical chewing by the termite, Coptotermes formosanus, which are critical for efficient cell wall digestion. Comparative studies on the termite-chewed and native (control) softwood tissues at the same size were conducted with the aid of advanced analytical techniques such as pyrolysis gas chromatography mass spectrometry, attenuated total reflectance Fourier transform infrared spectroscopy and thermogravimetry. The results strongly suggest a significant increase in the softwood cellulose enzymatic digestibility after termite chewing, accompanied with utilization of holocellulosic counterparts and an increase in the hydrolysable capacity of lignin collectively. In other words, the termite mechanical chewing process combines with specific biological pretreatment on the lignin counterpart in the plant cell wall, resulting in increased enzymatic cellulose digestibility in vitro. The specific lignin unlocking mechanism at this chewing stage comprises mainly of the cleavage of specific bonds from the lignin network and the modification and redistribution of functional groups in the resulting chewed plant tissue, which better expose the carbohydrate within the plant cell wall. Moreover, cleavage of the bond between the holocellulosic network and lignin molecule during the chewing process results in much better exposure of the biomass carbohydrate. Conclusion Collectively, these data indicate the participation of lignin-related enzyme(s) or polypeptide(s) and/or esterase(s), along with involvement of cellulases and hemicellulases in the chewing process of C. formosanus, resulting in an efficient pretreatment of biomass through a combination of mechanical and enzymatic processes. This pretreatment could be mimicked for industrial biomass conversion. PMID:22390274

A novel glutamine-rich putative transcriptional adaptor protein (TIG-1), preferentially expressed in placental and bone-marrow tissues.

PubMed

Abraham, S; Solomon, W B

2000-09-19

We used a subtractive hybridization protocol to identify novel expressed sequence tags (ESTs) corresponding to mRNAs whose expression was induced upon exposure of the human leukemia cell line K562 to the phorbol ester 12-O-tetradecanolyphorbol-13-acetate (TPA). The complete open reading frame of one of the novel ESTs, named TIG-1, was obtained by screening K562 cell and placental cDNA libraries. The deduced open reading frame of the TIG-1 cDNA encodes for a glutamine repeat-rich protein with a predicted molecular weight of 63kDa. The predicted open reading frame also contains a consensus bipartite nuclear localization signal, though no specific DNA-binding domain is found. The corresponding TIG-1 mRNA is ubiquitously expressed. Placental tissue expresses the TIG-1 mRNA 200 times more than the lowest expressing tissues such as kidney and lung. There is also preferential TIG-1 mRNA expression in cells of bone-marrow lineage.In-vitro transcription/translation of the TIG-1 cDNA yielded a polypeptide with an apparent molecular weight of 97kDa. Using polyclonal antibodies obtained from a rabbit immunized with the carboxy-terminal portion of bacterially expressed TIG-1 protein, a polypeptide with molecular weight of 97kDa was identified by Western blot analyses of protein lysates obtained from K562 cells. Cotransfection assays of K562 cells, using a GAL4-TIG-1 fusion gene and GAL4 operator-CAT, indicate that the TIG-1 protein may have transcriptional regulatory activity when tethered to DNA. We hypothesize that this novel glutamine-rich protein participates in a protein complex that regulates gene transcription. It has been demonstrated by Naar et al. (Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W.B., Tjian, R., 1999, Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828-830) that the amino acid sequences of peptide fragments obtained from a polypeptide found in a complex of proteins that alters chromatin structure (ARC) are identical to portions of the deduced open reading frame of TIG-1 mRNA.

Chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in rats.

PubMed

Han, Xun; Ran, Ye; Su, Min; Liu, Yinglu; Tang, Wenjing; Dong, Zhao; Yu, Shengyuan

2017-01-01

Background Preclinical experimental studies revealed an acute alteration of pituitary adenylate cyclase-activating polypeptide in response to a single activation of the trigeminovascular system, which suggests a potential role of pituitary adenylate cyclase-activating polypeptide in the pathogenesis of migraine. However, changes in pituitary adenylate cyclase-activating polypeptide after repeated migraine-like attacks in chronic migraine are not clear. Therefore, the present study investigated chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulations in the rat. Methods A rat model of chronic migraine was established by repeated chemical dural stimulations using an inflammatory soup for a different numbers of days. The pituitary adenylate cyclase-activating polypeptide levels were quantified in plasma, the trigeminal ganglia, and the trigeminal nucleus caudalis using radioimmunoassay and Western blotting in trigeminal ganglia and trigeminal nucleus caudalis tissues. Western blot analysis and real-time polymerase chain reaction were used to measure the protein and mRNA expression of pituitary adenylate cyclase-activating polypeptide-related receptors (PAC1, VPAC1, and VPAC2) in the trigeminal ganglia and trigeminal nucleus caudalis to identify changes associated with repetitive applications of chemical dural stimulations. Results All rats exhibited significantly decreased periorbital nociceptive thresholds to repeated inflammatory soup stimulations. Radioimmunoassay and Western blot analysis demonstrated significantly decreased pituitary adenylate cyclase-activating polypeptide levels in plasma and trigeminal ganglia after repetitive chronic inflammatory soup stimulation. Protein and mRNA analyses of pituitary adenylate cyclase-activating polypeptide-related receptors demonstrated significantly increased PAC1 receptor protein and mRNA expression in the trigeminal ganglia, but not in the trigeminal nucleus caudalis, and no significant differences were found in the expression of the VPAC1 and VPAC2 receptors. Conclusions This study demonstrated the chronic alteration of pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in the rat, which suggests the crucial involvement of pituitary adenylate cyclase-activating polypeptide in the development of migraine. The selective increase in pituitary adenylate cyclase-activating polypeptide-related receptors suggests that the PAC1 receptor pathway is a novel target for the treatment of migraine.

The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages

NASA Astrophysics Data System (ADS)

Peng, Zhou; Bu, Jiexun; McLuckey, Scott A.

2017-09-01

We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states. [Figure not available: see fulltext.

Inhibition and Ultraviolet-Induced Chemical Modification of UDP-Glucose:(1,3)-β-Glucan (Callose) Synthase by Chlorpromazine 1

PubMed Central

Harriman, Robert W.; Shao, Ai-Ping; Wasserman, Bruce P.

1992-01-01

UDP-glucose:(1,3)-β-glucan (callose) synthase (CS) from storage tissue of red beet (Beta vulgaris L.) was strongly inhibited by the phenothiazine drug chlorpromazine (CPZ). In the absence of ultraviolet irradiation, CPZ was a noncompetitive inhibitor with 50% inhibitory concentration values for plasma membrane and solubilized CS of 100 and 90 μm, respectively. Both the Ca2+- and Mg2+- stimulated components of CS activity were affected. CPZ inhibition was partially alleviated at saturating levels of Ca2+, but not Mg2+, suggesting that CPZ interferes with the Ca2+-binding site of CS. Binding experiments with [14C]CPZ, however, showed strong non-specific partitioning of CPZ into the plasma membrane, providing evidence that perturbation of the membrane environment is probably the predominant mode of inhibition. Ultraviolet irradiation at 254 nm markedly enhanced CPZ inhibition, with complete activity loss following exposure to 4 μm CPZ for 2 min. Inhibition followed a pseudo-first order mechanism with at least three CPZ binding sites per CS complex. Under these conditions, [3H]CPZ was covalently incorporated into plasma membrane preparations by a free radical mechanism; however, polypeptide labeling profiles showed labeling to be largely nonspecific, with many polypeptides labeled even at [3H]CPZ levels as low as 1 μm, and with boiled membranes. Although CPZ is one of the most potent known inhibitors of CS, its use as a photolabel will require a homogeneous CS complex or establishment of conditions that protect against the interaction of CPZ with specific binding sites located on various polypeptide components of the CS complex. PMID:16653219

Secretion of pancreatic polypeptide in patients with pancreatic endocrine tumors.

PubMed

Adrian, T E; Uttenthal, L O; Williams, S J; Bloom, S R

1986-07-31

Pancreatic polypeptide is often secreted by pancreatic endocrine tumors and is considered a marker for such tumors. To investigate the diagnostic value of this marker, we studied 323 patients with proved pancreatic endocrine tumors. We found plasma concentrations of pancreatic polypeptide to be elevated (more than 300 pmol per liter) in 144 patients (diagnostic sensitivity, 45 percent). However, plasma levels of pancreatic polypeptide can also be elevated in the absence of a pancreatic tumor. To ascertain whether the administration of atropine could distinguish between normal and tumor-associated polypeptide secretion, we studied 30 patients with pancreatic tumors and high plasma levels of pancreatic polypeptide, 18 patients without tumors who had elevated levels of pancreatic polypeptide, and eight normal controls. Polypeptide levels in the 18 patients without tumors were substantially lower than in the 30 patients with tumors. Atropine (1 mg intramuscularly) did not suppress polypeptide levels in patients with tumors, but did suppress plasma levels by more than 50 percent in all subjects without tumors. Thus, although its diagnostic sensitivity is low, pancreatic polypeptide appears to be a useful adjunctive marker of many pancreatic endocrine tumors, and the atropine suppression test can be used to distinguish normal from tumor-related secretion of the polypeptide. Identification of the type of pancreatic endocrine tumor still requires measurement of the hormone that is specific for the tumor.

Improvement of Learning and Memory Induced by Cordyceps Polypeptide Treatment and the Underlying Mechanism

PubMed Central

2018-01-01

Our previous research revealed that Cordyceps militaris can improve the learning and memory, and although the main active ingredient should be its polypeptide complexes, the underlying mechanism of its activity remains poorly understood. In this study, we explored the mechanisms by which Cordyceps militaris improves learning and memory in a mouse model. Mice were given scopolamine hydrobromide intraperitoneally to establish a mouse model of learning and memory impairment. The effects of Cordyceps polypeptide in this model were tested using the Morris water maze test; serum superoxide dismutase activity; serum malondialdehyde levels; activities of acetyl cholinesterase, Na+-k+-ATPase, and nitric oxide synthase; and gamma aminobutyric acid and glutamate contents in brain tissue. Moreover, differentially expressed genes and the related cellular signaling pathways were screened using an mRNA expression profile chip. The results showed that the genes Pik3r5, Il-1β, and Slc18a2 were involved in the effects of Cordyceps polypeptide on the nervous system of these mice. Our findings suggest that Cordyceps polypeptide may improve learning and memory in the scopolamine-induced mouse model of learning and memory impairment by scavenging oxygen free radicals, preventing oxidative damage, and protecting the nervous system. PMID:29736181

Changes in Gene Expression during Tomato Fruit Ripening 1

PubMed Central

Biggs, M. Scott; Harriman, Robert W.; Handa, Avtar K.

1986-01-01

Total proteins from pericarp tissue of different chronological ages from normally ripening tomato (Lycopersicon esculentum Mill. cv Rutgers) fruits and from fruits of the isogenic ripening-impaired mutants rin, nor, and Nr were extracted and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Analysis of the stained bands revealed increases in 5 polypeptides (94, 44, 34, 20, and 12 kilodaltons), decreases in 12 polypeptides (106, 98, 88, 76, 64, 52, 48, 45, 36, 28, 25, and 15 kilodaltons), and fluctuations in 5 polypeptides (85, 60, 26, 21, and 16 kilodaltons) as normal ripening proceeded. Several polypeptides present in ripening normal pericarp exhibited very low or undetectable levels in developing mutant pericarp. Total RNAs extracted from various stages of Rutgers pericarp and from 60 to 65 days old rin, nor, and Nr pericarp were fractionated into poly(A)+ and poly(A)− RNAs. Peak levels of total RNA, poly(A)+ RNA, and poly(A)+ RNA as percent of total RNA occurred between the mature green to breaker stages of normal pericarp. In vitro translation of poly(A)+ RNAs from normal pericarp in rabbit reticulocyte lysates revealed increases in mRNAs for 9 polypeptides (116, 89, 70, 42, 38, 33, 31, 29, and 26 kilodaltons), decreases in mRNAs for 2 polypeptides (41 and 35 kilodaltons), and fluctuations in mRNAs for 5 polypeptides (156, 53, 39, 30, and 14 kilodaltons) during normal ripening. Analysis of two-dimensional separation of in vitro translated polypeptides from poly(A)+ RNAs isolated from different developmental stages revealed even more extensive changes in mRNA populations during ripening. In addition, a polygalacturonase precursor (54 kilodaltons) was immunoprecipitated from breaker, turning, red ripe, and 65 days old Nr in vitro translation products. Images Fig. 1 Fig. 3 Fig. 5 Fig. 6 Fig. 7 PMID:16664828

Comparative assessment of the polypeptide profiles from lateral and primary roots of Phaseolus vulgaris L

NASA Technical Reports Server (NTRS)

Westberg, J.; Odom, W. R.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1994-01-01

In Phaseolus vulgaris, primary roots show gravitational sensitivity soon after emerging from the seed. In contrast, lateral roots are agravitropic during early development, and become gravitropic after several cm growth. Primary and lateral root tissues were examined by polyacrylamide gel electrophoresis, coupled with western blotting techniques, to compare proteins which may contribute to the acquisition of gravitational sensitivity. Root tips and zones of cell elongation were compared for each root type, using immunological probes for calmodulin, alpha-actin, alpha-tubulin, and proteins of the plastid envelope. Lateral roots contained qualitatively less calmodulin, and showed a slightly different pattern of actin-related epitope proteins, than did primary root tissues, suggesting that polypeptide differences may contribute to the gravitational sensitivity which these root types express.

Pancreatic polypeptide and calcitonin secretion from a pancreatic tumour-clinical improvement after hepatic artery embolization.

PubMed Central

Manche, A.; Wood, S. M.; Adrian, T. E.; Welbourn, R. B.; Bloom, S. R.

1983-01-01

We present a case in which plasma pancreatic polypeptide and calcitonin were found to be raised in association with an islet cell tumour of the pancreas and its hepatic metastases. In this patient, no specific endocrine syndrome was found. Therapeutic hepatic artery embolization improved the general health of the patient with no change in plasma pancreatic polypeptide, but a fall in calcitonin. PMID:6308585

Pancreatic polypeptide and calcitonin secretion from a pancreatic tumour-clinical improvement after hepatic artery embolization.

PubMed

Manche, A; Wood, S M; Adrian, T E; Welbourn, R B; Bloom, S R

1983-05-01

We present a case in which plasma pancreatic polypeptide and calcitonin were found to be raised in association with an islet cell tumour of the pancreas and its hepatic metastases. In this patient, no specific endocrine syndrome was found. Therapeutic hepatic artery embolization improved the general health of the patient with no change in plasma pancreatic polypeptide, but a fall in calcitonin.

Structure and pharmacological actions of phyllocaerulein, a caerulein-like nonapeptide: its occurrence in extracts of the skin of Phyllomedusa sauvagei and related Phyllomedusa species.

PubMed

Anastasi, A; Bertaccini, G; Cei, J M; De Caro, G; Erspamer, V; Impicciatore, M

1969-09-01

1. The South American amphibian Phyllomedusa sauvagei contains in its skin large amounts of a polypeptide closely resembling caerulein in its pharmacological actions. This polypeptide, called phyllocaerulein, was obtained in a pure form, and upon acid hydrolysis, enzymic digestion and end-group determination experiments it proved to be a nonapeptide of the following composition Pyr-Glu-Tyr(SO(3)H)-Thr-Gly-Trp-Met-Asp-Phe-NH(2)It may be seen that caerulein and phyllocaerulein have in common the C-terminal heptapeptide and the N-terminal pyroglutamyl residue.2. Phyllocaerulein is indistinguishable from caerulein even in parallel bioassay. However, the former polypeptide seems to be somewhat more potent than the latter on all the preparations tested.3. In different batches of Phyllomedusa sauvagei skin the phyllocaerulein content ranged between 150 and 600 mug/g of fresh tissue.Phyllocaerulein or similar polypeptides occur also in the skin of several other Phyllomedusa species, among which are Phyll. burmeisteri, Phyll. dachnicolor, Phyll, helenae, Phyll. annae, Phyll. callidryas and Phyll. bicolor.4. The qualitative identification and quantitative estimation of caerulein-like polypeptides in crude skin extracts may be complicated by the concomitant occurrence of other active polypeptides. These, however, are poorly effective on some test preparations which seem to respond selectively to caerulein.5. Like that of caerulein, the biological significance of phyllocaerulein is completely obscure.

Structure and pharmacological actions of phyllocaerulein, a caerulein-like nonapeptide

PubMed Central

Anastasi, A.; Bertaccini, G.; Cei, J. M.; De Caro, G.; Erspamer, V.; Impicciatore, M.

1969-01-01

1. The South American amphibian Phyllomedusa sauvagei contains in its skin large amounts of a polypeptide closely resembling caerulein in its pharmacological actions. This polypeptide, called phyllocaerulein, was obtained in a pure form, and upon acid hydrolysis, enzymic digestion and end-group determination experiments it proved to be a nonapeptide of the following composition Pyr-Glu-Tyr(SO3H)-Thr-Gly-Trp-Met-Asp-Phe-NH2 It may be seen that caerulein and phyllocaerulein have in common the C-terminal heptapeptide and the N-terminal pyroglutamyl residue. 2. Phyllocaerulein is indistinguishable from caerulein even in parallel bioassay. However, the former polypeptide seems to be somewhat more potent than the latter on all the preparations tested. 3. In different batches of Phyllomedusa sauvagei skin the phyllocaerulein content ranged between 150 and 600 μg/g of fresh tissue. Phyllocaerulein or similar polypeptides occur also in the skin of several other Phyllomedusa species, among which are Phyll. burmeisteri, Phyll. dachnicolor, Phyll, helenae, Phyll. annae, Phyll. callidryas and Phyll. bicolor. 4. The qualitative identification and quantitative estimation of caerulein-like polypeptides in crude skin extracts may be complicated by the concomitant occurrence of other active polypeptides. These, however, are poorly effective on some test preparations which seem to respond selectively to caerulein. 5. Like that of caerulein, the biological significance of phyllocaerulein is completely obscure. PMID:5824931

Nanoassemblies from homostructured polypeptides as efficient nanoplatforms for oral drug delivery.

PubMed

Jia, Yi; Tang, Yuan; He, Hongmei; Li, Shuhui; Che, Ling; Zhou, Xing; Dou, Yin; Zhang, Jianxiang; Li, Xiaohui

2013-04-01

The assembly of homostructured polypeptides bearing various side groups into well-defined nanostructures was presented, with their size and topology mainly dominated by the chemical structure and molecular weight of peptides. Pharmacokinetic and pharmacodynamic studies based on rat models suggested these newly constructed nanoassemblies with low cytotoxicity may function as novel nanoplatforms to efficiently and safely deliver therapeutics to achieve better efficacy but lower side effects. Other applications in biomedical fields, such as biotechnology, medical imaging, and tissue engineering, may also be expected. This research team investigated the assembly of homostructured polypeptides bearing various side groups into well-defined nanostructures, and demonstrated low cytotoxicity in rat disease models, suggesting that these novel nanoplatforms may safely and efficiently deliver therapeutics with low side effects. Copyright © 2013 Elsevier Inc. All rights reserved.

A Drug-Induced Hybrid Electrospun Poly-Capro-Lactone: Cell-Derived Extracellular Matrix Scaffold for Liver Tissue Engineering.

PubMed

Grant, Rhiannon; Hay, David C; Callanan, Anthony

2017-07-01

Liver transplant is the only treatment option for patients with end-stage liver failure, however, there are too few donor livers available for transplant. Whole organ tissue engineering presents a potential solution to the problem of rapidly escalating donor liver shortages worldwide. A major challenge for liver tissue engineers is the creation of a hepatocyte microenvironment; a niche in which liver cells can survive and function optimally. While polymers and decellularized tissues pose an attractive option for scaffold manufacturing, neither alone has thus far proved sufficient. This study exploited cell's native extracellular matrix (ECM) producing capabilities using two different histone deacetylase inhibitors, and combined these with the customizability and reproducibility of electrospun polymer scaffolds to produce a "best of both worlds" niche microenvironment for hepatocytes. The resulting hybrid poly-capro-lactone (PCL)-ECM scaffolds were validated using HepG2 hepatocytes. The hybrid PCL-ECM scaffolds maintained hepatocyte growth and function, as evidenced by metabolic activity and DNA quantitation. Mechanical testing revealed little significant difference between scaffolds, indicating that cells were responding to a biochemical and topographical profile rather than mechanical changes. Immunohistochemistry showed that the biochemical profile of the drug-derived and nondrug-derived ECMs differed in ratio of Collagen I, Laminin, and Fibronectin. Furthermore, the hybrid PCL-ECM scaffolds influence the gene expression profile of the HepG2s drastically; with expression of Albumin, Cytochrome P450 Family 1 Subfamily A Polypeptide 1, Cytochrome P450 Family 1 Subfamily A Polypeptide 2, Cytochrome P450 Family 3 Subfamily A Polypeptide 4, Fibronectin, Collagen I, and Collagen IV undergoing significant changes. Our results demonstrate that drug-induced hybrid PCL-ECM scaffolds provide a viable, translatable platform for creating a niche microenvironment for hepatocytes, supporting in vivo phenotype and function. These scaffolds offer great potential for tissue engineering and regenerative medicine strategies for whole organ tissue engineering.

Laser Tissue Welding - Distal Pancreatectomy Sealing Study

ClinicalTrials.gov

2018-04-20

Pancreatic Tumor, Benign; Pancreatic Neoplasms; Pancreatic Adenocarcinoma; Pancreatic Pseudocyst; Pancreatic Neuroendocrine Tumor; Pancreas; Insulinoma; Pancreatic Cyst; Pancreatic Teratoma; Pancreatic Polypeptide Tumor; Pancreatic Vipoma; Pancreatic Cystadenoma; Pancreas Injury; Pancreatic Gastrinoma; Pancreatic Glucagonoma

Tissue polypeptide-specific antigen (TPS) determinations before and during intermittent maximal androgen blockade in patients with metastatic prostatic carcinoma.

PubMed

Kil, P J M; Goldschmidt, H M J; Wieggers, B J A; Kariakine, O B; Studer, U E; Whelan, P; Hetherington, J; de Reijke, Th M; Hoekstra, J W; Collette, L

2003-01-01

To evaluate the prognostic significance of serially measured tissue polypeptide-specific antigen (TPS) levels in patients with metastatic prostatic carcinoma treated with intermittent maximal androgen blockade (MAB). To determine its value with respect to predicting response to treatment and time to clinical progression. Finally to compare TPS with prostate-specific antigen (PSA) measurements in terms of prognostic impact in patients with metastatic prostatic carcinoma. TPS and PSA measurements were performed before start of and monthly during intermittent MAB in 68 patients participating in EORTC protocol 30954. Both TPS and PSA were measured in serum. Fifty-six patients from eight centers were included in the final analysis because at least three TPS values were available. TPS and PSA values were correlated with clinical course of the disease. Median follow-up was 21.3 months. Three patient groups were defined on clinical grounds: (a) clinically progressive disease (n=18); (b) clinically stable disease (n=33); and (c) patients who did not reach a predefined nadir PSA value following 9 months of treatment (n=5). Pretreatment TPS was significantly higher in the clinically progressive patients than in the other patient groups (p=0.0041). When grouping patients according to their pretreatment TPS values (cut-off value of 100 U/l) the pretreatment TPS value (>100 U/l) proved to be a statistically significant prognostic factor with respect to time to progression: elevated TPS was associated with a 3.8 increased risk for progressive disease (p=0.0055). Pretreatment PSA (>100 ng/ml) was of no prognostic value for time to progression. In five patients increase of TPS coincided with or preceded clinical progression during treatment, whereas PSA remained normal. Additional value of pretreatment TPS measurements in metastatic prostate cancer patients is found in defining the patients with rapid clinical progression. Following MAB an increase in TPS signifies clinical progression even if PSA is found to remain normal.

A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions.

PubMed Central

Enrich, C; Bachs, O; Evans, W H

1988-01-01

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3214436

Synthesis of Globulins in Maize Embryos 1

PubMed Central

Kriz, Alan L.; Schwartz, Drew

1986-01-01

The two major components of the globulin fraction in Zea mays embryos are specified by the Prot gene. Pulse-chase analysis of protein synthesis in cultured, immature embryos indicates that the smaller Prot-specific polypeptide, PROT, is derived from the larger polypeptide, PROT'. These experiments also demonstrate that PROT' is derived from a short-lived precursor polypeptide, prePROT'. The primary Prot-specific translation product, as detected by in vitro translation of immature embryo RNA, is of a lower apparent molecular weight than pre-PROT', suggesting the involvement of co- and/or post-translational modification in the production of prePROT'. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16665136

Enzymes that cleave non-glycosidic ether bonds between lignins or derivatives thereof and saccharides

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

Kravit, Nancy G.; Schmidt, Katherine A.

The patent application relates to isolated polypeptides that specifically cleave non-glycosidic ether bonds between lignins or derivatives thereof and saccharides, and to cDNAs encoding the polypeptides. The patent application also relates to nucleic acid constructs, expression vectors and host cells comprising the cDNAs, as well as methods of producing and using the isolated polypeptides for treating pulp and biomass to increase soluble saccharide yield and enrich lignin fractions.

Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes.

PubMed

Lin, J C; Pagano, J S

1986-08-01

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.

Auxin-Regulated Polypeptide Changes at Different Stages of Strawberry Fruit Development 1

PubMed Central

Veluthambi, K.; Poovaiah, B. W.

1984-01-01

The pattern of polypeptides at different stages of strawberry (Fragaria ananassa Duch. cv Ozark Beauty) fruit development was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An 81,000-dalton polypeptide appeared between 5 and 10 days after pollination. Polypeptides with molecular weights of 76,000 and 37,000 daltons were formed after 10 days. The control exerted by auxin in the stage-specific formation of polypeptides was investigated by stopping fruit growth after removing the achenes and reinitiating fruit growth by the application of a synthetic auxin, α-naphthaleneacetic acid (NAA). When the achenes were removed from the 5- and 10-day-old fruits, the fruits failed to grow, the 81,000 dalton polypeptide was not formed between 5 and 10 days, and the 76,000- and 37,000-dalton polypeptides were not formed between 10 and 20 days. Application of NAA to fruits deprived of auxin by removal of achenes resulted in the resumption of growth and also in the appearance of these polypeptides. Removal of achenes of the 5- or 10-day-old fruits and growing them without auxin resulted in the formation of 52,000- and 57,000-dalton polypeptides. These two polypeptides were not formed when NAA was applied to fruits after removal of achenes. Supply of NAA to auxin-deprived fruits 5 days after removal of achenes resulted in resumption of growth and also in the disappearance of these two polypeptides, pointing out their possible relation to the inhibition of fruit growth. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16663624

Plasmodium falciparum polypeptides released during in vitro cultivation*

PubMed Central

Da Silva, L. Rodriguez; Loche, M.; Dayal, R.; Perrin, L. H.

1983-01-01

Synchronous cultures of Plasmodium falciparum were successively labelled with (35S)-methionine and both the supernatants and the pellets of infected red blood cells were collected. The release of TCA-precipitable material in the culture supernatants was low during the development of ring forms and trophozoites, increased during schizogony, and was maximum at the time of schizont rupture and merozoite reinvasion. Analysis of the supernatants by SDS — PAGE and autoradiography showed that both polypeptides common to the various developmental stages of the parasite and schizont/merozoite-specific polypeptides were released. Polypeptides of relative molecular mass 140 000, 82 000 and, to a lower degree, 41 000 were present in high amounts in the culture supernatants. These polypeptides have been shown to be the target of monoclonal antibodies that are able to inhibit the growth of P. falciparum cultures, and may be involved in protective immunity. The released polypeptides may also be used as target antigens in immunodiagnostic tests aiming at the detection of malaria infection. ImagesFig. 2AFig. 2BFig. 3 PMID:6340846

Aqueous cholesteric liquid crystals using uncharged rodlike polypeptides. Polypeptide vesicles by conformation-specific assembly. Ordered chiral macroporous hybrid silica-polypeptide composites

NASA Astrophysics Data System (ADS)

Bellomo, Enrico Giuseppe

2005-07-01

Aqueous cholesteric liquid crystals using uncharged rodlike polypeptides . The aqueous, lyotropic liquid-crystalline phase behavior of an alpha helical polypeptide, has been studied using optical microscopy and X-ray scattering. Solutions of optically pure polypeptide were found to form cholesteric liquid crystals at volume fractions that decreased with increasing average chain length. At very high volume fractions, the formation of a hexagonal mesophase was observed. The pitch of the cholesteric phase could be varied by a mixture of enantiomeric samples, where the pitch increased as the mixture approached equimolar. The cholesteric phases could be untwisted, using either magnetic field or shear flow, into nematic phases, which relaxed into cholesterics upon removal of field or shear. We have found that the phase diagram of this polypeptide in aqueous solution parallels that of poly(gamma-benzyl glutamate) in organic solvents, thus providing a useful system for liquid-crystal applications requiring water as solvent. Polypeptide vesicles by conformation-specific assembly. We have found that block copolymers composed of polypeptide segments provide significant advantages in controlling both the function and supramolecular structure of bioinspired self-assemblies. Incorporation of the stable chain conformations found in proteins into block copolymers was found to provide an additional element of control, beyond amphiphilicity and composition that defines self-assembled architecture. The abundance of functionality present in amino acids, and the ease by which they can be incorporated into these materials, also provides a powerful mechanism to impart block copolypeptides with function. This combination of structure and function work synergistically to enable significant advantages in the preparation of therapeutic agents as well as provide insight into design of self-assemblies beginning to approach the complexity of natural structures such as virus capsids. Ordered chiral macroporous hybrid silica-polypeptide composites. The mineralization of organic templates has been investigated as an effective way to control the size and structure of inorganic frameworks. Hybrid structures incorporating polypeptide with silica have been prepared and characterized using X-ray scattering, TGA, SEM and TEM. The results support the interaction between silica and polymer to form ordered chiral macroporous structures that can be easily controlled by polymer molecular weight and volume fraction.

Sodium-potassium-activated adenosine triphosphatase of electrophorus electric organ. X. Immunochemical properties of the Lubrol-solubilized enzume and its constituent polypeptides.

PubMed

Jean, D H; Albers, R W; Koval, G J

1975-02-10

Detergent (Lubrol WX)-solubilized sodium-potassium-activated adenosine triphosphatase ((Na+ + K+)-ATPase) of electrophorus electric organ contains two major constituent polypeptides with molecular weights of 96,000 and 58,000 which can be readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These two polypeptides can be clearly separated and can be obtained in milligram quantities by preparative sodium dodecyl sulfate gel electrophoresis. The separated polypeptides, after removal of sodium dodecyl sulfate, and Lubrol-solubilized (Na+ + K+)-ATPase activity to some degree. Moreover, the degree of inhibition is directly proportional to the increasing amounts of antisera. The inhibition is maximal 4 weeks after the first injection. Immunodiffusion in 1% agar gel indicated that only Lubrol-solubilized enzyme antiserum, but not 58,000-dalton or 96,00-dalton polypeptide antiserum, gives one major precipitin band. However, specific complex formation between each polypeptide antiserum and Lubrol-solubilized enzyme occurs. This was demonstrated indirectly. After incubating Lubrol-solubilized enzyme with increasing amounts of polypeptide antisera at 37 degrees for 15 min, they were placed in the side wells of an immunodiffusion plate with antiserum against Lubrol-solubilized enzyme in the central well. The intensity of the precipitin band decreased with increasing amounts of polypeptide antisera. Thus, the results indicate that both 96,000-dalton and 58,000-dalton polypeptides are integral subunits of (Na+ + K+)-ATPase.

Three-Dimensional Polypeptide Architectures Through Tandem Catalysis and Click Chemistry

NASA Astrophysics Data System (ADS)

Rhodes, Allison Jane

Rapid renal clearance, liver accumulation, proteolytic degradation and non-specificity are challenges small molecule drugs, peptides, proteins and nucleic acid therapeutics encounter en route to their intended destination within the body. Nanocarriers (i.e. dendritric polymers, vesicles, and micelles) of approximately 100 nm in diameter, shuttle small molecule drugs to their desired location through passive (EPR effect) and active (ligand-mediated) targeting, maximizing therapeutic efficiency. Polypeptide-based polymers are water-soluble, biocompatible, non-toxic and are therefore excellent candidates for nanocarriers. Dendritic polymers, including dendrimers, cylindrical brushes, and star polymers, are the newest class of nanomedicine drug delivery vehicles. The synthesis and characterization of dendritic polymers is challenging, with tedious and costly procedures. Dendritic polymers possess peripheral pendent functional groups that can potentially be used in ligand-mediated drug delivery vehicles and bioimaging applications. More specifically, cylindrical brushes are dendritic polymers where a single linear polymer (primary chain) has polymer chains (secondary chains) grafted to it. Recently, research groups have shown that cylindrical brush polymers are capable of nanoparticle and supramolecular structure self-assembly. The facile preparation of high-density brush copolypeptides by the "grafting from" approach will be discussed. This approach utilizes a novel, tandem catalytic methodology where alloc-alpha-aminoamide groups are installed within the side-chains of the alpha-amino-N-carboxyanhydride (NCA) monomer serving as masked initiators. These groups are inert during cobalt initiated NCA polymerization, and give alloc-alpha-aminoamide substituted polypeptide main-chains. The alloc-alpha-aminoamide groups are activated in situ using nickel to generate initiators for growth of side-chain brush segments. This method proves to be efficient, yielding well-defined, high-density brushes for applications in drug delivery and imaging. Here, we also report a method for the synthesis of soluble, well-defined, azido functionalized polypeptides in a straightforward, 3-step synthesis. Homo and diblock azidopolypeptides were prepared with controlled segment lengths via living polymerization using Co(PMe3)4 initiator. Through copper azide alkyne click chemistry (CuAAC) in organic solvent, azidopolypeptides were regioselectively and quantitatively modified with carboxylic acid (pH-responsive), amino acid and sugar functional groups. Finally, the advances towards well-defined hyperbranched polypeptides through alpha-amino-acid-N-thiocarboxyanhydrides (NTAs) will be discussed. Within the past 10 years, controlled NCA (alpha-amino acid-N-carboxyanhydride) ring-opening polymerization (ROP) has emerged, expanding the application of copolypeptide polymers in various drug delivery and tissue engineering motifs. Modification of NCA monomers to the corresponding alpha-amino-acid-N-thiocarboxyanhydride (NTA) will diversify ROP reactions, leading to more complex polypeptides (such as hyperbranched polymers), in addition to the possibility of performing these polymerizations under ambient conditions, which would greatly expand their potential utility. The project focuses on the preparation of hyperbranched polypeptides with well-defined architectures and controlled branching density in a one-pot reaction. This will be accomplished by taking advantage of the different selectivities of Co(PMe3)4 and depeNi(COD) polymerization initiators, and by exploiting the reactivity difference between NCA and the more stable NTA monomers.

Collagen in Human Tissues: Structure, Function, and Biomedical Implications from a Tissue Engineering Perspective

NASA Astrophysics Data System (ADS)

Balasubramanian, Preethi; Prabhakaran, Molamma P.; Sireesha, Merum; Ramakrishna, Seeram

The extracellular matrix is a complex biological structure encoded with various proteins, among which the collagen family is the most significant and abundant of all, contributing 30-35% of the whole-body protein. "Collagen" is a generic term for proteins that forms a triple-helical structure with three polypeptide chains, and around 29 types of collagen have been identified up to now. Although most of the members of the collagen family form such supramolecular structures, extensive diversity exists between each type of collagen. The diversity is not only based on the molecular assembly and supramolecular structures of collagen types but is also observed within its tissue distribution, function, and pathology. Collagens possess complex hierarchical structures and are present in various forms such as collagen fibrils (1.5-3.5 nm wide), collagen fibers (50-70 nm wide), and collagen bundles (150-250 nm wide), with distinct properties characteristic of each tissue providing elasticity to skin, softness of the cartilage, stiffness of the bone and tendon, transparency of the cornea, opaqueness of the sclera, etc. There exists an exclusive relation between the structural features of collagen in human tissues (such as the collagen composition, collagen fibril length and diameter, collagen distribution, and collagen fiber orientation) and its tissue-specific mechanical properties. In bone, a transverse collagen fiber orientation prevails in regions of higher compressive stress whereas longitudinally oriented collagen fibers correlate to higher tensile stress. The immense versatility of collagen compels a thorough understanding of the collagen types and this review discusses the major types of collagen found in different human tissues, highlighting their tissue-specific uniqueness based on their structure and mechanical function. The changes in collagen during a specific tissue damage or injury are discussed further, focusing on the many tissue engineering applications for which collagen scaffolds are currently being applied.

Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

DOEpatents

Freimuth, Paul I.

2010-04-06

The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

Expression of pituitary adenylate cyclase-activating polypeptide 1 and 2 receptor mRNA in gallbladder tissue of patients with gallstone or gallbladder polyps.

PubMed

Zhang, Zhen-Hai; Wu, Shuo-Dong; Gao, Hong; Shi, Gang; Jin, Jun-Zhe; Kong, Jing; Tian, Zhong; Su, Yang

2006-03-07

To detect the expression of pituitary adenylate cyclase-activating polypeptide receptor 1 (VPCAP1-R)and VPCAP2-R mRNA in gallbladder tissues of patients with gallstone or gallbladder polyps. The expression of VPCAP1-R and VPCAP2-R mRNA in gallbladder tissues was detected in 25 patients with gallstone,8 patients with gallbladder polyps and 7 donors of liver transplantation by reverse transcription polymerase chain reaction (RT-PCR). The VPCAP2-R mRNA expression level in the control group (1.09+/-0.58) was lower than that in the gallbladder polyp group (1.64+/-0.56) and the gallstone group (1.55+/-0.45) (P<0.05) while the VPCAP1-R mRNA expression level in the control group (1.15+/-0.23) was not apparently different from that in the gallbladder polyp group (1.28+/-0.56) and the gallstone group (1.27+/-0.38). The abnormal expression of VPCAP2-R mRNA in gallbladder tissue may play a role in the formation of gallbladder stone and gallbladder polyps.

[Insulin-like growth factor-1 (IGF-1) - structure and the role in the human body].

PubMed

Filus, Alicja; Zdrojewicz, Zygmunt

2015-01-01

In the recent years, managed to broadly explore the structure and role of insulin-like growth factors type 1 and 2 (IGF1 I 2). They belong to the structure of polypeptide hormones homologous to proinsulin. They are characterized by a wide range of activities. IGF-1 is a key mediator of most tissue effects of growth hormone (GH). In addition to effects on growth processes of the body, is also an important factor for cell homeostasis, is subject to both endocrine and tissue-specific auto- and paracrine regulation. In this paper, the current, general knowledge on the structure, function and mechanism of biological effects of IGF-1 in the human body was presented. Attention was also drawn to the directions of use of IGf-1 in the treatment of other diseases than the diseases of the hypothalamic-pituitary and growth disorders in children. © Polish Society for Pediatric Endocrinology and Diabetology.

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

PubMed

Hurst, H C; Masson, N; Jones, N C; Lee, K A

1990-12-01

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

Protease-mediated drug delivery

NASA Astrophysics Data System (ADS)

Dickson, Eva F.; Goyan, Rebecca L.; Kennedy, James C.; Mackay, M.; Mendes, M. A. K.; Pottier, Roy H.

2003-12-01

Drugs used in disease treatment can cause damage to both malignant and normal tissue. This toxicity limits the maximum therapeutic dose. Drug targeting is of high interest to increase the therapeutic efficacy of the drug without increasing systemic toxicity. Certain tissue abnormalities, disease processes, cancers, and infections are characterized by high levels of activity of specific extracellular and/or intracellular proteases. Abnormally high activity levels of specific proteases are present at sites of physical or chemical trauma, blood clots, malignant tumors, rheumatoid arthritis, inflammatory bowel disease, gingival disease, glomerulonerphritis, and acute pancreatitis. Abnormal protease activity is suspected in development of liver thrombosis, pulmonary emphysema, atherosclerosis, and muscular dystrophy. Inactiviating disease-associated proteases by the administration of appropriate protease inhibitors has had limited success. Instead, one could use such proteases to target drugs to treat the condition. Protease mediated drug delivery offers such a possibility. Solubilizing groups are attached to insoluble drugs via a polypeptide chain which is specifically cleavable by certian proteases. When the solubilized drug enounters the protease, the solubilizing moieties are cleaved, and the drug precipitates at the disease location. Thus, a smaller systemic dosage could result in a therapeutic drug concentration at the treatment site with less systemic toxicity.

Serum peptide expression and treatment responses in patients with advanced non-small-cell lung cancer

PubMed Central

An, Juan; Tang, Chuan-Hao; Wang, Na; Liu, Yi; Lv, Jin; Xu, Bin; Li, Xiao-Yan; Guo, Wan-Feng; Gao, Hong-Jun; He, Kun; Liu, Xiao-Qing

2018-01-01

Epidermal growth factor receptor (EGFR) mutation is an important predictor for response to personalized treatments of patients with advanced non-small-cell lung cancer (NSCLC). However its usage is limited due to the difficult of obtaining tissue specimens. A novel prediction system using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a perspective tool in European countries to identify patients who are likely to benefit from EGFR tyrosine kinase inhibitor (TKI) treatment. In the present study, MALDI-TOF MS was used on pretreatment serum samples of patients with advanced non-small-cell lung cancer to discriminate the spectra between disease control and disease progression groups in one cohort of Chinese patients. The candidate features for classification were subsequently validated in a blinded fashion in another set of patients. The correlation between plasma EGFR mutation status and the intensities of representative spectra for classification was evaluated. A total of 103 patients that were treated with EGFR-TKIs were included. It was determined that 8 polypeptides peaks were significant different between the disease control and disease progression group. A total of 6 polypeptides were established in the classification algorithm. The sensitivity of the algorithm to predict treatment responses was 76.2% (16/21) and the specificity was 81.8% (18/22). The accuracy rate of the algorithm was 79.1% (34/43). A total of 3 polypeptides were significantly correlated with EGFR mutations (P=0.04, P=0.03 and P=0.04, respectively). The present study confirmed that MALDI-TOF MS analysis can be used to predict responses to EGFR-TKI treatment of the Asian population where the EGFR mutation status differs from the European population. Furthermore, the expression intensities of the three polypeptides in the classification model were associated with EGFR mutation. PMID:29844828

A Bio-Inspired Two-Layer Sensing Structure of Polypeptide and Multiple-Walled Carbon Nanotube to Sense Small Molecular Gases

PubMed Central

Wang, Li-Chun; Su, Tseng-Hsiung; Ho, Cheng-Long; Yang, Shang-Ren; Chiu, Shih-Wen; Kuo, Han-Wen; Tang, Kea-Tiong

2015-01-01

In this paper, we propose a bio-inspired, two-layer, multiple-walled carbon nanotube (MWCNT)-polypeptide composite sensing device. The MWCNT serves as a responsive and conductive layer, and the nonselective polypeptide (40 mer) coating the top of the MWCNT acts as a filter into which small molecular gases pass. Instead of using selective peptides to sense specific odorants, we propose using nonselective, peptide-based sensors to monitor various types of volatile organic compounds. In this study, depending on gas interaction and molecular sizes, the randomly selected polypeptide enabled the recognition of certain polar volatile chemical vapors, such as amines, and the improved discernment of low-concentration gases. The results of our investigation demonstrated that the polypeptide-coated sensors can detect ammonia at a level of several hundred ppm and barely responded to triethylamine. PMID:25751078

Evidence of accelerated evolution and ectodermal-specific expression of presumptive BDS toxin cDNAs from Anemonia viridis.

PubMed

Nicosia, Aldo; Maggio, Teresa; Mazzola, Salvatore; Cuttitta, Angela

2013-10-30

Anemonia viridis is a widespread and extensively studied Mediterranean species of sea anemone from which a large number of polypeptide toxins, such as blood depressing substances (BDS) peptides, have been isolated. The first members of this class, BDS-1 and BDS-2, are polypeptides belonging to the β-defensin fold family and were initially described for their antihypertensive and antiviral activities. BDS-1 and BDS-2 are 43 amino acid peptides characterised by three disulfide bonds that act as neurotoxins affecting Kv3.1, Kv3.2 and Kv3.4 channel gating kinetics. In addition, BDS-1 inactivates the Nav1.7 and Nav1.3 channels. The development of a large dataset of A. viridis expressed sequence tags (ESTs) and the identification of 13 putative BDS-like cDNA sequences has attracted interest, especially as scientific and diagnostic tools. A comparison of BDS cDNA sequences showed that the untranslated regions are more conserved than the protein-coding regions. Moreover, the KA/KS ratios calculated for all pairwise comparisons showed values greater than 1, suggesting mechanisms of accelerated evolution. The structures of the BDS homologs were predicted by molecular modelling. All toxins possess similar 3D structures that consist of a triple-stranded antiparallel β-sheet and an additional small antiparallel β-sheet located downstream of the cleavage/maturation site; however, the orientation of the triple-stranded β-sheet appears to differ among the toxins. To characterise the spatial expression profile of the putative BDS cDNA sequences, tissue-specific cDNA libraries, enriched for BDS transcripts, were constructed. In addition, the proper amplification of ectodermal or endodermal markers ensured the tissue specificity of each library. Sequencing randomly selected clones from each library revealed ectodermal-specific expression of ten BDS transcripts, while transcripts of BDS-8, BDS-13, BDS-14 and BDS-15 failed to be retrieved, likely due to under-representation in our cDNA libraries. The calculation of the relative abundance of BDS transcripts in the cDNA libraries revealed that BDS-1, BDS-3, BDS-4, BDS-5 and BDS-6 are the most represented transcripts.

Systems for the expression of orthogonal translation components in eubacterial host cells

DOEpatents

Ryu, Youngha; Schultz, Peter G.

2013-01-22

The invention related to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural acids at genetically-programmed positions.

Tumor suppressor molecules and methods of use

DOEpatents

Welch, Peter J.; Barber, Jack R.

2004-09-07

The invention provides substantially pure tumor suppressor nucleic acid molecules and tumor suppressor polypeptides. The invention also provides hairpin ribozymes and antibodies selective for these tumor suppressor molecules. Also provided are methods of detecting a neoplastic cell in a sample using detectable agents specific for the tumor suppressor nucleic acids and polypeptides.

Systems for the expression of orthogonal translation components in eubacterial host cells

DOEpatents

Ryu, Youngha [San Diego, CA; Schultz, Peter G [La Jolla, CA

2011-06-14

The invention relates to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural amino acids at genetically-programmed positions.

Systems for the expression of orthogonal translation components eubacterial host cells

DOEpatents

Ryu, Youngha [San Diego, CA; Schultz, Peter G [La Jolla, CA

2012-06-12

The invention relates to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural amino acids at genetically-programmed positions.

Cysteine-containing peptide tag for site-specific conjugation of proteins

DOEpatents

Backer, Marina V.; Backer, Joseph M.

2008-04-08

The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety bound to the targeting moiety; the biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety. The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein, the adapter protein having a thiol group; the biological conjugate having a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein. The present invention is also directed to biological sequences employed in the above biological conjugates, as well as pharmaceutical preparations and methods using the above biological conjugates.

Cysteine-containing peptide tag for site-specific conjugation of proteins

DOEpatents

Backer, Marina V.; Backer, Joseph M.

2010-10-05

The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety bound to the targeting moiety; the biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety. The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein, the adapter protein having a thiol group; the biological conjugate having a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein. The present invention is also directed to biological sequences employed in the above biological conjugates, as well as pharmaceutical preparations and methods using the above biological conjugates.

Expression of Molecular Markers in Brain, Serum, and Lung Tissues Following Hypobaric Hypoxia

DTIC Science & Technology

2018-01-01

8 4.2 HIF-1α ELISA Results...9 4.3 Prolyl-4-hydroxylase Alpha Polypeptide I (P4Ha1) ELISA Results...10 4.4 Vascular Endothelial Growth Factor ELISA Results .......................................................12

Catalytic and reactive polypeptides and methods for their preparation and use

DOEpatents

Schultz, Peter

1994-01-01

Catalytic and reactive polypeptides include a binding site specific for a reactant or reactive intermediate involved in a chemical reaction of interest. The polypeptides further include at least one active functionality proximate the binding site, where the active functionality is capable of catalyzing or chemically participating in the chemical reaction in such a way that the reaction rate is enhanced. Methods for preparing the catalytic peptides include chemical synthesis, site-directed mutagenesis of antibody and enzyme genes, covalent attachment of the functionalities through particular amino acid side chains, and the like.

UV Spectrophotometric Method for Estimation of Polypeptide-K in Bulk and Tablet Dosage Forms

NASA Astrophysics Data System (ADS)

Kaur, P.; Singh, S. Kumar; Gulati, M.; Vaidya, Y.

2016-01-01

An analytical method for estimation of polypeptide-k using UV spectrophotometry has been developed and validated for bulk as well as tablet dosage form. The developed method was validated for linearity, precision, accuracy, specificity, robustness, detection, and quantitation limits. The method has shown good linearity over the range from 100.0 to 300.0 μg/ml with a correlation coefficient of 0.9943. The percentage recovery of 99.88% showed that the method was highly accurate. The precision demonstrated relative standard deviation of less than 2.0%. The LOD and LOQ of the method were found to be 4.4 and 13.33, respectively. The study established that the proposed method is reliable, specific, reproducible, and cost-effective for the determination of polypeptide-k.

Molecular biomimetics: nanotechnology through biology.

PubMed

Sarikaya, Mehmet; Tamerler, Candan; Jen, Alex K-Y; Schulten, Klaus; Baneyx, François

2003-09-01

Proteins, through their unique and specific interactions with other macromolecules and inorganics, control structures and functions of all biological hard and soft tissues in organisms. Molecular biomimetics is an emerging field in which hybrid technologies are developed by using the tools of molecular biology and nanotechnology. Taking lessons from biology, polypeptides can now be genetically engineered to specifically bind to selected inorganic compounds for applications in nano- and biotechnology. This review discusses combinatorial biological protocols, that is, bacterial cell surface and phage-display technologies, in the selection of short sequences that have affinity to (noble) metals, semiconducting oxides and other technological compounds. These genetically engineered proteins for inorganics (GEPIs) can be used in the assembly of functional nanostructures. Based on the three fundamental principles of molecular recognition, self-assembly and DNA manipulation, we highlight successful uses of GEPI in nanotechnology.

Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin.

PubMed

Keeley, Fred W; Bellingham, Catherine M; Woodhouse, Kimberley A

2002-02-28

Elastin is the major extracellular matrix protein of large arteries such as the aorta, imparting characteristics of extensibility and elastic recoil. Once laid down in tissues, polymeric elastin is not subject to turnover, but is able to sustain its mechanical resilience through thousands of millions of cycles of extension and recoil. Elastin consists of ca. 36 domains with alternating hydrophobic and cross-linking characteristics. It has been suggested that these hydrophobic domains, predominantly containing glycine, proline, leucine and valine, often occurring in tandemly repeated sequences, are responsible for the ability of elastin to align monomeric chains for covalent cross-linking. We have shown that small, recombinantly expressed polypeptides based on sequences of human elastin contain sufficient information to self-organize into fibrillar structures and promote the formation of lysine-derived cross-links. These cross-linked polypeptides can also be fabricated into membrane structures that have solubility and mechanical properties reminiscent of native insoluble elastin. Understanding the basis of the self-organizational ability of elastin-based polypeptides may provide important clues for the general design of self-assembling biomaterials.

Identification of HECT E3 ubiquitin ligase family genes involved in stem cell regulation and regeneration in planarians.

PubMed

Henderson, Jordana M; Nisperos, Sean V; Weeks, Joi; Ghulam, Mahjoobah; Marín, Ignacio; Zayas, Ricardo M

2015-08-15

E3 ubiquitin ligases constitute a large family of enzymes that modify specific proteins by covalently attaching ubiquitin polypeptides. This post-translational modification can serve to regulate protein function or longevity. In spite of their importance in cell physiology, the biological roles of most ubiquitin ligases remain poorly understood. Here, we analyzed the function of the HECT domain family of E3 ubiquitin ligases in stem cell biology and tissue regeneration in planarians. Using bioinformatic searches, we identified 17 HECT E3 genes that are expressed in the Schmidtea mediterranea genome. Whole-mount in situ hybridization experiments showed that HECT genes were expressed in diverse tissues and most were expressed in the stem cell population (neoblasts) or in their progeny. To investigate the function of all HECT E3 ligases, we inhibited their expression using RNA interference (RNAi) and determined that orthologs of huwe1, wwp1, and trip12 had roles in tissue regeneration. We show that huwe1 RNAi knockdown led to a significant expansion of the neoblast population and death by lysis. Further, our experiments showed that wwp1 was necessary for both neoblast and intestinal tissue homeostasis as well as uncovered an unexpected role of trip12 in posterior tissue specification. Taken together, our data provide insights into the roles of HECT E3 ligases in tissue regeneration and demonstrate that planarians will be a useful model to evaluate the functions of E3 ubiquitin ligases in stem cell regulation. Copyright © 2015 Elsevier Inc. All rights reserved.

A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

ERIC Educational Resources Information Center

Chang, Ming-Mei; Lovett, Janice

2011-01-01

Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

Two-dimensional sup 1 H nuclear magnetic resonance study of AaH IT, an anti-insect toxin from the scorpion Androctonus australis Hector. Sequential resonance assignments and folding of the polypeptide chain

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

Darbon, H.; Weber, C.; Braun, W.

1991-02-19

Sequence-specific nuclear magnetic resonance assignments for the polypeptide backbone and for most of the amino acid side-chain protons, as well as the general folding of AaH IT, are described. AaH IT is a neurotoxin purified from the venom of the scorpion Androctonus australis Hector and is specifically active on the insect nervous system. The secondary structure and the hydrogen-bonding patterns in the regular secondary structure elements are deduced from nuclear Overhauser effects and the sequence locations of the slowly exchanging amide protons. The backbone folding is determined by distance geometry calculations with the DISMAN program. The regular secondary structure includesmore » two and a half turns of {alpha}-helix running from residues 21 to 30 and a three-stranded antiparallel {beta}-sheet including peptides 3-5, 34-38, and 41-46. Two tight turns are present, one connecting the end of the {alpha}-helix to an external strand of the {beta}-sheet, i.e., turn 31-34, and another connecting this same strand to the central one, i.e., turn 38-41. The differences in the specificity of these related proteins, which are able to discriminate between mammalian and insect voltage-dependent sodium channels of excitable tissues, are most probably brought about by the position of the C-terminal peptide with regard to a hydrophobic surface common to all scorpion toxins examined thus far. Thus, the interaction of a given scorpion toxin with its receptor might well be governed by the presence of this solvent-exposed hydrophobic surface, whereas adjacent areas modulate the specificity of the interaction.« less

Engineering M13 for phage display.

PubMed

Sidhu, S S

2001-09-01

Phage display is achieved by fusing polypeptide libraries to phage coat proteins. The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA. Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA. The technology's success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms. Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions. Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms. These studies demonstrate that the M13 phage coat is extremely malleable, and this property can be used to engineer the phage particle specifically for phage display. These improvements expand the utility of phage display as a powerful tool in modern biotechnology.

A Novel Family in Medicago truncatula Consisting of More Than 300 Nodule-Specific Genes Coding for Small, Secreted Polypeptides with Conserved Cysteine Motifs1[w

PubMed Central

Mergaert, Peter; Nikovics, Krisztina; Kelemen, Zsolt; Maunoury, Nicolas; Vaubert, Danièle; Kondorosi, Adam; Kondorosi, Eva

2003-01-01

Transcriptome analysis of Medicago truncatula nodules has led to the discovery of a gene family named NCR (nodule-specific cysteine rich) with more than 300 members. The encoded polypeptides were short (60–90 amino acids), carried a conserved signal peptide, and, except for a conserved cysteine motif, displayed otherwise extensive sequence divergence. Family members were found in pea (Pisum sativum), broad bean (Vicia faba), white clover (Trifolium repens), and Galega orientalis but not in other plants, including other legumes, suggesting that the family might be specific for galegoid legumes forming indeterminate nodules. Gene expression of all family members was restricted to nodules except for two, also expressed in mycorrhizal roots. NCR genes exhibited distinct temporal and spatial expression patterns in nodules and, thus, were coupled to different stages of development. The signal peptide targeted the polypeptides in the secretory pathway, as shown by green fluorescent protein fusions expressed in onion (Allium cepa) epidermal cells. Coregulation of certain NCR genes with genes coding for a potentially secreted calmodulin-like protein and for a signal peptide peptidase suggests a concerted action in nodule development. Potential functions of the NCR polypeptides in cell-to-cell signaling and creation of a defense system are discussed. PMID:12746522

Antigenic Cross-reactivity among Haemonchus contortus, Oesophagostomum columbianum and Trichuris ovis of Goat.

PubMed

Jas, Ruma; Ghosh, Joydeb; DAS, Kinsuk

2016-01-01

Cross antigenicity is the major problem in developing a reliable tool for immunodiagnosis and immunoprophylaxis of parasitic diseases. Mixed infection due to different types of gastrointestinal parasites is more common than single species infection under field condition. The present study was undertaken to detect antigenic cross-reactivity among Haemonchus contortus, Oesophagostomum columbianum and Trichuris ovis of goats by SDS-PAGE and western blot analysis using hyperimmune sera (HIS) rose in rabbit separately against the antigens of the three nematode species. Thirteen, 16 and 14 polypeptides in crude somatic antigen (CSAg) of H. contortus (CSAg-Hc), O. columbianum (CSAg-Oc) and T. ovis (CSAg-To), respectively, were resolved in SDS PAGE analyses. It was revealed that 54 kDa peptide was shared by H.contortus and O. columbianum , whereas 47 kDa peptide was shared by O. columbianum and T. ovis . Western blot analyses revealed that three immunogenic polypeptides (MW 54, 49 and 42 kDa) in CSAg-Hc, five in CSAg-Oc (54, 47, 44, 38 and 35.5 kDa) and CSAg-To and five polypeptides (90, 51, 47, 39.5 and 31 kDa) in CSAg-To cross-reacted with the heterologous HIS. Four species-specific immunoreactive polypeptides (92, 85, 65 and 39 kDa) of H. contortus and two (72 & 26 kDa) in O. columbianum were also identified in the study. The shared polypeptides and species-specific polypeptides might be evaluated as protective antigen and subsequently exploitation for developing immunodiagnostic and for immunoprophylactic tools of for these common nematode species.

Antigenic Cross-reactivity among Haemonchus contortus, Oesophagostomum columbianum and Trichuris ovis of Goat

PubMed Central

JAS, Ruma; GHOSH, Joydeb; DAS, Kinsuk

2016-01-01

Background: Cross antigenicity is the major problem in developing a reliable tool for immunodiagnosis and immunoprophylaxis of parasitic diseases. Mixed infection due to different types of gastrointestinal parasites is more common than single species infection under field condition. Methods: The present study was undertaken to detect antigenic cross-reactivity among Haemonchus contortus, Oesophagostomum columbianum and Trichuris ovis of goats by SDS-PAGE and western blot analysis using hyperimmune sera (HIS) rose in rabbit separately against the antigens of the three nematode species. Results: Thirteen, 16 and 14 polypeptides in crude somatic antigen (CSAg) of H. contortus (CSAg-Hc), O. columbianum (CSAg-Oc) and T. ovis (CSAg-To), respectively, were resolved in SDS PAGE analyses. It was revealed that 54 kDa peptide was shared by H.contortus and O. columbianum, whereas 47 kDa peptide was shared by O. columbianum and T. ovis. Western blot analyses revealed that three immunogenic polypeptides (MW 54, 49 and 42 kDa) in CSAg-Hc, five in CSAg-Oc (54, 47, 44, 38 and 35.5 kDa) and CSAg-To and five polypeptides (90, 51, 47, 39.5 and 31 kDa) in CSAg-To cross-reacted with the heterologous HIS. Four species-specific immunoreactive polypeptides (92, 85, 65 and 39 kDa) of H. contortus and two (72 & 26 kDa) in O. columbianum were also identified in the study. Conclusion: The shared polypeptides and species-specific polypeptides might be evaluated as protective antigen and subsequently exploitation for developing immunodiagnostic and for immunoprophylactic tools of for these common nematode species. PMID:28127366

Purification of Growth Factor mRNA in Renal Tissues:bFGF-2, FGF-2, TGFα, and EGFR.

PubMed

Mydlo, J H

2001-01-01

Growth factors are polypeptides that induce cell mitogenicity, and thus play an important role in the etiology and progression of tumors (1). Fibroblast growth factors (FGF) constitute a family of structurally related polypeptides of 146 amino acids, which exhibit a wide spectrum of biologic activities, including angiogenesis or the formation of a vascular network. FGFs are mitogenic towards many mesodermal and ectodermal cell types, and can also induce and/or inhibit differentiation of cells (2). These heparin-binding factors are categorized as FGF-1 through FGF-10. Acidic FGF, or FGF-1, is found mostly in brain and other neural tissues. Basic FGF, or FGF- 2, a protein of 18 kDa mw, is one of the most ubiqitous growth factors. It is found in numerous benign and cancerous human and animal tissues, including kidney, prostate, and bladder (3-6). In some cases it has also been demonstrated to have potential as a tumor marker (7-11). One group reported greater recovery of both FGF-2 protein and FGF-2 mRNA from renal-cancer tissue compared to equal amounts of normal renal tissue (5). Furthermore, when purified FGF-2 from renal cell carcinoma (RCC) is added exogenously to other established renal tumorcell lines and endothelial cell lines, it demonstrates significant mitogenic activity (6). Thus, renal tumors may use FGF-2 in an autocrine manner to sustain themselves.

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

Cintron, C.; Hong, B.S.; Covington, H.I.

Whole neonate rabbit corneas and adult corneas containing 2-week-old scars were incubated in the presence of (/sup 14/C) glycine. Radiolabeled collagen extracted from the corneas and scar tissue were analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis and fluorography to determine the types and relative quantity of collagen polypeptides present and synthesized by these tissues. In addition to other collagen types, type III was found in both neonate cornea and scar tissue from adult cornea, albeit in relatively small quantities. Type III collagen in normal cornea was associated with the residue after pepsin digestion and formic acid extraction of the tissue, andmore » the same type of collagen was extracted from scar tissue after similar treatment. Type III collagen-specific monoclonal antibody bound to developing normal corneas and healing adult tissue sections, as determined by immunofluorescence. Antibody binding was localized to the endothelium and growing Descemet's membrane in fetal and neonate corneas, and restricted to the most posterior region of the corneal scar tissue. Although monoclonal antibody to keratan sulfate, used as a marker for stromal fibroblasts, bound to most of the scar tissue, the antibody failed to bind to the posterior scar tissue positive for type III collagen. We conclude that endothelial cells from fetal and neonate rabbit cornea and endothelium-derived fibroblasts from healing wounds of adult cornea synthesize and deposit type III collagen. Moreover, this collagen appears to be incorporated into the growing Descemet's membrane of normal corneas and narrow posterior portion of the scar tissue.« less

Human and porcine immunoreactive gastric inhibitory polypeptides (IR-GIP) are not identical.

PubMed

Bacarese-Hamilton, A J; Adrian, T E; Bloom, S R

1984-03-12

Immunoreactive gastric inhibitory polypeptide (IR-GIP) from human and porcine intestine was quantified by radioimmunoassay and the molecular forms characterised by gel permeation and reverse-phase high pressure liquid chromatography (HPLC). Gel filtration revealed two major immunoreactive peaks corresponding to the previously described 5-kDa and 8-kDa molecular forms, which appeared similar in both species. Isocratic reverse-phase HPLC revealed that the major immunoreactive GIP peak (5-kDa) in the human tissue eluted earlier than the corresponding porcine molecular form, indicating the latter to be less hydrophobic. These findings suggest significant species differences between human and porcine GIP.

Expression of pituitary adenylate cyclase-activating polypeptide 1 and 2 receptor mRNA in gallbladder tissue of patients with gallstone or gallbladder polyps

PubMed Central

Zhang, Zhen-Hai; Wu, Shuo-Dong; Gao, Hong; Shi, Gang; Jin, Jun-Zhe; Kong, Jing; Tian, Zhong; Su, Yang

2006-01-01

AIM: To detect the expression of pituitary adenylate cyclase-activating polypeptide receptor 1 (VPCAP1-R) and VPCAP2-R mRNA in gallbladder tissues of patients with gallstone or gallbladder polyps. METHODS: The expression of VPCAP1-R and VPCAP2-R mRNA in gallbladder tissues was detected in 25 patients with gallstone, 8 patients with gallbladder polyps and 7 donors of liver transplantation by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The VPCAP2-R mRNA expression level in the control group (1.09±0.58) was lower than that in the gallbladder polyp group (1.64 ± 0.56) and the gallstone group (1.55±0.45) (P < 0.05) while the VPCAP1-R mRNA expression level in the control group (1.15 ± 0.23) was not apparently different from that in the gallbladder polyp group (1.28±0.56) and the gallstone group (1.27 ± 0.38). CONCLUSION: The abnormal expression of VPCAP2-R mRNA in gallbladder tissue may play a role in the formation of gallbladder stone and gallbladder polyps. PMID:16552823

The prospective opportunities offered by magnetic scaffolds for bone tissue engineering: a review

PubMed Central

ORTOLANI, ALESSANDRO; BIANCHI, MICHELE; MOSCA, MASSIMILIANO; CARAVELLI, SILVIO; FUIANO, MARIO; MARCACCI, MAURILIO; RUSSO, ALESSANDRO

2016-01-01

Magnetic scaffolds are becoming increasingly attractive in tissue engineering, due to their ability to enhance bone tissue formation by attracting soluble factors, such as growth factors, hormones and polypeptides, directly to the implantation site, as well as their potential to improve the fixation and stability of the implant. Moreover, there is increasing evidence that the synergistic effects of magnetic scaffolds and magnetic fields can promote bone repair and regeneration. In this manuscript we review the recent innovations in bone tissue engineering that exploit magnetic biomaterials combined with static magnetic fields to enhance bone cell adhesion and proliferation, and thus bone tissue growth. PMID:28217659

The toxic effects of diethyl phthalate on the activity of glutamine synthetase in greater duckweed (Spirodela polyrhiza L.).

PubMed

Cheng, Tai-Sheng

2012-11-15

The toxic effects of diethyl phthalate (DEP), a potent allelochemical, on the enzyme activity and polypeptide accumulation of glutamine synthetase (GS) in greater duckweed were investigated. In our previous studies, DEP induced oxidative responses at concentrations from 0.5 to 2 mM in greater duckweed and the antioxidant enzymes played important roles in the defense strategy against DEP stress. In this study, DAB-H(2)O(2) and NBT stain for superoxide radicals (O(2)(·-)), lipid peroxidation, HSP70, and ammonia accumulation in DEP-treated duckweed tissues revealed adverse effect of DEP in plant growth. Biochemical analysis and physiological methods were combined to investigate GS activity and polypeptide accumulation under DEP-induced stress. The results showed that GS activity was reduced with the increasing concentration of DEP, indicative of enhanced toxic effect. Immunoblot analysis with chloroplast soluble fractions indicated that the chloroplastic GS (GS2) polypeptide from greater duckweed was degraded under DEP stress conditions. The response of GS2 to the DEP stress may be modulated by means of redox change in plant tissues, chloroplasts, and chloroplast lysates. The results suggest that DEP is toxic to the greater duckweed by inhibition of the GS isoenzymes in nitrogen assimilation and the GS2 plays important roles in the adaptation strategy against DEP toxicity. Copyright © 2012 Elsevier B.V. All rights reserved.

Redox changes accompanying storage protein mobilization in moist chilled and warm incubated walnut kernels prior to germination.

PubMed

Shahmoradi, Zeynab; Tamaskani, Fatemeh; Sadeghipour, Hamid Reza; Abdolzadeh, Ahmad

2013-01-01

Alterations in the redox state of storage proteins and the associated proteolytic processes were investigated in moist-chilled and warm-incubated walnut (Juglans regia L.) kernels prior to germination. The kernel total protein labeling with a thiol-specific fluorochrome i.e. monobromobimane (mBBr) revealed more reduction of 29-32 kDa putative glutelins, while in the soluble proteins, both putative glutelins and 41, 55 and 58 kDa globulins contained reduced disulfide bonds during mobilization. Thus, the in vivo more reduced disulfide bonds of storage proteins corresponds to greater solubility. After the in vitro reduction of walnut kernel proteins pre-treated by N-ethyl maleimide (NEM) with dithioerythrethiol (DTT) and bacterial thioredoxin, the 58 kDa putative globulin and a 6 kDa putative albumin were identified as disulfide proteins. Thioredoxin stimulated the reduction of the H(2)O(2)-oxidized 6 kDa polypeptide, but not the 58 kDa polypeptide by DTT. The solubility of 6 kDa putative albumin, 58 and 19-24 kDa putative globulins and glutelins, respectively, were increased by DTT. The in vitro specific mobilization of the 58 kDa polypeptide that occurred at pH 5.0 by the kernel endogenous protease was sensitive to the serine-protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and stimulated by DTT. The specific degradation of the 58 kDa polypeptide might be achieved through thioredoxin-mediated activation of a serine protease and/or reductive unfolding of its 58 kDa polypeptide substrate. As redox changes in storage proteins occurred equally in both moist chilled and warm incubated walnut kernels, the regulatory functions of thioredoxins in promoting seed germination may be due to other germination related processes. Copyright © 2012 Elsevier GmbH. All rights reserved.

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Study of the intercellular fluid of healthy Lupinus albus organs. Presence of a chitinase and a thaumatin-like protein.

PubMed Central

Regalado, A P; Ricardo, C P

1996-01-01

Proteins in the intercellular fluid (IF) of healthy Lupinus albus leaves were characterized. Silver staining of the proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed more than 30 polypeptides, with the major ones having a molecular mass lower than 36 kD. After amino-terminal amino acid sequence analysis, one of the major polypeptides, IF4, was shown to have no identity with any of the proteins present in the data bases. Two others, IF1 and IF3, showed identity with previously reported pathogenesis-related proteins, IF1 with an antifungal protein from Hordeum vulgare that belongs to the thaumatin family (PR-5 family), and IF3 with class III chitinase-lysozymes. IF3 was also present in the IF of stem and root and it represents the major polypeptide in the medium of L. albus cell-suspension cultures. The ubiquitous presence of this enzyme in healthy, nonstressed tissues of L. albus cannot be explained. PMID:8587984

Zonadhesin D3-Polypeptides Vary among Species but Are Similar in Equus Species Capable of Interbreeding1

PubMed Central

Tardif, Steve; Brady, Heidi A.; Breazeale, Kelly R.; Bi, Ming; Thompson, Leslie D.; Bruemmer, Jason E.; Bailey, Laura B.; Hardy, Daniel M.

2009-01-01

Zonadhesin is a rapidly evolving protein in the sperm acrosome that confers species specificity to sperm-zona pellucida adhesion. Though structural variation in zonadhesin likely contributes to its species-specific function, the protein has not previously been characterized in organisms capable of interbreeding. Here we compared properties of zonadhesin in several animals, including the horse (Equus caballus), donkey (E. asinus), and Grevy's zebra (E. grevyi) to determine if variation in zonadhesin correlates with ability of gametes to cross-fertilize. Zonadhesin localized to the apical acrosomes of spermatozoa from all three Equus species, similar to its localization in other animals. Likewise, in horse and donkey testis, zonadhesin was detected only in germ cells, first in the acrosomal granule of round spermatids and then in the developing acrosomes of elongating spermatids. Among non-Equus species, D3-domain polypeptides of mature, processed zonadhesin varied markedly in size and detergent solubility. However, zonadhesin D3-domain polypeptides in horse, donkey, and zebra spermatozoa exhibited identical electrophoretic mobility and detergent solubility. Equus zonadhesin D3-polypeptides (p110/p80 doublet) were most similar in size to porcine and bovine zonadhesin D3-polypeptides (p105). Sequence comparisons revealed that the horse zonadhesin precursor's domain content and arrangement are similar to those of zonadhesin from other large animals. Partial sequences of horse and donkey zonadhesin were much more similar to each other (>99% identity) than they were to orthologous sequences of human, pig, rabbit, and mouse zonadhesin (52%–72% identity). We conclude that conservation of zonadhesin D3-polypeptide properties correlates with ability of Equus species to interbreed. PMID:19794156

Reinventing Cell Penetrating Peptides Using Glycosylated Methionine Sulfonium Ion Sequences.

PubMed

Kramer, Jessica R; Schmidt, Nathan W; Mayle, Kristine M; Kamei, Daniel T; Wong, Gerard C L; Deming, Timothy J

2015-05-27

Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.

Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

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

Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.

2015-04-15

Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess lowmore » cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.« less

Purification and characterization of human pancreatic polypeptide expressed in E. coli.

PubMed

Griko, Y V; Kapanadze, M D

1995-08-04

The region of cDNA encoding human pancreatic polypeptide (hPP) was obtained by polymerase chain reaction (PCR) and subcloned into an expression vector. The pancreatic polypeptide gene was expressed in Escherichia coli in two versions: as a cleavable fusion protein with IgG-binding synthetic ZZ domains of protein A from Staphylococcus aureus or with the 1-48 fragment of lambda Cro repressor. Site-specific hydrolysis by hydroxylamine was used to cleave the fusion protein, releasing the human polypeptide. The structure of the obtained hPP has been studied by scanning microcalorimetry and circular dichroism spectrometry. It has been shown that hPP in solutions close to neutral has a compact and unique spatial structure with an extended hydrophobic core. This structure is stable at 20 degrees C and co-operatively breaks down upon heating from this temperature.

In situ detection of the activation of Rac1 and RalA small GTPases in mouse adipocytes by immunofluorescent microscopy following in vivo and ex vivo insulin stimulation.

PubMed

Takenaka, Nobuyuki; Nihata, Yuma; Ueda, Sho; Satoh, Takaya

2017-11-01

Rac1 has been implicated in insulin-dependent glucose uptake by mechanisms involving plasma membrane translocation of the glucose transporter GLUT4 in skeletal muscle. Although the uptake of glucose is also stimulated by insulin in adipose tissue, the role for Rac1 in adipocyte insulin signaling remains controversial. As a step to reveal the role for Rac1 in adipocytes, we aimed to establish immunofluorescent microscopy to detect the intracellular distribution of activated Rac1. The epitope-tagged Rac1-binding domain of a Rac1-specific target was utilized as a probe that specifically recognizes the activated form of Rac1. Rac1 activation in response to ex vivo and in vivo insulin stimulations in primary adipocyte culture and mouse white adipose tissue, respectively, was successfully observed by immunofluorescent microscopy. These Rac1 activations were mediated by phosphoinositide 3-kinase. Another small GTPase RalA has also been implicated in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Similarly to Rac1, immunofluorescent microscopy using an activated RalA-specific polypeptide probe allowed us to detect intracellular distribution of insulin-activated RalA in adipocytes. These novel approaches to visualize the activation status of small GTPases in adipocytes will largely contribute to the understanding of signal transduction mechanisms particularly for insulin action. Copyright © 2017 Elsevier Inc. All rights reserved.

DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers

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

Kim, Ki Hyung; Biomedical Research Institute and Pusan Cancer Center, Pusan National University Hospital, Busan; Kang, Yun-Jeong

Highlights: • Germ cell marker DDX4 was significantly increased in ovarian cancer. • Ovarian cancer stem cell marker CD133 was significantly increased in ovarian cancer. • DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. • CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4. • Germ cell marker DDX4 has the potential of ovarian cancer stem cell marker. - Abstract: DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondarymore » structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint. We investigated the relationship between DDX4 and ovarian cancer stem cells by analyzing the expression patterns of DDX4 and the cancer stem cell marker CD133 in ovarian cancers via tissue microarray. Both DDX4 and CD133 were significantly increased in ovarian cancer compared to benign tumors, and showed similar patterns of expression. In addition, DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. Furthermore, almost all CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4, suggesting a strong possibility that DDX4 plays an important role in cancer stem cells, and/or can be used as an ovarian cancer stem cell marker.« less

Organic Anion Transporting Polypeptide (OATP)2B1 Contributes to Gastrointestinal Toxicity of Anticancer Drug SN-38, Active Metabolite of Irinotecan Hydrochloride.

PubMed

Fujita, Daichi; Saito, Yoshimasa; Nakanishi, Takeo; Tamai, Ikumi

2016-01-01

Gastrointestinal toxicity, such as late-onset diarrhea, is a significant concern in irinotecan hydrochloride (CPT-11)-containing regimens. Prophylaxis of late-onset diarrhea has been reported with use of Japanese traditional (Kampo) medicine containing baicalin and with the antibiotic cefixime, and this has been explained in terms of inhibition of bacterial deconjugation of SN-38-glucuronide since unconjugated SN-38 (active metabolite of CPT-11) is responsible for the gastrointestinal toxicity. It is also prerequisite for SN-38 to be accumulated in intestinal tissues to exert toxicity. Based on the fact that liver-specific organic anion transporting polypeptide (OATP)1B1, a member of the same family as OATP2B1, is known to be involved in hepatic transport of SN-38, we hypothesized that intestinal transporter OATP2B1 contributes to the accumulation of SN-38 in gastrointestinal tissues, and its inhibition would help prevent associated toxicity. We found that uptake of SN-38 by OATP2B1-expressing Xenopus oocytes was significantly higher than that by control oocytes. OATP2B1-mediated uptake of SN-38 was saturable, pH dependent, and decreased in the presence of baicalin, cefixime, or fruit juices such as apple juice. In vivo gastrointestinal toxicity of SN-38 in mice caused by oral administration for consecutive 5 days was prevented by coingestion of apple juice. Thus, OATP2B1 contributes to the uptake of SN-38 by intestinal tissues, triggering gastrointestinal toxicity. So, in addition to the reported inhibition of bacterial β-glucuronidase by cefixime or baicalin, inhibition of OATP2B1 may also contribute to prevention of gastrointestinal toxicity. Apple juice may be helpful for prophylaxis of late-onset diarrhea observed in CPT-11 therapy without disturbance of the intestinal microflora. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Facilitating protein solubility by use of peptide extensions

DOEpatents

Freimuth, Paul I; Zhang, Yian-Biao; Howitt, Jason

2013-09-17

Expression vectors for expression of a protein or polypeptide of interest as a fusion product composed of the protein or polypeptide of interest fused at one terminus to a solubility enhancing peptide extension are provided. Sequences encoding the peptide extensions are provided. The invention further comprises antibodies which bind specifically to one or more of the solubility enhancing peptide extensions.

Transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene.

PubMed

Soria-Guerra, Ruth Elena; Rosales-Mendoza, Sergio; Márquez-Mercado, Crisóforo; López-Revilla, Rubén; Castillo-Collazo, Rosalba; Alpuche-Solís, Angel Gabriel

2007-07-01

A current priority of vaccinology is the development of multicomponent vaccines that protect against several pathogens. The diphtheria-pertussis-tetanus (DPT) vaccine prevents the symptoms of three serious and often fatal diseases due to the exotoxins produced by Corynebacterium diphteriae, Bordetella pertussis and Clostridium tetani. We are attempting to develop an edible DPT multicomponent vaccine in plants, based on the fusion of protective exotoxin epitopes encoded by synthetic genes. By means of Agrobacterium mediated transformation we generated transgenic tomatoes with a plant-optimised synthetic gene encoding a novel polypeptide containing two adjuvant and six DPT immunoprotective exotoxin epitopes joined by peptide linkers. In transformed tomato plants, integration of the synthetic DPT (sDPT) gene detected by PCR was confirmed by Southern blot, and specific transcripts of the expected molecular size were detected by RT-PCR. Expression of the putative polypeptide encoded by the sDPT gene was detected by immunoassay with specific antibodies to the diphtheria, pertussis and tetanus exotoxins. The sDPT gene is therefore integrated, transcribed and translated as the expected recombinant sDPT multiepitope polypeptide in transgenic tomatoes that constitute a potential edible vaccine.

Characterization of auxin-binding proteins from zucchini plasma membrane

NASA Technical Reports Server (NTRS)

Hicks, G. R.; Rice, M. S.; Lomax, T. L.

1993-01-01

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

Characterization of auxin-binding proteins from zucchini plasma membrane.

PubMed

Hicks, G R; Rice, M S; Lomax, T L

1993-01-01

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

Three polypeptides screened from phage display random peptide library may be the receptor polypeptide of Mycoplasma genitalium adhesion protein.

PubMed

Deng, Xiangying; Zhu, Youcong; Dai, Pei; Yu, Minjun; Chen, Liesong; Zhu, Cuiming; You, Xiaoxing; Li, Lingling; Zeng, Yanhua

2018-04-28

Mycoplasma genitalium adhesion protein (MgPa) is a major adhesin of M. genitalium, a human pathogen associated with a series of genitourinary tract diseases. MgPa plays a very important role in M. genitalium adhering to the host cells. However, the exact receptor peptides or proteins of MgPa are still poorly understood so far. Three polypeptides (V-H-W-D-F-R-Q-W-W-Q-P-S), (D-W-S-S-W-V -Y-R-D-P-Q-T) and (H-Y-I-D-F-R-W) were previously screened from a phage display random peptide library using recombinant MgPa (rMgPa) as a target molecule. In this study, three polypeptides were artificially synthesized and investigated as to whether they are potential receptors of MgPa. We found that rMgPa specifically bound to three synthesized polypeptides as determined via an indirect enzyme-linked immunosorbent assay (ELISA). Moreover, three polypeptides were further identified by indirect immunofluorescence microscopy (IFM). We confirmed that rMgPa and M. genitalium can adhere to SV-HUC-1 cells in vitro and that anti-rMgPa antibody and three synthesized polypeptides can partially inhibit the adherence of rMgPa and M. genitalium to SV-HUC-1 cells. In summary, these three polypeptides may be the essential receptor peptides of MgPa, and may aid in enhancing the understanding of biological function of MgPa and the possible pathogenic mechanism of M. genitalium. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effect of Aerobic Priming on the Response of Echinochloa crus-pavonis to Anaerobic Stress (Protein Synthesis and Phosphorylation).

PubMed Central

Zhang, F.; Lin, J. J.; Fox, T. C.; Mujer, C. V.; Rumpho, M. E.; Kennedy, R. A.

1994-01-01

Echinochloa species differ in their ability to germinate and grow in the absence of oxygen. Seeds of Echinochloa crus-pavonis (H.B.K.) Schult do not germinate under anoxia but remain viable for extended periods (at least 30 d) when incubated in an anaerobic environment. E. crus-pavonis can be induced to germinate and grow in an anaerobic environment if the seeds are first subjected to a short (1-18 h) exposure to aerobic conditions (aerobic priming). Changes in polypeptide patterns (constitutive and de novo synthesized) and protein phosphorylation induced by aerobic priming were investigated. In the absence of aerobic priming protein degradation was not evident under anaerobic conditions, although synthesis of a 20-kD polypeptide was induced. During aerobic priming, however, synthesis of 37- and 55-kD polypeptides was induced and persisted upon return of the seeds to anoxia. Furthermore, phosphorylation of two 18-kD polypeptides was observed only in those seeds that were labeled with 32PO4 during the aerobic priming period. Subsequent chasing in an anaerobic environment resulted in a decrease in phosphorylation of these polypeptides. Likewise, phosphorylation of the 18-kD polypeptides was not observed if the seeds were labeled in an anaerobic atmosphere. These results suggest that the regulated induction of the 20-, 37-, and 55- kD polypeptides may be important for anaerobic germination and growth of E. crus-pavonis and that the specific phosphorylation of the 18-kD polypeptides may be a factor in regulating this induction. PMID:12232272

Double-hydrophobic elastin-like polypeptides with added functional motifs: Self-assembly and cytocompatibility.

PubMed

Le, Duc H T; Tsutsui, Yoko; Sugawara-Narutaki, Ayae; Yukawa, Hiroshi; Baba, Yoshinobu; Ohtsuki, Chikara

2017-09-01

We have recently developed a novel double-hydrophobic elastin-like triblock polypeptide called GPG, designed after the uneven distribution of two different hydrophobic domains found in elastin, an extracellular matrix protein providing elasticity and resilience to tissues. Upon temperature trigger, GPG undergoes a sequential self-assembling process to form flexible beaded nanofibers with high homogeneity and excellent dispersibility in water. Given that GPG might be a potential elastin-mimetic material, we sought to explore the biological activities of this block polypeptide. Besides GPG, several functionalized derivatives were also constructed by fusing functional motifs such as KAAK or KAAKGRGDS at the C-terminal of GPG. Although the added motifs affected the kinetics of fiber formation and β-sheet contents, all three GPGs assembled into beaded nanofibers at the physiological temperature. The resulting GPG nanofibers preserved their beaded structures in cell culture medium; therefore, they were coated on polystyrene substrates to study their cytocompatibility toward mouse embryonic fibroblasts, NIH-3T3. Among the three polypeptides, GPG having the cell-binding motif GRGDS derived from fibronectin showed excellent cell adhesion and cell proliferation properties compared to other conventional materials, suggesting its promising applications as extracellular matrices for mammalian cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2475-2484, 2017. © 2017 Wiley Periodicals, Inc.

Absolute measurement of species differences in sodium taurocholate cotransporting polypeptide (NTCP/Ntcp) and its modulation in cultured hepatocytes.

PubMed

Qiu, Xi; Bi, Yi-An; Balogh, Larissa M; Lai, Yurong

2013-09-01

Species differences among membrane transporters can be remarkable and difficult to properly assess by conventional methods. Herein, we employed the first use of stable isotope labeling in mammals or stable isotope-labeled peptides combined with mass spectrometry to identify species differences in sodium taurocholate cotransporting polypeptide (NTCP/Ntcp) protein expression in liver tissue and to characterize the modulation of protein expression in sandwich-cultured human (SCHH) and rat hepatocytes (SCRH). The lower limit of quantification was established to be 5 fmol on column with a standard curve that was linear up to 2000 fmol. The accuracy and precision were evaluated with three quality control samples and known amounts of synthetic proteotypic peptides that were spiked into the membrane protein extracts. The overall relative error and coefficient of variation were less than 10%. The expression of Ntcp in mouse and rat was significant higher than that in human (five-fold) and monkey (two-fold) and ranked as mouse > rat > monkey > human. In the cultured hepatocytes, although significant downregulation of Ntcp expression in SCRH at day 5 after the culture was detected, NTCP expression in SCHH was comparable to the suspension hepatocytes. The results suggested that NTCP/Ntcp modulation in cultured hepatocytes is species specific. Copyright © 2013 Wiley Periodicals, Inc.

Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs

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

Hanecak, R.; Semler, B.L.; Anderson, C.W.

1982-07-01

Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH/sub 2/-coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins inmore » vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X.« less

The cell-wall glycoproteins of the green alga Scenedesmus obliquus. The predominant cell-wall polypeptide of Scenedesmus obliquus is related to the cell-wall glycoprotein gp3 of Chlamydomonas reinhardtii.

PubMed

Voigt, Jürgen; Stolarczyk, Adam; Zych, Maria; Malec, Przemysław; Burczyk, Jan

2014-02-01

The green alga Scenedesmus obliquus contains a multilayered cell wall, ultrastructurally similar to that of Chlamydomonas reinhardtii, although its proportion of hydroxyproline is considerably lower. Therefore, we have investigated the polypeptide composition of the insoluble and the chaotrope-soluble wall fractions of S. obliquus. The polypeptide pattern of the chaotrope-soluble wall fraction was strongly modified by chemical deglycosylation with anhydrous hydrogen fluoride (HF) in pyridine indicating that most of these polypeptides are glycosylated. Polypeptide constituents of the chaotrope-soluble cell-wall fraction with apparent molecular masses of 240, 270, 265, and 135 kDa cross-reacted with a polyclonal antibody raised against the 100 kDa deglycosylation product of the C. reinhardtii cell-wall glycoprotein GP3B. Chemical deglycosylation of the chaotrope-soluble wall fraction resulted in a 135 kDa major polypeptide and a 106 kDa minor component reacting with the same antibody. This antibody recognized specific peptide epitopes of GP3B. When the insoluble wall fraction of S. obliquus was treated with anhydrous HF/pyridine, three polypeptides with apparent molecular masses of 144, 135, and 65 kDa were solubilized, which also occured in the deglycosylated chaotrope-soluble wall fraction. These findings indicate that theses glycoproteins are cross-linked to the insoluble wall fraction via HF-sensitive bonds. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

The plant vacuolar Na+/H+ antiport.

PubMed

Barkla, B J; Apse, M P; Manolson, M F; Blumwald, E

1994-01-01

Salt stress imposes severe limitations on plant growth, however, the extent of growth reduction depends upon the soil salinity level and the plant species. One of the mechanisms employed by salt tolerant plants is the effective vacuolar compartmentalization of sodium. The sequestration of sodium into the vacuole occurs by the operation of a Na+/H+ antiport located at the tonoplast. Evidence for a plant vacuolar Na+/H+ antiport has been demonstrated in tissues, intact vacuoles and isolated tonoplast vesicles. In sugar beet cell suspensions, the activity of the vacuolar Na+/H+ antiport increased with increasing NaCl concentrations in the growth medium. This increased activity was correlated with the increased synthesis of a 170 kDa tonoplast polypeptide. In vivo labelling of tonoplast proteins showed the enhanced synthesis of the 170 kDa polypeptide not only upon exposure of the cells to salt, but also when the cells were grown in the presence of amiloride. Exposure of the cells to amiloride also resulted in increased vacuolar Na+/H+ antiport activity. Polyclonal antibodies raised against the 170 kDa polypeptide almost completely inhibited the antiport activity, suggesting the association of this protein with the plant vacuolar Na+/H+ antiport. Antibodies against the Na+/H+ antiport-associated polypeptide were used to screen a Beta lambda ZAP expression library. A partial clone of 1.65 kb was sequenced and found to encode a polypeptide with a putative transmembrane domain and a large hydrophilic C terminus. This clone showed no homology to any previously cloned gene at either the nucleic acid or the amino acid level.

Evaluation of tumour markers as differential diagnostic tool in patients with suspicion of liver metastases from breast cancer.

PubMed

Liska, Vaclav; Holubec, Lubos; Treska, Vladislav; Vrzalova, Jindra; Skalicky, Tomas; Sutnar, Alan; Kormunda, Stanislav; Bruha, Jan; Vycital, Ondrej; Finek, Jindrich; Pesta, Martin; Pecen, Ladislav; Topolcan, Ondrej

2011-04-01

The liver is the site of breast cancer metastasis in 50% of patients with advanced disease. Tumour markers have been demonstrated as being useful in follow-up of patients with breast cancer, in early detection of recurrence of breast cancer after radical surgical treatments, and in assessing oncologic therapy effect, but no study has been carried out on their usefullness in distinguishing benign liver lesions from breast cancer metastases. The aim of this study was therefore to evaluate the importance of tumour markers carcinoembryonic antigen (CEA), carbohydrate antigen CA19-9 (CA19-9), thymidine kinase (TK), tissue polypeptide antigen (TPA), tissue polypeptide-specific antigen (TPS) and cytokeratin 19 fragment (CYFRA 21-1) in differential diagnosis between benign liver lesions and liver metastases of breast cancer. The study includes 3 groups: 22 patients with liver metastases of breast cancer; 39 patients with benign liver lesions (hemangioma, focal nodular hyperplasia, liver cyst, hepatocellular adenoma); and 21 patients without any liver disease or lesion that were operated on for benign extrahepatic diseases (groin hernia, varices of lower limbs) as a control group. The serum levels of tumour markers were assessed by means of immunoanalytical methods. Preoperative serum levels of CYFRA 21-1, TPA, TPS and CEA were significantly higher in patients with liver metastases of breast cancer in contrast to healthy controls and patients with benign liver lesions (p-value<0.05). Serum levels of CA19-9 and TK were higher in patients with malignancy in comparison with benign liver disease and healthy controls but these differences were not statistically significant. Tumour markers CEA, CYFRA 21-1, TPA and TPS can be recommended as a good tool for differential diagnosis between liver metastases of breast cancer and benign liver lesions.

Peptides and polypeptides as scaffolds for optoelectronics and biomaterials applications

NASA Astrophysics Data System (ADS)

Charati, Manoj B.

Peptides and polypeptides are emerging as a new class of biomaterials due to their unique structural, physiochemical, mechanical, and biological properties. The development of peptide and protein-based biomaterials is driven by the convergence of convenient techniques for peptide/protein engineering and its importance in applications as smart biomaterials. The thesis is divided in two parts; the first part highlights the importance of incorporation of non-natural amino acids into peptides and proteins. In particular, incorporation on p-bromophenylalanine in short alpha-helical peptide templates to control the association of chromophores is discussed. In the second part, design of a multi-component, biocompatible polypeptide with superior elasticity is discussed. Part 1. Novel peptide templates to control association of chromophores. Tailor made peptide and protein materials have many versatile applications, as both conformation and functional group position can be controlled. Such control may have intriguing applications in the development of hybrid materials for electroactive applications. A critical need in fabricating devices from organic semiconducting materials is to achieve control over the conformation and distance between two conjugated chains. Controlling chromophore spacing and orientation with required precision over nanometer length scale poses a greater challenge. Here we propose a peptide based template to control the alignment of the methylstilbene and Oxa-PPV chromophores with desired orientations and spacing. The hybrid peptides were characterized via CD, exciton coupled CD, 1H NMR and photoluminescence experiments. It is observed that slight change in the orientation of molecules has pronounced effect on the photo-physical behavior of the molecules. Characterization of the hybrid peptides via circular dichroism (CD) confirmed the helical character of the designed peptides and indicated that inclusion of non-natural amino acids has significant effects on peptide conformation. pi-orbital interactions at the molecular level were observed to be very sensitive to intermolecular distance and orientation of the chromophores attached to the alpha-helical peptide templates. When the methylstilbene or Oxa-PPV molecules were arranged on the same side of the helix with intermolecular spacing of 6A, the chromophores interacted strongly with each other forming excimers. Such interactions were absent when the molecules were arranged on the opposite side of the helix. These peptide-templated systems therefore offer enormous opportunities for the elucidation of complex photophysical phenomena that occur in relatively aggregated morphologies of conjugated species, but under dilute solution conditions in which the number of chromphores in the aggregate can be manipulated. Part 2. Synthesis and characterization of biocompatible polypeptide elastomer. Lately, the significance of mechanical forces and biological cues involved in tissue remodeling are highly valued; thus the capacity of a biomaterial to present a fitting mechanical and biological environment for optimal tissue generation has become a key parameter for biomaterial design. In addition to having suitable mechanical properties, materials used for these applications need to be biologically active, i.e. trigger dynamic interactions with cells and stimulate explicit cell and tissue responses. Thus, we have designed a resilin-based modular biomaterial incorporating both mechanically and biologically active domains to sense and aptly respond to the bio-mechanical demand or changes in their environment. The use of resilin-like polypeptides offers access to a class of hydrophilic elastomers with excellent resilience and high frequency responsiveness, which can be used for encapsulating hydrophilic drugs like proteins for drug delivery, and provides hydrophilic extracellular matrix mimicking cell adhesive and enzyme degradable substrate for tissue engineering. Hence, we have designed a multi-component elastomeric polypeptide scaffold containing a resilin mimetic flexible domain that governs the mechanical properties, a cell adhesion domain, a proteolytic degradation site and a growth factor binding domain for applications in tissue regeneration. The modular protein was synthesized in an E. coli host and the purified protein, as analyzed via CD and FTIR exhibited mainly unordered conformation with slight contribution from type II beta-turns. The crosslinked protein is highly elastic in nature and support cell attachment. There materials could be used for tissue repair where high frequency responsiveness and superior fatigue resistance are important, such as scaffolds for vocal fold regenerations and as cardiovascular patches.

Variant forms of mitochondrial translation products in yeast: evidence for location of determinants on mitochondrial DNA.

PubMed

Douglas, M G; Butow, R A

1976-04-01

Products of mitochondrial protein synthesis in yeast have been labeled in vivo with 35SO42-. More than 20 polypeptide species fulfilling the criteria of mitochondrial translation products have been detected by analysis on sodium dodecyl sulfate-exponential polyacrylamide slab gels. A comparison of mitochondrial translation products in two wild-type strains has revealed variant forms of some polypeptide species which show genetic behavior consistent with the location of their structural genes on mtDNA. Our results demonstrate the feasibility of performing genetic analysis on putative gene products of mtDNA in wild-type yeast by direct examination of the segregation and recombination behavior of specific polypeptide species.

Differences in antigen presentation to MHC class I-and class II- restricted influenza virus-specific cytolytic T lymphocyte clones

PubMed Central

1986-01-01

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA- specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II- restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets. PMID:3485173

Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.

PubMed

Finka, Andrija; Goloubinoff, Pierre

2013-09-01

In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10% of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.

Recombinant soluble adenovirus receptor

DOEpatents

Freimuth, Paul I.

2002-01-01

Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

Functional domains of the T lymphocyte plasma membrane: characterization of the polypeptide composition.

PubMed

Szamel, M; Kaever, V; Resch, K

1987-01-01

Highly purified plasma membranes from calf thymocytes were fractionated by affinity chromatography on Concanavalin A-Sepharose into two subfractions, one eluting freely from the affinity column (MF1) and a second being specifically retained (MF2). SDS-polyacrylamide-gel-electrophoresis revealed different polypeptide patterns of the two plasma membrane subfractions. Polypeptides of apparent molecular weights of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2. In contrast, several proteins in the 55-65 kDa range were preferentially recovered in the non-adherent fraction. Five Five of the six polypeptides, preferentially recovered in MF2 proved to be glycoproteins, the 39 kDa peptide was non-glycosilated. The differences in the amounts of the polypeptides specifically enriched in the adherent fraction MF2 became even more clear-cut when plasma membranes solubilized with non-ionic detergents (lysolecithin, ET-18-2H, Triton-X-100) were separated by affinity chromatography on Concanavalin A-Sepharose. The non-glycosilated peptide of apparent molecular weight of 39 kDa was recovered together with several glycoproteins in the adherent fraction, MF2, suggesting that not single glycoproteins, but plasma membrane domains were separated by Concanavalin A-Sepharose. Although the glycoproteins of the non-adherent fraction MF1 bound significant amounts of Concanavalin A, the major Concanavalin A binding glycoproteins were recovered in the adherent fraction, MF2. The plasma membrane subfractions showed also different functional properties, the specific activities [Na+ + K+]AT-Pase, Ca2+ ATPase and lysolecithin acyltransferase were several-fold enriched in the adherent fraction, MF2, as compared to MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of thymocytes consisting of a different set of proteins, among others the major Concanavalin A binding glycoproteins with some membrane bound enzymes, probably implicated in the initiation of lymphocyte activation.

Revisiting structure-property relationship of pH-responsive polymers for drug delivery applications.

PubMed

Bazban-Shotorbani, Salime; Hasani-Sadrabadi, Mohammad Mahdi; Karkhaneh, Akbar; Serpooshan, Vahid; Jacob, Karl I; Moshaverinia, Alireza; Mahmoudi, Morteza

2017-05-10

pH-responsive polymers contain ionic functional groups as pendants in their structure. The total number of charged groups on polymer chains determines the overall response of the system to changes in the external pH. This article reviews various pH-responsive polymers classified as polyacids (e.g., carboxylic acid based polymers, sulfonamides, anionic polysaccharides, and anionic polypeptides) and polybases (e.g., polyamines, pyridine and imidazole containing polymers, cationic polysaccharides, and cationic polypeptides). We correlate the pH variations in the body at the organ level (e.g., gastrointestinal tract and vaginal environment), tissue level (e.g., cancerous and inflamed tissues), and cellular level (e.g., sub-cellular organelles), with the intrinsic properties of pH-responsive polymers. This knowledge could help to select more effective ('smart') polymeric systems based on the biological target. Considering the pH differences in the body, various drug delivery systems can be designed by utilizing smart biopolymeric compounds with the required pH-sensitivity. We also review the pharmaceutical application of pH-responsive polymeric carriers including hydrogels, polymer-drug conjugates, micelles, dendrimers, and polymersomes. © 2016.

Expression of VGF mRNA in developing neuroendocrine and endocrine tissues.

PubMed

Snyder, S E; Peng, B; Pintar, J E; Salton, S R J

2003-11-01

Analysis of knockout mice suggests that the neurotropin-inducible secreted polypeptide VGF (non-acronymic) plays an important role in the regulation of energy balance. VGF is synthesized by neurons in the central and peripheral nervous systems (CNS, PNS), as well as in the adult pituitary, adrenal medulla, endocrine cells of the stomach and pancreatic beta cells. Thus VGF, like cholecystokinin, leptin, ghrelin and other peptide hormones that have been shown to regulate feeding and energy expenditure, is synthesized in both the gut and the brain. Although detailed developmental studies of VGF localization in the CNS and PNS have been completed, little is known about the ontogeny of VGF expression in endocrine and neuroendocrine tIssues. Here, we report that VGF mRNA is detectable as early as embryonic day 15.5 in the developing rat gastrointestinal and esophageal lumen, pancreas, adrenal, and pituitary, and we further demonstrate that VGF mRNA is synthesized in the gravid rat uterus, together supporting possible functional roles for this polypeptide outside the nervous system and in the enteric plexus.

Structural specificity of mucosal-cell transport and metabolism of peptide drugs: implication for oral peptide drug delivery

NASA Technical Reports Server (NTRS)

Bai, J. P.; Amidon, G. L.

1992-01-01

The brush border membrane of intestinal mucosal cells contains a peptide carrier system with rather broad substrate specificity and various endo- and exopeptidase activities. Small peptide (di-/tripeptide)-type drugs with or without an N-terminal alpha-amino group, including beta-lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors, are transported by the peptide transporter. Polypeptide drugs are hydrolyzed by brush border membrane proteolytic enzymes to di-/tripeptides and amino acids. Therefore, while the intestinal brush border membrane has a carrier system facilitating the absorption of di-/tripeptide drugs, it is a major barrier limiting oral availability of polypeptide drugs. In this paper, the specificity of peptide transport and metabolism in the intestinal brush border membrane is reviewed.

Immunogenic Antigen Selective Cancer Immunotherapy | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Institute on Aging working on cancer immunotherapy and detection report the use of SPANX-B polypeptides in the treatment and identification of cancer. Specific human malignancies targeted for the treatments disclosed include melanoma and lung, colon, renal, ovarian and breast carcinomas. The NIA seeks parties interested in licensing or collaborative research to further develop, evaluate, or commercialize SPANX-B polypeptides in the treatment and identification of cancer.

N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride: An immune-enhancing adjuvant for hepatitis E virus recombinant polypeptide vaccine in mice.

PubMed

Tao, Wei; Zheng, Hai-Qun; Fu, Ting; He, Zhuo-Jing; Hong, Yan

2017-08-03

Adjuvants are essential for enhancing vaccine potency by improving the humoral and/or cell-mediated immune response to vaccine antigens. This study was performed to evaluate the immuno-enhancing characteristic of N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride (HTCC), the cationically modified chitosan, as an adjuvant for hepatitis E virus (HEV) recombinant polypeptide vaccine. Animal experiments showed that HTCC provides adjuvant activity when co-administered with HEV recombinant polypeptide vaccine by intramuscularly route. Vaccination using HTCC as an adjuvant was associated with increases of the serum HEV-specific IgG antibodies, splenocytes proliferation and the growths of CD4 + CD8 - T lymphocytes and IFN-γ-secreting T lymphocytes in peripheral blood. These findings suggested that HTCC had strong immuno-enhancing effect. Our findings are the first to demonstrate that HTCC is safe and effective in inducing a good antibody response and stimulating Th1-biased immune responses for HEV recombinant polypeptide vaccine.

High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides.

PubMed

Bahniuk, Markian S; Alshememry, Abdullah K; Unsworth, Larry D

2016-12-01

The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields of 250-660 mg per liter of cells, depending on the specific construct. This was considerably greater than previously reported yields for similar ELPs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to be purify these peptides.

Nuclear Involvement in the Appearance of a Chloroplast-Encoded 32,000 Dalton Thylakoid Membrane Polypeptide Integral to the Photosystem II Complex 1

PubMed Central

Leto, Kenneth J.; Keresztes, Aron; Arntzen, Charles J.

1982-01-01

The genetic locus for the high chlorophyll fluorescent photosystem II-deficient maize mutant hcf*-3 has been definitively located to the nuclear genome. Fluorography of lamellar polypeptides labeled with [35S]methionine in vivo revealed the specific loss of a heavily labeled 32,000 dalton thylakoid membrane polypeptide as well as its chloroplast encoded precursor species at 34,000 daltons. Examination of freeze-fractured mesophyll and bundle sheath thylakoids from hcf*-3 revealed that both plastid types lacked the large EFs particles believed to consist of the photosystem II reaction center-core complex and associated light harvesting chlorophyll-proteins. The present evidence suggests that the synthesis or turnover/integration of the chloroplast-encoded 34,000 to 32,000 dalton polypeptide is under nuclear control, and that these polyipeptides are integral components of photosystem II which may be required for the assembly or structural stabilization of newly formed photosystem II reaction centers in both mesophyll and bundle sheath chloroplasts. Images PMID:16662421

Isolation of pheromone precursor genes of Magnaporthe grisea.

PubMed

Shen, W C; Bobrowicz, P; Ebbole, D J

1999-01-01

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia. Copyright 1999 Academic Press.

Multiple polypeptides immunologically related to beta-poly(L-malate) hydrolase (polymalatase) in the plasmodium of the slime mold Physarum polycephalum.

PubMed

Karl, M; Holler, E

1998-01-15

Plasmodia of Physarum polycephalum contain large amounts of the cell-type-specific polyanion beta-poly(L-malate) and of a corresponding specific hydrolase (polymalatase), both expressed in the plasmodial form of the organism. We have partially purified polymalatase, the preparation consisting of several polypeptides, which could not be separated without destroying the hydrolase activity. Polypeptides of 68 kDa and 97 kDa were identified as polymalatases. Both were glycosylated, the 68-kDa form giving rise to a 54-kDa form when deglycosylated, and the 97-kDa form giving rise to an 88-kDa polypeptide that was indistinguishable from an 88-kDa inactive species also contained in the enzyme preparation. Antisera against each of these proteins were used to detect the intracellular distribution of the proteins. We found that the antisera crossreacted with the three proteins and, furthermore, with a multiplicity of polypeptides ubiquitously distributed over the plasmodium. Results of a two-dimensional non-denaturing in the first dimension and SDS-denaturing polyacrylamide gel electrophoresis in the second dimension suggested that the proteins were derived from a 200-kDa 'precursor' protein by proteolytic fragmentation. Polymalatase activity could be generated from a high molecular-mass precursor. According to several pieces of evidence, the proteolytic nicking occurred within plasmodia. The fragments were sticky and gave rise to preferred sizes of nicked macromolecules. The observed multiplicity varied as a function of the age of the cultures. The cellular distribution and the intracellular pH value were not compatible with an in situ polymalatase activity and suggested other, presently unknown, function(s) such as in the transportation of beta-poly(L-malate) from the nucleus to the culture medium.

Biogenesis of protein bodies during legumin accumulation in developing olive (Olea europaea L.) seed.

PubMed

Jimenez-Lopez, Jose C; Zienkiewicz, Agnieszka; Zienkiewicz, Krzysztof; Alché, Juan D; Rodríguez-García, Maria I

2016-03-01

Much of our current knowledge about seed development and differentiation regarding reserves synthesis and accumulation come from monocot (cereals) plants. Studies in dicotyledonous seeds differentiation are limited to a few species and in oleaginous species are even scarcer despite their agronomic and economic importance. We examined the changes accompanying the differentiation of olive endosperm and cotyledon with a focus on protein bodies (PBs) biogenesis during legumin protein synthesis and accumulation, with the aim of getting insights and a better understanding of the PBs' formation process. Cotyledon and endosperm undergo differentiation during seed development, where an asynchronous time-course of protein synthesis, accumulation, and differential PB formation patterns was found in both tissues. At the end of seed maturation, a broad population of PBs, particularly in cotyledon cells, was distinguishable in terms of number per cell and morphometric and cytochemical features. Olive seed development is a tissue-dependent process characterized by differential rates of legumin accumulation and PB formation in the main tissues integrating seed. One of the main features of the impressive differentiation process is the specific formation of a broad group of PBs, particularly in cotyledon cells, which might depend on selective accumulation and packaging of proteins and specific polypeptides into PBs. The nature and availability of the major components detected in the PBs of olive seed are key parameters in order to consider the potential use of this material as a suitable source of carbon and nitrogen for animal or even human use.

An immunohistochemical screening of neurochemical markers in fungiform papillae and taste buds of the anterior rat tongue.

PubMed

Astbäck, J; Arvidson, K; Johansson, O

1997-02-01

The occurrence and distribution of several neurochemical markers were investigated. Numerous nerve fibres were shown, using antibodies to protein gene product (PGP) 9.5, neurone-specific enolase, calcitonin gene-related peptide (CGRP), substance P. neurokinin A or protein S-100. The presence of vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine amide (PHI), neuropeptide tyrosine, dopamine-beta-hydroxylase (DBH), cholecystokinin/gastrin, glutamate and galanin was more scarce. Nerve fibres containing these above-mentioned markers were found at several locations, i.e. in the epithelium, connective tissue, and around blood vessels. In the taste buds, numerous PGP 9.5, neurone-specific enolase-, CGRP-, substance P-, neurokinin A- and protein S-100-containing structures were found, but few VIP and galanin ones. No immunoreactivity was found with antibodies against somatostatin, bombesin, enkephalin or dynorphin. These findings extend knowledge about the general as well as the neurochemical messenger-based innervation of rat fungiform papillae, forming a firm basis for future functional investigations of normal, experimental and also clinical materials.

Optical imaging of gastric cancer with near-infrared heptamethine carbocyanine fluorescence dyes.

PubMed

Zhao, Ningning; Zhang, Caiqin; Zhao, Yong; Bai, Bing; An, Jiaze; Zhang, Hai; Wu, Jason Boyang; Shi, Changhong

2016-08-30

Near-infrared fluorescence (NIRF) imaging agents are promising tools for noninvasive cancer imaging. Here, we explored the tumor-specific targeting ability of NIRF heptamethine carbocyanine MHI-148 dye in cultured gastric cancer cells, gastric cancer cell-derived and patient-derived tumor xenograft (PDX) models. We show that the NIRF dye specifically accumulated in tumor regions of both xenograft models, suggesting the potential utility of the dye for tumor-specific imaging and targeting in gastric cancer. We also demonstrated significant correlations between NIRF signal intensity and tumor volume in PDX models. Mechanistically, the higher cellular uptake of MHI-148 in gastric cancer cells than in normal cells was stimulated by hypoxia and activation of a group of organic anion-transporting polypeptide (OATP) genes. Importantly, this NIRF dye was not retained in inflammatory stomach tissues induced by gastric ulcer in mice. In addition, fresh clinical gastric tumor specimens, when perfused with NIR dye, exhibited increased uptake of NIR dye in situ. Together, these results show the possibility of using NIRF dyes as novel candidate agents for clinical imaging and detection of gastric cancer.

Growth factors in urologic tissues: detection, characterization, and clinical applications.

PubMed

Mydlo, J H; Macchia, R J

1992-12-01

During the last two decades, enormous strides have been made in understanding cellular and molecular biology. The direction of treatment of many neoplasms and other diseases are starting at the microscopic level. Growth factors are polypeptides that play a part in the development and maintenance of living tissues. We, as well as others, have investigated the role that growth factors play particularly in urologic tissues, both benign and malignant. We review several well-known growth factors and their function in prostate, kidney, and bladder tissues, as well as their functions in other regulating processes of the human body, and also the use of growth factors as tumor markers, and antibodies to growth factors as possible treatment of disease.

Human elastin polypeptides improve the biomechanical properties of three-dimensional matrices through the regulation of elastogenesis.

PubMed

Boccafoschi, Francesca; Ramella, Martina; Sibillano, Teresa; De Caro, Liberato; Giannini, Cinzia; Comparelli, Roberto; Bandiera, Antonella; Cannas, Mario

2015-03-01

The replacement of diseased tissues with biological substitutes with suitable biomechanical properties is one of the most important goal in tissue engineering. Collagen represents a satisfactory choice for scaffolds. Unfortunately, the lack of elasticity represents a restriction to a wide use of collagen for several applications. In this work, we studied the effect of human elastin-like polypeptide (HELP) as hybrid collagen-elastin matrices. In particular, we studied the biomechanical properties of collagen/HELP scaffolds considering several components involved in ECM remodeling (elastin, collagen, fibrillin, lectin-like receptor, metalloproteinases) and cell phenotype (myogenin, myosin heavy chain) with particular awareness for vascular tissue engineering applications. Elastin and collagen content resulted upregulated in collagen-HELP matrices, even showing an improved structural remodeling through the involvement of proteins to a ECM remodeling activity. Moreover, the hybrid matrices enhanced the contractile activity of C2C12 cells concurring to improve the mechanical properties of the scaffold. Finally, small-angle X-ray scattering analyses were performed to enable a very detailed analysis of the matrices at the nanoscale, comparing the scaffolds with native blood vessels. In conclusion, our work shows the use of recombinant HELP, as a very promising complement able to significantly improve the biomechanical properties of three-dimensional collagen matrices in terms of tensile stress and elastic modulus. © 2014 Wiley Periodicals, Inc.

Rate limit of protein elastic response is tether dependent.

PubMed

Berkovich, Ronen; Hermans, Rodolfo I; Popa, Ionel; Stirnemann, Guillaume; Garcia-Manyes, Sergi; Berne, Bruce J; Fernandez, Julio M

2012-09-04

The elastic restoring force of tissues must be able to operate over the very wide range of loading rates experienced by living organisms. It is surprising that even the fastest events involving animal muscle tissues do not surpass a few hundred hertz. We propose that this limit is set in part by the elastic dynamics of tethered proteins extending and relaxing under a changing load. Here we study the elastic dynamics of tethered proteins using a fast force spectrometer with sub-millisecond time resolution, combined with Brownian and Molecular Dynamics simulations. We show that the act of tethering a polypeptide to an object, an inseparable part of protein elasticity in vivo and in experimental setups, greatly reduces the attempt frequency with which the protein samples its free energy. Indeed, our data shows that a tethered polypeptide can traverse its free-energy landscape with a surprisingly low effective diffusion coefficient D(eff) ~ 1,200 nm(2)/s. By contrast, our Molecular Dynamics simulations show that diffusion of an isolated protein under force occurs at D(eff) ~ 10(8) nm(2)/s. This discrepancy is attributed to the drag force caused by the tethering object. From the physiological time scales of tissue elasticity, we calculate that tethered elastic proteins equilibrate in vivo with D(eff) ~ 10(4)-10(6) nm(2)/s which is two to four orders magnitude smaller than the values measured for untethered proteins in bulk.

Tunable mechanical stability and deformation response of a resilin-based elastomer.

PubMed

Li, Linqing; Teller, Sean; Clifton, Rodney J; Jia, Xinqiao; Kiick, Kristi L

2011-06-13

Resilin, the highly elastomeric protein found in specialized compartments of most arthropods, possesses superior resilience and excellent high-frequency responsiveness. Enabled by biosynthetic strategies, we have designed and produced a modular, recombinant resilin-like polypeptide bearing both mechanically active and biologically active domains to create novel biomaterial microenvironments for engineering mechanically active tissues such as blood vessels, cardiovascular tissues, and vocal folds. Preliminary studies revealed that these recombinant materials exhibit promising mechanical properties and support the adhesion of NIH 3T3 fibroblasts. In this Article, we detail the characterization of the dynamic mechanical properties of these materials, as assessed via dynamic oscillatory shear rheology at various protein concentrations and cross-linking ratios. Simply by varying the polypeptide concentration and cross-linker ratios, the storage modulus G' can be easily tuned within the range of 500 Pa to 10 kPa. Strain-stress cycles and resilience measurements were probed via standard tensile testing methods and indicated the excellent resilience (>90%) of these materials, even when the mechanically active domains are intercepted by nonmechanically active biological cassettes. Further evaluation, at high frequencies, of the mechanical properties of these materials were assessed by a custom-designed torsional wave apparatus (TWA) at frequencies close to human phonation, indicating elastic modulus values from 200 to 2500 Pa, which is within the range of experimental data collected on excised porcine and human vocal fold tissues. The results validate the outstanding mechanical properties of the engineered materials, which are highly comparable to the mechanical properties of targeted vocal fold tissues. The ease of production of these biologically active materials, coupled to their outstanding mechanical properties over a range of compositions, suggests their potential in tissue regeneration applications.

Stable expression of hepatitis delta virus antigen in a eukaryotic cell line.

PubMed

Macnaughton, T B; Gowans, E J; Reinboth, B; Jilbert, A R; Burrell, C J

1990-06-01

The gene encoding the hepatitis delta virus structural antigen (HDAg) was linked to a neomycin resistance gene in a retrovirus expression vector, and human HepG2 cells were transfected with the recombinant plasmid. A stable cell line was cloned that expressed HDAg in the nuclei of 100% of cells, in a pattern indicating a close relationship with cell nucleoli. Analysis of partially purified recombinant HDAg by HPLC showed an Mr in the range of 7 x 10(5) to 2 x 10(6), which appeared to contain conformation-dependent epitopes, whereas the density of the antigen was 1.19 g/ml by equilibrium centrifugation in caesium chloride, and in rate zonal centrifugation it sedimented with a value of 50S, close to that of particulate hepatitis B virus surface antigen. Immunoblotting demonstrated a single polypeptide with an Mr of 24K which corresponded to the smaller of the two HDAg-specific polypeptides present in infected sera. The recombinant HDAg polypeptide was shown to be a RNA-binding protein with specificity for both genomic and antigenomic species of hepatitis delta virus RNA.

Changes in the pattern of protein synthesis during zoospore germination in Blastocladiella emersonii.

PubMed Central

Silva, A M; Maia, J C; Juliani, M H

1987-01-01

Using two-dimensional gel electrophoresis, we analyzed the pattern of proteins synthesized during Blastocladiella emersonii zoospore germination in an inorganic solution, in both the presence and absence of actinomycin D. During the transition from zoospore to round cells (the first 25 min), essentially no qualitative differences were noticeable, indicating that the earliest stages of germination are entirely preprogrammed with stored RNA. Later in germination (after 25 min), however, changes in the pattern of protein synthesis were found. Some of these proteins (a total of 6 polypeptides) correspond possibly to a selective translation of stored messages, whereas the majority of the changed proteins (22 polypeptides) corresponds to newly synthesized mRNA. Thus, multiple levels of protein synthesis regulation seem to occur during zoospore germination, involving both transcriptional and translational controls. We also analyzed the pattern of protein synthesis during germination in a nutrient medium; synthesis of specific polypeptides occurred during late germination. During early germination posttranslational control was also observed, several labeled proteins from zoospores being specifically degraded or charge modified. Images PMID:3571161

An efficient route to bispecific antibody production using single-reactor mammalian co-culture

PubMed Central

Shatz, Whitney; Ng, Domingos; Dutina, George; Wong, Athena W.; Sonoda, Junichiro; Scheer, Justin M.

2016-01-01

ABSTRACT Bispecific antibodies have shown promise in the clinic as medicines with novel mechanisms of action. Lack of efficient production of bispecific IgGs, however, has limited their rapid advancement. Here, we describe a single-reactor process using mammalian cell co-culture production to efficiently produce a bispecific IgG with 4 distinct polypeptide chains without the need for parallel processing of each half-antibody or additional framework mutations. This method resembles a conventional process, and the quality and yield of the monoclonal antibodies are equal to those produced using parallel processing methods. We demonstrate the application of the approach to diverse bispecific antibodies, and its suitability for production of a tissue specific molecule targeting fibroblast growth factor receptor 1 and klotho β that is being developed for type 2 diabetes and other obesity-linked disorders. PMID:27680183

Review: history of the amyloid fibril.

PubMed

Sipe, J D; Cohen, A S

2000-06-01

Rudolph Virchow, in 1854, introduced and popularized the term amyloid to denote a macroscopic tissue abnormality that exhibited a positive iodine staining reaction. Subsequent light microscopic studies with polarizing optics demonstrated the inherent birefringence of amyloid deposits, a property that increased intensely after staining with Congo red dye. In 1959, electron microscopic examination of ultrathin sections of amyloidotic tissues revealed the presence of fibrils, indeterminate in length and, invariably, 80 to 100 A in width. Using the criteria of Congophilia and fibrillar morphology, 20 or more biochemically distinct forms of amyloid have been identified throughout the animal kingdom; each is specifically associated with a unique clinical syndrome. Fibrils, also 80 to 100 A in width, have been isolated from tissue homogenates using differential sedimentation or solubility. X-ray diffraction analysis revealed the fibrils to be ordered in the beta pleated sheet conformation, with the direction of the polypeptide backbone perpendicular to the fibril axis (cross beta structure). Because of the similar dimensions and tinctorial properties of the fibrils extracted from amyloid-laden tissues and amyloid fibrils in tissue sections, they have been assumed to be identical. However, the spatial relationship of proteoglycans and amyloid P component (AP), common to all forms of amyloid, to the putative protein only fibrils in tissues, has been unclear. Recently, it has been suggested that, in situ, amyloid fibrils are composed of proteoglycans and AP as well as amyloid proteins and thus resemble connective tissue microfibrils. Chemical and physical definition of the fibrils in tissues will be needed to relate the in vitro properties of amyloid protein fibrils to the pathogenesis of amyloid fibril formation in vivo. Copyright 2000 Academic Press.

Design and synthesis of nonionic copolypeptide hydrogels with reversible thermoresponsive and tunable physical properties.

PubMed

Zhang, Shanshan; Alvarez, Daniel J; Sofroniew, Michael V; Deming, Timothy J

2015-04-13

Polypeptide-based formulations that undergo liquid to hydrogel transitions upon change in temperature have become desirable targets since they can be mixed with cells or injected into tissues as liquids, and subsequently transform into rigid scaffolds or depots. Such materials have been challenging to prepare using synthetic polypeptides, especially when reversible gelation and tunable physical properties are desired. Here, we designed and prepared new nonionic diblock copolypeptide hydrogels (DCH) containing hydrophilic poly(γ-[2-(2-methoxyethoxy)ethyl]-rac-glutamate) and hydrophobic poly(l-leucine) segments, named DCHEO, and also further incorporated copolypeptide domains into DCHEO to yield unprecedented thermoresponsive DCH, named DCHT. Although previous attempts to prepare nonionic hydrogels composed solely of synthetic polypeptides have been unsuccessful, our designs yielded materials with highly reversible thermal transitions and tunable properties. Nonionic, thermoresponsive DCHT were found to support the viability of suspended mesenchymal stem cells in vitro and were able to dissolve and provide prolonged release of both hydrophilic and hydrophobic molecules. The versatility of these materials was further demonstrated by the independent molecular tuning of DCHT liquid viscosity at room temperature and DCHT hydrogel stiffness at elevated temperature, as well as the DCHT liquid to hydrogel transition temperature itself.

Preclinical Vaccine Study of Plasmodium vivax Circumsporozoite Protein Derived-Synthetic Polypeptides Formulated in Montanide ISA 720 and Montanide ISA 51 Adjuvants

PubMed Central

Arévalo-Herrera, Myriam; Vera, Omaira; Castellanos, Angélica; Céspedes, Nora; Soto, Liliana; Corradin, Giampietro; Herrera, Sócrates

2011-01-01

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate previously assessed in animals and humans. Here, combinations of three synthetic polypeptides corresponding to amino (N), central repeat (R), and carboxyl (C) regions of the CS protein formulated in Montanide ISA 720 or Montanide ISA 51 adjuvants were assessed for immunogenicity in rodents and primates. BALB/c mice and Aotus monkeys were divided into test and control groups and were immunized three times with doses of 50 and 100 μg of vaccine or placebo. Antigen-specific antimalarial antibodies were determined by enzyme-linked immunosorbent assay, immunofluorescent antibody test, and IFN-γ responses by enzyme-linked immunosorbent spot (ELIspot). Both vaccine formulations were highly immunogenic in both species. Mice developed better antibody responses against C and R polypeptides, whereas the N polypeptide was more immunogenic in monkeys. Anti-peptide antibodies remained detectable for several months and recognized native proteins on sporozoites. Differences between Montanide ISA 720 and Montanide ISA 51 formulations were not significant. PMID:21292874

Polypeptide N-acetylgalactosaminyltransferase-6 expression in gastric cancer

PubMed Central

Guo, Yan; Shi, Jingjing; Zhang, Jun; Li, Haixin; Liu, Ben; Guo, Hua

2017-01-01

Gastric cancer (GC) is one of the leading causes of cancer-related deaths, with limited improvement in its clinical outcome worldwide. Aberrant mucin-type O-glycosylation is a critical event widespread in the development of GC. Polypeptide N-acetylgalactosaminyltransferases (GALNTs) regulate the initial step and determine the sites of mucin-type O-glycoprotein bio-synthesis. GALNT6 has considerable potential as a biomarker in various cancers. The roles of GALNT6 in GC were analyzed, and the results showed that GALNT6 expression markedly increased in GC tissues compared with those in adjacent gastric tissues. High intratumoral GALNT6 density was associated with the clinicopathological parameters of TNM stage and distant metastasis. GALNT6 was identified as an independent prognosticator for the poor prognosis of GC patients. Moreover, the high expression level of GALNT6 was significantly associated with the low expression levels of E-cadherin and β-catenin and the high expression levels of MMP9. These findings indicated that GALNT6 could provide new insights into the characterization of GC as well as contribute to the development of an efficient prognostic indicator and novel therapeutic modalities for GC. PMID:28744137

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

PubMed Central

Juhász, Tamás; Szentléleky, Eszter; Szűcs Somogyi, Csilla; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

2015-01-01

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load. PMID:26230691

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures.

PubMed

Juhász, Tamás; Szentléleky, Eszter; Somogyi, Csilla Szűcs; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

2015-07-29

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.

Analysis of a Viral Agent Isolated from Multiple Sclerosis Brain Tissue: Characterization as a Parainfluenzavirus Type 1

PubMed Central

Lewandowski, L. J.; Lief, F. S.; Verini, M. A.; Pienkowski, M. M.; ter Meulen, V.; Koprowski, H.

1974-01-01

A virus originally isolated from cell cultures obtained by lysolecithin-induced fusion of human multiple sclerosis brain cells with CV-1 cells has been analyzed for its antigenic, RNA, and polypeptide compositions, and for selective biological properties. Our findings establish that this isolate, designated 6/94 virus, contains a 50S RNA genome and is, as yet, indistinguishable from Sendai virus in its antigenic and total polypeptide compositions. Despite these similarities, the 6/94 and Sendai viruses differ in certain phenotypic properties. 6/94 virus is markedly less cytocidal for chick fibroblasts, especially at 37 C and, after β-propiolactone inactivation, it possesses a greater capacity for cell fusion and a lower toxicity than does comparably treated Sendai virus. In addition, 6/94 virus shows greater hemolytic activity. Images PMID:4363249

Cellobiohydrolase I enzymes

DOEpatents

Adney, William S; Himmel, Michael E; Decker, Stephen R; Knoshaug, Eric P; Nimlos, Mark R; Crowley, Michael F; Jeoh, Tina

2014-01-28

Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.

Specific Binding of Protoporphyrin IX to a Membrane-Bound 63 Kilodalton Polypeptide in Cucumber Cotyledons Treated with Diphenyl Ether-Type Herbicides.

PubMed

Sato, R; Oshio, H; Koike, H; Inoue, Y; Yoshida, S; Takahashi, N

1991-06-01

Porphyrin accumulation in excised cucumber cotyledons (Cucumis sativus L.) treated with a N-phenylimide S-23142 (N-[4-chloro-2-fluoro-5-propargyloxyphenyl]-3,4,5,6- tetrahydrophthalimide) and a diphenylether acifluorfen-ethyl (ethyl-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitro benzoic acid) was studied. Most of the accumulated porphyrins were found in the membrane fractions of 6,000g and 30,000g pellets, forming a complex with a membrane polypeptide. The complex was solubilized with 1% n-dodecyl beta-d-maltoside and its molecular mass was estimated to be 63,000 and 66,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation high performance liquid chromatography (HPLC), respectively. The polypeptide also existed in untreated cotyledons but had little protoporphyrin IX. The complex was also formed in vitro by mixing the 30,000g pellets from untreated cotyledons and authentic protoporphyrin IX. However, protoporphyrin IX formed the complex specifically with the 63,000 dalton polypeptide and not with the other proteins both in vivo and in vitro. At least four fluorescent porphyrins, including protoporphyrin IX, were found in the acetone extract of the cotyledons by HPLC using a reversed phase column. Protoporphyrin IX was one of the two porphyrins that formed the complex. These results suggest that S-23142 and acifluorfenethyl enhance the accumulation of protoporphyrin IX, which forms the complex with the membrane protein.

Immunological Evidence for the Existence of a Carrier Protein for Sucrose Transport in Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Root Storage Tissue.

PubMed Central

Getz, H. P.; Grosclaude, J.; Kurkdjian, A.; Lelievre, F.; Maretzki, A.; Guern, J.

1993-01-01

Monoclonal antibodies were raised in mice against a highly purified tonoplast fraction from isolated red beet (Beta vulgaris L. ssp. conditiva) root vacuoles. Positive hybridoma clones and sub-clones were identified by prescreening using an enzyme-linked immunosorbent assay (ELISA) and by postscreening using a functional assay. This functional assay consisted of testing the impact of hybridoma supernatants and antibody-containing ascites fluids on basal and ATP-stimulated sugar uptake in vacuoles, isolated from protoplasts, as well as in tonoplast vesicles, prepared from tissue homogenates of red beet roots. Antibodies from four clones were particularly positive in ELISAs and they inhibited sucrose uptake significantly. These antibodies were specific inhibitors of sucrose transport, but they exhibited relatively low membrane and species specificity since uptake into red beet root protoplasts and sugarcane tonoplast vesicles was inhibited as well. Fast protein liquid chromatography assisted size exclusion chromatography on Superose 6 columns yielded two major peaks in the 55 to 65-kD regions and in the 110- to 130-kD regions of solubilized proteins from red beet root tonoplasts, which reacted positively in immunoglobulin-M(IgM)-specific ELISAs with anti-sugarcane tonoplast monoclonal IgM antibodies. Only reconstituted proteoliposomes containing polypeptides from the 55- to 65-kD band took up [14C]-sucrose with linear rates for 2 min, suggesting that this fraction contains the tonoplast sucrose carrier. PMID:12231863

Differential expression of diacylglycerol acyltransferase (DGAT) genes in olive tissues.

PubMed

Giannoulia, K; Haralampidis, K; Poghosyan, Z; Murphy, D J; Hatzopoulos, P

2000-12-01

Fatty acids are accumulated in triacylglycerols (TAGs), in specialized organelles of seeds named oil bodies. The major site of TAG accumulation is detected in developing seed and mesocarp of certain species. We have isolated two cDNAs encoding DGAT enzymes from olives. The deduced polypeptides differ by 26 amino acids in size. However, they have high homology and almost identical hydropathy profiles. The DGAT gene is expressed in all tissues that synthesize TAGs. However, higher levels of DGAT transcripts have been detected in seed tissues of developing olive drupe. DGAT expression and mRNA accumulation in drupe tissues is developmentally regulated. Each DGAT transcript shows a distinct profile of accumulation. The existence of two different DGAT transcripts might reflect two different enzymes with discrete function and/or localization.

Photoaffinity labeling of protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides.

PubMed

Camadro, J M; Matringe, M; Thome, F; Brouillet, N; Mornet, R; Labbe, P

1995-05-01

Diphenylether-type herbicides are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane-bound enzyme involved in the heme and chlorophyll biosynthesis pathways. Tritiated acifluorfen and a diazoketone derivative of tritiated acifluorfen were specifically bound to a single class of high-affinity binding sites on yeast mitochondrial membranes with apparent dissociation constants of 7 nM and 12.5 nM, respectively. The maximum density of specific binding sites, determined by Scatchard analysis, was 3 pmol.mg-1 protein. Protoporphyrinogen oxidase specific activity was estimated to be 2500 nmol protoporphyrinogen oxidized h-1.mol-1 enzyme. The diazoketone derivative of tritiated acifluorfen was used to specifically photolabel yeast protoporphyrinogen oxidase. The specifically labeled polypeptide in wild-type mitochondrial membranes had an apparent molecular mass of 55 kDa, identical to the molecular mass of the purified enzyme. This photolabeled polypeptide was not detected in a protoporphyrinogen-oxidase-deficient yeast strain, but the membranes contained an equivalent amount of inactive immunoreactive protoporphyrinogen oxidase protein.

Catalytic and reactive polypeptides and methods for their preparation and use

DOEpatents

Schultz, Peter

1994-01-01

Catalytic and reactive polypeptides include a binding site specific for a reactant or reactive intermediate involved in a chemical reaction of interest. The polypeptides further include at least one active functionality proximate the binding site, where the active functionality is capable of catalyzing or chemically participating in the chemical reaction in such a way that the reaction rate is enhanced. Methods for preparing the catalytic peptides include chemical synthesis, site-directed mutagenesis of antibody and enzyme genes, covalent attachment of the functionalities through particular amino acid side chains, and the like. This invention was made with Government support under Grant Contract No. AI-24695, awarded by the Department of health and Human Services, and under Grant Contract No. N 00014-87-K-0256, awarded by the Office of Naval Research. The Government has certain rights in this invention.

Rapid measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides.

PubMed

Barnwal, Ravi Pratap; Rout, Ashok K; Chary, Kandala V R; Atreya, Hanudatta S

2007-12-01

We present two NMR experiments, (3,2)D HNHA and (3,2)D HNHB, for rapid and accurate measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides based on the principle of G-matrix Fourier transform NMR spectroscopy and quantitative J-correlation. These experiments, which facilitate fast acquisition of three-dimensional data with high spectral/digital resolution and chemical shift dispersion, will provide renewed opportunities to utilize them for sequence specific resonance assignments, estimation/characterization of secondary structure with/without prior knowledge of resonance assignments, stereospecific assignment of prochiral groups and 3D structure determination, refinement and validation. Taken together, these experiments have a wide range of applications from structural genomics projects to studying structure and folding in polypeptides.

Role of Monomer Sequence, Hydrogen Bonding and Mesoscale Architecture in Marine Antifouling Coatings

NASA Astrophysics Data System (ADS)

Segalman, Rachel

Polypeptoids are non-natural, sequence specific polymers that offer the opportunity to probe the effect of monomer sequence, chirality, and chain shape on self-assembly and surface properties. Additionally, polypeptoid synthesis is more scaleable than traditional polypeptides suggesting their utility in large area applications. We have designed efficient marine anti-fouling coatings by using triblock copolymer scaffolds to which polypeptoids are tethered in order to tune both the modulus and surface energies with great precision. Surprisingly, when short sequences are tethered to a polymer backbone, polypeptoids consistently outperform analogous polypeptides in antifouling properties. We hypothesize that the hydrogen bonding inherent to the polypeptide backbone drives the observed differences in performance. We also find that the polymer scaffold housing the polypeptoids also plays a crucial role in directing surface presentation and therefore the overall coating properties.

Brassicaceae Express Multiple Isoforms of Biotin Carboxyl Carrier Protein in a Tissue-Specific Manner1

PubMed Central

Thelen, Jay J.; Mekhedov, Sergei; Ohlrogge, John B.

2001-01-01

Plastidial acetyl-coenzyme A carboxylase from most plants is a multi-enzyme complex comprised of four different subunits. One of these subunits, the biotin carboxyl carrier protein (BCCP), was previously proposed to be encoded by a single gene in Arabidopsis. We report and characterize here a second Arabidopsis BCCP (AtBCCP2) cDNA with 42% amino acid identity to AtBCCP1 and 75% identity to a class of oilseed rape (Brassica napus) BCCPs. Both Arabidopsis BCCP isoforms were expressed in Escherichia coli and found to be biotinylated and supported carboxylation activity when reconstituted with purified, recombinant Arabidopsis biotin carboxylase. In vitro translated AtBCCP2 was competent for import into pea (Pisum sativum) chloroplasts and processed to a 25-kD polypeptide. Extracts of Arabidopsis seeds contained biotinylated polypeptides of 35 and 25 kD, in agreement with the masses of recombinant AtBCCP1 and 2, respectively. AtBCCP1 protein was present in developing tissues from roots, leaves, flowers, siliques, and seeds, whereas AtBCCP2 protein was primarily expressed in 7 to 10 d-after-flowering seeds at levels approximately 2-fold less abundant than AtBCCP1. AtBCCP1 transcript reflected these protein expression profiles present in all developing organs and highest in 14-d leaves and siliques, whereas AtBCCP2 transcript was present in flowers and siliques. In protein blots, four different BCCP isoforms were detected in developing seeds from oilseed rape. Of these, a 35-kD BCCP was detected in immature leaves and developing seeds, whereas developing seeds also contained 22-, 25-, and 37-kD isoforms highly expressed 21 d after flowering. These data indicate that oilseed plants in the family Brassicaceae contain at least one to three seed-up-regulated BCCP isoforms, depending upon genome complexity. PMID:11299381

Machine learning classifier for identification of damaging missense mutations exclusive to human mitochondrial DNA-encoded polypeptides.

PubMed

Martín-Navarro, Antonio; Gaudioso-Simón, Andrés; Álvarez-Jarreta, Jorge; Montoya, Julio; Mayordomo, Elvira; Ruiz-Pesini, Eduardo

2017-03-07

Several methods have been developed to predict the pathogenicity of missense mutations but none has been specifically designed for classification of variants in mtDNA-encoded polypeptides. Moreover, there is not available curated dataset of neutral and damaging mtDNA missense variants to test the accuracy of predictors. Because mtDNA sequencing of patients suffering mitochondrial diseases is revealing many missense mutations, it is needed to prioritize candidate substitutions for further confirmation. Predictors can be useful as screening tools but their performance must be improved. We have developed a SVM classifier (Mitoclass.1) specific for mtDNA missense variants. Training and validation of the model was executed with 2,835 mtDNA damaging and neutral amino acid substitutions, previously curated by a set of rigorous pathogenicity criteria with high specificity. Each instance is described by a set of three attributes based on evolutionary conservation in Eukaryota of wildtype and mutant amino acids as well as coevolution and a novel evolutionary analysis of specific substitutions belonging to the same domain of mitochondrial polypeptides. Our classifier has performed better than other web-available tested predictors. We checked performance of three broadly used predictors with the total mutations of our curated dataset. PolyPhen-2 showed the best results for a screening proposal with a good sensitivity. Nevertheless, the number of false positive predictions was too high. Our method has an improved sensitivity and better specificity in relation to PolyPhen-2. We also publish predictions for the complete set of 24,201 possible missense variants in the 13 human mtDNA-encoded polypeptides. Mitoclass.1 allows a better selection of candidate damaging missense variants from mtDNA. A careful search of discriminatory attributes and a training step based on a curated dataset of amino acid substitutions belonging exclusively to human mtDNA genes allows an improved performance. Mitoclass.1 accuracy could be improved in the future when more mtDNA missense substitutions will be available for updating the attributes and retraining the model.

Crystallization of calcium oxalates is controlled by molecular hydrophilicity and specific polyanion-crystal interactions.

PubMed

Grohe, Bernd; Taller, Adam; Vincent, Peter L; Tieu, Long D; Rogers, Kem A; Heiss, Alexander; Sørensen, Esben S; Mittler, Silvia; Goldberg, Harvey A; Hunter, Graeme K

2009-10-06

To gain more insight into protein structure-function relationships that govern ectopic biomineralization processes in kidney stone formation, we have studied the ability of urinary proteins (Tamm-Horsfall protein, osteopontin (OPN), prothrombin fragment 1 (PTF1), bikunin, lysozyme, albumin, fetuin-A), and model compounds (a bikunin fragment, recombinant-, milk-, bone osteopontin, poly-L-aspartic acid (poly asp), poly-L-glutamic acid (poly glu)) in modulating precipitation reactions of kidney stone-related calcium oxalate mono- and dihydrates (COM, COD). Combining scanning confocal microscopy and fluorescence imaging, we determined the crystal faces of COM with which these polypeptides interact; using scanning electron microscopy, we characterized their effects on crystal habits and precipitated volumes. Our findings demonstrate that polypeptide adsorption to COM crystals is dictated first by the polypeptide's affinity for the crystal followed by its preference for a crystal face: basic and relatively hydrophobic macromolecules show no adsorption, while acidic and more hydrophilic polypeptides adsorb either nonspecifically to all faces of COM or preferentially to {100}/{121} edges and {100} faces. However, investigating calcium oxalates grown in the presence of these polypeptides showed that some acidic proteins that adsorb to crystals do not affect crystallization, even if present in excess of physiological concentrations. These proteins (albumin, bikunin, PTF1, recombinant OPN) have estimated total hydrophilicities from 200 to 850 kJ/mol and net negative charges from -9 to -35, perhaps representing a "window" in which proteins adsorb and coat urinary crystals (support of excretion) without affecting crystallization. Strongest effects on crystallization were observed for polypeptides that are either highly hydrophilic (>950 kJ/mol) and highly carboxylated (poly asp, poly glu), or else highly hydrophilic and highly phosphorylated (native OPN isoforms), suggesting that highly hydrophilic proteins strongly affect precipitation processes in the urinary tract. Therefore, the level of hydrophilicity and net charge is a critical factor in the ability of polypeptides to affect crystallization and to regulate biomineralization processes.

Evidence against vasoactive intestinal polypeptide (VIP) as a dilator and in favour of substance P as a constrictor in airway neurogenic responses.

PubMed

Karlsson, J A; Persson, C G

1983-07-01

Propranolol-resistant neurogenic relaxation persisted in (carbachol-contracted) guinea-pig tracheae already relaxed by supramaximal concentrations of vasoactive intestinal polypeptide (VIP). Also, VIP relaxed preparations that were under neurogenic inhibition. In hilus bronchi, about 60% of a neurogenic contraction was atropine-resistant. (Arg5, D-Trp7.9) SP 5-11 specifically antagonized this contraction and those produced by exogenous substance P. Substance P, but not VIP, seems to be involved in nerve-mediated effects on guinea-pig airway tone.

The cytoplasmic domain of the Drosophila cell adhesion molecule neuroglian is not essential for its homophilic adhesive properties in S2 cells.

PubMed

Hortsch, M; Wang, Y M; Marikar, Y; Bieber, A J

1995-08-11

Drosophila neuroglian is a transmembrane glycoprotein that has strong structural and sequence homology to the vertebrate L1 gene family of cell adhesion molecules (Bieber, A.J., Snow, P.M., Hortsch, M., Patel, N.H., Jacobs, J.R., Traquina, Z.R., Schilling, J., and Goodman, C.S. (1989) Cell 59, 447-460. Two different neuroglian protein forms that are generated by a differential splicing process are expressed in a tissue-specific fashion by embryonic and larval cells (Hortsch, M., Bieber, A.J., Patel, N.H., and Goodman, C.S. (1990) Neuron 4, 697-709). The two neuroglial polypeptides differ only in their cytoplasmic domains. Both of these neuroglian species, when transfected into the expressed in Drosophila S2 cells, induce the calcium-independent, homophilic aggregation of transformed cells. A third artificial neuroglian protein form was constructed by substituting the neuroglian transmembrane segment and cytoplasmic domains with the glycosyl phosphatidylinositol attachment signal of the Drosophila fasciclin I protein. This cDNA construct generates a glycosyl phosphatidylinositol-anchored form of neuroglian, which retains the ability to induce homophilic cell aggregation when expressed in S2 cells, and was able to interact with both of the two naturally occurring neuroglian polypeptides. These results demonstrate that neuroglian mediates a calcium-independent, homophilic cell adhesion activity and that neither cytoplasmic neuroglian domains nor a direct interaction with cytoskeletal elements is essential for this property.

Molecular Properties of neurotoxin receptors sites associated with sodium channels from mammalian brain.

PubMed

Catterall, W A; Hartshorne, R P; Beneski, D A

1982-01-01

Neurotoxins that act at specific receptor sites on voltage-sensitive sodium channels have been used as molecular probes to identify and purify protein components of sodium channels from mammalian brain. Photoreactive derivatives of scorpion toxin have been prepared and used to covalently label sodium channels in intact synaptosomes. Two polypeptides, alpha with Mr approximately 270,000 and beta with Mr approximately 38,000, are specifically labeled indicating that they are components of the scorpion toxin receptor site on the sodium channel. The sodium channel can be solubilized with retention of specific binding of [3H] saxitoxin using nonionic detergents such as Triton X-100. The solubilized saxitoxin receptor has molecular weight of 316,000 +/- 63,000 and binds 0.9 g of Triton X-100 and phospholipid per g of protein. The solubilized receptor can be purified 750-fold by ion exchange chromatography, wheat germ lectin/Sepharose chromatography and sucrose gradient sedimentation to a final specific activity of 1488 pmol/mg. Analysis of the polypeptide chain composition of the most highly purified fractions indicates that alpha and beta comprise 65% of the protein of these fractions and are only the polypeptides whose presence correlates with saxitoxin binding activity. These studies lead to a working hypothesis of sodium channel structure in which the intact channel is comprised of a complex with Mr of approximately 316,000 containing one mole of alpha (Mr approximately 270,000) and one to three moles of beta (Mr approximately 38,000).

Isolation and characterization of spinach photosystem II membrane-associated catalase and polyphenol oxidase.

PubMed

Sheptovitsky, Y G; Brudvig, G W

1996-12-17

Photosystem II (PSII) membranes exhibit catalase and polyphenol oxidase (PPO) activities. Mild heat treatment of PSII membranes for 90 min at 30 degrees C releases most of these enzyme activities into the supernatant, accompanied by a 7-fold activation of PPO. In contrast, mild heat treatment of thylakoid membranes does not release significant amounts of either activity, indicating that both enzymes are bound to the luminal surface of the thylakoid membrane. The heat-released PSII membrane-associated catalase and PPO have been purified and characterized. Catalase activity was correlated with a 63 kDa polypeptide which was purified by batch adsorption to anion-exchange beads followed by gel filtration. The PSII membrane-associated catalase is unstable in solution, probably due to irreversible aggregation. The enzyme was characterized in terms of molecular and subunit size, amino-acid composition, UV-visible absorption, heme content, pH optimum, inhibitor sensitivity, and K(m) value for H2O2. Its properties indicate that the PSII membrane-associated catalase is a luminal thylakoid membrane-bound heme enzyme that has not been identified previously. The residual catalase activity of PSII membranes after mild heat treatment is irreversibly inhibited with 3-amino-1,2,4-triazole, a specific inhibitor of heme catalases, without inhibition of O2-evolution activity. This result indicates that little, if any, of the catalase activity from PSII membranes in the dark is catalyzed by the O2-evolving center of PSII. PPO activity was correlated with a 48 kDa polypeptide. However, the 48 kDa polypeptide and another heat-released polypeptide of 72 kDa have the same N-terminal sequence, which is also identical to that of a known 64 kDa protein [Hind, G., Marshak, D. R., & Coughlan, S. J. (1995) Biochemistry 34, 8157-8164]. During heat treatment of PSII membranes and further manipulations it was found that the 72 kDa polypeptide was largely converted into the 48 kDa polypeptide. Thus, the 72 kDa polypeptide appears to be a latent precursor of the active 48 kDa PPO. The PSII membrane-associated PPO was purified by anion-exchange chromatography and was characterized in terms of substrate specificity, pH optimum, inhibitor sensitivity and native molecular weight. The heat-released PPO appears to be identical to the enzyme previously isolated from spinach thylakoid membranes [Golbeck, J. H., & Cammarata, K. V. (1981) Plant Physiol. 67, 977-984].

Statistical thermodynamics of protein folding: Comparison of a mean-field theory with Monte Carlo simulations

NASA Astrophysics Data System (ADS)

Hao, Ming-Hong; Scheraga, Harold A.

1995-01-01

A comparative study of protein folding with an analytical theory and computer simulations, respectively, is reported. The theory is based on an improved mean-field formalism which, in addition to the usual mean-field approximations, takes into account the distributions of energies in the subsets of conformational states. Sequence-specific properties of proteins are parametrized in the theory by two sets of variables, one for the energetics of mean-field interactions and one for the distribution of energies. Simulations are carried out on model polypeptides with different sequences, with different chain lengths, and with different interaction potentials, ranging from strong biases towards certain local chain states (bond angles and torsional angles) to complete absence of local conformational preferences. Theoretical analysis of the simulation results for the model polypeptides reveals three different types of behavior in the folding transition from the statistical coiled state to the compact globular state; these include a cooperative two-state transition, a continuous folding, and a glasslike transition. It is found that, with the fitted theoretical parameters which are specific for each polypeptide under a different potential, the mean-field theory can describe the thermodynamic properties and folding behavior of the different polypeptides accurately. By comparing the theoretical descriptions with simulation results, we verify the basic assumptions of the theory and, thereby, obtain new insights about the folding transitions of proteins. It is found that the cooperativity of the first-order folding transition of the model polypeptides is determined mainly by long-range interactions, in particular the dipolar orientation; the local interactions (e.g., bond-angle and torsion-angle potentials) have only marginal effect on the cooperative characteristic of the folding, but have a large impact on the difference in energy between the folded lowest-energy structure and the unfolded conformations of a protein.

Passive immunization against highly pathogenic Avian Influenza Virus (AIV) strain H7N3 with antiserum generated from viral polypeptides protect poultry birds from lethal viral infection

PubMed Central

Shahzad, Mirza Imran; Naeem, Khalid; Mukhtar, Muhammad; Khanum, Azra

2008-01-01

Our studies were aimed at developing a vaccination strategy that could provide protection against highly pathogenic avian influenza virus (AIV), H7N3 or its variants outbreaks. A purified viral stock of highly pathogenic H7N3 isolate was lysed to isolate viral proteins by electrophresing on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by their elution from gel through trituration in phosphate buffered saline (PBS). Overall, five isolated viral polypeptides/proteins upon characterization were used to prepare hyperimmune monovalent serum against respective polypeptides independently and a mixture of all five in poultry birds, and specificity confirmation of each antiserum through dot blot and Western blotting. Antiserum generated from various group birds was pooled and evaluated in 2-week old broiler chicken, for its protection against viral challenge. To evaluate in-vivo protection of each antiserum against viral challenges, six groups of 2-week old broiler chicken were injected with antiserum and a seventh control group received normal saline. Each group was exposed to purified highly pathogenic AIV H7N3 strain at a dose 105 embryo lethal dose (ELD50). We observed that nucleoprotein (NP) antiserum significantly protected birds from viral infection induced morbidity, mortality and lowered viral shedding compared with antiserum from individual viral proteins or mixed polypeptides/proteins inclusive of NP component. The capability of individual viral polypeptide specific antisera to protect against viral challenges in decreasing order was nucleoprotein (NP) > hemagglutinin (HA) > neuraminidase (NA) > viral proteins mix > viral polymerase (PM) > non-structural proteins (NS). Our data provide proof of concept for potential utilization of passive immunization in protecting poultry industry during infection outbreaks. Furthermore conserved nature of avian NP makes it an ideal candidate to produce antiserum protective against viral infection. PMID:19040734

Polypeptide Synthesis in Simian Virus 5-Infected Cells

PubMed Central

Peluso, Richard W.; Lamb, Robert A.; Choppin, Purnell W.

1977-01-01

Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F0) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F1 and F2. No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus replication is discussed. Images PMID:196101

A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis.

PubMed

Song, Jun; Braun, Gordon; Bevis, Eric; Doncaster, Kristen

2006-08-01

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.

Effect of supramolecular organization of a cartilaginous tissue on thermal stability of collagen II

NASA Astrophysics Data System (ADS)

Ignat'eva, N. Yu.; Averkiev, S. V.; Lunin, V. V.; Grokhovskaya, T. E.; Obrezkova, M. V.

2006-08-01

The thermal stability of collagen II in various cartilaginous tissues was studied. It was found that heating a tissue of nucleus pulposus results in collagen II melting within a temperature range of 60-70°C; an intact tissue of hyaline cartilage (of nasal septum and cartilage endplates) is a thermally stable system, where collagen II is not denatured completely up to 100°C. It was found that partial destruction of glycosaminoglycans in hyaline cartilage leads to an increase in the degree of denaturation of collagen II upon heating, although a significant fraction remains unchanged. It was shown that electrostatic interactions of proteoglycans and collagen only slightly affect the thermal stability of collagen II in the tissues. Evidently, proteoglycan aggregates play a key role: they create topological hindrances for moving polypeptide chains, thereby reducing the configurational entropy of collagen macromolecules in the state of a random coil.

Rate limit of protein elastic response is tether dependent

PubMed Central

Berkovich, Ronen; Hermans, Rodolfo I.; Popa, Ionel; Stirnemann, Guillaume; Garcia-Manyes, Sergi; Berne, Bruce J.; Fernandez, Julio M.

2012-01-01

The elastic restoring force of tissues must be able to operate over the very wide range of loading rates experienced by living organisms. It is surprising that even the fastest events involving animal muscle tissues do not surpass a few hundred hertz. We propose that this limit is set in part by the elastic dynamics of tethered proteins extending and relaxing under a changing load. Here we study the elastic dynamics of tethered proteins using a fast force spectrometer with sub-millisecond time resolution, combined with Brownian and Molecular Dynamics simulations. We show that the act of tethering a polypeptide to an object, an inseparable part of protein elasticity in vivo and in experimental setups, greatly reduces the attempt frequency with which the protein samples its free energy. Indeed, our data shows that a tethered polypeptide can traverse its free-energy landscape with a surprisingly low effective diffusion coefficient Deff ∼ 1,200 nm2/s. By contrast, our Molecular Dynamics simulations show that diffusion of an isolated protein under force occurs at Deff ∼ 108 nm2/s. This discrepancy is attributed to the drag force caused by the tethering object. From the physiological time scales of tissue elasticity, we calculate that tethered elastic proteins equilibrate in vivo with Deff ∼ 104–106 nm2/s which is two to four orders magnitude smaller than the values measured for untethered proteins in bulk. PMID:22895787

Quantitative assessments of the distinct contributions of polypeptide backbone amides versus sidechain groups to chain expansion via chemical denaturation

PubMed Central

Holehouse, Alex S.; Garai, Kanchan; Lyle, Nicholas; Vitalis, Andreas; Pappu, Rohit V.

2015-01-01

In aqueous solutions with high concentrations of chemical denaturants such as urea and guanidinium chloride (GdmCl) proteins expand to populate heterogeneous conformational ensembles. These denaturing environments are thought to be good solvents for generic protein sequences because properties of conformational distributions align with those of canonical random coils. Previous studies showed that water is a poor solvent for polypeptide backbones and therefore backbones form collapsed globular structures in aqueous solvents. Here, we ask if polypeptide backbones can intrinsically undergo the requisite chain expansion in aqueous solutions with high concentrations of urea and GdmCl. We answer this question using a combination of molecular dynamics simulations and fluorescence correlation spectroscopy. We find that the degree of backbone expansion is minimal in aqueous solutions with high concentrations denaturants. Instead, polypeptide backbones sample conformations that are denaturant-specific mixtures of coils and globules, with a persistent preference for globules. Therefore, typical denaturing environments cannot be classified as good solvents for polypeptide backbones. How then do generic protein sequences expand in denaturing environments? To answer this question, we investigated the effects of sidechains using simulations of two archetypal sequences with amino acid compositions that are mixtures of charged, hydrophobic, and polar groups. We find that sidechains lower the effective concentration of backbone amides in water leading to an intrinsic expansion of polypeptide backbones in the absence of denaturants. Additional dilution of the effective concentration of backbone amides is achieved through preferential interactions with denaturants. These effects lead to conformational statistics in denaturing environments that are congruent with those of canonical random coils. Our results highlight the role of sidechain-mediated interactions as determinants of the conformational properties of unfolded states in water and in influencing chain expansion upon denaturation. PMID:25664638

Targeting prostate cancer cells with hybrid elastin-like polypeptide/liposome nanoparticles

PubMed Central

Zhang, Wei; Song, Yunmei; Eldi, Preethi; Guo, Xiuli; Hayball, John D; Garg, Sanjay; Albrecht, Hugo

2018-01-01

Prostate cancer cells frequently overexpress the gastrin-releasing peptide receptor, and various strategies have been applied in preclinical settings to target this receptor for the specific delivery of anticancer compounds. Recently, elastin-like polypeptide (ELP)-based self-assembling micelles with tethered GRP on the surface have been suggested to actively target prostate cancer cells. Poorly soluble chemotherapeutics such as docetaxel (DTX) can be loaded into the hydrophobic cores of ELP micelles, but only limited drug retention times have been achieved. Herein, we report the generation of hybrid ELP/liposome nanoparticles which self-assembled rapidly in response to temperature change, encapsulated DTX at high concentrations with slow release, displayed the GRP ligand on the surface, and specifically bound to GRP receptor expressing PC-3 cells as demonstrated by flow cytometry. This novel type of drug nanocarrier was successfully used to reduce cell viability of prostate cancer cells in vitro through the specific delivery of DTX. PMID:29391790

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

PubMed Central

Milhausen, M; Gill, P R; Parker, G; Agabian, N

1982-01-01

Immunoprecipitation of Caulobacter crescentus polyribosomes with antiflagellin antibody provided RNA for the synthesis of cDNA probes that were used to identify three specific EcoRI restriction fragments (6.8, 10, and 22 kilobases) in genomic digests of Caulobacter DNA. The RNA was present only in polyribosomes isolated from a time interval in the Caulobacter cell cycle that was coincident with flagellin polypeptide synthesis. The structural gene for Mr 27,500 flagellin polypeptide was assigned to a region of the 10-kilobase EcoRI restriction fragment by DNA sequence analysis. Analysis of mutants defective in motility further established a correlation between the Mr 27,500 flagellin gene and the flaE gene locus [Johnson, R. C. & Ely, B. (1979) J. Bacteriol. 137, 627-634]. The other EcoRI fragments that hybridize with the immunoprecipitated polyribosome-derived cDNA probe are also temporally regulated and have features that suggest they encode other polypeptides associated with the flagellum. Modifications were required to adapt the procedure of immunoprecipitation of polyribosomes for use with Caulobacter and should be applicable to the production of specific structural gene probes from other prokaryotic systems. Images PMID:6294658

Structural properties of matrix metalloproteinases.

PubMed

Bode, W; Fernandez-Catalan, C; Tschesche, H; Grams, F; Nagase, H; Maskos, K

1999-04-01

Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation. Their proteolytic activity must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumour growth and metastasis. Knowledge of the tertiary structures of the proteins involved is crucial for understanding their functional properties and interference with associated dysfunctions. Within the last few years, several three-dimensional MMP and MMP-TIMP structures became available, showing the domain organization, polypeptide fold and main specificity determinants. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure-based design and improvement of high-affinity ligands, which might be elaborated into drugs. A multitude of reviews surveying work done on all aspects of MMPs have appeared in recent years, but none of them has focused on the three-dimensional structures. This review was written to close the gap.

Induction, immunochemical identity and immunofluorescence localization of an 80 000-molecular-weight peroxisome-proliferation-associated polypeptide (polypeptide PPA-80) and peroxisomal enoyl-CoA hydratase of mouse liver and renal cortex.

PubMed

Lalwani, N D; Reddy, M K; Mangkornkanok-Mark, M; Reddy, J K

1981-07-15

The hypolipidaemic drugs methyl clofenapate, BR-931, Wy-14643 and procetofen induced a marked proliferation of peroxisomes in the parenchymal cells of liver and the proximal-convoluted-tubular epithelium of mouse kidney. The proliferation of peroxisomes was associated with 6-12-fold increase in the peroxisomal palmitoyl-CoA oxidizing capacity of the mouse liver. Enhanced activity of the peroxisomal palmitoyl-CoA oxidation system was also found in the renal-cortical homogenates of hypolipidaemic-drug-treated mice. The activity of enoyl-CoA hydratase in the mouse liver increased 30-50-fold and in the kidney cortex 3-5-fold with hypolipidaemic-drug-induced peroxisome proliferation in these tissues, and over 95% of this induced activity was found to be heat-labile peroxisomal enzyme in both organs. Sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis of large-particle and microsomal fractions obtained from the liver and kidney cortex of mice treated with hypolipidaemic peroxisome proliferators demonstrated a substantial increase in the quantity of an 80000-mol.wt. peroxisome-proliferation-associated polypeptide (polypeptide PPA-80). The heat-labile peroxisomal enoyl-CoA hydratase was purified from the livers of mice treated with the hypolipidaemic drug methyl clofenapate; the antibodies raised against this electrophoretically homogeneous protein yielded a single immunoprecipitin band with purified mouse liver enoyl-CoA hydratase and with liver and kidney cortical extracts of normal and hypolipidaemic-drug-treated mice. These anti-(mouse liver enoyl-CoA hydratase) antibodies also cross-reacted with purified rat liver enoyl-CoA hydratase and with the polypeptide PPA-80 obtained from rat and mouse liver. Immunofluorescence studies with anti-(polypeptide PPA-80) and anti-(peroxisomal enoyl-CoA hydratase) provided visual evidence for the localization and induction of polypeptide PPA-80 and peroxisomal enoyl-CoA hydratase in the liver and kidney respectively of normal and hypolipidaemic-drug-treated mice. In the kidney, the distribution of these two proteins is identical and limited exclusively to the cytoplasm of proximal-convoluted-tubular epithelium. The immunofluorescence studies clearly complement the biochemical and ultrastructural observations of peroxisome induction in the liver and kidney cortex of mice fed on hypolipidaemic drugs. In addition, preliminary ultrastructural studies with the protein-A-gold-complex technique demonstrate that the heat-labile hepatic enoyl-CoA hydratase is localized in the peroxisome matrix.

Pancreatic Polypeptide Cell Proliferation in the Pancreas and Duodenum Coexisting in a Patient With Pancreatic Adenocarcinoma Treated With a GLP-1 Analog.

PubMed

Talmon, Geoffrey A; Wren, J David; Nguyen, Christophe L; Pour, Parviz M

2017-07-01

A partial pancreaticogastrodudenectomy was performed on a 66-year old man with type 2 diabetes mellitus because of an invasive, moderately differentiated adenocarcinoma in the head of the pancreas. In the adjacent grossly normal tissue of the uncinate process, there was a massive proliferation of pancreatic polypeptide (PP) cells confined to this region and showed invasive pattern. Strikingly, in the heaped area of his duodenum, there was a strikingly large number of PP, glucagon, a few insulin cells in a mini-islet-like patterns composed of glucagon and insulin cells. Among the etiological factors, the possible long-lasting effects of the GLP-1 analog, with which the patient was treated, are discussed. This is the first report in the literature of both the coexistence of a pancreatic adenocarcinoma and invasive PPoma and the occurrence of PP and insulin cells in human duodenal mucosa.

Improvement of physical properties of calcium phosphate cement by elastin-like polypeptide supplementation.

PubMed

Jang, Ji-Hyun; Shin, Sumi; Kim, Hyun-Jung; Jeong, Jinyoung; Jin, Hyo-Eon; Desai, Malav S; Lee, Seung-Wuk; Kim, Sun-Young

2018-03-26

Calcium phosphate cements (CPCs) are synthetic bioactive cements widely used as hard tissue substitutes. Critical limitations of use include their poor mechanical properties and poor anti-washout behaviour. To address those limitations, we combined CPC with genetically engineered elastin-like polypeptides (ELPs). We investigated the effect of the ELPs on the physical properties and biocompatibility of CPC by testing ELP/CPC composites with various liquid/powder ratios. Our results show that the addition of ELPs improved the mechanical properties of the CPC, including the microhardness, compressive strength, and washout resistance. The biocompatibility of ELP/CPC composites was also comparable to that of the CPC alone. However, supplementing CPC with ELPs functionalized with octaglutamate as a hydroxyapatite binding peptide increased the setting time of the cement. With further design and modification of our biomolecules and composites, our research will lead to products with diverse applications in biology and medicine.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G: a possible role in the resistance to HIV of HIV-exposed seronegative individuals.

PubMed

Biasin, Mara; Piacentini, Luca; Lo Caputo, Sergio; Kanari, Yasuyoshi; Magri, Giuliana; Trabattoni, Daria; Naddeo, Valentina; Lopalco, Lucia; Clivio, Alberto; Cesana, Eugenio; Fasano, Francesca; Bergamaschi, Cristina; Mazzotta, Francesco; Miyazawa, Masaaki; Clerici, Mario

2007-04-01

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), a human cytidine deaminase, is a potent inhibitor of HIV replication. To explore a possible role of this protein in modulating in vivo susceptibility to HIV infection, we analyzed APOBEC3G expression in HIV-exposed seronegative individuals, HIV-seropositive patients, and healthy control subjects. The results showed that the expression of APOBEC3G is significantly increased in peripheral blood mononuclear cells (PBMCs)--mainly CD14(+) cells--and in cervical tissues of HIV-exposed seronegative individuals. Higher APOBEC3G expression correlated with a reduced susceptibility of PBMCs to in vitro infection with the HIV-1(Ba-L) R5 strain. APOBEC3G could be important in modulating in vivo susceptibility to sexually transmitted HIV infection.

Spermine stimulation of a nuclear NII kinase from pea plumules and its role in the phosphorylation of a nuclear polypeptide

NASA Technical Reports Server (NTRS)

Datta, N.; Schell, M. B.; Roux, S. J.

1987-01-01

We have previously demonstrated that spermine stimulates the phosphorylation of a 47 kilodalton nuclear polypeptide from pea plumules (N Datta, LK Hardison, SJ Roux 1986 Plant Physiol 82: 681-684). In this paper we report that spermine stimulates the activity of a cyclic AMP independent casein kinase, partially purified from a chromatin fraction of pea plumule nuclei. This effect of spermine was substrate specific; i.e. with casein as substrate, spermine stimulated the kinase activity, and with phosvitin as substrate, spermine completely inhibited the activity. The stimulation by spermine of the casein kinase was, in part, due to the lowering of the Mg2+ requirement of the kinase. Heparin could partially inhibit this casein kinase activity and spermine completely overcame this inhibition. By further purification of the casein kinase extract on high performance liquid chromatography, we fractionated it into an NI and an NII kinase. Spermine stimulated the NII kinase by 5- to 6-fold but had no effect on the NI kinase. Using [gamma-32P]GTP, we have shown that spermine promotes the phosphorylation of the 47 kilodalton polypeptide(s) in isolated nuclei, at least in part by stimulating an NII kinase.

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy.

PubMed

Fields, Mark; Spencer, Nicholas; Dudhia, Jayesh; McMillan, Paul F

2017-06-01

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple-helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ∼60-80°C for hydrated samples. We report high temperature Raman spectra for freeze-dried cartilage samples that show an increase in laser-excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H 2 O is retained within the freeze-dried tissue. This is confirmed by thermogravimetric analysis that show 5-7 wt% H 2 O remaining within freeze-dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re-hydration following recovery indicate that the capacity of the denatured collagen to re-absorb water is reduced. Our results are important for revealing the presence of bound H 2 O within the collagen component of connective tissue even after freeze-drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure. © 2017 Wiley Periodicals, Inc.

Mechanistic background and clinical applications of indocyanine green fluorescence imaging of hepatocellular carcinoma.

PubMed

Ishizawa, Takeaki; Masuda, Koichi; Urano, Yasuteru; Kawaguchi, Yoshikuni; Satou, Shouichi; Kaneko, Junichi; Hasegawa, Kiyoshi; Shibahara, Junji; Fukayama, Masashi; Tsuji, Shingo; Midorikawa, Yutaka; Aburatani, Hiroyuki; Kokudo, Norihiro

2014-02-01

Although clinical applications of intraoperative fluorescence imaging of liver cancer using indocyanine green (ICG) have begun, the mechanistic background of ICG accumulation in the cancerous tissues remains unclear. In 170 patients with hepatocellular carcinoma cells (HCC), the liver surfaces and resected specimens were intraoperatively examined by using a near-infrared fluorescence imaging system after preoperative administration of ICG (0.5 mg/kg i.v.). Microscopic examinations, gene expression profile analysis, and immunohistochemical staining were performed for HCCs, which showed ICG fluorescence in the cancerous tissues (cancerous-type fluorescence), and HCCs showed fluorescence only in the surrounding non-cancerous liver parenchyma (rim-type fluorescence). ICG fluorescence imaging enabled identification of 273 of 276 (99%) HCCs in the resected specimens. HCCs showed that cancerous-type fluorescence was associated with higher cancer cell differentiation as compared with rim-type HCCs (P < 0.001). Fluorescence microscopy identified the presence of ICG in the canalicular side of the cancer cell cytoplasm, and pseudoglands of the HCCs showed a cancerous-type fluorescence pattern. The ratio of the gene and protein expression levels in the cancerous to non-cancerous tissues for Na(+)/taurocholate cotransporting polypeptide (NTCP) and organic anion-transporting polypeptide 8 (OATP8), which are associated with portal uptake of ICG by hepatocytes that tended to be higher in the HCCs that showed cancerous-type fluorescence than in those that showed rim-type fluorescence. Preserved portal uptake of ICG in differentiated HCC cells by NTCP and OATP8 with concomitant biliary excretion disorders causes accumulation of ICG in the cancerous tissues after preoperative intravenous administration. This enables highly sensitive identification of HCC by intraoperative ICG fluorescence imaging.

Heat Increases the Editing Efficiency of Human Papillomavirus E2 Gene by Inducing Upregulation of APOBEC3A and 3G.

PubMed

Yang, Yang; Wang, Hexiao; Zhang, Xinrui; Huo, Wei; Qi, Ruiqun; Gao, Yali; Zhang, Gaofeng; Song, Bing; Chen, Hongduo; Gao, Xinghua

2017-04-01

Apolipoprotein B mRNA-editing catalytic polypeptide (APOBEC) 3 proteins have been identified as potent viral DNA mutators and have broad antiviral activity. In this study, we demonstrated that apolipoprotein B mRNA-editing catalytic polypeptide 3A (A3A) and A3G expression levels were significantly upregulated in human papillomavirus (HPV)-infected cell lines and tissues. Heat treatment resulted in elevated expression of A3A and A3G in a temperature-dependent manner in HPV-infected cells. Correspondingly, HPV-infected cells heat-treated at 44 °C showed accumulated G-to-A or C-to-T mutation in HPV E2 gene. Knockdown of A3A or A3G could promote cell viability, along with the lower frequency of A/T in HPV E2 gene. In addition, regressing genital viral warts also harbored high G-to-A or C-to-T mutation in HPV E2 gene. Taken together, we demonstrate that apolipoprotein B mRNA-editing catalytic polypeptide 3 expression and editing function was heat sensitive to a certain degree, partly explaining the mechanism of action of local hyperthermia to treat viral warts. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Proteomics of a new esophageal cancer cell line established from Persian patient.

PubMed

Moghanibashi, Mehdi; Jazii, Ferdous Rastgar; Soheili, Zahra-Soheila; Zare, Maryam; Karkhane, Aliasghar; Parivar, Kazem; Mohamadynejad, Parisa

2012-05-25

Although the highest incidence of esophageal squamous cell carcinoma (ESCC) has repeatedly been reported from Persia (Iran), nevertheless the so far proteomic published reports were limited to one study on tissue specimens. Here we report the proteome of a newly established cell line from Persian ESCC patients and compare it with the normal primary cell proteome. Among polypeptides, whose expression was different in cell line sixteen polypeptides were identified by MALDI/TOF/TOF spectrometry. S100-A8 protein, annexin A1, annexin A2, regulatory subunit of calpain, subunit alpha type-3 of proteasome and glutamate dehydrogenase 1 were proteins down-regulated in cell line while peroxiredoxin-5, non-muscle myosin light polypeptide 6, keratin 1, annexin A4, keratin 8, tropomyosin 3, stress-induced-phosphoprotein 1 and albumin were found to be subject of up-regulation in cell line compared to the primary normal cells. The proteomic results were further verified by western blotting and RT-PCR on annexin A1 and keratin 8. In addition, among the aforementioned proteins, glutamate dehydrogenase 1, regulatory subunit of calpain, subunit alpha of type-3 proteasome and annexin A4 are proteins whose deregulation in ESCC is reported for the first time by this study. Copyright © 2012 Elsevier B.V. All rights reserved.

Differential effect of genetic variants of Na(+)-taurocholate co-transporting polypeptide (NTCP) and organic anion-transporting polypeptide 1B1 (OATP1B1) on the uptake of HMG-CoA reductase inhibitors.

PubMed

Choi, Min-Koo; Shin, Ho Jung; Choi, Young-Lim; Deng, Jian-Wei; Shin, Jae-Gook; Song, Im-Sook

2011-01-01

The purpose of this study was to investigate the effect of genetic variations in organic anion-transporting polypeptide 1B1 (OATP1B1) and Na(+)/taurocholate co-transporting polypeptide (NTCP) on the uptake of various statins having different affinities for these transporters. The functional activities and simultaneous expression of NTCP and OATP1B1 were confirmed by the uptake of taurocholate and estrone-3-sulphate as representative substrates for NTCP and OATP1B1, respectively, and by an immunofluorescence analysis. The substrate specificities of NTCP and OATP1B1 for statins and the effects of genetic variations on the uptake of rosuvastatin, pitavastatin, and atorvastatin were measured. Based on the K(m) values and intrinsic clearances of the three statins, pitavastatin was taken up more efficiently than rosuvastatin and atorvastatin by OATP1B1. Consequently, the cellular accumulation of pitavastatin was modulated according to the genetic variation of OATP1B1 (OATP1B1*15), rather than NTCP*2. In contrast, NTCP*2 displayed greater transport of atorvastatin and rosuvastatin, compared with NTCP wild type. Thus, the measurements of decreased rosuvastatin and atorvastatin transport by OATP1B1*15 were confounded by the presence of NTCP and its genetic variant, NTCP*2. In conclusion, the functional consequences of genetic variants of NTCP and OATP1B1 may be different for various statins, depending on the substrate specificity of the OATP1B1 and NTCP transporters.

A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest–specific gene product

PubMed Central

Rupp, Gerald; Porter, Mary E.

2003-01-01

The dynein regulatory complex (DRC) is an important intermediate in the pathway that regulates flagellar motility. To identify subunits of the DRC, we characterized a Chlamydomonas motility mutant obtained by insertional mutagenesis. The pf2-4 mutant displays an altered waveform that results in slow swimming cells. EM analysis reveals defects in DRC structure that can be rescued by reintroduction of the wild-type PF2 gene. Immunolocalization studies show that the PF2 protein is distributed along the length of the axoneme, where it is part of a discrete complex of polypeptides. PF2 is a coiled-coil protein that shares significant homology with a mammalian growth arrest–specific gene product (Gas11/Gas8) and a trypanosome protein known as trypanin. PF2 and its homologues appear to be universal components of motile axonemes that are required for DRC assembly and the regulation of flagellar motility. The expression of Gas8/Gas11 transcripts in a wide range of tissues may also indicate a potential role for PF2-related proteins in other microtubule-based structures. PMID:12847082

A Developmentally Regulated Chaperone Complex for the Endoplasmic Reticulum of Male Haploid Germ Cells

PubMed Central

van Lith, Marcel; Karala, Anna-Riikka; Bown, Dave; Gatehouse, John A.; Ruddock, Lloyd W.; Saunders, Philippa T.K.

2007-01-01

Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Δ-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility. PMID:17507649

Intrinsic, Functional, and Structural Properties of β-Thymosins and β-Thymosin/WH2 Domains in the Regulation and Coordination of Actin Self-Assembly Dynamics and Cytoskeleton Remodeling.

PubMed

Renault, L

2016-01-01

β-Thymosins are a family of heat-stable multifunctional polypeptides that are expressed as small proteins of about 5kDa (~45 amino acids) almost exclusively in multicellular animals. They were first isolated from the thymus. As full-length or truncated polypeptides, they appear to stimulate a broad range of extracellular activities in various signaling pathways, including tissue repair and regeneration, inflammation, cell migration, and immune defense. However, their cell surface receptors and structural mechanisms of regulations in these multiple pathways remain still poorly understood. Besides their extracellular activities, they belong to a larger family of small, intrinsically disordered actin-binding domains called WH2/β-thymosin domains that have been identified in more than 1800 multidomain proteins found in different taxonomic domains of life and involved in various actin-based motile processes including cell morphogenesis, motility, adhesions, tissue development, intracellular trafficking, or pathogen infections. This review briefly surveys the main recent findings to understand how these small, intrinsically disordered but functional domains can interact with many unrelated partners and can thus integrate and coordinate various intracellular activities in actin self-assembly dynamics and cell signaling pathways linked to their cytoskeleton remodeling. © 2016 Elsevier Inc. All rights reserved.

Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea.

PubMed Central

Brown, D P; Idler, K B; Katz, L

1990-01-01

The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF. The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family. The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages. A proposal for the function of this polypeptide is presented. The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks. Images FIG. 2 FIG. 3 PMID:2180909

Antibody engineering of a cytotoxic monoclonal antibody 84 against human embryonic stem cells: Investigating the effects of multivalency on cytotoxicity.

PubMed

Klement, Maximilian; Zheng, Jiyun; Liu, Chengcheng; Tan, Heng-Liang; Wong, Victor Vai Tak; Choo, Andre Boon-Hwa; Lee, Dong-Yup; Ow, Dave Siak-Wei

2017-02-10

Antibody fragments have shown targeted specificity to their antigens, but only modest tissue retention times in vivo and in vitro. Multimerization has been used as a protein engineering tool to increase the number of binding units and thereby enhance the efficacy and retention time of antibody fragments. In this work, we explored the effects of valency using a series of self-assembling polypeptides based on the GCN4 leucine zipper multimerization domain fused to a single-chain variable fragment via an antibody upper hinge sequence. Four engineered antibody fragments with a valency from one to four antigen-binding units of a cytotoxic monoclonal antibody 84 against human embryonic stem cells (hESC) were constructed. We hypothesized that higher cytotoxicity would be observed for fragments with increased valency. Flow cytometry analysis revealed that the trimeric and tetrameric engineered antibody fragments resulted in the highest degree of cytotoxicity to the undifferentiated hESC, while the engineered antibody fragments were observed to have improved tissue penetration into cell clusters. Thus, a trade off was made for the trimeric versus tetrameric fragment due to improved tissue penetration. These results have direct implications for antibody-mediated removal of undifferentiated hESC during regenerative medicine and cell therapy. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Molecular cloning, characterization and in vitro expression of SERPIN B1 of bighorn sheep (Ovis canadensis) and domestic sheep (Ovis aries), and comparison with that of other species.

PubMed

Subramaniam, Renuka; Dassanayake, Rohana P; Norimine, Junzo; Brown, Wendy C; Knowles, Donald P; Srikumaran, Subramaniam

2010-10-15

Mannheimia haemolytica infection results in enhanced PMN-mediated tissue damage in the lungs of bighorn sheep (BHS) compared to that of domestic sheep (DS). SERPIN B1 is an inhibitor of PMN-derived serine proteases. It prevents lung tissue injury by inhibiting the serine proteases released as a result of PMN lysis and degranulation. It is conceivable that PMNs of BHS exhibit decreased quantity and/or activity of SERPIN B1 which results in enhanced tissue injury and decreased bacterial clearance in pneumonic lungs of BHS. The objective of this study was to clone and express SERPIN B1 of BHS and DS, and develop antibodies to facilitate quantification of SERPIN B1. The 1,134bp cDNA of SERPIN B1 of BHS and DS encodes a polypeptide of 377 amino acids. SERPIN B1 of BHS and DS exhibits 100% identity at the nucleotide and amino acid levels. The amino acid sequence of ovine (BHS/DS) SERPIN B1 displays 69%, 71%, 74%, 78% and 80% identity with that of rats, dogs, mice, humans and horses, respectively. Ovine SERPIN B1 expressed in Escherichia coli was used to develop polyclonal antibodies in mice. Western blot analysis revealed the specificity of these antibodies for ovine rSERPIN B1. (c) 2010 Elsevier B.V. All rights reserved.

Targeted polypeptide degradation

DOEpatents

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

2008-05-13

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

Evidence against vasoactive intestinal polypeptide (VIP) as a dilator and in favour of substance P as a constrictor in airway neurogenic responses.

PubMed Central

Karlsson, J. A.; Persson, C. G.

1983-01-01

Propranolol-resistant neurogenic relaxation persisted in (carbachol-contracted) guinea-pig tracheae already relaxed by supramaximal concentrations of vasoactive intestinal polypeptide (VIP). Also, VIP relaxed preparations that were under neurogenic inhibition. In hilus bronchi, about 60% of a neurogenic contraction was atropine-resistant. (Arg5, D-Trp7.9) SP 5-11 specifically antagonized this contraction and those produced by exogenous substance P. Substance P, but not VIP, seems to be involved in nerve-mediated effects on guinea-pig airway tone. PMID:6197124

Convergent synthesis of proteins by kinetically controlled ligation

DOEpatents

Kent, Stephen; Pentelute, Brad; Bang, Duhee; Johnson, Erik; Durek, Thomas

2010-03-09

The present invention concerns methods and compositions for synthesizing a polypeptide using kinetically controlled reactions involving fragments of the polypeptide for a fully convergent process. In more specific embodiments, a ligation involves reacting a first peptide having a protected cysteyl group at its N-terminal and a phenylthioester at its C-terminal with a second peptide having a cysteine residue at its N-termini and a thioester at its C-termini to form a ligation product. Subsequent reactions may involve deprotecting the cysteyl group of the resulting ligation product and/or converting the thioester into a thiophenylester.

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

PubMed Central

Ross, G S; Wegrzyn, T; MacRae, E A; Redgwell, R J

1994-01-01

A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening. PMID:7991682

Hydrogenase polypeptide and methods of use

DOEpatents

Adams, Michael W.W.; Hopkins, Robert C.; Jenney, JR, Francis E.; Sun, Junsong

2016-02-02

Provided herein are polypeptides having hydrogenase activity. The polypeptide may be multimeric, and may have hydrogenase activity of at least 0.05 micromoles H.sub.2 produced min.sup.-1 mg protein.sup.-1. Also provided herein are polynucleotides encoding the polypeptides, genetically modified microbes that include polynucleotides encoding one or more subunits of the multimeric polypeptide, and methods for making and using the polypeptides.

Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA.

PubMed Central

Mariottini, P; Chomyn, A; Riley, M; Cottrell, B; Doolittle, R F; Attardi, G

1986-01-01

In previous work, antibodies prepared against chemically synthesized peptides predicted from the DNA sequence were used to identify the polypeptides encoded in three of the eight unassigned reading frames (URFs) of human mitochondrial DNA (mtDNA). In the present study, this approach has been extended to other human mtDNA URFs. In particular, antibodies directed against the NH2-terminal octapeptide of the putative URF2 product specifically precipitated component 11 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative URF4 product reacted specifically with components 4 and 5, and antibodies against a COOH-terminal heptapeptide of the presumptive URF4L product reacted specifically with component 26. Antibodies against the NH2-terminal heptapeptide of the putative product of URF5 reacted with component 1, but only to a marginal extent; however, the results of a trypsin fingerprinting analysis of component 1 point strongly to this component as being the authentic product of URF5. The polypeptide assignments to the mtDNA URFs analyzed here are supported by the relative electrophoretic mobilities of proteins 11, 4-5, 26, and 1, which are those expected for the molecular weights predicted from the DNA sequence for the products of URF2, URF4, URF4L, and URF5, respectively. With the present assignment, seven of the eight human mtDNA URFs have been shown to be expressed in HeLa cells. Images PMID:3456601

Texas Red transport across rat and dogfish shark (Squalus acanthias) choroid plexus.

PubMed

Reichel, Valeska; Miller, David S; Fricker, Gert

2008-10-01

Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na(+)-replacement, but was not affected by a 10-fold increase in buffer K(+). In shark CP, Na(+)-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K(+) medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the Mrp1 inhibitor MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals.

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

Li, YanHua, E-mail: liyanhua.1982@aliyun.com; Li, AiHua; Yang, Z.Q.

Cell death-inducing DNA fragmentation factor-α-like effector b (CIDEb) is a member of the CIDE family of apoptosis-inducing factors, CIDEa and CIDEc have been reported to be Lipid droplets (LDs)-associated proteins that promote atypical LD fusion in adipocytes, and responsible for liver steatosis under fasting and obese conditions, whereas CIDEb promotes lipid storage under normal diet conditions [1], and promotes the formation of triacylglyceride-enriched VLDL particles in hepatocytes [2]. Here, we report the gene cloning, chromosome mapping, tissue distribution, genetic expression analysis, and identification of a novel splicing variant of the porcine CIDEb gene. Sequence analysis shows that the open readingmore » frame of the normal porcine CIDEb isoform covers 660bp and encodes a 219-amino acid polypeptide, whereas its alternative splicing variant encodes a 142-amino acid polypeptide truncated at the fourth exon and comprised of the CIDE-N domain and part of the CIDE-C domain. The deduced amino acid sequence of normal porcine CIDEb shows an 85.8% similarity to the human protein and 80.0% to the mouse protein. The CIDEb genomic sequence spans approximately 6KB comprised of five exons and four introns. Radiation hybrid mapping demonstrated that porcine CIDEb is located at chromosome 7q21 and at a distance of 57cR from the most significantly linked marker, S0334, regions that are syntenic with the corresponding region in the human genome. Tissue expression analysis indicated that normal CIDEb mRNA is ubiquitously expressed in many porcine tissues. It was highly expressed in white adipose tissue and was observed at relatively high levels in the liver, lung, small intestine, lymphatic tissue and brain. The normal version of CIDEb was the predominant form in all tested tissues, whereas the splicing variant was expressed at low levels in all examined tissues except the lymphatic tissue. Furthermore, genetic expression analysis indicated that CIDEb mRNA levels were significantly higher in the white adipose tissue of lean pigs than their obese counterparts, in contrast to porcine CIDEa and CIDEc [3]. We therefore speculate that CIDEb may play a contrary role to the other CIDEs. The basic molecular information we provide here will be useful for further investigations of the physiological function of the gene, which will be helpful in better understanding the role of the CIDE family in lipid metabolism in pig models.« less

Gene and genon concept: coding versus regulation

PubMed Central

2007-01-01

We analyse here the definition of the gene in order to distinguish, on the basis of modern insight in molecular biology, what the gene is coding for, namely a specific polypeptide, and how its expression is realized and controlled. Before the coding role of the DNA was discovered, a gene was identified with a specific phenotypic trait, from Mendel through Morgan up to Benzer. Subsequently, however, molecular biologists ventured to define a gene at the level of the DNA sequence in terms of coding. As is becoming ever more evident, the relations between information stored at DNA level and functional products are very intricate, and the regulatory aspects are as important and essential as the information coding for products. This approach led, thus, to a conceptual hybrid that confused coding, regulation and functional aspects. In this essay, we develop a definition of the gene that once again starts from the functional aspect. A cellular function can be represented by a polypeptide or an RNA. In the case of the polypeptide, its biochemical identity is determined by the mRNA prior to translation, and that is where we locate the gene. The steps from specific, but possibly separated sequence fragments at DNA level to that final mRNA then can be analysed in terms of regulation. For that purpose, we coin the new term “genon”. In that manner, we can clearly separate product and regulative information while keeping the fundamental relation between coding and function without the need to introduce a conceptual hybrid. In mRNA, the program regulating the expression of a gene is superimposed onto and added to the coding sequence in cis - we call it the genon. The complementary external control of a given mRNA by trans-acting factors is incorporated in its transgenon. A consequence of this definition is that, in eukaryotes, the gene is, in most cases, not yet present at DNA level. Rather, it is assembled by RNA processing, including differential splicing, from various pieces, as steered by the genon. It emerges finally as an uninterrupted nucleic acid sequence at mRNA level just prior to translation, in faithful correspondence with the amino acid sequence to be produced as a polypeptide. After translation, the genon has fulfilled its role and expires. The distinction between the protein coding information as materialised in the final polypeptide and the processing information represented by the genon allows us to set up a new information theoretic scheme. The standard sequence information determined by the genetic code expresses the relation between coding sequence and product. Backward analysis asks from which coding region in the DNA a given polypeptide originates. The (more interesting) forward analysis asks in how many polypeptides of how many different types a given DNA segment is expressed. This concerns the control of the expression process for which we have introduced the genon concept. Thus, the information theoretic analysis can capture the complementary aspects of coding and regulation, of gene and genon. PMID:18087760

Polypeptides having laccase activity and polynucleotides encoding same

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

Liu, Ye; Tang, Lan; Duan, Junxin

The present invention relates to isolated polypeptides having laccase activity and polynucleotides encoding the polypeptides and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies.

PubMed

Beck, P; Nicholas, H

1975-03-01

1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G,and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly 125I-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as little as 450 ng of polypeptide hormone-antibody protein. An additional advantage of the method is that a single iodination of the readily available antibodies to immunoglobulin G allows the establishemnt of several polypeptide hormone assays

The Role of ARX in Human Pancreatic Endocrine Specification

PubMed Central

Gage, Blair K.; Asadi, Ali; Baker, Robert K.; Webber, Travis D.; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J.

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs. PMID:26633894

The Role of ARX in Human Pancreatic Endocrine Specification.

PubMed

Gage, Blair K; Asadi, Ali; Baker, Robert K; Webber, Travis D; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.

Monosaccharide transporter of the human erythrocyte. Characterization of an improved preparation.

PubMed

Baldwin, S A; Baldwin, J M; Lienhard, G E

1982-08-03

The human erythrocyte monosaccharide transporter has been purified through the use of the dialyzable detergent octyl glucoside. It was found that the transporter denatures in the detergent and that the rate of this process could be reduced by increasing the ratio of phospholipid to detergent. The transporter was obtained in higher yield and with a higher specific activity for cytochalasin B binding than has been previously reported. Scatchard plot analysis of cytochalasin B binding to the reconstituted preparations gave a dissociation constant of 1.5 X 10(-7) M, and there were found to be 15.3 nmol of sites/mg of protein. On the basis of a value of 46 000 for the molecular weight of the polypeptide, this specific activity corresponds to 0.70 site/polypeptide chain; and there are reasons to believe that the value of the stoichiometry may be one site per functional transporter polypeptide. The complete amino acid composition and the N- and C-terminal residues of the transporter have been determined. Both the intact transporter and transporter that had been partially depleted of carbohydrate by treatment with endo-beta-galactosidase were found to migrate anomalously upon sodium dodecyl sulfate--polyacrylamide gel electrophoresis, relative to the behavior of standard proteins.

Structural analysis of photosystem I polypeptides using chemical crosslinking

NASA Technical Reports Server (NTRS)

Armbrust, T. S.; Odom, W. R.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1994-01-01

Thylakoid membranes, obtained from leaves of 14 d soybean (Glycine max L. cv. Williams) plants, were treated with the chemical crosslinkers glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to investigate the structural organization of photosystem I. Polypeptides were resolved using lithium dodecyl sulfate polyacrylamide gel electrophoresis, and were identified by western blot analysis using a library of polyclonal antibodies specific for photosystem I subunits. An electrophoretic examination of crosslinked thylakoids revealed numerous crosslinked products, using either glutaraldehyde or EDC. However, only a few of these could be identified by western blot analysis using subunit-specific polyclonal antibodies. Several glutaraldehyde dependent crosslinked species were identified. A single band was identified minimally composed of PsaC and PsaD, documenting the close interaction between these two subunits. The most interesting aspect of these studies was a crosslinked species composed of the PsaB subunit observed following EDC treatment of thylakoids. This is either an internally crosslinked species, which will provide structural information concerning the topology of the complex PsaB protein, a linkage with a polypeptide for which we do not yet have an immunological probe, or a masking of epitopes by the EDC linkage at critical locations in the peptide which is linked to PsaB.

Characterization of violaxanthin de-epoxidase purified in the presence of Tween 20: effects of dithiothreitol and pepstatin A.

PubMed

Kuwabara, T; Hasegawa, M; Kawano, M; Takaichi, S

1999-11-01

Violaxanthin de-epoxidase (VDE) was purified from thylakoid membranes of spinach by conventional column chromatography in the presence of Tween 20. The neutral detergent was necessary to prevent non-specific interaction of VDE with column resins. In anion-exchange chromatography on Mono Q, VDE appeared in two peaks. Both peaks exhibited a polypeptide of 41 kDa when fully reduced with 5 mM dithiothreitol. Re-chromatography of either peak gave rise to both peaks, suggesting that the two forms of VDE are interconvertible. VDE characteristically changed its electrophoretic mobility depending on the concentration of dithiothreitol with which the protein was treated. When non-reduced, it showed two polypeptides of 43 and 42 kDa. These polypeptides moved down to the position of 40 kDa, and then up to the position of 41 kDa, along with the increase in the dithiothreitol concentration from 0 to 2 mM. These findings suggest that VDE has more than one disulfide bond and takes multiple forms depending on the extent of the reduction. Studies with various types of protein-modifying reagent revealed that VDE is sensitive to pepstatin A, a specific inhibitor of aspartic protease. This finding suggests that the reaction center of VDE contains a reactive aspartic acid residue(s).

Islet amyloid polypeptide (IAPP):cDNA cloning and identification of an amyloidogenic region associated with the species-specific occurrence of age-related diabetes mellitus.

PubMed

Betsholtz, C; Svensson, V; Rorsman, F; Engström, U; Westermark, G T; Wilander, E; Johnson, K; Westermark, P

1989-08-01

We have cloned and sequenced a human islet amyloid polypeptide (IAPP) cDNA. A secretory 89 amino acid IAPP protein precursor is predicted from which the 37 amino acid IAPP molecule is formed by amino- and carboxyterminal proteolytic processing. The IAPP peptide is 43-46% identical in amino acid sequence to the two members of the calcitonin gene-related peptide (CGRP) family. Evolutionary conserved proteolytic processing sites indicate that similar proteases are involved in the maturation of IAPP and CGRP and that the IAPP amyloid polypeptide is identical to the normal proteolytic product of the IAPP precursor. A synthetic peptide corresponding to a carboxyteminal fragment of human IAPP is shown to spontaneously form amyloid-like fibrils in vitro. Antibodies against this peptide cross-react with IAPP from species that develop amyloid in pancreatic islets in conjunction with age-related diabetes mellitus (human, cat, racoon), but do not cross-react with IAPP from other tested species (mouse, rat, guinea pig, dog). Thus, a species-specific structural motif in the putative amyloidogenic region of IAPP is associated with both amyloid formation and the development of age-related diabetes mellitus. This provides a new molecular clue to the pathogenesis of this disease.

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

PubMed Central

Dayal, R; Hurlimann, J; Suard, Y M; Kraehenbuhl, J P

1982-01-01

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6177316

Interdependence, Reflexivity, Fidelity, Impedance Matching, and the Evolution of Genetic Coding

PubMed Central

Carter, Charles W; Wills, Peter R

2018-01-01

Abstract Genetic coding is generally thought to have required ribozymes whose functions were taken over by polypeptide aminoacyl-tRNA synthetases (aaRS). Two discoveries about aaRS and their interactions with tRNA substrates now furnish a unifying rationale for the opposite conclusion: that the key processes of the Central Dogma of molecular biology emerged simultaneously and naturally from simple origins in a peptide•RNA partnership, eliminating the epistemological utility of a prior RNA world. First, the two aaRS classes likely arose from opposite strands of the same ancestral gene, implying a simple genetic alphabet. The resulting inversion symmetries in aaRS structural biology would have stabilized the initial and subsequent differentiation of coding specificities, rapidly promoting diversity in the proteome. Second, amino acid physical chemistry maps onto tRNA identity elements, establishing reflexive, nanoenvironmental sensing in protein aaRS. Bootstrapping of increasingly detailed coding is thus intrinsic to polypeptide aaRS, but impossible in an RNA world. These notions underline the following concepts that contradict gradual replacement of ribozymal aaRS by polypeptide aaRS: 1) aaRS enzymes must be interdependent; 2) reflexivity intrinsic to polypeptide aaRS production dynamics promotes bootstrapping; 3) takeover of RNA-catalyzed aminoacylation by enzymes will necessarily degrade specificity; and 4) the Central Dogma’s emergence is most probable when replication and translation error rates remain comparable. These characteristics are necessary and sufficient for the essentially de novo emergence of a coupled gene–replicase–translatase system of genetic coding that would have continuously preserved the functional meaning of genetically encoded protein genes whose phylogenetic relationships match those observed today. PMID:29077934

The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities.

PubMed

Zhang, Y; LeRoy, G; Seelig, H P; Lane, W S; Reinberg, D

1998-10-16

Histone acetylation and deacetylation were found to be catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription. ATP-dependent nucleosome remodeling, mediated by different multisubunit complexes, was thought to be involved only in transcription activation. Here we report the isolation of a protein complex that contains both histone deacetylation and ATP-dependent nucleosome remodeling activities. The complex contains the histone deacetylases HDAC1/2, histone-binding proteins, the dermatomyositis-specific autoantigen Mi2beta, a polypeptide related to the metastasis-associated protein 1, and a novel polypeptide of 32 kDa. Patients with dermatomyositis have a high rate of malignancy. The finding that Mi2beta exists in a complex containing histone deacetylase and nucleosome remodeling activities suggests a role for chromatin reorganization in cancer metastasis.

Identification of the Bile Acid Transporter Slco1a6 as a Candidate Gene That Broadly Affects Gene Expression in Mouse Pancreatic Islets

PubMed Central

Tian, Jianan; Keller, Mark P.; Oler, Angie T.; Rabaglia, Mary E.; Schueler, Kathryn L.; Stapleton, Donald S.; Broman, Aimee Teo; Zhao, Wen; Kendziorski, Christina; Yandell, Brian S.; Hagenbuch, Bruno; Broman, Karl W.; Attie, Alan D.

2015-01-01

We surveyed gene expression in six tissues in an F2 intercross between mouse strains C57BL/6J (abbreviated B6) and BTBR T+ tf/J (abbreviated BTBR) made genetically obese with the Leptinob mutation. We identified a number of expression quantitative trait loci (eQTL) affecting the expression of numerous genes distal to the locus, called trans-eQTL hotspots. Some of these trans-eQTL hotspots showed effects in multiple tissues, whereas some were specific to a single tissue. An unusually large number of transcripts (∼8% of genes) mapped in trans to a hotspot on chromosome 6, specifically in pancreatic islets. By considering the first two principal components of the expression of genes mapping to this region, we were able to convert the multivariate phenotype into a simple Mendelian trait. Fine mapping the locus by traditional methods reduced the QTL interval to a 298-kb region containing only three genes, including Slco1a6, one member of a large family of organic anion transporters. Direct genomic sequencing of all Slco1a6 exons identified a nonsynonymous coding SNP that converts a highly conserved proline residue at amino acid position 564 to serine. Molecular modeling suggests that Pro564 faces an aqueous pore within this 12-transmembrane domain-spanning protein. When transiently overexpressed in HEK293 cells, BTBR organic anion transporting polypeptide (OATP)1A6-mediated cellular uptake of the bile acid taurocholic acid (TCA) was enhanced compared to B6 OATP1A6. Our results suggest that genetic variation in Slco1a6 leads to altered transport of TCA (and potentially other bile acids) by pancreatic islets, resulting in broad gene regulation. PMID:26385979

Integrin-binding elastin-like polypeptide as an in situ gelling delivery matrix enhances the therapeutic efficacy of adipose stem cells in healing full-thickness cutaneous wounds.

PubMed

Choi, Seong-Kyoon; Park, Jin-Kyu; Kim, Jung-Hee; Lee, Kyeong-Min; Kim, Enjoo; Jeong, Kyu-Shik; Jeon, Won Bae

2016-09-10

One crucial issue in stem cell therapy used for tissue repair is often the lack of selective carriers to deliver stem cells to the site of injury where the native extracellular matrix is pathologically damaged or lost. Therefore, it is necessary to develop a biomaterial that is permissive to stem cells and is suitable to replace injured or missing matrix. The major aim of this study is to investigate the potential of an RGD-containing elastin-like polypeptide (REP) with the structure TGPG[VGRGD(VGVPG)6]20WPC to engraft adipose stem cells (ASC) to full-thickness excisional wounds in mice. We implanted REP into the wound defects via body temperature-induced in situ aggregation. Engrafted REP exhibited a half-life of 2.6days in the wounds and did not elicit any pathological immune responses. REP itself significantly accelerated wound closure and reepithelialization and upregulated the expression of dermal tissue components. A combined administration of REP and ASC formed a hydrogel-like ASC/REP composite, which provided better neovascularization than the use of ASCs alone and increased the viability of transplanted ASC, improving overall wound healing. In vitro and in vivo mechanistic investigations suggested that REP enhances ASC survival at least in part via the Fak/Src adhesion-induced upregulation of Mek/Erk and PI3K/Akt survival pathways. We conclude that REP is a promising therapeutic agent for the improvement of stem cell-based therapy for enhanced tissue regeneration and repair. Copyright © 2016 Elsevier B.V. All rights reserved.

Elastin-like polypeptide matrices for enhancing adeno-associated virus-mediated gene delivery to human neural stem cells.

PubMed

Kim, J-S; Chu, H S; Park, K I; Won, J-I; Jang, J-H

2012-03-01

The successful development of efficient and safe gene delivery vectors continues to be a major obstacle to gene delivery in stem cells. In this study, we have developed an elastin-like polypeptide (ELP)-mediated adeno-associated virus (AAV) delivery system for transducing fibroblasts and human neural stem cells (hNSCs). AAVs have significant promise as therapeutic vectors because of their safety and potential for use in gene targeting in stem cell research. ELP has been recently employed as a biologically inspired 'smart' biomaterial that exhibits an inverse temperature phase transition, thereby demonstrating promise as a novel drug carrier. The ELP that was investigated in this study was composed of a repetitive penta-peptide with [Val-Pro-Gly-Val-Gly]. A novel AAV variant, AAV r3.45, which was previously engineered by directed evolution to enhance transduction in rat NSCs, was nonspecifically immobilized onto ELPs that were adsorbed beforehand on a tissue culture polystyrene surface (TCPS). The presence of different ELP quantities on the TCPS led to variations in surface morphology, roughness and wettability, which were ultimately key factors in the modulation of cellular transduction. Importantly, with substantially reduced viral quantities compared with bolus delivery, ELP-mediated AAV delivery significantly enhanced delivery efficiency in fibroblasts and hNSCs, which have great potential for use in tissue engineering applications and neurodegenerative disorder treatments, respectively. The enhancement of cellular transduction in stem cells, as well as the feasibility of ELPs for utilization in three-dimensional scaffolds, will contribute to the advancement of gene therapy for stem cell research and tissue regenerative medicine.

Pyroelectricity in globular protein lysozyme films

NASA Astrophysics Data System (ADS)

Stapleton, A.; Noor, M. R.; Haq, E. U.; Silien, C.; Soulimane, T.; Tofail, S. A. M.

2018-03-01

Pyroelectricity is the ability of certain non-centrosymmetric materials to generate an electric charge in response to a change in temperature and finds use in a range of applications from burglar alarms to thermal imaging. Some biological materials also exhibit pyroelectricity but the examples of the effect are limited to fibrous proteins, polypeptides, and tissues and organs of animals and plants. Here, we report pyroelectricity in polycrystalline aggregate films of lysozyme, a globular protein.

Primary light-harvesting system: phycobilisomes and associated membranes. Progress report, January 1, 1981-December 31, 1981

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

Gantt, E.

1981-01-01

Phycobilisomes, serving as primary light harvesting complexes in cyanobacteria and red algae, were investigated. Structurally the phycobilisomes of both groups have the same fundamental phycobiliprotein arrangement. Allophycocyanin is in the center near the thylakoid. Stacked rods composed of phycocyanin, or phycocyanin-phycoerythrin radiate peripherally from the allophycocyanin core. Phycobilisomes of Nostoc sp. and Fremyella diplosiphon, after separation into separate allophycocyanin and phycoerythrin-phycocyanin fractions have been associated in vitro. Hybrid phycobilisomes, derived from mixtures of phycobiliprotein from these species were also obtained. The interaction is specific since reassociation was not obtained with phycobiliprotein complexes of some other algae. Phycobilisomes, whether native, ormore » associated in vitro, were similar in their sedimentation, absorption, fluorescence excitation, fluorescence emission, and by electron microscopy. Furthermore, many of the colorless polypeptides were also highly similar between Nostoc and Fremyella. The similarity formed may reflect an evolutionary relationship between the two species. The polypeptide composition of Porphyridium cruentum phycobilisomes is the most complex of any thus far examined. The phycobiliprotein containing polypeptides comprised 84% of the total stainable protein, while the remaining were colorless. Most of the colorless polypeptides occurred in a pelletable fraction, which was enriched in allophycocyanin and phycocyanin, it is probable that some are involved in the linking of these phycobiliproteins.« less

Purification and chemical characterisation of a cell wall-associated β-galactosidase from mature sweet cherry (Prunus avium L.) fruit.

PubMed

Gerardi, Carmela; Blando, Federica; Santino, Angelo

2012-12-01

Using four different chromatographic steps, β-galactosidase was purified from the ripe fruit of sweet cherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunus avium β-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of four different active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and β-galactosidase-activity staining. The active polypeptides were individually excised from the gel and subjected to SDS-PAGE. Each of the four native enzymes showing β-galactosidase activity was composed of two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjected to N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry β-galactosidase showed a 43% identity with the 44 kDa subunit of persimmon and apple β-galactosidases and the 48 kDa subunit of carambola galactosidase I. The sweet cherry β-galactosidase exhibited a strict specificity towards p-nitrophenyl β-D-galactopyranoside, a pH optimum of 4.0 and K(m) and V(max) values of 0.42 mM and 4.12 mmol min(-1) mg(-1) of protein respectively with this substrate. The enzyme was also active towards complex glycans. Taken together the results of this study prompted a role for this class of enzymes on sweet cherry fruit ripening and softening. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Diagnostic potential of low molecular weight excretory secretory proteins of Paramphistomum epiclitum for caprine amphistomosis.

PubMed

Jaiswal, Amit Kumar; Shanker, Daya; Sudan, Vikrant; Singh, Amit

2018-06-15

In the present study, the 75% alcoholic fractionation of excretory-secretory (ES) antigen isolated from 200 to 300 live P. epiclitum was assessed for its diagnostic potential for the detection of caprine amphistomosis by using antibody detection enzyme immunoassay. Prior to enzyme immunoassay, 75% alcoholic fractionation of excretory-secretory (ES) antigen was subjected to SDS- PAGE and western blot analysis for the presence of immunoreactive polypeptides. SDS-PAGE analysis of ES antigen resolved a total 7 polypeptides bands of size 56, 27, 25, 22.5, 12, 11 and 10 kDa. Western blot analysis revealed only two immunoreactive polypeptides (11 kDa and 12 kDa) when polypeptides resolved in SDS-PAGE were probed with known positive pooled serum. None of the polypeptides showed reactions with pooled known negative serum. The working dilutions of antigen, sera and conjugates were determined by checkerboard titration for employing ELISA and cut-off O.D. was calculated 0.616 by using the mean absorbance value of 11 negative kid sera. The sensitivity and specificity of ELISA was found to be 100% and 86.76%, respectively. As per kappa value estimation, the strength of agreement was found to be good. Antibodies to 75% alcoholic fractionation of ES antigen was detected in 20% goats (n = 160) of either sex, although faecal examination detected 10.6% goats to be infected with amphistomosis. The study confirmed that 75% alcoholic fractionation of ES antigen of P. epiclitum based ELISA had good value for serodiagnosis of caprine amphistomosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides.

PubMed

Hermand, P; Mouro, I; Huet, M; Bloy, C; Suyama, K; Goldstein, J; Cartron, J P; Bailly, P

1993-07-15

Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA-encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33-45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side-out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen expression at the cell surface requires correct transport and/or folding of the Rh proteins, possibly as a complex with one-membrane proteins of the Rh cluster that are lacking in Rhnull cells.

Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins

PubMed Central

Jung, Huihun; Pena-Francesch, Abdon; Saadat, Alham; Sebastian, Aswathy; Kim, Dong Hwan; Hamilton, Reginald F.; Albert, Istvan; Allen, Benjamin D.; Demirel, Melik C.

2016-01-01

Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility. PMID:27222581

Knock Down of Heat Shock Protein 27 (HspB1) Induces Degradation of Several Putative Client Proteins

PubMed Central

Gibert, Benjamin; Eckel, Bénédicte; Fasquelle, Lydie; Moulin, Maryline; Bouhallier, Frantz; Gonin, Vincent; Mellier, Gregory; Simon, Stéphanie; Kretz-Remy, Carole; Arrigo, André-Patrick; Diaz-Latoud, Chantal

2012-01-01

Hsp27 belongs to the heat shock protein family and displays chaperone properties in stress conditions by holding unfolded polypeptides, hence avoiding their inclination to aggregate. Hsp27 is often referenced as an anti-cancer therapeutic target, but apart from its well-described ability to interfere with different stresses and apoptotic processes, its role in non-stressed conditions is still not well defined. In the present study we report that three polypeptides (histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3) were degraded in human cancerous cells displaying genetically decreased levels of Hsp27. In addition, these proteins interacted with Hsp27 complexes of different native size. Altogether, these findings suggest that HDAC6, STAT2 and procaspase-3 are client proteins of Hsp27. Hence, in non stressed cancerous cells, the structural organization of Hsp27 appears to be a key parameter in the regulation by this chaperone of the level of specific polypeptides through client-chaperone type of interactions. PMID:22238643

Peptide Beacons: A New Design for Polypeptide-Based Optical Biosensors

PubMed Central

Oh, Kenneth J.; Cash, Kevin J.; Hugenberg, Verena; Plaxco, Kevin W.

2008-01-01

Phage display and other in vitro selection techniques produce short polypeptides that tightly and specifically bind to any of a wide range of macromolecular targets. Here we demonstrate a potentially general means of converting such polypeptides into optical biosensors. The sensing architecture we have developed, termed peptide beacons, is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed to their target. Using this effect to segregate a long-lived fluorophore from an electron transfer-based contact quencher, both covalently attached to the peptide, we have produced a robust optical sensor for anti-HIV antibodies. The binding-induced segregation of the fluorophore-quencher pair produces a six-fold increase in sensor emission, thus allowing us to readily detect as low as ∼250 pM of the target antibody. Because the sensor is based on binding-induced folding and a visible-light fluorophore, it is sufficiently selective to work directly in complex, contaminant-ridden samples such as saliva and blood. PMID:17461545

Silencing of GSTP1 gene by CpG island DNA hypermethylation in HBV-associated hepatocellular carcinomas.

PubMed

Zhong, Sheng; Tang, Mandy W; Yeo, Winnie; Liu, Cuiling; Lo, Y M Dennis; Johnson, Philip J

2002-04-01

Glutathione S-transferases, enzymes that defend cells against damage mediated by oxidant and electrophilic carcinogens, may be critical determinants of cancer pathogenesis. In this report, we assess the role of epigenetic silencing of the GSTP1 gene, a gene encoding the pi-class glutathione S-transferase, in the pathogenesis of hepatitis B virus (HBV)-associated hepatocellular carcinomas (HCC). The cell lines Hep3B, HepG2, and a cohort of 43 HBV-associated HCC tissue specimens and corresponding nontumor tissues were subjected to analysis for GSTP1 epigenetic alteration and expression. GSTP1 "CpG" island DNA hypermethylation in the liver cell lines, and the tissue specimens were determined by methylation-specific PCR and correlated with expression of the gene using reverse-transcription PCR, immunoblotting, and immunohistochemistry. GSTP1 CpG island DNA hypermethylation was detected in 28 of 43 (65.1%) HCC tissues and 4 of 40 (10%) corresponding nontumor tissues. GSTP1 protein was absent in those cases showing hypermethylation of the gene. Similarly, DNA from Hep3B and HepG2 cell lines displayed complete GSTP1 hypermethylation in the CpG island, and they failed to express GSTP1 mRNA and the corresponding protein product. Treatment of the cell lines with the DNA methyltransferase inhibitor 5-aza-deoxycytidine reversed the hypermethylation, and restored GSTP1 mRNA and polypeptide expression. These data indicate that epigenetic silencing of GSTP1 gene expression by CpG island DNA hypermethylation is common in human HBV-associated HCC. In addition, somatic GSTP1 inactivation via CpG island hypermethylation may contribute to the pathogenesis of this malignancy.

Proline-poor hydrophobic domains modulate the assembly and material properties of polymeric elastin.

PubMed

Muiznieks, Lisa D; Reichheld, Sean E; Sitarz, Eva E; Miao, Ming; Keeley, Fred W

2015-10-01

Elastin is a self-assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline-poor and, as an isolated polypeptide, is susceptible to formation of amyloid-like structures comprised of stacked β-sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin-like polypeptides designed with different numbers of proline-poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline-poor hydrophobic sequences to self-assembly through characterization of phase separation, and to the tensile properties of cross-linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form β-structure, both affecting polypeptide chain flexibility and cross-link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence-structure-function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties. © 2015 Wiley Periodicals, Inc.

Resilin-like polypeptide-poly(ethylene gylcol) hybrid hydrogels for mechanically-demanding tissue engineering applications

NASA Astrophysics Data System (ADS)

McGann, Christopher Leland

Technological progress in the life sciences and engineering has combined with important insights in the fields of biology and material science to make possible the development of biological substitutes which aim to restore function to damaged tissue. Numerous biomimetic hydrogels have been developed with the purpose of harnessing the regenerative capacity of cells and tissue through the rational deployment of biological signals. Aided by recombinant DNA technology and protein engineering methods, a new class of hydrogel precursor, the biosynthetic protein polymer, has demonstrated great promise towards the development of highly functional tissue engineering materials. In particular, protein polymers based upon resilin, a natural protein elastomer, have demonstrated outstanding mechanical properties that would have great value in soft tissue applications. This dissertation introduces hybrid hydrogels composed of recombinant resilin-like polypeptides (RLPs) cross-linked with multi-arm PEG macromers. Two different chemical strategies were employed to form RLP-PEG hydrogels: one utilized a Michael-type addition reaction between the thiols of cysteine residues present within the RLP and vinyl sulfone moieties functionalized on a multi-arm PEG macromer; the second system cross-links a norbornene-functionalized RLP with a thiol-functionalized multi-arm PEG macromer via a photoinitiated thiol-ene step polymerization. Oscillatory rheology and tensile testing confirmed the formation of elastic, resilient hydrogels in the RLP-PEG system cross-linked via Michael-type addition. These hydrogels supported the encapsulation and culture of both human aortic adventitial fibroblasts and human mesenchymal stem cells. Additionally, these RLP-PEG hydrogels exhibited phase separation behavior during cross-linking that led to the formation of a heterogeneous microstructure. Degradation could be triggered through incubation with matrix metalloproteinase. Photocross-linking was conferred to RLPs through the successful conjugation of norbornene acid to the protein. Oscillatory rheology characterized the gelation and subsequent mechanical properties of the photoreactive RLP-PEG hydrogels while the cytocompatibility was confirmed via the successful encapsulation and culture of human mesenchymal stem cells. Both strategies demonstrate the utility of hybrid materials that combine biosynthetic proteins with synthetic polymers. As resilient and cytocompatible materials, RLP-PEG hybrid hydrogels offer an exciting strategy towards the development of biomimetic tissue engineering scaffolds for mechanically-demanding applications.

moxFG region encodes four polypeptides in the methanol-oxidizing bacterium Methylobacterium sp. strain AM1

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

Anderson, D.J.; Lidstrom, M.E.

The polypeptides encoded by a putative methanol oxidation (mox) operon of Methylobacterium sp. strain AM1 were expressed in Escherichia coli, using a coupled in vivo T7 RNA polymerase/promoter gene expression system. Two mox genes had been previously mapped to this region: moxF, the gene encoding the methanol dehydrogenase (MeDH) polypeptide; and moxG, a gene believed to encode a soluble type c cytochrome, cytochrome c/sub L/. In this study, four polypeptides of M/sub r/, 60,000, 30,000, 20,000, and 12,000 were found to be encoded by the moxFG region and were tentatively designated moxF, -J, -G, and -I, respectively. The arrangement ofmore » the genes (5' to 3') was found to be moxFJGI. The identities of three of the four polypeptides were determined by protein immunoblot analysis. The product of moxF, the M/sub r/-60,000 polypeptide, was confirmed to be the MeDH polypeptide. The product of moxG, the M/sub r/-20,000 polypeptide, was identified as mature cytochrome c/sub L/, and the product of moxI, the M/sub r/-12,000 polypeptide, was identified as a MeDH-associated polypeptide that copurifies with the holoenzyme. The identity of the M/sub r/-30,000 polypeptide (the moxJ gene product) could not be determined. The function of the M/sub r/-12,000 MeDH-associated polypeptide is not yet clear. However, it is not present in mutants that lack the M/sub r/-60,000 MeDH subunit, and it appears that the stability of the MeDH-associated polypeptide is dependent on the presence of the M/sub r/-60,000 MeDH polypeptide. Our data suggest that both the M/sub r/-30,000 and -12,000 polypeptides are involved in methanol oxidation, which would bring to 12 the number of mox genes in Methylobacterium sp. strain AM1.« less

Molecular characterization, tissue distribution and feeding related changes of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti).

PubMed

Lin, Fangjun; Zhou, Chaowei; Chen, Hu; Wu, Hongwei; Xin, Zhiming; Liu, Ju; Gao, Yundi; Yuan, Dengyue; Wang, Tao; Wei, Rongbin; Chen, Defang; Yang, Shiyong; Wang, Yan; Pu, Yundan; Li, Zhiqiong

2014-02-25

The protein nucleobindin-2 (NUCB2) was identified over a decade ago and recently raised great interest as its derived peptide nesfatin-1 was shown to reduce food intake and body weight in rodents. However, the involvement of NUCB2 in feeding behavior has not well been studied in fish. In the present study, we characterized the structure, distribution, and meal responsive of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti) for the first time. The full length cDNA of Ya-fish was 2140base pair (bp), which encoded a polypeptide of 487 amino acid residues including a 23 amino acid signal peptide. A high conservation in NUCB2 sequences was found in vertebrates, however the proposed propeptide cleavage site (Arg-Arg) conserved among other species is not present in Ya-fish NUCB2A sequence. Tissue distribution analysis revealed that Ya-fish NUCB2A mRNA was ubiquitously expressed in all test tissues, and abundant expression was detected in several regions including the hypothalamus, hepatopancreas, ovary and intestines. NUCB2A mRNA expression respond to feeding status change may vary and be tissue specific. NUCB2A mRNA levels significantly increased (P<0.05) in the hypothalamus and intestines after feeding and substantially decreased (P<0.01) during a week food deprivation in the hypothalamus. Meanwhile, NUCB2A mRNA in the hepatopancreas was significantly elevated (P<0.001) during food deprivation, and a similar increase was also found after short-time fasting. This points toward a potential hepatopancreas specific local role for NUCB2A in the regulation of metabolism during food deprivation. Collectively, these results provide the molecular and functional evidence to support potential anorectic and metabolic roles for NUCB2A in Ya-fish. Copyright © 2013 Elsevier B.V. All rights reserved.

[Collagens: why such a structural complexity?].

PubMed

Borel, J P; Monboisse, J C

1993-01-01

The collagens are a family of extracellular fibrillar proteins, characterized by the presence of one or several domains termed "triple helix", that are made of three polypeptide chains folded around each other. They elicit a huge worldwide research activity, marked every year by the publishing of dozens of books and thousands of papers. This family is presently represented by more than 16 individualized types, all differing by their molecular structure and by the way helical and globular domains are arranged. In any case, however, at least one triple helical domain exists. It is formed by the association of three polypeptide chains, each of them containing a glycine every three residues and many proline or hydroxyproline residues, and attests for the belonging of the protein to the collagen group. These multiple molecular forms and their specific architecture raise questions that remain unsolved. Why is this triple helix structure adopted in the case of collagens? Is it because the simple alpha helix of protein cannot extend over more than a few nanometers and is not solid enough? Why not a double helix like that of DNA? It would probably not be rigid enough. Why are there many globular domains interspersed between fibrillar ones? Probably these domains are useful for the association of peptide chains in register prior to their folding, then they participate in the transport of the elementary molecules from the synthesizing cells to their final place in the connective tissue and, finally, they insert the molecules into their specific place inside the growing fibrils. Collagen fibres as they are evidenced by histological methods, for instance in tendons, are of complex structure. Most of their constituting sub-units are type I tropocollagen molecules but they also contain in their center a filament of type V collagen that seems to serve as a guide during their edification. On the surface of the fibres are molecules of type III collagen that limit the growth in diameter and also type XII molecules that serve to bind the fibres to the surrounding substances. The collagen type multiplicity is explained by their various functions (mechanical role for tendons and ligaments, functions of wrapping around muscle cells, basement membrane role as a support for endothelial cells, function of glomerular filter, etc.). The fact that every collagen type contains several different polypeptide chains remains poorly explained. It may serve for the orientation of every elementary molecule inside the complex array of the polymer.(ABSTRACT TRUNCATED AT 400 WORDS)

Genome-wide identification and comparative expression analysis of LEA genes in watermelon and melon genomes.

PubMed

Celik Altunoglu, Yasemin; Baloglu, Mehmet Cengiz; Baloglu, Pinar; Yer, Esra Nurten; Kara, Sibel

2017-01-01

Late embryogenesis abundant (LEA) proteins are large and diverse group of polypeptides which were first identified during seed dehydration and then in vegetative plant tissues during different stress responses. Now, gene family members of LEA proteins have been detected in various organisms. However, there is no report for this protein family in watermelon and melon until this study. A total of 73 LEA genes from watermelon ( ClLEA ) and 61 LEA genes from melon ( CmLEA ) were identified in this comprehensive study. They were classified into four and three distinct clusters in watermelon and melon, respectively. There was a correlation between gene structure and motif composition among each LEA groups. Segmental duplication played an important role for LEA gene expansion in watermelon. Maximum gene ontology of LEA genes was observed with poplar LEA genes. For evaluation of tissue specific expression patterns of ClLEA and CmLEA genes, publicly available RNA-seq data were analyzed. The expression analysis of selected LEA genes in root and leaf tissues of drought-stressed watermelon and melon were examined using qRT-PCR. Among them, ClLEA - 12 - 17 - 46 genes were quickly induced after drought application. Therefore, they might be considered as early response genes for water limitation conditions in watermelon. In addition, CmLEA - 42 - 43 genes were found to be up-regulated in both tissues of melon under drought stress. Our results can open up new frontiers about understanding of functions of these important family members under normal developmental stages and stress conditions by bioinformatics and transcriptomic approaches.

Quantification of VGF- and pro-SAAS-derived peptides in endocrine tissues and the brain, and their regulation by diet and cold stress.

PubMed

Chakraborty, Tandra R; Tkalych, Oleg; Nanno, Daniela; Garcia, Angelo L; Devi, Lakshmi A; Salton, Stephen R J

2006-05-17

Two novel granin-like polypeptides, VGF and pro-SAAS, which are stored in and released from secretory vesicles and are expressed widely in nervous, endocrine, and neuroendocrine tissues, play roles in the regulation of body weight, feeding, and energy expenditure. Both VGF and pro-SAAS are cleaved into peptide fragments, several of which are biologically active. We utilized a highly sensitive and specific radioimmunoassay (RIA) to immunoreactive, pro-SAAS-derived PEN peptides, developed another against immunoreactive, VGF-derived AQEE30 peptides, and quantified these peptides in various mouse tissues and brain regions. Immunoreactive AQEE30 was most abundant in the pituitary, while brain levels were highest in hypothalamus, striatum, and frontal cortex. Immunoreactive PEN levels were highest in the pancreas and spinal cord, and in brain, PEN was most abundant in striatum, hippocampus, pons and medulla, and cortex. Since both peptides were expressed in hypothalamus, a region of the brain that controls feeding and energy expenditure, double label immunofluorescence studies were employed. These demonstrated that 42% of hypothalamic arcuate neurons coexpress VGF and SAAS peptides, and that the intracellular distributions of these peptides in arcuate neurons differed. By RIA, cold stress increased immunoreactive AQEE30 and PEN peptide levels in female but not male hypothalamus, while a high fat diet increased AQEE30 and PEN peptide levels in female but not male hippocampus. VGF and SAAS-derived peptides are therefore widely expressed in endocrine, neuroendocrine, and neural tissues, can be accurately quantified by RIA, and are differentially regulated in the brain by diet and cold stress.

Method to determine transcriptional regulation pathways in organisms

DOEpatents

Gardner, Timothy S.; Collins, James J.; Hayete, Boris; Faith, Jeremiah

2012-11-06

The invention relates to computer-implemented methods and systems for identifying regulatory relationships between expressed regulating polypeptides and targets of the regulatory activities of such regulating polypeptides. More specifically, the invention provides a new method for identifying regulatory dependencies between biochemical species in a cell. In particular embodiments, provided are computer-implemented methods for identifying a regulatory interaction between a transcription factor and a gene target of the transcription factor, or between a transcription factor and a set of gene targets of the transcription factor. Further provided are genome-scale methods for predicting regulatory interactions between a set of transcription factors and a corresponding set of transcriptional target substrates thereof.

Free energy landscapes of short peptide chains using adaptively biased molecular dynamics

NASA Astrophysics Data System (ADS)

Karpusenka, Vadzim; Babin, Volodymyr; Roland, Christopher; Sagui, Celeste

2009-03-01

We present the results of a computational study of the free energy landscapes of short polypeptide chains, as a function of several reaction coordinates meant to distinguish between several known types of helices. The free energy landscapes were calculated using the recently developed adaptively biased molecular dynamics method followed up with equilibrium ``umbrella correction'' runs. Specific polypeptides investigated include small chains of pure and mixed alanine, glutamate, leucine, lysine and methionine (all amino acids with strong helix-forming propensities), as well as glycine, proline(having a low helix forming propensities), tyrosine, serine and arginine. Our results are consistent with the existing experimental and other theoretical evidence.

Methods for engineering polypeptide variants via somatic hypermutation and polypeptide made thereby

DOEpatents

Tsien, Roger Y; Wang, Lei

2015-01-13

Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

Characterization of plasma membrane domains of mouse EL4 lymphoma cells obtained by affinity chromatography on concanavalin A-Sepharose.

PubMed

Szamel, M; Goppelt, M; Resch, K

1985-12-19

Purified plasma membranes of mouse EL4 lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including gamma-glutamyl transpeptidase, 5'-nucleotidase and Mg2+-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL4 lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.

Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.

PubMed

Gradišar, Helena; Božič, Sabina; Doles, Tibor; Vengust, Damjan; Hafner-Bratkovič, Iva; Mertelj, Alenka; Webb, Ben; Šali, Andrej; Klavžar, Sandi; Jerala, Roman

2013-06-01

Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil-forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

The radioimmunoassay and physiology of somatostatin in the pancreas and gastrointestinal tract.

PubMed

McIntosh, C; Arnold, R

1978-05-01

Radioimmunoassays for somatostain have demonstrated that high concentrations of the polypeptide are present in the pancreas and gastrointestinal tract of a number of species. Although measurement in tissue extracts is relatively unproblematic, detection and characterization of somatostatin-like material in plasma has proved technically difficult. Studies of pancreatic somatostatin release in vitro suggest a possible function in the regulation of islet hormone secretion, but the mode of action remains to be elucidated. Although, at present, no clinical relevance can be attributed to the somatostain radioimmunoassay reports of somatostatin secreting tumors and changes in stomach tissue content in patients with ulcer disease indicate a contributory role in the pathophysiology of certain disease states.

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

Ghosh, Koushik; Balog, Eva Rose M.; Sista, Prakash

We report a method for creating hybrid organic-inorganic “nanoflowers” using calcium or copper ions as the inorganic component and a recombinantly expressed elastin-like polypeptide (ELP) as the organic component. Polypeptides provide binding sites for the dynamic coordination with metal ions, and then such noncovalent complexes become nucleation sites for primary crystals of metal phosphates. We have shown that the interaction between the stimuli-responsive ELP and Ca{sup 2+} or Cu{sup 2+}, in the presence of phosphate, leads to the growth of micrometer-sized particles featuring nanoscale patterns shaped like flower petals. The morphology of these flower-like composite structures is dependent upon themore » temperature of growth and has been characterized by scanning electron microscopy. The composition of nanoflowers has also been analyzed by energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The temperature-dependent morphologies of these hybrid nanostructures, which arise from the controllable phase transition of ELPs, hold potential for morphological control of biomaterials in emerging applications such as tissue engineering and biocatalysis.« less

Effects of pituitary adenylate cyclase activating polypeptide in the urinary system, with special emphasis on its protective effects in the kidney.

PubMed

Reglodi, Dora; Kiss, Peter; Horvath, Gabriella; Lubics, Andrea; Laszlo, Eszter; Tamas, Andrea; Racz, Boglarka; Szakaly, Peter

2012-04-01

Pituitary adenylate cyclase activating polypeptide (PACAP) is a widespread neuropeptide with diverse effects in the nervous system and peripheral organs. One of the most well-studied effects of PACAP is its cytoprotective action, against different harmful stimuli in a wide variety of cells and tissues. PACAP occurs in the urinary system, from the kidney to the lower urinary tract. The present review focuses on the nephroprotective effects of PACAP and summarizes data obtained regarding the protective effects of PACAP in different models of kidney pathologies. In vitro data show that PACAP protects tubular cells against oxidative stress, myeloma light chain, cisplatin, cyclosporine-A and hypoxia. In vivo data provide evidence for its protective effects in ischemia/reperfusion, cisplatin, cyclosporine-A, myeloma kidney injury, diabetic nephropathy and gentamicin-induced kidney damage. Results accumulated on the renoprotective effects of PACAP suggest that PACAP is an emerging candidate for treatment of human kidney pathologies. Copyright © 2011 Elsevier Ltd. All rights reserved.

rFN/Cad-11-Modified Collagen Type II Biomimetic Interface Promotes the Adhesion and Chondrogenic Differentiation of Mesenchymal Stem Cells

PubMed Central

Guo, Hongfeng; Zhang, Yuan; Li, Zhengsheng; Kang, Fei; Yang, Bo; Kang, Xia; Wen, Can; Yan, Yanfei; Jiang, Bo; Fan, Yujiang

2013-01-01

Properties of the cell-material interface are determining factors in the successful function of cells for cartilage tissue engineering. Currently, cell adhesion is commonly promoted through the use of polypeptides; however, due to their lack of complementary or modulatory domains, polypeptides must be modified to improve their ability to promote adhesion. In this study, we utilized the principle of matrix-based biomimetic modification and a recombinant protein, which spans fragments 7–10 of fibronectin module III (heterophilic motif ) and extracellular domains 1–2 of cadherin-11 (rFN/Cad-11) (homophilic motif ), to modify the interface of collagen type II (Col II) sponges. We showed that the designed material was able to stimulate cell proliferation and promote better chondrogenic differentiation of rabbit mesenchymal stem cells (MSCs) in vitro than both the FN modified surfaces and the negative control. Further, the Col II/rFN/Cad-11-MSCs composite stimulated cartilage formation in vivo; the chondrogenic effect of Col II alone was much less significant. These results suggested that the rFN/Cad-11-modified collagen type II biomimetic interface has dual biological functions of promoting adhesion and stimulating chondrogenic differentiation. This substance, thus, may serve as an ideal scaffold material for cartilage tissue engineering, enhancing repair of injured cartilage in vivo. PMID:23919505

Polypeptides having catalase activity and polynucleotides encoding same

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

Liu, Ye; Duan, Junxin; Zhang, Yu

Provided are isolated polypeptides having catalase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

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

Liu, Ye; Duan, Junxin; Zhang, Yu

Provided are isolated polypeptides having beta-glucosidase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

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

Zhang, Yu; Liu, Ye; Duan, Junxin

Provided are isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-xylosidase activity and polynucleotides encoding same

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

Liu, Ye; Tang, Lan; Zhang, Yu

Provided are isolated polypeptides having beta-xylosidase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Shaghasi, Tarana

2016-11-01

The present invention provides hybrid polypeptides having cellobiohydrolase activity. The present invention also provides polynucleotides encoding the hybrid polypeptides; nucleic acid constructs, vectors and host cells comprising the polynucleotides; and processes of using the hybrid polypeptides.

Folding and self-assembly of polypeptides: Dynamics and thermodynamics from molecular simulation

NASA Astrophysics Data System (ADS)

Fluitt, Aaron Michael

Empowered by their exquisite three-dimensional structures, or "folds," proteins carry out biological tasks with high specificity, efficiency, and fidelity. The fold that optimizes biological function represents a stable configuration of the constituent polypeptide molecule(s) under physiological conditions. Proteins and polypeptides are not static, however: battered by thermal motion, they explore a distribution of folds that is determined by the sequence of amino acids, the presence and identity of other molecules, and the thermodynamic conditions. In this dissertation, we apply molecular simulation techniques to the study of two polypeptides that have unusually diffuse distributions of folds under physiological conditions: polyglutamine (polyQ) and islet amyloid polypeptide (IAPP). Neither polyQ nor IAPP adopts a predominant fold in dilute aqueous solution, but at sufficient concentrations, both are prone to self-assemble into stable, periodic, and highly regular aggregate structures known as amyloid. The appearance of amyloid deposits of polyQ in the brain, and of IAPP in the pancreas, are associated with Huntington's disease and type 2 diabetes, respectively. A molecular view of the mechanism(s) by which polyQ and IAPP fold and self-assemble will enhance our understanding of disease pathogenesis, and it has the potential to accelerate the development of therapeutics that target early-stage aggregates. Using molecular simulations with spatial and temporal resolution on the atomic scale, we present analyses of the structural distributions of polyQ and IAPP under various conditions, both in and out of equilibrium. In particular, we examine amyloid fibers of polyQ, the IAPP dimer in solution, and single IAPP fragments at a lipid bilayer. We also benchmark the molecular models, or "force fields," available for such studies, and we introduce a novel simulation algorithm.

Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity.

PubMed

Brito-Argáez, Ligia; Tamayo-Sansores, José A; Madera-Piña, Dianeli; García-Villalobos, Francisco J; Moo-Puc, Rosa E; Kú-González, Ángela; Villanueva, Marco A; Islas-Flores, Ignacio

2016-12-01

The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr ∼7.57 kDa and pI ∼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the ∼7.57 kDa polypeptide, immunodetected an ∼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the ∼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the ∼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the ∼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same

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

Liu, Ye; Shaghasi, Tarana

The present invention relates to hybrid polypeptides having cellobiohydrolase activity. The present invention also relates to polynucleotides encoding the hybrid polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and processes of using the hybrid polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

2015-06-09

Provided are isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Duan, Junxin; Tang, Lan

2015-09-22

The present invention provides isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cell comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activitiy and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan; Duan, Junxin

2015-12-15

The present invention provides isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Isolation of Polypeptide Sample and Measurement of Its Concentration.

ERIC Educational Resources Information Center

Beanan, Maureen J.

2000-01-01

Introduces a laboratory experiment that isolates a bacterial polypeptide sample and measures the concentration of polypeptides in the sample. Uses Escherichia coli strain MM294 and performs a bio-rad assay to determine the concentration of polypeptides. (YDS)

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan

2015-07-14

The present invention provides isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Down-regulation of NTCP expression by cyclin D1 in hepatitis B virus-related hepatocellular carcinoma has clinical significance

PubMed Central

Kang, Jingting; Wang, Jie; Cheng, Jin; Cao, Zhiliang; Chen, Ran; Li, Huiyu; Liu, Shuang; Chen, Xiangmei; Sui, Jianhua; Lu, Fengmin

2017-01-01

The sodium-dependent taurocholate cotransporter polypeptide (NTCP) has been identified as a liver specific functional receptor for the hepatitis B virus (HBV). Previous studies indicated that the expression of NTCP may be associated with the proliferation status of hepatocytes. However, the involvement of NTCP in hepatocellular carcinoma (HCC) cells proliferation remains unclear. In this study, we confirmed that NTCP was down-regulated in HCC tumor tissues compared with that in the adjacent non-tumor tissues (P < 0.0001). Clinically, lower expression of NTCP was correlated with poor post-surgery survival rate (P = 0.0009) and larger tumor tissue mass (P = 0.003) of HCC patients. This was supported by the finding that ectopic expression of NTCP in both HepG2 and Huh-7 cells could significantly suppress hepatocytes growth by arresting cells in G0/G1 phase. We also discovered that cyclin D1 could transcriptionally suppress NTCP expression by inhibiting the activity of NTCP promoter, while arresting HCC cells in G0/G1 phase by serum starvation could upregulate NTCP mRNA levels. This is the first study to report that the transcriptional inhibition of NTCP expression during cell cycle progression was mediated by cyclin D1. The down-regulated NTCP expression was associated with poor prognosis and lower HBV cccDNA level in HCC patients. Therefore, NTCP expression levels might serve as a novel prognostic predictive marker for post-surgery survival rate of HCC patients. PMID:28915572

Down-regulation of NTCP expression by cyclin D1 in hepatitis B virus-related hepatocellular carcinoma has clinical significance.

PubMed

Kang, Jingting; Wang, Jie; Cheng, Jin; Cao, Zhiliang; Chen, Ran; Li, Huiyu; Liu, Shuang; Chen, Xiangmei; Sui, Jianhua; Lu, Fengmin

2017-08-22

The sodium-dependent taurocholate cotransporter polypeptide (NTCP) has been identified as a liver specific functional receptor for the hepatitis B virus (HBV). Previous studies indicated that the expression of NTCP may be associated with the proliferation status of hepatocytes. However, the involvement of NTCP in hepatocellular carcinoma (HCC) cells proliferation remains unclear. In this study, we confirmed that NTCP was down-regulated in HCC tumor tissues compared with that in the adjacent non-tumor tissues ( P < 0.0001). Clinically, lower expression of NTCP was correlated with poor post-surgery survival rate ( P = 0.0009) and larger tumor tissue mass ( P = 0.003) of HCC patients. This was supported by the finding that ectopic expression of NTCP in both HepG2 and Huh-7 cells could significantly suppress hepatocytes growth by arresting cells in G0/G1 phase. We also discovered that cyclin D1 could transcriptionally suppress NTCP expression by inhibiting the activity of NTCP promoter, while arresting HCC cells in G0/G1 phase by serum starvation could upregulate NTCP mRNA levels. This is the first study to report that the transcriptional inhibition of NTCP expression during cell cycle progression was mediated by cyclin D1. The down-regulated NTCP expression was associated with poor prognosis and lower HBV cccDNA level in HCC patients. Therefore, NTCP expression levels might serve as a novel prognostic predictive marker for post-surgery survival rate of HCC patients.

Sequence Directionality Dramatically Affects LCST Behavior of Elastin-Like Polypeptides.

PubMed

Li, Nan K; Roberts, Stefan; Quiroz, Felipe Garcia; Chilkoti, Ashutosh; Yingling, Yaroslava G

2018-04-30

Elastin-like polypeptides (ELP) exhibit an inverse temperature transition or lower critical solution temperature (LCST) transition phase behavior in aqueous solutions. In this paper, the thermal responsive properties of the canonical ELP, poly(VPGVG), and its reverse sequence poly(VGPVG) were investigated by turbidity measurements of the cloud point behavior, circular dichroism (CD) measurements, and all-atom molecular dynamics (MD) simulations to gain a molecular understanding of mechanism that controls hysteretic phase behavior. It was shown experimentally that both poly(VPGVG) and poly(VGPVG) undergo a transition from soluble to insoluble in aqueous solution upon heating above the transition temperature ( T t ). However, poly(VPGVG) resolubilizes upon cooling below its T t , whereas the reverse sequence, poly(VGPVG), remains aggregated despite significant undercooling below the T t . The results from MD simulations indicated that a change in sequence order results in significant differences in the dynamics of the specific residues, especially valines, which lead to extensive changes in the conformations of VPGVG and VGPVG pentamers and, consequently, dissimilar propensities for secondary structure formation and overall structure of polypeptides. These changes affected the relative hydrophilicities of polypeptides above T t , where poly(VGPVG) is more hydrophilic than poly(VPGVG) with more extended conformation and larger surface area, which led to formation of strong interchain hydrogen bonds responsible for stabilization of the aggregated phase and the observed thermal hysteresis for poly(VGPVG).

Human LDL Structural Diversity Studied by IR Spectroscopy

PubMed Central

Fernández-Higuero, José A.; Salvador, Ana M.; Martín, Cesar; Milicua, José Carlos G.; Arrondo, José L. R.

2014-01-01

Lipoproteins are responsible for cholesterol traffic in humans. Low density lipoprotein (LDL) delivers cholesterol from liver to peripheral tissues. A misleading delivery can lead to the formation of atherosclerotic plaques. LDL has a single protein, apoB-100, that binds to a specific receptor. It is known that the failure associated with a deficient protein-receptor binding leads to plaque formation. ApoB-100 is a large single lipid-associated polypeptide difficulting the study of its structure. IR spectroscopy is a technique suitable to follow the different conformational changes produced in apoB-100 because it is not affected by the size of the protein or the turbidity of the sample. We have analyzed LDL spectra of different individuals and shown that, even if there are not big structural changes, a different pattern in the intensity of the band located around 1617 cm−1 related with strands embedded in the lipid monolayer, can be associated with a different conformational rearrangement that could affect to a protein interacting region with the receptor. PMID:24642788

Immunoprevention of Basal Cell Carcinomas with Recombinant Hedgehog-interacting Protein

PubMed Central

Vogt, Annika; Chuang, Pao-Tien; Hebert, Jennifer; Hwang, Jimmy; Lu, Ying; Kopelovich, Levy; Athar, Mohammad; Bickers, David R.; Epstein, Ervin H.

2004-01-01

Basal cell carcinomas (BCCs) are driven by abnormal hedgehog signaling and highly overexpress several hedgehog target genes. We report here our use of one of these target genes, hedgehog-interacting protein (Hip1), as a tumor-associated antigen for immunoprevention of BCCs in Ptch1+/− mice treated with ionizing radiation. Hip1 mRNA is expressed in adult mouse tissues at levels considerably lower than those in BCCs. Immunization with either of two large recombinant Hip1 polypeptides was well tolerated in Ptch1+/− mice, induced B and T cell responses detectable by enzyme-linked immunosorbent assay, Western blot, delayed type hypersensitivity, and enzyme-linked immunospot assay, and reduced the number of BCCs by 42% (P < 0.001) and 32% (P < 0.01), respectively. We conclude that immunization with proteins specifically up-regulated by hedgehog signaling may hold promise as a preventive option for patients such as those with the basal cell nevus syndrome who are destined to develop large numbers of BCCs. PMID:15024045

Genetic expansion of chaperonin-containing TCP-1 (CCT/TRiC) complex subunits yields testis-specific isoforms required for spermatogenesis in planarian flatworms.

PubMed

Counts, Jenna T; Hester, Tasha M; Rouhana, Labib

2017-12-01

Chaperonin-containing Tail-less complex polypeptide 1 (CCT) is a highly conserved, hetero-oligomeric complex that ensures proper folding of actin, tubulin, and regulators of mitosis. Eight subunits (CCT1-8) make up this complex, and every subunit has a homolog expressed in the testes and somatic tissue of the planarian flatworm Schmidtea mediterranea. Gene duplications of four subunits in the genomes of S. mediterranea and other planarian flatworms created paralogs to CCT1, CCT3, CCT4, and CCT8 that are expressed exclusively in the testes. Functional analyses revealed that each CCT subunit expressed in the S. mediterranea soma is essential for homeostatic integrity and survival, whereas sperm elongation defects were observed upon knockdown of each individual testis-specific paralog (Smed-cct1B; Smed-cct3B; Smed-cct4A; and Smed-cct8B), regardless of potential redundancy with paralogs expressed in both testes and soma (Smed-cct1A; Smed-cct3A; Smed-cct4B; and Smed-cct8A). Yet, no detriment was observed in the number of adult somatic stem cells (neoblasts) that maintain differentiated tissue in planarians. Thus, expression of all eight CCT subunits is required to execute the essential functions of the CCT complex. Furthermore, expression of the somatic paralogs in planarian testes is not sufficient to complete spermatogenesis when testis-specific paralogs are knocked down, suggesting that the evolution of chaperonin subunits may drive changes in the development of sperm structure and that correct CCT subunit stoichiometry is crucial for spermiogenesis. © 2017 Wiley Periodicals, Inc.

Texas Red transport across rat and dogfish shark (Squalus acanthias) choroid plexus

PubMed Central

Reichel, Valeska; Miller, David S.; Fricker, Gert

2008-01-01

Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na+-replacement, but was not affected by a 10-fold increase in buffer K+. In shark CP, Na+-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K+ medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the Mrp1 inhibitor MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals. PMID:18650317

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

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

Liu, Ye; Tang, Lan; Duan, Junxin

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

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

Spodsberg, Nikolaj

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

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

Lopez de Leon, Alfredo; Rey, Michael

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

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

Spodsberg, Nikolaj

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

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

Zhang, Yu; Liu, Ye; Duan, Junxin

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2012-09-18

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2010-12-14

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Harris, Paul [Carnation, WA; Lopez de Leon, Alfredo [Davis, CA; Rey, Micheal [Davis, CA; Ding, Hanshu [Davis, CA; Vlasenko, Elena [Davis, CA

2012-02-21

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2016-06-28

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2016-05-31

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2015-02-10

The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2016-02-23

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Ding, Hanshu

2013-04-30

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Hanshu, Ding

2012-10-30

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan

2015-11-20

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2015-01-27

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2014-10-21

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2015-03-10

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2017-05-02

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2015-03-31

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2015-07-14

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Brown, Kimberly [Elk Grove, CA; Harris, Paul [Carnation, WA; Lopez De Leon, Alfredo [Davis, CA; Merino, Sandra [West Sacremento, CA

2007-05-22

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Harris, Paul; Tang, Lan; Wu, Wenping

2013-11-19

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Morant, Marc D.; Harris, Paul

2015-10-13

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan; Harris, Paul; Wu, Wenping

2012-10-02

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Methods for using polypeptides having cellobiohydrolase activity

DOEpatents

Morant, Marc D; Harris, Paul

2016-08-23

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polynucleotides encoding polypeptides having beta-glucosidase activity

DOEpatents

Harris, Paul; Golightly, Elizabeth

2010-03-02

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2013-06-18

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

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

Spodsberg, Nikolaj

2016-12-13

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2014-10-14

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Tang, Lan; Henriksen, Svend Hostgaard Bang

2016-05-17

The present invention provides isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Nano polypeptide particles reinforced polymer composite fibers.

PubMed

Li, Jiashen; Li, Yi; Zhang, Jing; Li, Gang; Liu, Xuan; Li, Zhi; Liu, Xuqing; Han, Yanxia; Zhao, Zheng

2015-02-25

Because of the intensified competition of land resources for growing food and natural textile fibers, there is an urgent need to reuse and recycle the consumed/wasted natural fibers as regenerated green materials. Although polypeptide was extracted from wool by alkaline hydrolysis, the size of the polypeptide fragments could be reduced to nanoscale. The wool polypeptide particles were fragile and could be crushed down to nano size again and dispersed evenly among polymer matrix under melt extrusion condition. The nano polypeptide particles could reinforce antiultraviolet capability, moisture regain, and mechanical properties of the polymer-polypeptide composite fibers.

Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

PubMed

Yegnasubramanian, Srinivasan; Lin, Xiaohui; Haffner, Michael C; DeMarzo, Angelo M; Nelson, William G

2006-02-09

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

Immunogold staining procedure for the localisation of regulatory peptides.

PubMed

Varndell, I M; Tapia, F J; Probert, L; Buchan, A M; Gu, J; De Mey, J; Bloom, S R; Polak, J M

1982-01-01

The use of protein A- and IgG-conjugated colloidal gold staining methods for the immuno-localisation of peptide hormones and neurotransmitters at light- and electron microscope level are described and discussed. Bright-field and dark-ground illumination modes have been used to visualise the gold-labelled antigenic sites at the light microscope level. Immunogold staining procedures at the ultrastructural level using region-specific antisera have been adopted to localise specific molecular forms of peptides including gastrin (G17 and G34), glucagon and pro-glucagon, insulin and pro-insulin, in normal tissue and in tumours of the gastroenteropancreatic system. Similar methods have been used to demonstrate the heterogeneity of p-type nerves in the enteric nervous system. Vasoactive intestinal polypeptide (VIP) has been localised to granular sites (mean +/- S.D. granule diameter = 98 +/- 19 nm) in nerve terminals of the enteric plexuses and in tumour cells of diarrhoeogenic VIP-producing neoplasias (mean +/- S.D. granule diameter = 126 +/- 37 nm) using immunogold procedures applied to ultraviolet-cured ultrathin sections. Co-localisation of amines and peptides in carotid body type I cells and in chromaffin cells of normal adrenal medulla and phaeochromocytomas has also been demonstrated. Advantages of the immunogold procedures over alternative immunocytochemical techniques are discussed.

Differential DNA methylation of the meiosis-specific gene FKBP6 in testes of yak and cattle-yak hybrids.

PubMed

Li, B; Luo, H; Weng, Q; Wang, S; Pan, Z; Xie, Z; Wu, W; Liu, H; Li, Q

2016-12-01

FK506-binding protein 6 (FKBP6) is essential for meiosis during mammalian spermatogenesis. However, the molecular regulation of FKBP6 during spermatogenesis remains unclear. In the present study, we performed molecular characterization of the meiosis-specific gene FKBP6 in yak testes. Yak FKBP6 encodes a polypeptide of 295 amino acid residues with an FK506-binding domain (FKBP_C) and three tetratricopeptide repeat domains. The methylation level of the FKBP6 promoter in testes was significantly higher in cattle-yak with male sterility than in yak, and the FKBP6 promoter was methylated in liver tissues in which FKBP6 is not expressed. FKBP6 promoter activity was significantly decreased after treatment with the M.SssI methyltransferase in vitro. Furthermore, the FKBP6 gene was remarkably activated in bovine mammary epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine. Taken together, our results demonstrate for the first time that the FKBP6 promoter is differentially methylated in testes; together with the functional promoter analysis, this suggests that methylation of this promoter may contribute to cattle-yak male infertility. © 2016 Blackwell Verlag GmbH.

Bioinspired Star-Shaped Poly(l-lysine) Polypeptides: Efficient Polymeric Nanocarriers for the Delivery of DNA to Mesenchymal Stem Cells.

PubMed

Walsh, David P; Murphy, Robert D; Panarella, Angela; Raftery, Rosanne M; Cavanagh, Brenton; Simpson, Jeremy C; O'Brien, Fergal J; Heise, Andreas; Cryan, Sally-Ann

2018-05-07

The field of tissue engineering is increasingly recognizing that gene therapy can be employed for modulating in vivo cellular response thereby guiding tissue regeneration. However, the field lacks a versatile and biocompatible gene delivery platform capable of efficiently delivering transgenes to mesenchymal stem cells (MSCs), a cell type often refractory to transfection. Herein, we describe the extensive and systematic exploration of three architectural variations of star-shaped poly(l-lysine) polypeptide (star-PLL) with varying number and length of poly(l-lysine) arms as potential nonviral gene delivery vectors for MSCs. We demonstrate that star-PLL vectors are capable of self-assembling with pDNA to form stable, cationic nanomedicines. Utilizing high content screening, live cell imaging, and mechanistic uptake studies we confirm the intracellular delivery of pDNA by star-PLLs to MSCs is a rapid process, which likely proceeds via a clathrin-independent mechanism. We identify a star-PLL composition with 64 poly(l-lysine) arms and five l-lysine subunits per arm as a particularly efficient vector that is capable of delivering both reporter genes and the therapeutic transgenes bone morphogenetic protein-2 and vascular endothelial growth factor to MSCs. This composition facilitated a 1000-fold increase in transgene expression in MSCs compared to its linear analogue, linear poly(l-lysine). Furthermore, it demonstrated comparable transgene expression to the widely used vector polyethylenimine using a lower pDNA dose with significantly less cytotoxicity. Overall, this study illustrates the ability of the star-PLL vectors to facilitate efficient, nontoxic nucleic acid delivery to MSCs thereby functioning as an innovative nanomedicine platform for tissue engineering applications.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

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

Schnorr, Kirk; Kramer, Randall

2017-08-08

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

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

Tang, Lan; Liu, Ye; Duan, Junxin

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-xylosidase activity and polynucleotides encoding same

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

Zhang, Yu; Liu, Ye; Duan, Junxin

The present invention relates to isolated polypeptides having beta-xylosidase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo [Davis, CA; Ding, Hanshu [Davis, CA; Brown, Kimberly [Elk Grove, CA

2011-10-25

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Harris, Paul; Golightly, Elizabeth

2012-11-27

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

2016-06-14

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

2016-11-22

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan [Beijing, CN; Liu, Ye [Beijing, CN; Duan, Junxin [Beijing, CN; Zhang, Yu [Beijing, CN; Jorgensen, Christian Isak [Bagsvaerd, DK; Kramer, Randall [Lincoln, CA

2012-04-03

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Duan, Junxin [Beijing, CN; Liu, Ye [Beijing, CN; Tang, Lan [Beijing, CN; Wu, Wenping [Beijing, CN; Quinlan, Jason [Albany, CA; Kramer, Randall [Lincoln, CA

2012-03-27

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Joergensen, Christian; Kramer, Randall

2016-11-29

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Joergensen, Christian; Kramer, Randall

2014-09-16

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding the same

DOEpatents

Spodsberg, Nikolaj [Bagsvaed, DK

2014-01-07

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The inventino also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Wu, Wenping; Kramer, Randall

2014-10-21

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Schnorr, Kirk; Kramer, Randall

2016-04-05

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Harris, Paul [Carnation, WA; Golightly, Elizabeth [Reno, NV

2007-07-17

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

2013-10-29

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Harris, Paul [Carnation, WA; Golightly, Elizabeth [Reno, NV

2011-06-14

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

2013-04-16

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Duan, Junxin; Tang, Lan; Liu, Ye; Wu, Wenping; Quinlan, Jason; Kramer, Randall

2013-06-18

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Schnorr, Kirk; Kramer, Randall

2016-08-09

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

The Expanding World of Tissue Engineering: The Building Blocks and New Applications of Tissue Engineered Constructs

PubMed Central

Zorlutuna, Pinar; Vrana, Nihal Engin; Khademhosseini, Ali

2013-01-01

The field of tissue engineering has been growing in the recent years as more products have made it to the market and as new uses for the engineered tissues have emerged, motivating many researchers to engage in this multidisciplinary field of research. Engineered tissues are now not only considered as end products for regenerative medicine, but also have emerged as enabling technologies for other fields of research ranging from drug discovery to biorobotics. This widespread use necessitates a variety of methodologies for production of tissue engineered constructs. In this review, these methods together with their non-clinical applications will be described. First, we will focus on novel materials used in tissue engineering scaffolds; such as recombinant proteins and synthetic, self assembling polypeptides. The recent advances in the modular tissue engineering area will be discussed. Then scaffold-free production methods, based on either cell sheets or cell aggregates will be described. Cell sources used in tissue engineering and new methods that provide improved control over cell behavior such as pathway engineering and biomimetic microenvironments for directing cell differentiation will be discussed. Finally, we will summarize the emerging uses of engineered constructs such as model tissues for drug discovery, cancer research and biorobotics applications. PMID:23268388

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2012-10-16

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Dotson, William D.; Greenier, Jennifer; Ding, Hanshu

2007-09-18

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated nucleic acids encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acids as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Wu, Wenping; Kramer, Randall

2013-11-19

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2014-09-30

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2017-09-05

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2010-06-22

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding the same

DOEpatents

Brown, Kimberly; Harris, Paul

2013-12-17

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2016-08-09

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

2013-12-24

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Cellulases, nucleic acids encoding them and methods for making and using them

DOEpatents

Blum, David; Gemsch Cuenca, Joslin; Dycaico, Mark

2013-04-23

This invention relates to molecular and cellular biology and biochemistry. In one aspect, the invention provides polypeptides having cellulase activity, e.g., endoglucanase, cellobiohydrolase, mannanase and/or .beta.-glucosidase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides cellulase activity, e.g., endoglucanase, cellobiohydrolase, mannanase and/or .beta.-glucosidase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts.

Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

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

Wogulis, Mark; Sweeney, Matthew; Heu, Tia

The present invention relates to chimeric GH61 polypeptides having cellulolytic enhancing activity. The present invention also relates to polynucleotides encoding the chimeric GH61 polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the chimeric GH61 polypeptides.

Recombinant host cells and nucleic acid constructs encoding polypeptides having cellulolytic enhancing activity

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

Schnorr, Kirk; Kramer, Randall

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

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

Semelr, B.L.; Anderson, C.W.; Hanecak, R.

1982-02-01

A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNA polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNAmore » sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNA replication complex.« less

Response to reactive nitrogen intermediates in Mycobacterium tuberculosis: induction of the 16-kilodalton alpha-crystallin homolog by exposure to nitric oxide donors.

PubMed

Garbe, T R; Hibler, N S; Deretic, V

1999-01-01

In contrast to the apparent paucity of Mycobacterium tuberculosis response to reactive oxygen intermediates, this organism has evolved a specific response to nitric oxide challenge. Exposure of M. tuberculosis to NO donors induces the synthesis of a set of polypeptides that have been collectively termed Nox. In this work, the most prominent Nox polypeptide, Nox16, was identified by immunoblotting and by N-terminal sequencing as the alpha-crystallin-related, 16-kDa small heat shock protein, sHsp16. A panel of chemically diverse donors of nitric oxide, with the exception of nitroprusside, induced sHsp16 (Nox16). Nitroprusside, a coordination complex of Fe2+ with a nitrosonium (NO+) ion, induced a 19-kDa polypeptide (Nox19) homologous to the nonheme bacterial ferritins. We conclude that the NO response in M. tuberculosis is dominated by increased synthesis of the alpha-crystallin homolog sHsp16, previously implicated in stationary-phase processes and found in this study to be a major M. tuberculosis protein induced upon exposure to reactive nitrogen intermediates.

Mechanically Controlled Electron Transfer in a Single-Polypeptide Transistor

NASA Astrophysics Data System (ADS)

Sheu, Sheh-Yi; Yang, Dah-Yen

2017-01-01

Proteins are of interest in nano-bio electronic devices due to their versatile structures, exquisite functionality and specificity. However, quantum transport measurements produce conflicting results due to technical limitations whereby it is difficult to precisely determine molecular orientation, the nature of the moieties, the presence of the surroundings and the temperature; in such circumstances a better understanding of the protein electron transfer (ET) pathway and the mechanism remains a considerable challenge. Here, we report an approach to mechanically drive polypeptide flip-flop motion to achieve a logic gate with ON and OFF states during protein ET. We have calculated the transmission spectra of the peptide-based molecular junctions and observed the hallmarks of electrical current and conductance. The results indicate that peptide ET follows an NC asymmetric process and depends on the amino acid chirality and α-helical handedness. Electron transmission decreases as the number of water molecules increases, and the ET efficiency and its pathway depend on the type of water-bridged H-bonds. Our results provide a rational mechanism for peptide ET and new perspectives on polypeptides as potential candidates in logic nano devices.

Ozone-Induced Alterations in the Accumulation of Newly Synthesized Proteins in Leaves of Maize.

PubMed Central

Pino, M. E.; Mudd, J. B.; Bailey-Serres, J.

1995-01-01

We examined the response of leaves of 3-week-old maize (Zea mays L.) to short-term (5 h) fumigation with O3-enriched air (0, 0.12, 0.24, or 0.36 [mu]L/L). Older leaves and leaf tissue developed more severe visible damage at higher external O3 concentrations. To investigate the immediate effect of O3 exposure on the accumulation of newly synthesized leaf proteins, leaves were labeled with [35S]methionine after 2 h and fumigated for an additional 3 h. O3-induced alterations of leaf proteins were observed in a concentration-dependent manner. There was a significant decrease in [35S]methionine incorporation into protein at the highest O3 concentration. Developmental differences in accumulation of de novo-synthesized leaf proteins were observed when the leaf tip, middle, and basal sections were labeled under 0 [mu]L/L O3, and additional changes were apparent upon exposure to increasing O3 concentrations. Changes in leaf protein synthesis were observed in the absence of visible leaf injury. Subcellular fractionation revealed O3-induced alterations in soluble and membrane-associated proteins. A number of thylakoid membrane-associated proteins showed specific increases in response to O3 fumigation. In contrast, the synthesis of a 32-kD polypeptide associated with thylakoid membranes was reduced in response to O3 fumigation in parallel with reduced incorporation of [35S]methionine into protein. Immunoprecipitation identified this polypeptide as the D1 protein of photosystem II. A reduction in the accumulation of newly synthesized D1 could have consequences for the efficiency of photosynthesis and other cellular processes. PMID:12228510

Mutations in TUBB8 and Human Oocyte Meiotic Arrest

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

Feng, Ruizhi; Sang, Qing; Kuang, Yanping

BACKGROUND: We present that human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, earlymore » embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.« less

Mutations in TUBB8 and Human Oocyte Meiotic Arrest

DOE PAGES

Feng, Ruizhi; Sang, Qing; Kuang, Yanping; ...

2016-01-21

BACKGROUND: We present that human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, earlymore » embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.« less

Glycosphingolipid-facilitated membrane insertion and internalization of cobra cardiotoxin. The sulfatide.cardiotoxin complex structure in a membrane-like environment suggests a lipid-dependent cell-penetrating mechanism for membrane binding polypeptides.

PubMed

Wang, Chia-Hui; Liu, Jyung-Hurng; Lee, Shao-Chen; Hsiao, Chwan-Deng; Wu, Wen-Guey

2006-01-06

Cobra cardiotoxins, a family of basic polypeptides having lipid- and heparin-binding capacities similar to the cell-penetrating peptides, induce severe tissue necrosis and systolic heart arrest in snakebite victims. Whereas cardiotoxins are specifically retained on the cell surface via heparan sulfate-mediated processes, their lipid binding ability appears to be responsible, at least in part, for cardiotoxin-induced membrane leakage and cell death. Although the exact role of lipids involved in toxin-mediated cytotoxicity remains largely unknown, monoclonal anti-sulfatide antibody O4 has recently been shown to inhibit the action of CTX A3, the major cardiotoxin from Taiwan cobra venom, on cardiomyocytes by preventing cardiotoxin-induced membrane leakage and CTX A3 internalization into mitochondria. Here, we show that anti-sulfatide acts by blocking the binding of CTX A3 to the sulfatides in the plasma membrane to prevent sulfatide-dependent CTX A3 membrane pore formation and internalization. We also describe the crystal structure of a CTX A3-sulfatide complex in a membrane-like environment at 2.3 angstroms resolution. The unexpected orientation of the sulfatide fatty chains in the structure allows prediction of the mode of toxin insertion into the plasma membrane. CTX A3 recognizes both the headgroup and the ceramide interfacial region of sulfatide to induce a lipid conformational change that may play a key role in CTX A3 oligomerization and cellular internalization. This proposed lipid-mediated toxin translocation mechanism may also shed light on the cellular uptake mechanism of the amphiphilic cell-penetrating peptides known to involve multiple internalization pathways.

Determination of the Gene Sequence of Poliovirus with Pactamycin

PubMed Central

Summers, D. F.; Maizel, J. V.

1971-01-01

By examination of the virus-specific polypeptides formed after the addition of pactamycin, an inhibitor of protein chain initiation, to infected cells, it has been possible to tentatively locate the virus coat proteins at the amino terminus of the large, virus-specific protein precursor, and, therefore, to assign the coat protein cistron to the 5′ end of the RNA. PMID:4330946

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

PubMed

Crusius, Kerstin; Finster, Silke; McClary, John; Xia, Wei; Larsen, Brent; Schneider, Douglas; Lu, Hong-Tao; Biancalana, Sara; Xuan, Jian-Ai; Newton, Alicia; Allen, Debbie; Bringmann, Peter; Cobb, Ronald R

2006-10-01

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

Multifunctional quantum dot-polypeptide hybrid nanogel for targeted imaging and drug delivery

NASA Astrophysics Data System (ADS)

Yang, Jie; Yao, Ming-Hao; Wen, Lang; Song, Ji-Tao; Zhang, Ming-Zhen; Zhao, Yuan-Di; Liu, Bo

2014-09-01

A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron microscopy, and dynamic light scattering measurements. Hydrophobic and hydrophilic drugs can be simultaneously loaded in a QD-polypeptide nanogel. In vitro drug release of drug-loaded QD-polypeptide nanogels varies strongly with temperature, pH, and competitors. A drug-loaded QD-polypeptide nanogel with an arginine-glycine-aspartic acid (RGD) motif exhibited efficient receptor-mediated endocytosis in αvβ3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy and flow cytometry. In contrast, non-targeted QD-polypeptide nanogels revealed minimal binding and uptake in HeLa cells. Compared with the original QDs, the QD-polypeptide nanogels showed lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Furthermore, the cytotoxicity of the targeted QD-polypeptide nanogel was lower for normal NIH 3T3 cells than that for HeLa cancer cells. These results demonstrate that the integration of imaging and drug delivery functions in a single QD-polypeptide nanogel has the potential for application in cancer diagnosis, imaging, and therapy.A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron microscopy, and dynamic light scattering measurements. Hydrophobic and hydrophilic drugs can be simultaneously loaded in a QD-polypeptide nanogel. In vitro drug release of drug-loaded QD-polypeptide nanogels varies strongly with temperature, pH, and competitors. A drug-loaded QD-polypeptide nanogel with an arginine-glycine-aspartic acid (RGD) motif exhibited efficient receptor-mediated endocytosis in αvβ3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy and flow cytometry. In contrast, non-targeted QD-polypeptide nanogels revealed minimal binding and uptake in HeLa cells. Compared with the original QDs, the QD-polypeptide nanogels showed lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Furthermore, the cytotoxicity of the targeted QD-polypeptide nanogel was lower for normal NIH 3T3 cells than that for HeLa cancer cells. These results demonstrate that the integration of imaging and drug delivery functions in a single QD-polypeptide nanogel has the potential for application in cancer diagnosis, imaging, and therapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03058c

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc

2014-01-14

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase, or beta-glucosidase activity and isolated polynucleotides encoding polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Synthesis of antireflective silica coatings through the synergy of polypeptide layer-by-layer assemblies and biomineralization

NASA Astrophysics Data System (ADS)

Lee, Yung-Lun; Lin, Ting-Xuan; Hsu, Feng-Ming; Jan, Jeng-Shiung

2016-01-01

We report a versatile approach to synthesize silica coatings with antireflective (AR) characteristics through the combination of a layer-by-layer (LbL) assembly technique and biomineralization. LbL assembled decanoyl-modified poly(l-lysine)/poly(l-glutamic acid) (PLL-g-Dec/PLGA) multilayer films were used as templates for silica mineralization, followed by calcination. The specific deposition of silica onto the LbL polypeptide assemblies through amine-catalyzed polycondensation resulted in silica coatings that exhibited the transcription of the nano-/microstructured polypeptide films and their film thickness and porosity can be tuned by varying the number of bilayers, degree of substitution, and PLL molecular weight. AR silica coatings exhibiting more than 6% increase in transmittance in the near UV/visible spectral range can be obtained at an optimized refractive index, thickness, and surface roughness. The abrasion test showed that the silica coatings exhibited sufficient structural durability due to continuous silica nanostructures and low surface roughness. This study demonstrated that nanostructured thin films can be synthesized for AR coatings using the synergy between the LbL assembly technique and biomineralization.We report a versatile approach to synthesize silica coatings with antireflective (AR) characteristics through the combination of a layer-by-layer (LbL) assembly technique and biomineralization. LbL assembled decanoyl-modified poly(l-lysine)/poly(l-glutamic acid) (PLL-g-Dec/PLGA) multilayer films were used as templates for silica mineralization, followed by calcination. The specific deposition of silica onto the LbL polypeptide assemblies through amine-catalyzed polycondensation resulted in silica coatings that exhibited the transcription of the nano-/microstructured polypeptide films and their film thickness and porosity can be tuned by varying the number of bilayers, degree of substitution, and PLL molecular weight. AR silica coatings exhibiting more than 6% increase in transmittance in the near UV/visible spectral range can be obtained at an optimized refractive index, thickness, and surface roughness. The abrasion test showed that the silica coatings exhibited sufficient structural durability due to continuous silica nanostructures and low surface roughness. This study demonstrated that nanostructured thin films can be synthesized for AR coatings using the synergy between the LbL assembly technique and biomineralization. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06948c

Moisture absorption and retention properties, and activity in alleviating skin photodamage of collagen polypeptide from marine fish skin.

PubMed

Hou, Hu; Li, Bafang; Zhang, Zhaohui; Xue, Changhu; Yu, Guangli; Wang, Jingfeng; Bao, Yuming; Bu, Lin; Sun, Jiang; Peng, Zhe; Su, Shiwei

2012-12-01

Collagen polypeptides were prepared from cod skin. Moisture absorption and retention properties of collagen polypeptides were determined at different relative humidities. In addition, the protective effects of collagen polypeptide against UV-induced damage to mouse skin were evaluated. Collagen polypeptides had good moisture absorption and retention properties and could alleviate the damage induced by UV radiation. The action mechanisms of collagen polypeptide mainly involved enhancing immunity, reducing the loss of moisture and lipid, promoting anti-oxidative properties, inhibiting the increase of glycosaminoglycans, repairing the endogenous collagen and elastin protein fibres, and maintaining the ratio of type III to type I collagen. Copyright © 2012 Elsevier Ltd. All rights reserved.

Expression of PACAP and PAC1 Receptor in Normal Human Thyroid Gland and in Thyroid Papillary Carcinoma.

PubMed

Bardosi, Sebastian; Bardosi, Attila; Nagy, Zsuzsanna; Reglodi, Dora

2016-10-01

Pituitary adenylate cyclase activating polypeptide (PACAP) belongs to the vasoactive intestinal peptide-secretin-glucagon peptide family, isolated first from ovine hypothalamus. The diverse physiological effects of PACAP are known mainly from animal experiments, including several actions in endocrine glands. Alteration of PACAP expression has been shown in several tumors, but changes in expression of PACAP and its specific PAC1 receptor in human thyroid gland pathologies have not yet been investigated. Therefore, the aim of the present study was to investigate expression of PACAP and its PAC1 receptor in human thyroid papillary carcinoma, the most common endocrine malignant tumor. PACAP and PAC1 receptor expressions were investigated from thyroid gland samples of patients with papillary carcinomas. The staining intensity of follicular epithelial cells and thyroid colloid of tumor tissue was compared to that of tumor-free tissue in the same thyroid glands in a semi-quantitative way. Our results reveal that both PACAP(-like) and PAC1 receptor(-like) immunoreactivities are altered in papillary carcinoma. Stronger PACAP immunoreactivity was observed in active follicles. Colloidal PACAP immunostaining was either lacking or very weak, and more tumorous cells displayed strong apical immunoreactivity. Regarding PAC1 receptor, cells of the normal thyroid tissue showed strong granular expression, which was lacking in the tumor cells. The cytoplasm of tumor cells displayed weak, minimal staining, while in a few tumor cells we observed strong PAC1 receptor expression. This pattern was similar to that observed in the PACAP expression, but fewer in number. In summary, we showed alteration of PACAP and PAC1 receptor expression in human thyroid papillary carcinoma, indicating that PACAP regulation is disturbed in tumorous tissue of the thyroid gland. The exact role of PACAP in thyroid tumor growth should be further explored.

Polypeptide having or assisting in carbohydrate material degrading activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

2016-02-16

The invention relates to a polypeptide which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having beta-glucosidase activity and uses thereof

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

Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well asmore » the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.« less

Polypeptide having swollenin activity and uses thereof

DOEpatents

Schoonneveld-Bergmans, Margot Elizabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica D; Damveld, Robbertus Antonius

2015-11-04

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having beta-glucosidase activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel; Damveld, Robbertus Antonius

2015-09-01

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having cellobiohydrolase activity and uses thereof

DOEpatents

Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Roubos, Johannes Andries; Los, Alrik Pieter

2015-09-15

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having acetyl xylan esterase activity and uses thereof

DOEpatents

Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

2015-10-20

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having carbohydrate degrading activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica Diana; Damveld, Robbertus Antonius

2015-08-18

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Mosaic HIV envelope immunogenic polypeptides

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

Korber, Bette T. M.; Gnanakaran, S.; Perkins, Simon

Disclosed herein are mosaic HIV envelope (Env) polypeptides that can elicit an immune response to HIV (such as cytotoxic T cell (CTL), helper T cell, and/or humoral responses). Also disclosed are sets of the disclosed mosaic Env polypeptides, which include two or more (for example, three) of the polypeptides. Also disclosed herein are methods for treating or inhibiting HIV in a subject including administering one or more of the disclosed immunogenic polypeptides or compositions to a subject infected with HIV or at risk of HIV infection. In some embodiments, the methods include inducing an immune response to HIV in amore » subject comprising administering to the subject at least one (such as two, three, or more) of the immunogenic polypeptides or at least one (such as two, three, or more) nucleic acids encoding at least one of the immunogenic polypeptides disclosed herein.« less

Toxicity study of isolated polypeptide from wool hydrolysate.

PubMed

Li, Jiashen; Li, Yi; Zhang, Yu; Liu, Xuan; Zhao, Zheng; Zhang, Jing; Han, Yanxia; Zhou, Dangxia

2013-07-01

The cytotoxicity of wool polypeptide has been evaluated by both cell and animal models. Wool was dissolved in sodium hydroxide solution, the pH value of the solution was adjusted to 5.55 and the precipitate was harvested as wool polypeptide. The spray-dried polypeptide was collected as powders and characterized by SEM, FTIR and TG-DSC. The cell culturing results showed that wool polypeptide had no obvious negative effect on cell viability in vitro. Both acute oral toxicity and subacute 30-day oral toxicology studies showed that wool polypeptide had no influence on body weight, feed consumption, blood chemistry, and hematology at any dose levels. There were no treatment related findings on gross or detailed necroscopy, organ weights, organ/body weight ratios and histology. Our study indicated the absence of toxicity in wool polypeptide and supported its safe use as a food ingredient or drug carrier. Copyright © 2013 Elsevier Ltd. All rights reserved.

Eukaryotic polypeptide elongation system and its sensitivity to the inhibitory substances of plant origin.

PubMed

Gałasiński, W

1996-05-01

The structural and functional characteristics of the elongation system (ribosomes and elongation factors) are presented. The immunochemical and diagnostic meaning of the ribosome investigations is considered. Evidence of the participation of ribosomes in the first step of protein glycosylation is presented. The heterogeneous elongation factor eEF-1, isolated from Guerin epithelioma, can be separated into three fractions: one of them functionally corresponds to EF-1 alpha, the second on to EF-1 beta gamma, and the third is an unidentified, active aggregate named EF-1B, which contains the subunit forms EF-1 alpha and EF-1 beta gamma, and other polypeptides showing protein kinase activity. The aggregate EF-1B can be autophosphorylated, while the subunit forms EF-1 alpha and EF-1 beta gamma can neither become autophosphorylated nor phosphorylate other polypeptides. The subunit form EF-beta gamma consists from two polypeptides of 32 and 51 kDa, corresponding to other eukaryotic beta and gamma polypeptides, respectively. EF-1 beta gamma is thermostable and protects against thermal inactivation of EF-1 alpha in the EF-1 alpha-EF-1 beta gamma complex. Pure eEF-2 preparations isolated from normal and neoplastic tissues show different structural features. The existence of eEF-2 in multiple forms, differing in molecular mass, have been found. The eEF-2 with molecular weight of about 100 kDa can be phosphorylated, while eEF-2 of about 65 kDa was not phosphorylated by protein kinase eEF-2. The phosphorylated eEF-2 lost its activity, and this effect was reversed by dephosphorylation. The eEF-2 (65 kDa) was isolated from the active polyribosomes, and it may directly participate in the translocation step of the peptide elongation. It was noted that the components of elongation system can be inhibited, in separate steps, by the substances isolated from various sources of plant origin. Alkaloids emetine and cepheline, cardiac remedy digoxin, saponin glycoside, and its aglycon directly inactivated ribosomes. Quercetin inhibited eEF-1 activity by directly influencing its subunit form EF-1 alpha. eEF-2 was shown to be a target site of the inhibitory action of the glycoside isolated from Melissa officinalis leaves.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc Dominique

2014-10-14

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Thermal and acid tolerant beta-xylosidases, genes encoding, related organisms, and methods

DOEpatents

Thompson, David N [Idaho Falls, ID; Thompson, Vicki S [Idaho Falls, ID; Schaller, Kastli D [Ammon, ID; Apel, William A [Jackson, WY; Lacey, Jeffrey A [Idaho Falls, ID; Reed, David W [Idaho Falls, ID

2011-04-12

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof are provided. Further provided are methods of at least partially degrading xylotriose and/or xylobiose using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof.

Brachytherapy Using Elastin-Like Polypeptides with (131)I Inhibit Tumor Growth in Rabbits with VX2 Liver Tumor.

PubMed

Liu, Xinpei; Shen, Yiming; Zhang, Xuqian; Lin, Rui; Jia, Qiang; Chang, Yixiang; Liu, Wenge; Liu, Wentian

2016-10-01

Brachytherapy is a targeted type of radiotherapy utilized in the treatment of cancers. Elastin-like polypeptides are a unique class of genetically engineered peptide polymers that have several attractive properties for brachytherapy. To explore the feasibility and application of brachytherapy for VX2 liver tumor using elastin-like polypeptides with (131)I so as to provide reliable experimental evidence for a new promising treatment of liver cancer. Elastin-like polypeptide as carrier was labeled with (131)I using the iodogen method. Ten eligible rabbits with VX2 liver tumor were randomly divided into the treatment group (n = 5) and control group (n = 5). The treatment group received brachytherapy using elastin-like polypeptide with (131)I, and in the control group, elastin-like polypeptide was injected into the VX2 liver tumor as a control. Periodic biochemical and imaging surveillances were required to assess treatment efficacy. The stability of elastin-like polypeptide with (131)I in vitro was maintained at over 96.8 % for 96 h. Biochemistry and imaging indicated brachytherapy using elastin-like polypeptide with (131)I for liver tumor can improve liver function and inhibit tumor growth (P < 0.05). Elastin-like polypeptide can be an ideal carrier of (131)I and have high labeling efficiency, radiochemical purity and stability. Brachytherapy using elastin-like polypeptide with (131)I for liver tumor is a useful therapy that possesses high antitumor efficacy advantages.

Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating.

PubMed

Nishi, Naoto; Miyamoto, Takuya; Waku, Tomonori; Tanaka, Naoki; Hagiwara, Yoshimichi

2016-01-01

The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1-5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure.

Topography of succinate dehydrogenase in the mitochondrial inner membrane. A study using limited proteolysis and immunoblotting.

PubMed Central

Clarkson, G H; Neagle, J; Lindsay, J G

1991-01-01

The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest that this polypeptide interacts with the matrix side of the inner membrane via two distinct domains; these are detected as stable membrane-associated fragments of 32,000 Mr and 27,000 Mr after treatment of submitochondrial particles with papain or proteinase K, although the 27,000-Mr fragment can be degraded further to low-Mr peptides with trypsin or alpha-chymotrypsin. A stable 32,000-34,000-Mr fragment is generated by a variety of specific and non-specific proteinases, indicating that it may be embedded largely within the lipid bilayer, or is inaccessible to proteolytic attack owing to its proximity to the surface of the intact membrane, possibly interacting with the hydrophobic membrane anchoring polypeptides of the succinate: ubiquinone reductase complex. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1996968

Mucinous colorectal adenocarcinoma with signet-ring cells: immunohistochemical and ultrastructural study.

PubMed

Seretis, E; Konstantinidou, A; Arnogiannakis, N; Xinopoulos, D; Voloudakis-Baltatzis, I E

2010-12-01

A primary mucinous colorectal adenocarcinoma tissue with signet-ring cells, as revealed after histological evaluation, was examined ultrastructurally. The authors also analyzed the immunohistochemical data of the tissue for serotonin, vasoactive intestinal polypeptide (VIP), bombesin, somatostatin, and glucagon, using the peroxidase anti-peroxidase (PAP) method and the immunogold labeling method for light and electron microscope, respectively. Electron microscopically mucinous adenocarcinoma was characterized by the formation of small lumen. Adenocarcinoma cells were full of mucous granules of varying electron density, providing a good environment for the tumor cells to grow. They also exhibited a significant loss of microvilli and intracytoplasmic junctions, which could allow the cells to disseminate. Signet-ring cells were located in the basal site of the ducts or in the lamina propria and appeared neoplastic, with mucin accumulation intracellularly and an eccentric crescent-shaped nucleus. The cytoplasmic organelles were decreased and at the periphery of the cell. The PAP method demonstrated that these cells were strongly positive for bombesin and also positive for vasointestinal polypeptide (VIP). The immunogold method detected bombesin immunoreactivity in the vacuoles as well as in other cytoplasmic membranes, whereas VIP was localized mainly in the plasma membrane. The location of signet-ring cells combined with the immunoreactivity for bombesin and VIP indicated that signet-ring cells were of neuroendocrine origin and probably dedifferentiated enterochromaffin-like endocrine cells. These findings have implications for understanding the biological behavior of these composite malignant tumors and could help in the knowledge of the origin of signet-ring cells.

Imaging mass spectrometry of endogenous polypeptides and secondary metabolites from galls induced by root-knot nematodes in tomato roots.

PubMed

Barbosa, Eder Alves; Bonfim Junior, Mauro Ferreira; Bloch, Carlos; Rocha, Thales Lima; Engler, Gilbert; de Almeida Engler, Janice

2018-04-17

Nematodes are devastating pests that infect most cultivated plant species and cause considerable agricultural losses worldwide. The understanding of metabolic adjustments induced during plant-nematode interaction is crucial to generate resistant plants or to select more efficient molecules to fight against this pest. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used herein for in situ detection and mapping endogenous polypeptides and secondary metabolites from nematode-induced gall tissue. One of the major critical features of this technique is sample preparation, mainly the generation of intact sections of plant cells with their rigid cell walls and vacuolated cytoplasm. Our experimental settings allowed us to obtain sections without contamination of exogenous ions or diffusion of molecules and to map the differential presence of low and high molecular weight ions in uninfected roots compared to nematode-induced galls. We predict the presence of lipids in both uninfected roots and galls, which was validated by MALDI-TOF-MS/MS and high-resolution mass spectrometry analysis of lipid extracts. Based on the isotopic ion distribution profile, both esters and glycerophospholipids were predicted compounds and may be playing an important role in gall development. Our results indicate that the MALDI-MSI technology is a promising tool to identify secondary metabolites as well as peptides and proteins in complex plant tissues like galls to decipher molecular processes responsible for infection and maintenance of these feeding sites during nematode parasitism.

Multitriggered Tumor-Responsive Drug Delivery Vehicles Based on Protein and Polypeptide Coassembly for Enhanced Photodynamic Tumor Ablation.

PubMed

Zhang, Ning; Zhao, Fenfang; Zou, Qianli; Li, Yongxin; Ma, Guanghui; Yan, Xuehai

2016-11-01

Tumor-responsive nanocarriers are highly valuable and demanded for smart drug delivery particularly in the field of photodynamic therapy (PDT), where a quick release of photosensitizers in tumors is preferred. Herein, it is demonstrated that protein-based nanospheres, prepared by the electrostatic assembly of proteins and polypeptides with intermolecular disulfide cross-linking and surface polyethylene glycol coupling, can be used as versatile tumor-responsive drug delivery vehicles for effective PDT. These nanospheres are capable of encapsulation of various photosensitizers including Chlorin e6 (Ce6), protoporphyrin IX, and verteporfin. The Chlorin e6-encapsulated nanospheres (Ce6-Ns) are responsive to changes in pH, redox potential, and proteinase concentration, resulting in multitriggered rapid release of Ce6 in an environment mimicking tumor tissues. In vivo fluorescence imaging results indicate that Ce6-Ns selectively accumulate near tumors and the quick release of Ce6 from Ce6-Ns can be triggered by tumors. In tumors the fluorescence of released Ce6 from Ce6-Ns is observed at 0.5 h postinjection, while in normal tissues the fluorescence appeared at 12 h postinjection. Tumor ablation is demonstrated by in vivo PDT using Ce6-Ns and the biocompatibility of Ce6-Ns is evident from the histopathology imaging, confirming the enhanced in vivo PDT efficacy and the biocompatibility of the assembled drug delivery vehicles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

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

Takegami, T.; Semler, B.L.; Anderson, C.W.

1983-01-01

The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have alsomore » revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 andf P3 regions of the polyprotein. Sequence analysis by stepwise Edman degradation show that these proteins are 3b/9 (M/sub r/77,000) and X/9 (M/sub r/50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9.« less

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

PubMed Central

Aleksunes, Lauren M.

2010-01-01

Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting β polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) α and β] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory factors that influence transporter expression and function, including transcriptional activation and post-translational modifications as well as subcellular trafficking. Sex differences, ontogeny, and pharmacological and toxicological regulation of transporters are also addressed. Transporters are important transmembrane proteins that mediate the cellular entry and exit of a wide range of substrates throughout the body and thereby play important roles in human physiology, pharmacology, pathology, and toxicology. PMID:20103563

Polypeptides having xylanase activity and polynucleotides encoding same

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

Spodsberg, Nikolaj; Shaghasi, Tarana

The present invention relates to polypeptides having xylanase activity, catalytic domains, and carbohydrate binding domains, and polynucleotides encoding the polypeptides, catalytic domains, and carbohydrate binding domains. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, and carbohydrate binding domains.

Polypeptides having endoglucanase activity and polynucleotides encoding same

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

Spodsberg, Nikolaj; Shagasi, Tarana

The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj; Shagasi, Tarana

2015-06-30

The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

Thermal and acid tolerant beta xylosidases, arabinofuranosidases, genes encoding, related organisms, and methods

DOEpatents

Thompson, David N; Thompson, Vicki S; Schaller, Kastli D; Apel, William A; Reed, David W; Lacey, Jeffrey A

2013-04-30

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof are provided. Further provided are methods of at least partially degrading xylotriose, xylobiose, and/or arabinofuranose-substituted xylan using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof.

Polypeptides having beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc Dominique

2014-05-06

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

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

Stringer, Mary Ann; McBrayer, Brett

2016-11-29

The present invention relates to isolated polypeptides having cellobiohydrolase activity, catalytic domains, and cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains, and cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, or cellulose binding domains.

Polypeptides having beta-glucosidase activity and beta-xylosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc Dominique

2014-05-06

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and beta-xylosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc Dominique

2014-04-29

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Thermally targeted delivery of chemotherapeutics and anti-cancer peptides by elastin-like polypeptide.

PubMed

Raucher, Drazen; Massodi, Iqbal; Bidwell, Gene L

2008-03-01

Current chemotherapy treatment of solid tumors is limited due to a lack of specific delivery of the drugs to the tumor, leading to systemic toxicity. Therefore, it is necessary to develop targeted cancer therapies and tumor-targeted drug carriers. The authors review the development of elastin-like polypeptide (ELP) as a potential carrier for thermally targeted delivery of therapeutics. The authors searched Medline for articles concerning the application of ELP as a drug delivery vector for small molecule drugs and therapeutic peptides. ELP has been demonstrated to be a promising thermally targeted carrier. Further examination of the in vivo biodistribution and efficacy will provide the necessary data to advance ELP technology toward the ultimate goal of human therapeutics.

Oral immunogenicity of tomato-derived sDPT polypeptide containing Corynebacterium diphtheriae, Bordetella pertussis and Clostridium tetani exotoxin epitopes.

PubMed

Soria-Guerra, Ruth E; Rosales-Mendoza, Sergio; Moreno-Fierros, Leticia; López-Revilla, Rubén; Alpuche-Solís, Angel G

2011-03-01

DPT vaccine, designed to immunize against diphtheria, pertussis, and tetanus, has been shown to be effective in humans. Nevertheless, dissatisfaction with the whole-cell preparations is due to the reactogenicity, which has to lead to the development of new safer formulations. Previously, we described the expression in tomato of a plant-optimized synthetic gene encoding the recombinant polypeptide sDPT, containing mainly immunoprotective epitopes of the diphtheria, pertussis and tetanus exotoxins and two adjuvants. In this study, we examined whether the ingestion of tomato-derived sDPT protein induces specific antibodies in mice after three weekly doses scheme. A positive group immunized with DPT toxoids was included. Specific antibody levels were assessed in serum, gut and lung. Sera tested for IgG antibody response to pertussis, tetanus and diphtheria toxin showed responses to the foreign antigens; interestingly, the response to diphtheria epitope was similar to those observed in the positive group. We found higher IgG1 than IgG2a responses in serum. A modest IgG response was observed in the tracheopulmonary fluid. High response of IgA against tetanus toxin was evident in gut, which was statistically comparable to that obtained in the positive group. The levels of response in these groups were higher than those in mice that received wild-type tomato. These findings support the concept of using transgenic tomatoes expressing sDPT polypeptide as model for edible vaccine against diphtheria, pertussis, and tetanus.

Immunochemical Proof that a Novel Rearranging Gene Encodes the T Cell Receptor δ Subunit

NASA Astrophysics Data System (ADS)

Band, Hamid; Hochstenbach, Frans; McLean, Joanne; Hata, Shingo; Krangel, Michael S.; Brenner, Michael B.

1987-10-01

The T cell receptor (TCR) δ protein is expressed as part of a heterodimer with TCR γ , in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR δ was produced that binds specifically to the surface of several TCR γ δ cell lines and immunoprecipitates the TCR γ δ as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR δ subunit alone after chain separation. A candidate human TCR δ complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR γ δ cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR δ . This complementary DNA clone thus corresponds to the gene that encodes the TCR δ subunit.

Apoptosis induced by islet amyloid polypeptide soluble oligomers is neutralized by diabetes-associated specific antibodies

PubMed Central

Bram, Yaron; Frydman-Marom, Anat; Yanai, Inbal; Gilead, Sharon; Shaltiel-Karyo, Ronit; Amdursky, Nadav; Gazit, Ehud

2014-01-01

Soluble oligomeric assemblies of amyloidal proteins appear to act as major pathological agents in several degenerative disorders. Isolation and characterization of these oligomers is a pivotal step towards determination of their pathological relevance. Here we describe the isolation of Type 2 diabetes-associated islet amyloid polypeptide soluble cytotoxic oligomers; these oligomers induced apoptosis in cultured pancreatic cells, permeated model lipid vesicles and interacted with cell membranes following complete internalization. Moreover, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, were exclusively identified in diabetic patients and were shown to neutralize the apoptotic effect induced by these oligomers. Our findings support the notion that human IAPP peptide can form highly toxic oligomers. The presence of antibodies identified in the serum of diabetic patients confirms the pathological relevance of the oligomers. In addition, the newly identified structural epitopes may also provide new mechanistic insights and a molecular target for future therapy. PMID:24589570

Structural basis of substrate specificity in the serine proteases.

PubMed Central

Perona, J. J.; Craik, C. S.

1995-01-01

Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system. PMID:7795518

Kinetics of a Collagen-Like Polypeptide Fragmentation after Mid-IR Free-Electron Laser Ablation

PubMed Central

Zavalin, Andrey; Hachey, David L.; Sundaramoorthy, Munirathinam; Banerjee, Surajit; Morgan, Steven; Feldman, Leonard; Tolk, Norman; Piston, David W.

2008-01-01

Tissue ablation with mid-infrared irradiation tuned to collagen vibrational modes results in minimal collateral damage. The hypothesis for this effect includes selective scission of protein molecules and excitation of surrounding water molecules, with the scission process currently favored. In this article, we describe the postablation infrared spectral decay kinetics in a model collagen-like peptide (Pro-Pro-Gly)10. We find that the decay is exponential with different decay times for other, simpler dipeptides. Furthermore, we find that collagen-like polypeptides, such as (Pro-Pro-Gly)10, show multiple decay times, indicating multiple scission locations and cross-linking to form longer chain molecules. In combination with data from high-resolution mass spectrometry, we interpret these products to result from the generation of reactive intermediates, such as free radicals, cyanate ions, and isocyanic acid, which can form cross-links and protein adducts. Our results lead to a more complete explanation of the reduced collateral damage resulting from infrared laser irradiation through a mechanism involving cross-linking in which collagen-like molecules form a network of cross-linked fibers. PMID:18441025

Vasoactive intestinal polypeptide (VIP) innervation of the human eyelid glands.

PubMed

Seifert, P; Spitznas, M

1999-06-01

This study was conducted to obtain morphological proof of innervating nerve fibres in the glands of the human eyelid (accessory lacrimal glands of Wolfring, meibomian glands, goblet cells, glands of Zeis, glands of Moll, sweat glands, glands of lanugo hair follicles) and identification of the secretomotorically active neuropeptide vasoactive intestinal polypeptide (VIP) as a common transmitter. Epoxy-embedded ultrathin sections of tissue samples from human eyelids were studied using electron microscopy. Paraffin sections fixed in Bouin-Hollande solution were immunostained with rabbit antiserum against VIP. With the electron microscope we were able to identify nerves in the glandular stroma of all the glands examined with the exception of goblet cells. Intraepithelial single axons were only seen in the parenchyma of Wolfring glands. The morphological findings corresponded with the immunological finding of VIP-positive, nerve-like structures in the same locations, with the exception of lanugo hair follicle glands, and goblet cells. Our findings indicate that the glands of the eyelids and main lacrimal gland represent a functional unit with VIP as a possible common stimulating factor. Copyright 1999 Academic Press.

The Regulation of Steroid Action by Sulfation and Desulfation

PubMed Central

Mueller, Jonathan W.; Gilligan, Lorna C.; Idkowiak, Jan; Arlt, Wiebke

2015-01-01

Steroid sulfation and desulfation are fundamental pathways vital for a functional vertebrate endocrine system. After biosynthesis, hydrophobic steroids are sulfated to expedite circulatory transit. Target cells express transmembrane organic anion-transporting polypeptides that facilitate cellular uptake of sulfated steroids. Once intracellular, sulfatases hydrolyze these steroid sulfate esters to their unconjugated, and usually active, forms. Because most steroids can be sulfated, including cholesterol, pregnenolone, dehydroepiandrosterone, and estrone, understanding the function, tissue distribution, and regulation of sulfation and desulfation processes provides significant insights into normal endocrine function. Not surprisingly, dysregulation of these pathways is associated with numerous pathologies, including steroid-dependent cancers, polycystic ovary syndrome, and X-linked ichthyosis. Here we provide a comprehensive examination of our current knowledge of endocrine-related sulfation and desulfation pathways. We describe the interplay between sulfatases and sulfotransferases, showing how their expression and regulation influences steroid action. Furthermore, we address the role that organic anion-transporting polypeptides play in regulating intracellular steroid concentrations and how their expression patterns influence many pathologies, especially cancer. Finally, the recent advances in pharmacologically targeting steroidogenic pathways will be examined. PMID:26213785

Requirement of Xmsx-1 in the BMP-triggered ventralization of Xenopus embryos.

PubMed

Yamamoto, T S; Takagi, C; Ueno, N

2000-03-01

Signaling triggered by polypeptide growth factors leads to the activation of their target genes. Several homeobox genes are known to be induced in response to polypeptide growth factors in early Xenopus development. In particular, Xmsx-1, an amphibian homologue of vertebrate Msx-1, is well characterized as a target gene of bone morphogenetic protein (BMP). Here, using a dominant-negative form of Xmsx-1 (VP-Xmsx-1), which is a fusion protein made with the virus-derived VP16 activation domain, we have examined whether Xmsx-1 activity is required in the endogenous ventralizing pathway. VP-Xmsx-1 induced a secondary body axis, complete with muscle and neural tissues, when overexpressed in ventral blastomeres, suggesting that Xmsx-1 activity is necessary for both mesoderm and ectoderm to be ventralized. We have also examined the epistatic relationship between Xmsx-1 and another ventralizing homeobox protein, Xvent-1, and show that Xmsx-1 is likely to be acting upstream of Xvent-1. We propose that Xmsx-1 is required in the BMP-stimulated ventralization pathway that involves the downstream activation of Xvent-1.

The isolation, purification and amino-acid sequence of insulin from the teleost fish Cottus scorpius (daddy sculpin).

PubMed

Cutfield, J F; Cutfield, S M; Carne, A; Emdin, S O; Falkmer, S

1986-07-01

Insulin from the principal islets of the teleost fish, Cottus scorpius (daddy sculpin), has been isolated and sequenced. Purification involved acid/alcohol extraction, gel filtration, and reverse-phase high-performance liquid chromatography to yield nearly 1 mg pure insulin/g wet weight islet tissue. Biological potency was estimated as 40% compared to porcine insulin. The sculpin insulin crystallised in the absence of zinc ions although zinc is known to be present in the islets in significant amounts. Two other hormones, glucagon and pancreatic polypeptide, were copurified with the insulin, and an N-terminal sequence for pancreatic polypeptide was determined. The primary structure of sculpin insulin shows a number of sequence changes unique so far amongst teleost fish. These changes occur at A14 (Arg), A15 (Val), and B2 (Asp). The B chain contains 29 amino acids and there is no N-terminal extension as seen with several other fish. Presumably as a result of the amino acid substitutions, sculpin insulin does not readily form crystals containing zinc-insulin hexamers, despite the presence of the coordinating B10 His.

Identification of dehydrin-like proteins responsive to chilling in floral buds of blueberry (Vaccinium, section Cyanococcus).

PubMed

Muthalif, M M; Rowland, L J

1994-04-01

The level of three major polypeptides of 65, 60, and 14 kD increased in response to chilling unit accumulation in floral buds of a woody perennial, blueberry (Vaccinium, section Cynaococcus). The level of the polypeptides increased most dramatically within 300 h of chilling and decreased to the prechilling level with the initiation of budbreak. Cold-hardiness levels were assessed for dormant buds of Vaccinium corymbosum and Vaccinium ashei after different chilling treatments until the resumption of growth. These levels coincided with the level of the chilling-responsive polypeptides. Like some other previously described cold-induced proteins in annual plants, the level of the chilling-induced polypeptides also increased in leaves in response to cold treatment; the chilling-induced polypeptides were heat stable, resisting aggregation after incubation at 95 degrees C for 15 min. By fractionating bud proteins first by isoelectric point (pI) and then by molecular mass, the pI values of the 65- and 60-kD polypeptides were found to be 7.5 to 8.0 and the pI value of the 14-kD polypeptide was judged to be 8.5. Purification of the 65- and 60-kD polypeptides, followed by digestion with endoproteinase Lys-C and sequencing of selected fragments, revealed similarities in amino acid composition between the 65- and 60-kD polypeptides and dehydrins. Indeed, antiserum to the lysine-rich consensus sequence EKKGIMDKIKEKLPG of dehydrin proteins cross-reacted to all three of the major chilling-responsive polypeptides of blueberry, identifying these as dehydrins or dehydrin-like proteins.

Identification of dehydrin-like proteins responsive to chilling in floral buds of blueberry (Vaccinium, section Cyanococcus).

PubMed Central

Muthalif, M M; Rowland, L J

1994-01-01

The level of three major polypeptides of 65, 60, and 14 kD increased in response to chilling unit accumulation in floral buds of a woody perennial, blueberry (Vaccinium, section Cynaococcus). The level of the polypeptides increased most dramatically within 300 h of chilling and decreased to the prechilling level with the initiation of budbreak. Cold-hardiness levels were assessed for dormant buds of Vaccinium corymbosum and Vaccinium ashei after different chilling treatments until the resumption of growth. These levels coincided with the level of the chilling-responsive polypeptides. Like some other previously described cold-induced proteins in annual plants, the level of the chilling-induced polypeptides also increased in leaves in response to cold treatment; the chilling-induced polypeptides were heat stable, resisting aggregation after incubation at 95 degrees C for 15 min. By fractionating bud proteins first by isoelectric point (pI) and then by molecular mass, the pI values of the 65- and 60-kD polypeptides were found to be 7.5 to 8.0 and the pI value of the 14-kD polypeptide was judged to be 8.5. Purification of the 65- and 60-kD polypeptides, followed by digestion with endoproteinase Lys-C and sequencing of selected fragments, revealed similarities in amino acid composition between the 65- and 60-kD polypeptides and dehydrins. Indeed, antiserum to the lysine-rich consensus sequence EKKGIMDKIKEKLPG of dehydrin proteins cross-reacted to all three of the major chilling-responsive polypeptides of blueberry, identifying these as dehydrins or dehydrin-like proteins. PMID:8016270

Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same

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

Morant, Marc

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Extracellular secretion of recombinant proteins

DOEpatents

Linger, Jeffrey G.; Darzins, Aldis

2014-07-22

Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A [San Diego, CA; Zhao, Lishan [Emeryville, CA; Cayouette, Michelle H [San Diego, CA

2012-01-24

The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Pituitary adenylate cyclase-activating polypeptide: a novel peptide with protean implications.

PubMed

Pisegna, Joseph R; Oh, David S

2007-02-01

The purpose of this review is to highlight the importance of pituitary adenylate cyclase-activating polypeptide in physiological processes and to describe how this peptide is becoming increasingly recognized as having a major role in the body. Since its discovery in 1989, investigators have sought to determine the site of biological activity and the function of pituitary adenylate cyclase-activating polypeptide in maintaining homeostasis. Since its discovery, pituitary adenylate cyclase-activating polypeptide appears to play an important role in the regulation of processes within the central nervous system and gastrointestinal tract, as well in reproductive biology. Pituitary adenylate cyclase-activating polypeptide has been shown to regulate tumor cell growth and to regulate immune function through its effects on T lympocytes. These discoveries suggest the importance of pituitary adenylate cyclase-activating polypeptide in neuronal development, neuronal function, gastrointestinal tract function and reproduction. Future studies will examine more closely the role of pituitary adenylate cyclase-activating polypeptide in regulation of malignantly transformed cells, as well as in regulation of immune function.

Unimpaired postprandial pancreatic polypeptide secretion in Parkinson's disease and REM sleep behavior disorder.

PubMed

Unger, Marcus M; Ekman, Rolf; Björklund, Anna-Karin; Karlsson, Gösta; Andersson, Chatarina; Mankel, Katharina; Bohne, Katharina; Tebbe, Johannes J; Stiasny-Kolster, Karin; Möller, Jens C; Mayer, Geert; Kann, Peter H; Oertel, Wolfgang H

2013-04-01

Pancreatic polypeptide is released immediately after food ingestion. The release is operated by vagal-abdominal projections and has therefore been suggested as a test for vagal nerve integrity. Pathoanatomical and clinical studies indicate vagal dysfunction in early Parkinson's disease (PD). We assessed the postprandial secretion of pancreatic polypeptide and motilin in healthy controls (n = 18) and patients with idiopathic rapid-eye-movement sleep behavior disorder (iRBD, n = 10), a potential premotor stage of PD, as well as in drug-naive (n = 19) and treated (n = 19) PD patients. The postprandial pancreatic polypeptide secretion showed a physiological pattern in all groups and even an enhanced response in drug-naive PD and iRBD. Motilin concentrations correlated with pancreatic polypeptide concentrations. Postprandial pancreatic polypeptide secretion is not a suitable test for vagal nerve integrity in PD. The unimpaired pancreatic polypeptide response in iRBD and PD might be explained by partially intact vagal-abdominal projections or compensatory mechanisms substituting a defective neuronal brain-gut axis. Copyright © 2012 Movement Disorders Society.

A de novo designed 11 kDa polypeptide: model for amyloidogenic intrinsically disordered proteins.

PubMed

Topilina, Natalya I; Ermolenkov, Vladimir V; Sikirzhytski, Vitali; Higashiya, Seiichiro; Lednev, Igor K; Welch, John T

2010-07-01

A de novo polypeptide GH(6)[(GA)(3)GY(GA)(3)GE](8)GAH(6) (YE8) has a significant number of identical weakly interacting beta-strands with the turns and termini functionalized by charged amino acids to control polypeptide folding and aggregation. YE8 exists in a soluble, disordered form at neutral pH but is responsive to changes in pH and ionic strength. The evolution of YE8 secondary structure has been successfully quantified during all stages of polypeptide fibrillation by deep UV resonance Raman (DUVRR) spectroscopy combined with other morphological, structural, spectral, and tinctorial characterization. The YE8 folding kinetics at pH 3.5 are strongly dependent on polypeptide concentration with a lag phase that can be eliminated by seeding with a solution of folded fibrillar YE8. The lag phase of polypeptide folding is concentration dependent leading to the conclusion that beta-sheet folding of the 11-kDa amyloidogenic polypeptide is completely aggregation driven.

Design and preparation of beta-sheet forming repetitive and block-copolymerized polypeptides.

PubMed

Higashiya, Seiichiro; Topilina, Natalya I; Ngo, Silvana C; Zagorevskii, Dmitri; Welch, John T

2007-05-01

The design and rapid construction of libraries of genes coding beta-sheet forming repetitive and block-copolymerized polypeptides bearing various C- and N-terminal sequences are described. The design was based on the assembly of DNA cassettes coding for the (GA)3GX amino acid sequence where the (GAGAGA) sequences would constitute the beta-strand units of a larger beta-sheet assembly. The edges of this beta-sheet would be functionalized by the turn-inducing amino acids (GX). The polypeptides were expressed in Escherichia coli using conventional vectors and were purified by Ni-nitriloacetic acid (NTA) chromatography. The correlation of polymer structure with molecular weight was investigated by gel electrophoresis and mass spectrometry. The monomer sequences and post-translational chemical modifications were found to influence the mobility of the polypeptides over the full range of polypeptide molecular weights while the electrophoretic mobility of lower molecular weight polypeptides was more susceptible to C- and N-termini polypeptide modifications.

Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere

NASA Astrophysics Data System (ADS)

Munegumi, Toratane; Tanikawa, Naoya

2017-09-01

Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.

Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere.

PubMed

Munegumi, Toratane; Tanikawa, Naoya

2017-09-01

Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.

Drug Delivery to the Ischemic Brain

PubMed Central

Thompson, Brandon J.; Ronaldson, Patrick T.

2014-01-01

Cerebral ischemia occurs when blood flow to the brain is insufficient to meet metabolic demand. This can result from cerebral artery occlusion that interrupts blood flow, limits CNS supply of oxygen and glucose, and causes an infarction/ischemic stroke. Ischemia initiates a cascade of molecular events inneurons and cerebrovascular endothelial cells including energy depletion, dissipation of ion gradients, calcium overload, excitotoxicity, oxidative stress, and accumulation of ions and fluid. Blood-brain barrier (BBB) disruption is associated with cerebral ischemia and leads to vasogenic edema, a primary cause of stroke-associated mortality. To date, only a single drug has received US Food and Drug Administration (FDA) approval for acute ischemic stroke treatment, recombinant tissue plasminogen activator (rt-PA). While rt-PA therapy restores perfusion to ischemic brain, considerable tissue damage occurs when cerebral blood flow is re-established. Therefore, there is a critical need for novel therapeutic approaches that can “rescue” salvageable brain tissue and/or protect BBB integrity during ischemic stroke. One class of drugs that may enable neural cell rescue following cerebral ischemia/reperfusion injury is the HMG-CoA reductase inhibitors (i.e., statins). Understanding potential CNS drug delivery pathways for statins is critical to their utility in ischemic stroke. Here, we review molecular pathways associated with cerebral ischemia and novel approaches for delivering drugs to treat ischemic disease. Specifically, we discuss utility of endogenous BBB drug uptake transporters such as organic anion transporting polypeptides (OATPs/Oatps) and nanotechnology-based carriers for optimization of CNS drug delivery. Overall, this chapter highlights state-of-the-art technologies that may improve pharmacotherapy of cerebral ischemia. PMID:25307217

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

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

Thompson, David N.; Apel, William A.; Thompson, Vicki S.

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

2015-06-02

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

2013-10-15

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N [Idaho Falls, ID; Apel, William A [Jackson, WY; Thompson, Vicki S [Idaho Falls, ID; Reed, David W [Idaho Falls, ID; Lacey, Jeffrey A [Idaho Falls, ID; Henriksen, Emily D [Idaho Falls, ID

2012-06-19

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

2013-04-23

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

2010-12-28

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

2013-07-30

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

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

Thompson, David N; Apel, William A; Thompson, Vicki S

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

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

Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.

Nitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH 3) to nitrite (NO 2 ₋) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH 4 +-dependent O 2uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide usingmore » a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA.« less

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

PubMed Central

Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris

2016-01-01

Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2−) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, and de novo protein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA. PMID:26826234

Tracking polypeptide folds on the free energy surface: effects of the chain length and sequence.

PubMed

Brukhno, Andrey V; Ricchiuto, Piero; Auer, Stefan

2012-07-26

Characterization of the folding transition in polypeptides and assessing the thermodynamic stability of their structured folds are of primary importance for approaching the problem of protein folding. We use molecular dynamics simulations for a coarse grained polypeptide model in order to (1) obtain the equilibrium conformation diagram of homopolypeptides in a broad range of the chain lengths, N = 10, ..., 100, and temperatures, T (in a multicanonical ensemble), and (2) determine free energy profiles (FEPs) projected onto an optimal, so-called "natural", reaction coordinate that preserves the height of barriers and the diffusion coefficients on the underlying free energy hyper-surface. We then address the following fundamental questions. (i) How well does a kinetically determined free energy landscape of a single chain represent the polypeptide equilibrium (ensemble) behavior? In particular, under which conditions might the correspondence be lost, and what are the possible implications for the folding processes? (ii) How does the free energy landscape depend on the chain length (homopolypeptides) and the monomer interaction sequence (heteropolypeptides)? Our data reveal that at low T values equilibrium structures adopted by relatively short homopolypeptides (N < 60) are dominated by α-helical folds which correspond to the primary and secondary minima of the FEP. In contrast, longer homopolypeptides (N > 70), upon quasi-equilibrium cooling, fold preferentially in β-bundles with small helical portions, while the FEPs exhibit no distinct global minima. Moreover, subject to the choice of the initial configuration, at sufficiently low T, essentially metastable structures can be found and prevail far from the true thermodynamic equilibrium. We also show that, by sequence-enabling the polypeptide model, it is possible to restrict the chain to a very specific part of the configuration space, which results in substantial simplification and smoothing of the free energy landscape as compared to the case of the corresponding homopolypeptide.

Keto-isovalerate decarboxylase enzymes and methods of use thereof

DOEpatents

McElvain, Jessica; O'Keefe, Daniel P.; Paul, Brian James; Payne, Mark S.; Rothman, Steven Cary; He, Hongxian

2016-01-19

Provided herein are polypeptides and polynucleotides encoding such polypeptides which have ketoisovalerate decarboxylase activity. Also provided are recombinant host cells comprising such polypeptides and polynucleotides and methods of use thereof.

Changes in the Polypeptide Patterns of Barley Seedlings Exposed to Jasmonic Acid and Salinity 1

PubMed Central

Maslenkova, Liliana Todorova; Miteva, Tania Simeonova; Popova, Losanka P.

1992-01-01

Soluble and thylakoid membrane proteins of jasmonic acid (JA)-treated and salt-stressed barley (Hordeum vulgare L.) seedlings were investigated using 15% sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. High JA concentrations induced marked quantitative and qualitative changes in polypeptide profiles concerning mainly the proteins with approximately equal mobility, as in NaCl-stressed plants. The most obvious increase in thylakoid polypeptide band intensity was at 55 to 57 kilodaltons (kD). The relative share of some polypeptides with apparent molecular masses above 66 kD and of polypeptides with lower molecular masses in the region of 20.5 to 15 kD was enhanced. At the same time, one new band at 31 to 31.5 kD was well expressed at 25 and 250 micromolar JA concentrations and became discernible in the 100 micromolar NaCl-treated plants. The intensity of some polypeptides of soluble proteins (molecular masses of 60, 47, 37, 30, and 23.4 kD) increased with increasing JA concentration, whereas the intensities of other polypeptide bands (55, 21.4, and 15 kD) decreased. Enhanced levels of 60-, 47-, 34-, and 30-kD polypeptides and reduced levels of 55- and 15-kD polypeptides were present in NaCl-treated plants. The appearance of one new polypeptide, of 25.1 kD, was observed only in NaCl-treated plants. At 100 millimolar NaCl, an eightfold increase in proline content was observed while at 250 micromolar JA, the proline content was threefold over the control. It is hypothesized that exogenously applied jasmonates act as stress agents. As such, they provoke alterations in the proline content and they can modulate typical stress responses by induction of stress proteins. ImagesFigure 1Figure 4Figure 5 PMID:16668698

Bioresorbable polypeptide-based comb-polymers efficiently improves the stability and pharmacokinetics of proteins in vivo.

PubMed

Turabee, Md Hasan; Thambi, Thavasyappan; Lym, Jae Seung; Lee, Doo Sung

2017-03-28

Stimuli-responsive polypeptides are a promising class of biomaterials due to their tunable physicochemical and biological properties. Herein, a series of novel pH- and thermo-responsive block copolymers based on polypeptides were synthesized by ring-opening polymerization of γ-benzyl-l-glutamate-N-carboxyanhydride in the presence of poly(ethylene glycol)-diamine macroinitiator followed by aminolysis. The resulting polypeptide-based triblock copolymer, poly[(2-(dibutylamino)ethyl-l-glutamate)-co-(γ-benzyl-l-glutamate)]-poly(ethylene glycol)-b-poly[(2-(dibutylamino)ethyl-l-glutamate)-co-(γ-benzyl-l-glutamate)] (PNLG-co-PBLG-b-PEG-b-PBLG-co-PNLG), exists as a low viscous sol at low pH and temperature (≤pH 6.4, 25 °C) but it transforms to a soft gel under physiological conditions (pH 7.4 and 37 °C). The physical properties of the polypeptide gel can be tuned by controlling the ratio between hydrophobic PBLG and pH-sensitive PNLG blocks. The polypeptide-based copolymer did not show any noticeable cytotoxicity to fibroblast cells in vitro. It was found that subcutaneous injection of the polypeptide copolymer solution into the dorsal region of Sprague-Dawley (SD) rats formed a gel instantly without major inflammation. The gels were completely biodegraded in six weeks and found to be bioresorbable. Human growth hormone (hGH)-loaded polypeptide-based biodegradable copolymer sols readily formed a viscoelastic gel that inhibited an initial burst and prolonged the hGH release for one week. Overall, due to their bioresorbable and sustained release protein characteristics, polypeptide hydrogels may serve as viable platforms for therapeutic protein delivery and the surface tunable properties of polypeptide hydrogels can be exploited for other potential therapeutic proteins.

Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide.

PubMed

Wang, Hong-Xia; Song, Ziyuan; Lao, Yeh-Hsing; Xu, Xin; Gong, Jing; Cheng, Du; Chakraborty, Syandan; Park, Ji Sun; Li, Mingqiang; Huang, Dantong; Yin, Lichen; Cheng, Jianjun; Leong, Kam W

2018-05-08

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications. Copyright © 2018 the Author(s). Published by PNAS.

Identification and characterization of the reptilian GnRH-II gene in the leopard gecko, Eublepharis macularius, and its evolutionary considerations.

PubMed

Ikemoto, Tadahiro; Park, Min Kyun

2003-10-16

To elucidate the molecular phylogeny and evolution of a particular peptide, one must analyze not the limited primary amino acid sequences of the low molecular weight mature polypeptide, but rather the sequences of the corresponding precursors from various species. Of all the structural variants of gonadotropin-releasing hormone (GnRH), GnRH-II (chicken GnRH-II, or cGnRH-II) is remarkably conserved without any sequence substitutions among vertebrates, but its precursor sequences vary considerably. We have identified and characterized the full-length complementary DNA (cDNA) encoding the GnRH-II precursor and determined its genomic structure, consisting of four exons and three introns, in a reptilian species, the leopard gecko Eublepharis macularius. This is the first report about the GnRH-II precursor cDNA/gene from reptiles. The deduced leopard gecko prepro-GnRH-II polypeptide had the highest identities with the corresponding polypeptides of amphibians. The GnRH-II precursor mRNA was detected in more than half of the tissues and organs examined. This widespread expression is consistent with the previous findings in several species, though the roles of GnRH outside the hypothalamus-pituitary-gonadal axis remain largely unknown. Molecular phylogenetic analysis combined with sequence comparison showed that the leopard gecko is more similar to fishes and amphibians than to eutherian mammals with respect to the GnRH-II precursor sequence. These results strongly suggest that the divergence of the GnRH-II precursor sequences seen in eutherian mammals may have occurred along with amniote evolution.

Toward a unified model of passive drug permeation II: the physiochemical determinants of unbound tissue distribution with applications to the design of hepatoselective glucokinase activators.

PubMed

Ghosh, Avijit; Maurer, Tristan S; Litchfield, John; Varma, Manthema V; Rotter, Charles; Scialis, Renato; Feng, Bo; Tu, Meihua; Guimaraes, Cris R W; Scott, Dennis O

2014-10-01

In this work, we leverage a mathematical model of the underlying physiochemical properties of tissues and physicochemical properties of molecules to support the development of hepatoselective glucokinase activators. Passive distribution is modeled via a Fick-Nernst-Planck approach, using in vitro experimental data to estimate the permeability of both ionized and neutral species. The model accounts for pH and electrochemical potential across cellular membranes, ionization according to Henderson-Hasselbalch, passive permeation of the neutral species using Fick's law, and passive permeation of the ionized species using the Nernst-Planck equation. The mathematical model of the physiochemical system allows derivation of a single set of parameters governing the distribution of drug molecules across multiple conditions both in vitro and in vivo. A case study using this approach in the development of hepatoselective glucokinase activators via organic anion-transporting polypeptide-mediated hepatic uptake and impaired passive distribution to the pancreas is described. The results for these molecules indicate the permeability penalty of the ionized form is offset by its relative abundance, leading to passive pancreatic exclusion according to the Nernst-Planck extension of Fickian passive permeation. Generally, this model serves as a useful construct for drug discovery scientists to understand subcellular exposure of acids or bases using specific physiochemical properties. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

The Dual Role of TGFβ in Human Cancer: From Tumor Suppression to Cancer Metastasis

PubMed Central

Lebrun, Jean-Jacques

2012-01-01

The transforming growth factor-beta (TGFβ) superfamily encompasses widespread and evolutionarily conserved polypeptide growth factors that regulate and orchestrate growth and differentiation in all cell types and tissues. While they regulate asymmetric cell division and cell fate determination during early development and embryogenesis, TGFβ family members play a major regulatory role in hormonal and immune responses, cell growth, cell death and cell immortalization, bone formation, tissue remodeling and repair, and erythropoiesis throughout adult life. The biological and physiological functions of TGFβ, the founding member of this family, and its receptors are of central importance to human diseases, particularly cancer. By regulating cell growth, death, and immortalization, TGFβ signaling pathways exert tumor suppressor effects in normal cells and early carcinomas. Thus, it is not surprising that a high number of human tumors arise due to mutations or deletions in the genes coding for the various TGFβ signaling components. As tumors develop and progress, these protective and cytostatic effects of TGFβ are often lost. TGFβ signaling then switches to promote cancer progression, invasion, and tumor metastasis. The molecular mechanisms underlying this dual role of TGFβ in human cancer will be discussed in depth in this paper, and it will highlight the challenge and importance of developing novel therapeutic strategies specifically aimed at blocking the prometastatic arm of the TGFβ signaling pathway without affecting its tumor suppressive effects. PMID:27340590

Highly expressed EGFR in pearl sac may facilitate the pearl formation in the pearl oyster, Pinctada fucata.

PubMed

Zhu, Wenjie; Fan, Sigang; Huang, Guiju; Zhang, Dongling; Liu, Baosuo; Bi, Xiaomin; Yu, Dahui

2015-07-25

Epidermal growth factor receptor (EGFR) plays an important role in cell growth, proliferation, differentiation and migration. Yet whether it functions in pearl formation or not is not reported. In this study, EGFR was cloned from the pearl oyster Pinctada fucata (named as Pf-EGFR) and its expression profiles were investigated. The cDNA was 2156bp long with an ORF of 1017bp encoding 338 amino acid residues. The deduced polypeptide contained an L domain and a cysteine-rich domain, consistent with the characteristics of ErbB family. The sequence of Pf-EGFR shared 22.78-56.71% identity with other EGFRs. The genomic sequence of Pf-EGFR consisted of six exons and five introns, being 5190bp in total length, and expressed in all the tested tissues with a higher expression level in the pearl sac (P<0.05). In situ hybridization showed that Pf-EGFR was specifically expressed on both the inner side of the outer fold and the outer side of the inner fold of the mantle, as well as on the whole pearl sac including the connective tissues. In addition, Pf-EGFR was markedly increased at larval metamorphosis, significantly higher than other development periods (P<0.05). These results indicated that the Pf-EGFR may facilitate pearl formation as well as larval metamorphosis. Copyright © 2015 Elsevier B.V. All rights reserved.

MALDI Mass Spectrometry Imaging: A Novel Tool for the Identification and Classification of Amyloidosis.

PubMed

Winter, Martin; Tholey, Andreas; Kristen, Arnt; Röcken, Christoph

2017-11-01

Amyloidosis is a group of diseases caused by extracellular accumulation of fibrillar polypeptide aggregates. So far, diagnosis is performed by Congo red staining of tissue sections in combination with polarization microscopy. Subsequent identification of the causative protein by immunohistochemistry harbors some difficulties regarding sensitivity and specificity. Mass spectrometry based approaches have been demonstrated to constitute a reliable method to supplement typing of amyloidosis, but still depend on Congo red staining. In the present study, we used matrix-assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI-IMS MSI) to investigate amyloid deposits in formalin-fixed and paraffin-embedded tissue samples. Utilizing a novel peptide filter method, we found a universal peptide signature for amyloidoses. Furthermore, differences in the peptide composition of ALλ and ATTR amyloid were revealed and used to build a reliable classification model. Integrating the peptide filter in MALDI-IMS MSI analysis, we developed a bioinformatics workflow facilitating the identification and classification of amyloidosis in a less time and sample-consuming experimental setup. Our findings demonstrate also the feasibility to investigate the amyloid's protein composition, thus paving the way to establish classification models for the diverse types of amyloidoses and to shed further light on the complex process of amyloidogenesis. © 2017 The Authors, Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ultrastructural preservation of tissues and their reaction to the infection with trichinella in the El Plomo mummy: Muscle fiber ultrastructure and trichinosis/mummy of the Cerro El Plomo.

PubMed

Héctor, Rodríguez; Omar, Espinoza-Navarro; González, Mercedes; Mario, Castro

2017-08-01

The El Plomo mummy was a pre-Columbian Incan child who was found mummified in the Andes Mountains above an altitude of 17,700 feet. In the environment, natural mummification occurred due to low temperatures and strong winds. Dating measurements (relative dating) by experts from the National Museum of Natural History of Chile established that the mummified body corresponds the Inca period (1,450 to 1,500 AD). In 2003, the body was transferred to the University of Chile Medical School for exhaustive medical examination. Tissue samples from the right quadriceps muscle were extracted and fixed in glutaraldehyde and postfixed in osmium tetroxide to obtain ultrathin sections to be observed by transmission electron microscope. Images were recorded on photographic paper, digitalized and analyzed by experts on morphology. Results showed a preservation of cell boundaries in striated muscle cells, but specific subcellular organelles or contractile sarcomeric units (actin and myosin) were unable to be recognized. However, the classical ultrastructural morphology of the polypeptide collagen type I was preserved intact both in primary and secondary organization. Therefore, we concluded that the process of natural mummification by freezing and strong winds is capable of damaging the ultrastructure of muscle cells and preserving collagen type I intact. © 2017 Wiley Periodicals, Inc.

Muscular innervation of the proximal duodenum of the guinea pig.

PubMed

Iino, S

2000-10-01

We investigated the muscular structure and innervation of the gastroduodenal junction in the guinea pig. In the gastroduodenal junction, the innermost layer of the circular muscle contained numerous nerve fibers and terminals. Since this nerve network continued onto the deep muscular plexus (DMP) of the duodenum, we surmised that the numerous nerve fibers in the gastroduodenal junction were specialized DMP in the most proximal part of the duodenum. The innermost layer containing many nerve fibers was about 1,000 microm in length and 100 microm in thickness in the proximal duodenum. This layer contained numerous connective tissue fibers composed of collagen and elastic fibers. Five to 30 smooth muscle cells lay in contact with each other and were surrounded by fine connective tissue. The nerve fibers in the proximal duodenum contained nerve terminals immunoreactive for choline acetyltransferase, dynorphin, enkephalin, galanin, gastrin-releasing peptide, nitric oxide synthase, substance P, and vasoactive intestinal polypeptide. Adrenergic fibers which contained tyrosine hydroxylase immunoreactivity were rare in the proximal duodenum. In the innermost layer of the proximal duodenum, there were numerous c-Kit immunopositive cells that were in contact with nerve terminals. This study allowed us to clarify the specific architecture of the most proximal portion of the duodenum. The functional significance of the proximal duodenum in relation to the electrical connection and neural cooperation of the musculature between the antrum and the duodenum is also discussed.

Versatile platform for nanotechnology based on circular permutations of chaperonin protein

NASA Technical Reports Server (NTRS)

McMillan, R. Andrew (Inventor); Kagawa, Hiromi (Inventor); Paavola, Chad D. (Inventor); Chan, Suzanne L. (Inventor); Li, Yi-Fen (Inventor); Trent, Jonathan D. (Inventor)

2010-01-01

The present invention provides chaperonin polypeptides which are modified to include N-terminal and C-terminal ends that are relocated from the central pore region to various different positions in the polypeptide which are located on the exterior of the folded modified chaperonin polypeptide. In the modified chaperonin polypeptide, the naturally-occurring N-terminal and C-terminal ends are joined together directly or with an intervening linker peptide sequence. The relocated N-terminal or C-terminal ends can be covalently joined to, or bound with another molecule such as a nucleic acid molecule, a lipid, a carbohydrate, a second polypeptide, or a nanoparticle. The modified chaperonin polypeptides can assemble into double-ringed chaperonin structures. Further, the chaperonin structures can organize into higher order structures such as nanofilaments or nanoarrays which can be used to produce nanodevices and nanocoatings.

A versatile expression vector for the growth and amplification of unmodified phage display polypeptides.

PubMed

Winton, Alexander J; Baptiste, Janae L; Allen, Mark A

2018-09-01

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains. Copyright © 2018. Published by Elsevier Inc.

A majority of mice show long-term expression of a human. beta. -globin gene after retrovirus transfer into hematopoietic stem cells

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

Bender, M.A.; Gelinas, R.E.; Miller

1989-04-01

Murine bone marrow was infected with a high-titer retrovirus vector containing the human {beta}-globin and neomycin phosphotransferase genes. Anemic W/W/sup v/ mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human {beta}-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human {beta}-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human {beta}-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide.more » DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active {beta}-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.« less

Prediction of the aggregation propensity of proteins from the primary sequence: aggregation properties of proteomes.

PubMed

Castillo, Virginia; Graña-Montes, Ricardo; Sabate, Raimon; Ventura, Salvador

2011-06-01

In the cell, protein folding into stable globular conformations is in competition with aggregation into non-functional and usually toxic structures, since the biophysical properties that promote folding also tend to favor intermolecular contacts, leading to the formation of β-sheet-enriched insoluble assemblies. The formation of protein deposits is linked to at least 20 different human disorders, ranging from dementia to diabetes. Furthermore, protein deposition inside cells represents a major obstacle for the biotechnological production of polypeptides. Importantly, the aggregation behavior of polypeptides appears to be strongly influenced by the intrinsic properties encoded in their sequences and specifically by the presence of selective short regions with high aggregation propensity. This allows computational methods to be used to analyze the aggregation properties of proteins without the previous requirement for structural information. Applications range from the identification of individual amyloidogenic regions in disease-linked polypeptides to the analysis of the aggregation properties of complete proteomes. Herein, we review these theoretical approaches and illustrate how they have become important and useful tools in understanding the molecular mechanisms underlying protein aggregation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Exploration of the immunoreactivity of the Traditional Chinese medicine Shenrouyangzhentang to vasoactive intestinal polypeptide

PubMed Central

Gu, Yu-Chun; Chen, De-Zhen

1997-01-01

AIM: To study the immunoreactivity of the Chinese medicine Shenrouyangzhentang to vasoactive intestinal polypeptide (VIP) and its therapeutic mechanism. METHODS: The immunoreactivity of the Chinese medicine Shenrouyangzhentang to VIP was detected in the plasma of 20 normal people and 20 patients with Piyinxu (Spleen Yin deficiency) using the radioimmunoassay (RIA) method. RESULTS: The maximum binding rate B0/T was 53.29%, the non-specific binding rate N0/T was 1.170%, and the VIP standard curve was Y = 0.81983 + 0.44319X - 0.28927X2, R2 = 0.990. The VIP content in Shenrouyangzhentang was 106.6 ng/L ± 20 ng/L), while it was 90.16 ng/L ± 15 ng/L in normal human plasma and 63.25 ng/L ± 11 ng/L in the plasma of Pixinxu patients. The difference between normal plasma and Pixinxu patient plasma was statistically significant (P < 0.05). CONCLUSION: The Chinese medicine Shenrouyangzhentang demonstrated VIP immunoreactivity similar to that of normal plasma. The (vasoactive intestinal polypeptide) VIP content in Pixinxu patient plasma was lower than that in healthy subjects (P < 0.05). PMID:27041949

The influence of the side-chain sequence on the structure-activity correlations of immunomodulatory branched polypeptides. Synthesis and conformational analysis of new model polypeptides.

PubMed

Mezö, G; Hudecz, F; Kajtár, J; Szókán, G; Szekerke, M

1989-10-01

New branched polypeptides were synthesized for a detailed study of the influence of the side-chain structure on the conformation and biological properties. The first subset of polypeptides were prepared by coupling of tetrapeptides to poly[L-Lys]. These polymers contain either DL-Ala3-X [poly[Lys-(X-DL-Ala3)n

Use of linalool synthase in genetic engineering of scent production

DOEpatents

Pichersky, E.

1998-12-15

A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed. 5 figs.

Use of linalool synthase in genetic engineering of scent production

DOEpatents

Pichersky, Eran

1998-01-01

A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

Chemically modified carbonic anhydrases useful in carbon capture systems

DOEpatents

Novick, Scott; Alvizo, Oscar

2013-01-15

The present disclosure relates to chemically modified carbonic anhydrase polypeptides and soluble compositions, homogenous liquid formulations comprising them. The chemically modified carbonic anhydrase polypeptides have improved properties relative to the same carbonic anhydrase polypeptide that is not chemically modified including the improved properties of increased activity and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides methods of preparing the chemically modified polypeptides and methods of using the chemically modified polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering.

Chemically modified carbonic anhydrases useful in carbon capture systems

DOEpatents

Novick, Scott J; Alvizo, Oscar

2013-10-29

The present disclosure relates to chemically modified carbonic anhydrase polypeptides and soluble compositions, homogenous liquid formulations comprising them. The chemically modified carbonic anhydrase polypeptides have improved properties relative to the same carbonic anhydrase polypeptide that is not chemically modified including the improved properties of increased activity and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides methods of preparing the chemically modified polypeptides and methods of using the chemically modified polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering.

Novozymes, Inc.

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

Spodsberg, Nikolaj

The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Ketol-acid reductoisomerase enzymes and methods of use

DOEpatents

Govindarajan, Sridhar; Li, Yougen; Liao, Der-Ing; O'Keefe, Daniel P.; Minshull, Jeremy Stephen; Rothman, Steven Cary; Tobias, Alexander Vincent

2015-10-27

Provided herein are polypeptides having ketol-aid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

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

Zhang, Yu; Tang, Lan; Henriksen, Svend Hostgaard Bang

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Glycation induces formation of amyloid cross-beta structure in albumin.

PubMed

Bouma, Barend; Kroon-Batenburg, Loes M J; Wu, Ya-Ping; Brünjes, Bettina; Posthuma, George; Kranenburg, Onno; de Groot, Philip G; Voest, Emile E; Gebbink, Martijn F B G

2003-10-24

Amyloid fibrils are components of proteinaceous plaques that are associated with conformational diseases such as Alzheimer's disease, transmissible spongiform encephalopathies, and familial amyloidosis. Amyloid polypeptides share a specific quarternary structure element known as cross-beta structure. Commonly, fibrillar aggregates are modified by advanced glycation end products (AGE). In addition, AGE formation itself induces protein aggregation. Both amyloid proteins and protein-AGE adducts bind multiligand receptors, such as receptor for AGE, CD36, and scavenger receptors A and B type I, and the serine protease tissue-type plasminogen activator (tPA). Based on these observations, we hypothesized that glycation induces refolding of globular proteins, accompanied by formation of cross-beta structure. Using transmission electron microscopy, we demonstrate here that glycated albumin condensates into fibrous or amorphous aggregates. These aggregates bind to amyloid-specific dyes Congo red and thioflavin T and to tPA. In contrast to globular albumin, glycated albumin contains amino acid residues in beta-sheet conformation, as measured with circular dichroism spectropolarimetry. Moreover, it displays cross-beta structure, as determined with x-ray fiber diffraction. We conclude that glycation induces refolding of initially globular albumin into amyloid fibrils comprising cross-beta structure. This would explain how glycated ligands and amyloid ligands can bind to the same multiligand "cross-beta structure" receptors and to tPA.

[Comparison of TPS, CEA, CYFRA21-1 and STNFR in diagnosis of lung cancer].

PubMed

Liao, Qiulin; Li, Lianhua; Chen, Mingsheng

2005-08-20

In recent years, new progress has been made in research of tumor markers. And namely tissue polypeptide specific antigen (TPS), cytokeratin 19-fragments (CYFRA21-1) and soluble tumor necrosis factor receptor (STNFR) are new tumor markers that have been used in clinical application. The aim of this study is to determine and compare the diagnostic value of 4 kinds of tumor markers, TPS, carcinoembryonic antigen (CEA), CYFRA21-1 and STNFR in patients with lung cancer. The serum levels of TPS, CEA, CYFRA21-1 and STNFR were determined in 72 patients with lung cancer, 54 patients with pulmonary benign diseases and 32 healthy adults by enzyme-linked immunosorbent assay (ELISA). The levels of the four tumor markers in lung cancer group were significantly higher than those in benign disease group (P < 0.005) and healthy control group (P < 0.001). Among the four markers, STNFR had the highest sensitivity (81.9%), CYFRA21-1 had the highest specificity (91.5%) and TPS had the highest accuracy (83.5%). TPS, CYFRA21-1 and STNFR can be used as very useful and sensitive tumor markers in the diagnosis of lung cancer, in which CYFRA21-1 may be the most useful tumor marker for clinical application.

Proteomic Analysis of Pathogenic and Attenuated Alcelaphine Herpesvirus 1▿

PubMed Central

Dry, Inga; Haig, David M.; Inglis, Neil F.; Imrie, Lisa; Stewart, James P.; Russell, George C.

2008-01-01

The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in susceptible ungulates but infects its natural host, wildebeest, without obvious clinical signs. In tissue culture, AlHV-1 is initially predominantly cell associated and virulent but on extended culture becomes cell-free and attenuated. We wanted to determine what changes in protein composition had taken place during the transition from virulent to attenuated virus in culture. Purified virus preparations were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. Peptides were identified in serial gel slices by using MASCOT software to interrogate virus-specific and nonredundant sequence databases. Twenty-three AlHV-1-encoded proteins and six cellular proteins were identified in the attenuated and virulent viruses. Two polypeptides were detected in only the virulent virus preparations, while one other protein was found in only the attenuated virus. Two of these virus-specific proteins were identified by a single peptide, suggesting that these may be low-abundance virion proteins rather than markers of attenuation or pathogenesis. The results suggest that attenuation of AlHV-1 is not the result of gross changes in the composition of the virus particle but probably due to altered viral gene expression in the infected cell. PMID:18353942

Production of immunologically active surface antigens of hepatitis B virus by Escherichia coli.

PubMed Central

MacKay, P; Pasek, M; Magazin, M; Kovacic, R T; Allet, B; Stahl, S; Gilbert, W; Schaller, H; Bruce, S A; Murray, K

1981-01-01

Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value of an in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals. PMID:6170067

Deoxypyrimidine kinases of herpes simplex viruses types 1 and 2: comparison of serological and structural properties.

PubMed

Thouless, M E; Wildy, P

1975-02-01

The kinetics of formation, the stability at 40 degrees C and the serological properties of thymidine kinase and deoxycytidine kinase activities induced by herpes simplex virus have been examined. The results are consistent with the hypothesis that both activities are carried on the same molecule-a deoxypyrimidine kinase. Mutants deficient in deoxypyrimidine kinase have been used to produce, by absorption of general antisera, deoxypyrimidine kinase-specific antisera. Using immunoprecipitation and SDS-polyacrylamide gel electrophoresis, only one size of polypeptide (mol. wt. 42400 plus or minus 200) has been found, constituting the type 2 enzyme. This is close to published values for the type i enzyme but co-electrophoresis demonstrated that the polypeptide of the type i enzyme was slightly bigger.

Molecular analysis of a phytohemagglutinin-defective cultivar of Phaseolus vulgaris L.

PubMed

Vitale, A; Ceriotti, A; Bollini, R

1985-10-01

The seeds of Phaseolus vulgaris cv. Pinto III are known to lack detectable amounts of phytohemagglutinin (PHA) and to accumulate very reduced levels of PHA mRNA compared with normal cultivars. Using PHA complementary-DNA clones and monospecific antibodies we analyzed cv. Pinto III genomic DNA and cotyledonary proteins synthesized both in vitro and in vivo. We detected genomic DNA sequences that hybridize with complementary-DNA clones for the two different classes of PHA polypeptides (PHA-E and PHA-L), at levels comparable to a normal bean cultivar. This indicates that the cv. Pinto III phenotype is not the result of a large deletion of the PHA structural genes. Messenger RNA isolated from cv. Pinto III developing cotyledons synthesizes in vitro very small amounts of a protein which is recognized by antibodies specific for PHA, and gives, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single band with molecular weight similar but not identical to that of PHA-L polypeptides. This protein is also synthesized in vivo at a very reduced level, less than 1% compared with PHA in normal cultivars, and has mitogenic activity comparable to that of the PHA-L subunit, while it shows very weak erythroagglutinating activity. The initial steps in the synthesis and processing of this protein are identical to those already identified for PHA polypeptides. The cv. Pinto III protein could be either a PHA-L polypeptide whose synthesis is not affected by the mutation or a PHA-like lectin present normally at low levels in P. vulgaris.

Isolated nucleic acids encoding antipathogenic polypeptides and uses thereof

DOEpatents

Altier, Daniel J.; Crane, Virginia C.; Ellanskaya, Irina; Ellanskaya, Natalia; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K.; Schepers, Eric J.; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

2010-04-20

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from fungal fermentation broths. Nucleic acids that encode the antipathogenic polypeptides are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

Antifungal polypeptides

DOEpatents

Altier, Daniel J.; Dahlbacka, Glen; Ellanskaya, legal representative, Natalia; Herrmann, Rafael; Hunter-Cevera, Jennie; McCutchen, Billy F.; Presnail, James K.; Rice, Janet A.; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser; Ellanskaya, deceased, Irina

2007-12-11

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

Antifungal polypeptides

DOEpatents

Altier, Daniel J.; Dahlbacka, Glen; Elleskaya, Irina; Ellanskaya, legal representative; Natalia; Herrmann, Rafael; Hunter-Cevera, Jennie; McCutchen, Billy F.; Presnail, James K.; Rice, Janet A.; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

2010-08-10

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

Antifungal polypeptides

DOEpatents

Altier, Daniel J [Waukee, IA; Dahlbacka, Glen [Oakland, CA; Elleskaya, Irina [Kyiv, UA; Ellanskaya, legal representative, Natalia; Herrmann, Rafael [Wilmington, DE; Hunter-Cevera, Jennie [Elliott City, MD; McCutchen, Billy F [College Station, IA; Presnail, James K [Avondale, PA; Rice, Janet A [Wilmington, DE; Schepers, Eric [Port Deposit, MD; Simmons, Carl R [Des Moines, IA; Torok, Tamas [Richmond, CA; Yalpani, Nasser [Johnston, IA

2011-04-12

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

Antifungal polypeptides

DOEpatents

Altier, Daniel J [Granger, IA; Dahlbacka, Glen [Oakland, CA; Ellanskaya, Irina [Kyiv, UA; Ellanskaya, legal representative, Natalia; Herrmann, Rafael [Wilmington, DE; Hunter-Cevera, Jennie [Elliott City, MD; McCutchen, Billy F [College Station, TX; Presnail, James K [Avondale, PA; Rice, Janet A [Wilmington, DE; Schepers, Eric [Port Deposit, MD; Simmons, Carl R [Des Moines, IA; Torok, Tamas [Richmond, CA; Yalpani, Nasser [Johnston, IA

2012-04-03

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

[beta]-Glucan Synthesis in the Cotton Fiber (III. Identification of UDP-Glucose-Binding Subunits of [beta]-Glucan Synthases by Photoaffinity Labeling with [[beta]-32P]5[prime]-N3-UDP-Glucose.

PubMed Central

Li, L.; Drake, R. R.; Clement, S.; Brown, R. M.

1993-01-01

Using differential product entrapment and photolabeling under specifying conditions, we identifIed a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Gossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring [beta]-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of [beta]-1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring [beta]-1,3-glucan synthesis, is coincident with [beta]-1,3-glucan synthase activity, and is calcium dependent. The possible involvement of other polypeptides in the synthesis of [beta]-1,3-glucan is discussed. PMID:12231766

Chirality-selected phase behaviour in ionic polypeptide complexes

DOE PAGES

Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; ...

2015-01-14

In this study, polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with amore » β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation.« less

Localization of arginine decarboxylase in tobacco plants.

PubMed

Bortolotti, Cristina; Cordeiro, Alexandra; Alcázar, Rubén; Borrell, Antoni; Culiañez-Macià, Francisco A.; Tiburcio, Antonio F.; Altabella, Teresa

2004-01-01

The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli, and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types.

Synthesis and processing of equine herpesvirus 1 glycoprotein D.

PubMed

Flowers, C C; Flowers, S P; Jennings, S R; O'Callaghan, D J

1995-04-01

Previous studies (C. C. Flowers and D. J. O'Callaghan, 1992, Virology 190, 307-315) employed peptide-specific antibodies to identify the product of the glycoprotein D (gD) gene of equine herpesvirus 1 strain Kentucky A (KyA). gD polypeptides of 55 and 58 kDa were detected in EHV-1-infected L-M cells, and the 58-kDa protein was observed in the membrane fraction of EHV-1 virions. In this report, the kinetics of synthesis and processing of gD polypeptides are described. One-hour pulse-labeling of EHV-1-infected L-M cells revealed that gD proteins are first detected at 6 hr after infection and that maximal synthesis of gD occurs between 5 and 8 hr postinfection. gD polypeptides accumulate progressively with time of infection as shown by immunoprecipitation analysis of gD proteins. Pulse-chase analysis of gD revealed that the 55-kDa protein is a precursor to the 58-kDa species and that processing of all pulse-labeled precursor protein requires approximately 2.5 hr. Analysis of the carbohydrate content of gD proteins, as judged by their sensitivity to digestion with endoglycosidases, revealed that the 55-kDa gD precursor contains high-mannose N-linked oligosaccharides, while the 58-kDa gD mature polypeptide possesses complex type oligosaccharides. Expression of the mature form of gD on the cell surface, as determined by fluorescent flow cytometric analysis, is delayed compared to the accumulation of the mature form of gD within the cell. The gD ORF encodes a potential protein of 442 amino acids but analysis of the translated sequence of gD indicated that the gD polypeptide is 392 amino acids, a size predicted by previous mapping of the transcription start site of the gD mRNA. Coupled in vitro transcription/translation of a pGEM-3Z construct containing the 392-amino-acid gD ORF, in the absence or presence of canine pancreatic microsomes, demonstrated that the 43-kDa gD polypeptide undergoes processing in vitro. These studies demonstrate that the EHV-1 strain KyA gD is processed in a fashion similar to that of the gD proteins of other alphaherpesviruses.

The expression of 11β-hydroxysteroid dehydrogenase type 1 and 2 in nasal polyp-derived epithelial cells and its possible contribution to glucocorticoid activation in nasal polyp.

PubMed

Kook, Jin Ho; Kim, Hyun Jin; Kim, Kyung Won; Park, Se Jin; Kim, Tae Hoon; Lim, Sae Hee; Kang, Sung Hoon; Lee, Sang Hag

2015-01-01

The actions of glucocorticoids in target tissues depend on the local metabolism of glucocorticoids catalyzed by 11β hydroxysteroid dehydrogenase (HSD) 1 and 2. Glucocorticoids are the most effective anti-inflammatory drugs in the treatment of nasal polyps. However, the mechanisms that underlie the anti-inflammatory effects are unclear. The present study analyzed the expression of 11β-HSD1, 11β-HSD2, and steroidogenic enzymes (cytochrome P450, family 11, subfamily B, polypeptide 1 [CYP11B1]; cytochrome P450, family 11, subfamily A, polypeptide 1 [CYP11A1]) in nasal polyp tissues, and endogenous cortisol levels in nasal polyp-derived epithelial cells. The expression levels and distribution pattern of 11β-HSD1, 11β-HSD2, CYP11B1, and CYP11A1 were determined in nasal polyp tissues or nasal polyp-derived epithelial cells by using real-time polymerase chain reaction, Western blot, and immunohistochemistry testing. The expression levels of cortisol by using enzyme-linked immunosorbent assay were determined in cultured polyp-derived epithelial cells treated with adrenocorticotrophic hormone (ACTH), 11β-HSD1 inhibitor, or small interfering ribonucleic acid technique. The effect of glucocorticoids on the expression levels of these enzymes was investigated in cultured cells. Expressed in nasal polyp tissues and nasal polyp-derived epithelial cells were 11β-HSD1, 11β-HSD2, CYP11B1, and CYP11A1. Cortisol production in cultured epithelial cells was decreased in cells treated with 11β-HSD1 small interfering ribonucleic acid or inhibitor, compared with nontreated cells. Cultured cells treated with adrenocorticotropic hormone induced increased cortisol production. 11β-HSD1 expression levels were upregulated in cells treated with glucocorticoid. Analysis of these results indicated that 11β-HSD1 expressed in polyp-derived epithelial cells may be involved in the anti-inflammatory function of glucocorticoid in the treatment of nasal polyps, which contributes to increased levels of endogenous cortisol.

Postnatal Migration of Cerebellar Interneurons

PubMed Central

Galas, Ludovic; Bénard, Magalie; Lebon, Alexis; Komuro, Yutaro; Schapman, Damien; Vaudry, Hubert; Vaudry, David; Komuro, Hitoshi

2017-01-01

Due to its continuing development after birth, the cerebellum represents a unique model for studying the postnatal orchestration of interneuron migration. The combination of fluorescent labeling and ex/in vivo imaging revealed a cellular highway network within cerebellar cortical layers (the external granular layer, the molecular layer, the Purkinje cell layer, and the internal granular layer). During the first two postnatal weeks, saltatory movements, transient stop phases, cell-cell interaction/contact, and degradation of the extracellular matrix mark out the route of cerebellar interneurons, notably granule cells and basket/stellate cells, to their final location. In addition, cortical-layer specific regulatory factors such as neuropeptides (pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin) or proteins (tissue-type plasminogen activator (tPA), insulin growth factor-1 (IGF-1)) have been shown to inhibit or stimulate the migratory process of interneurons. These factors show further complexity because somatostatin, PACAP, or tPA have opposite or no effect on interneuron migration depending on which layer or cell type they act upon. External factors originating from environmental conditions (light stimuli, pollutants), nutrients or drug of abuse (alcohol) also alter normal cell migration, leading to cerebellar disorders. PMID:28587295

Epitope mapping of commercial antibodies that detect myocilin.

PubMed

Patterson-Orazem, Athéna C; Hill, Shannon E; Fautsch, Michael P; Lieberman, Raquel L

2018-05-09

The presence of myocilin is often used in the process of validating trabecular meshwork (TM) cells and eye tissues, but the antibody reagents used for detection are poorly characterized. Indeed, for over a century, researchers have been using antibodies to track proteins of interest in a variety of biological contexts, but many antibodies remain ill-defined at the molecular level and in their target epitope. Such issues have prompted efforts from major funding agencies to validate reagents and combat reproducibility issues across biomedical sciences. Here we characterize the epitopes recognized by four commercial myocilin antibodies, aided by structurally and biochemically characterized myocilin fragments. All four antibodies recognize enriched myocilin secreted from human TM cell media. The detection of myocilin fragments by ELISA and Western blot reveal a variety of epitopes across the myocilin polypeptide chain. A more precise understanding of myocilin antibody targets, including conformational specificity, should aid the community in standardizing protocols across laboratories and in turn, lead to a better understanding of eye physiology and disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Characterization of a heparin-binding growth factor from adenocarcinoma of the kidney.

PubMed

Mydlo, J H; Heston, W D; Fair, W R

1988-12-01

A polypeptide isolated from tissue extracts of renal adenocarcinoma was mitogenic for BALB/c 3T3 cells and human umbilical vein (HUV) cells in culture. It also demonstrated angiogenic ability using the chorioallantoic membrane bioassay. Using heparin-sepharose affinity chromatography the purified protein eluted with a NaCl concentration between 1.4 and 1.8 M and demonstrated a molecular weight of approximately 17,000 daltons based on SDS polyacrylamide gel electrophoresis. Half maximal stimulation of tritiated thymidine incorporation into BALB/c 3T3 cells was achieved by 1.6 ng./ml. of the heparin binding material. Western blot analysis using antibodies specific to basic fibroblast growth factor (bFGF) only or acidic FGF (aFGF) only demonstrated that the purified protein binds to the former and not the latter. The characteristics of this material, in effect the elution profile off heparin-Sepharose, the molecular weight, angiogenic activity and the results of western blot analysis, suggest that this growth factor is similar to the family of basic fibroblast growth factors.

Are non-muscle actin isoforms functionally equivalent?

PubMed

Simiczyjew, Aleksandra; Pietraszek-Gremplewicz, Katarzyna; Mazur, Antonina Joanna; Nowak, Dorota

2017-11-01

Actin is highly conserved and it is the most widespread protein in eukaryotic cells. One of the most important features of actin, which allows it to have many different functions, is its ability to polymerize and interact with many other proteins. Actins are the major constituent of the actin cytoskeleton, which is an important system that is involved in various aspects of cell function, including cell motility, structure, integrity, regulation of signal transduction and transcription. Six mammal actin isoforms are highly conserved and share common functions. Two of them, β and γ non-muscle actin isoforms, which differ only by four amino acids located at the N-terminus of the polypeptide chain, are required for survival and proper cell functioning. We also summarized data about actbl2, which is suggested to be a newly discovered isoactin. Here, we review the current knowledge about tissue-specific expression of the non-muscle actin isoforms and possible functional differences between them. We also discuss molecular tools, which in recent years have allowed for a better understanding of the role of these proteins in cell functioning.

Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome.

PubMed Central

Farza, H; Hadchouel, M; Scotto, J; Tiollais, P; Babinet, C; Pourcel, C

1988-01-01

We have sought to address the problem of the host and tissue specificity of the hepatitis B virus (HBV) by using transgenic mice obtained after injection of head-to-tail dimers of the HBV genome. Viral DNA replication and protein synthesis were obtained in one of nine transgenic mice containing integrated HBV DNA. The RNAs encoding the HBV surface antigen and the core antigen were synthesized in the liver, the kidney, and the heart. In these organs, DNA replicative intermediates similar to those found during normal infection were associated with corelike structures. Large amounts of core polypeptides and capsids were detected in the nuclei in the absence of any pathological effect. These results show that the different steps of HBV multiplication can take place in nonliver nonhuman cells once the problem of entry into the host cell is overcome. In the absence of a small laboratory animal infectable by HBV, such transgenic mice should be helpful for the study of many aspects of viral multiplication. Images PMID:2845128

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

Okita, T.W.; Nakata, P.A.; Anderson, J.M.

ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tubermore » subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.« less

Amino acid sequence of bovine muzzle epithelial desmocollin derived from cloned cDNA: a novel subtype of desmosomal cadherins.

PubMed

Koch, P J; Goldschmidt, M D; Walsh, M J; Zimbelmann, R; Schmelz, M; Franke, W W

1991-05-01

Desmosomes are cell-type-specific intercellular junctions found in epithelium, myocardium and certain other tissues. They consist of assemblies of molecules involved in the adhesion of specific cell types and in the anchorage of cell-type-specific cytoskeletal elements, the intermediate-size filaments, to the plasma membrane. To explore the individual desmosomal components and their functions we have isolated DNA clones encoding the desmosomal glycoprotein, desmocollin, using antibodies and a cDNA expression library from bovine muzzle epithelium. The cDNA-deduced amino-acid sequence of desmocollin (presently we cannot decide to which of the two desmocollins, DC I or DC II, this clone relates) defines a polypeptide with a calculated molecular weight of 85,000, with a single candidate sequence of 24 amino acids sufficiently long for a transmembrane arrangement, and an extracellular aminoterminal portion of 561 amino acid residues, compared to a cytoplasmic part of only 176 amino acids. Amino acid sequence comparisons have revealed that desmocollin is highly homologous to members of the cadherin family of cell adhesion molecules, including the previously sequenced desmoglein, another desmosome-specific cadherin. Using riboprobes derived from cDNAs for Northern-blot analyses, we have identified an mRNA of approximately 6 kb in stratified epithelia such as muzzle epithelium and tongue mucosa but not in two epithelial cell culture lines containing desmosomes and desmoplakins. The difference may indicate drastic differences in mRNA concentration or the existence of cell-type-specific desmocollin subforms. The molecular topology of desmocollin(s) is discussed in relation to possible functions of the individual molecular domains.

Ordered biological nanostructures formed from chaperonin polypeptides

NASA Technical Reports Server (NTRS)

Trent, Jonathan D. (Inventor); McMillan, R. Andrew (Inventor); Paavola, Chad D. (Inventor); Kagawa, Hiromi (Inventor)

2010-01-01

The following application relates to nanotemplates, nanostructures, nanoarrays and nanodevices formed from wild-type and mutated chaperonin polypeptides, methods of producing such compositions, methods of using such compositions and particular chaperonin polypeptides that can be utilized in producing such compositions.

pH-sensitive interaction of HMG-CoA reductase inhibitors (statins) with organic anion transporting polypeptide 2B1.

PubMed

Varma, Manthena V; Rotter, Charles J; Chupka, Jonathan; Whalen, Kevin M; Duignan, David B; Feng, Bo; Litchfield, John; Goosen, Theunis C; El-Kattan, Ayman F

2011-08-01

The human organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) is ubiquitously expressed and may play an important role in the disposition of xenobiotics. The present study aimed to examine the role of OATP2B1 in the intestinal absorption and tissue uptake of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitors (statins). We first investigated the functional affinity of statins to the transporter as a function of extracellular pH, using OATP2B1-transfeced HEK293 cells. The results indicate that OATP2B1-mediated transport is significant for rosuvastatin, fluvastatin and atorvastatin, at neutral pH. However, OATP2B1 showed broader substrate specificity as well as enhanced transporter activity at acidic pH. Furthermore, uptake at acidic pH was diminished in the presence of proton ionophore, suggesting proton gradient as the driving force for OATP2B1 activity. Notably, passive transport rates are predominant or comparable to active transport rates for statins, except for rosuvastatin and fluvastatin. Second, we studied the effect of OATP modulators on statin uptake. At pH 6.0, OATP2B1-mediated transport of atorvastatin and cerivastatin was not inhibitable, while rosuvastatin transport was inhibited by E-3-S, rifamycin SV and cyclosporine with IC(50) values of 19.7 ± 3.3 μM, 0.53 ± 0.2 μM and 2.2 ± 0.4 μM, respectively. Rifamycin SV inhibited OATP2B1-mediated transport of E-3-S and rosuvastatin with similar IC(50) values at pH 6.0 and 7.4, suggesting that the inhibitor affinity is not pH-dependent. Finally, we noted that OATP2B1-mediated transport of E-3-S, but not rosuvastatin, is pH sensitive in intestinal epithelial (Caco-2) cells. However, uptake of E-3-S and rosuvastatin by Caco-2 cells was diminished in the presence of proton ionophore. The present results indicate that OATP2B1 may be involved in the tissue uptake of rosuvastatin and fluvastatin, while OATP2B1 may play a significant role in the intestinal absorption of several statins due to their transporter affinity at acidic pH.

P43/pro-EMAPII: A Potential Biomarker for Discriminating Traumatic Versus Ischemic Brain Injury

PubMed Central

Yao, Changping; Williams, Anthony J.; Ottens, Andrew K.; Lu, X.-C. May; Liu, Ming Cheng; Hayes, Ronald L.; Wang, Kevin K.; Tortella, Frank C.

2009-01-01

Abstract To gain additional insights into the pathogenic cellular and molecular mechanisms underlying different types of brain injury (e.g., trauma versus ischemia), recently attention has focused on the discovery and study of protein biomarkers. In previous studies, using a high-throughput immunoblotting (HTPI) technique, we reported changes in 29 out of 998 proteins following acute injuries to the rat brain (penetrating traumatic versus focal ischemic). Importantly, we discovered that one protein, endothelial monocyte-activating polypeptide II precursor (p43/pro-EMAPII), was differentially expressed between these two types of brain injury. Among other functions, p43/pro-EMAPII is a known pro-inflammatory cytokine involved in the progression of apoptotic cell death. Our current objective was to verify the changes in p43/pro-EMAPII expression, and to evaluate the potentially important implications that the differential regulation of this protein has on injury development. At multiple time points following either a penetrating ballistic-like brain injury (PBBI), or a transient middle cerebral artery occlusion (MCAo) brain injury, tissue samples (6–72 h), CSF samples (24 h), and blood samples (24 h) were collected from rats for analysis. Changes in protein expression were assessed by Western blot analysis and immunohistochemistry. Our results indicated that p43/pro-EMAPII was significantly increased in brain tissues, CSF, and plasma following PBBI, but decreased after MCAo injury compared to their respective sham control samples. This differential expression of p43/pro-EMAPII may be a useful injury-specific biomarker associated with the underlying pathologies of traumatic versus ischemic brain injury, and provide valuable information for directing injury-specific therapeutics. PMID:19317603

Stage-specific expression of DDX4 and c-kit at different developmental stages of the porcine testis.

PubMed

Lee, Ran; Lee, Won-Young; Park, Hyun-Jung; Ha, Woo-Tae; Woo, Jae-Seok; Chung, Hak-Jae; Lee, Ji-Heon; Hong, Kwonho; Song, Hyuk

2018-03-01

Spermatogenesis begins with spermatogonial stem cells (SSCs), which are located in the basement membrane of the adult testes. Previous studies have described specific biomarkers for undifferentiated porcine spermatogonia or SSCs; however, these markers are not sufficient to understand spermatogenesis at different developmental stages. The objective of this study was characterize the expression of DEAD-Box polypeptide 4 (DDX4, also known as VASA) and tyrosine-protein kinase kit (c-kit), as potential markers of male germ cells in the porcine testis. In porcine testis tissue at prepubertal stages (5, 30, and 60 days), DDX4 and c-kit protein expression was detected in the most undifferentiated spermatogonia, which also express protein gene product 9.5 (PGP9.5). However, in porcine testis tissues from pubertal and postpubertal stages (90, 120, and 150 days), DDX4 and c-kit were not detected in PGP9.5-positive undifferentiated spermatogonia. The DDX4 expression pattern was similar to that of c-kit in the porcine testis. In adult porcine testes, DDX4-expressing cells were located on the lumenal side, compared to synaptonemal complex protein 3-positive primary spermatocytes, but DDX-4 was not co-expressed with acrosin, a known acrosome marker. In addition, DDX4 was detected in PGP9.5-expressing porcine SSCs in culture. Based on our results, we suggest that DDX4 and c-kit are putative markers of undifferentiated spermatogonia in the prepubertal porcine testis. While in the postpubertal porcine testis, they are markers of differentiated spermatocytes. These findings may facilitate future studies of porcine spermatogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Coexpression patterns of vimentin and glial filament protein with cytokeratins in the normal, hyperplastic, and neoplastic breast.

PubMed Central

Gould, V. E.; Koukoulis, G. K.; Jansson, D. S.; Nagle, R. B.; Franke, W. W.; Moll, R.

1990-01-01

The authors studied by immunohistochemistry the intermediate filament (IF) protein profile of 66 frozen samples of breast tissue, including normal parenchyma, all variants of fibrocystic disease (FCD), fibroadenomas, cystosarcoma phylloides, and ductal and lobular carcinomas. Monoclonal antibodies (MAbs) to cytokeratins included MAb KA 1, which binds to polypeptide 5 in a complex with polypeptide 14 and recognizes preferentially myoepithelial cells; MAb KA4, which binds to polypeptides 14, 15, 16 and 19; individual MAbs to polypeptides 7, 13, and 16, 17, 18, and 19, and the MAb mixture AE1/AE3. The authors also applied three MAbs to vimentin (Vim), and three MAbs to glial filament protein (GFP). Selected samples were studied by double-label immunofluorescence microscopy and by staining sequential sections with some of the said MAbs, an MAb to alpha-smooth muscle actin, and well-characterized polyclonal antibodies for the possible coexpression of diverse types of cytoskeletal proteins. Gel electrophoresis and immunoblot analysis also were performed. All samples reacted for cytokeratins with MAbs AE1/AE3, although the reaction did not involve all cells. Monoclonal antibody KA4 stained preferentially the luminal-secretory cells in the normal breast and in FCD, whereas it stained the vast majority of cells in all carcinomas. Monoclonal antibody KA1 stained preferentially the basal-myoepithelial cells of the normal breast and FCD while staining tumor cell subpopulations in 4 of 31 carcinomas. Vimentin-positive cells were found in 8 of 12 normal breasts and in 12 of 20 FCD; in most cases, Vim-reactive cells appeared to be myoepithelial, but occasional luminal cells were also stained. Variable subpopulations of Vim-positive cells were noted in 9 of 20 ductal and in 1 of 7 lobular carcinomas. Glial filament protein-reactive cells were found in normal breast lobules and ducts and in 15 of 20 cases of FCD; with rare exceptions, GFP-reactivity was restricted to basally located, myoepithelial-appearing cells. Occasional GFP-reactive cells were found in 3 of 31 carcinomas. Evaluation of sequential sections and double-label immunofluorescence microscopy showed the coexpression of certain cytokeratins (possibly including polypeptides 14 and 17) with vimentin and alpha-smooth muscle actin together with GFP in some myoepithelial cells. The presence of GFP in myoepithelial cells was confirmed by gel electrophoresis and immunoblotting. Our results indicate that coexpression of cytokeratin with vimentin and/or GFP is comparatively frequent in normal basal-myoepithelial cells of the breast.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1700618

A Bacillus subtilis Secreted Protein with a Role in Endospore Coat Assembly and Function

PubMed Central

Serrano, Mónica; Zilhão, Rita; Ricca, Ezio; Ozin, Amanda J.; Moran, Charles P.; Henriques, Adriano O.

1999-01-01

Bacterial endospores are encased in a complex protein coat, which confers protection against noxious chemicals and influences the germination response. In Bacillus subtilis, over 20 polypeptides are organized into an amorphous undercoat, a lamellar lightly staining inner structure, and an electron-dense outer coat. Here we report on the identification of a polypeptide of about 30 kDa required for proper coat assembly, which was extracted from spores of a gerE mutant. The N-terminal sequence of this polypeptide matched the deduced product of the tasA gene, after removal of a putative 27-residue signal peptide, and TasA was immunologically detected in material extracted from purified spores. Remarkably, deletion of tasA results in the production of asymmetric spores that accumulate misassembled material in one pole and have a greatly expanded undercoat and an altered outer coat structure. Moreover, we found that tasA and gerE mutations act synergistically to decrease the efficiency of spore germination. We show that tasA is the most distal member of a three-gene operon, which also encodes the type I signal peptidase SipW. Expression of the tasA operon is enhanced 2 h after the onset of sporulation, under the control of ςH. When tasA transcription is uncoupled from sipW expression, a presumptive TasA precursor accumulates, suggesting that its maturation depends on SipW. Mature TasA is found in supernatants of sporulating cultures and intracellularly from 2 h of sporulation onward. We suggest that, at an early stage of sporulation, TasA is secreted to the septal compartment. Later, after engulfment of the prespore by the mother cell, TasA acts from the septal-proximal pole of the spore membranes to nucleate the organization of the undercoat region. TasA is the first example of a polypeptide involved in coat assembly whose production is not mother cell specific but rather precedes its formation. Our results implicate secretion as a mechanism to target individual proteins to specific cellular locations during the assembly of the bacterial endospore coat. PMID:10368135

Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

DOEpatents

Young, Travis; Schultz, Peter G

2013-12-17

Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

DOEpatents

Young, Travis; Schultz, Peter G

2014-01-28

Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

DOEpatents

Young, Travis; Schultz, Peter G.

2015-08-18

Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

Tunable drug loading and release from polypeptide multilayer nanofilms

PubMed Central

Jiang, Bingbing; Li, Bingyun

2009-01-01

Polypeptide multilayer nanofilms were prepared using electrostatic layer-by-layer self-assembly nanotechnology. Small charged drug molecules (eg, cefazolin, gentamicin, and methylene blue) were loaded in polypeptide multilayer nanofilms. Their loading and release were found to be pH-dependent and could also be controlled by changing the number of film layers and drug incubation time, and applying heat-treatment after film formation. Antibioticloaded polypeptide multilayer nanofilms showed controllable antibacterial properties against Staphylococcus aureus. The developed biodegradable polypeptide multilayer nanofilms are capable of loading both positively- and negatively-charged drug molecules and promise to serve as drug delivery systems on biomedical devices for preventing biomedical device-associated infection, which is a significant clinical complication for both civilian and military patients. PMID:19421369

Gamma-carboxylation and fragmentation of osteocalcin in human serum defined by mass spectrometry

USDA-ARS?s Scientific Manuscript database

Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is n...

Purification of Xenopus laevis mitochondrial RNA polymerase and identification of a dissociable factor required for specific transcription

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

Bogenhagen, D.F.; Insdorf, N.F.

1988-07-01

The Xenopus laevis mitochondrial RNA (mtRNA) polymerase was purified to near homogeneity with an overall yield approaching 50%. The major polypeptides in the final fraction were a doublet of proteins of approximately 140 kilodaltons that copurified with the mtRNA polymerase activity. It appeared likely that the smaller polypeptide is a breakdown product of the larger one. The highly purified polymerase was active in nonspecific transcription but required a dissociable factor for specific transcription of X. laevis mtDNA. The factor could be resolved from mtRNA polymerase by hydrophobic chromatography and had a sedimentation coefficient of 3.0 S. The transcription factor elutedmore » from both the hydrophobic column and a Mono Q anion-exchange column as a single symmetrical peak. The mtRNA polymerase and this factor together are necessary and sufficient for active transcription from four promoters located in a noncoding region of the mtDNA genome between the gene for tRNA/sup Phe/ and the displacement loop.« less

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

Fogh, R.H.; Mabbutt, B.C.; Kem, W.R.

Sequence-specific assignments are reported for the 500-MHz H nuclear magnetic resonance (NMR) spectrum of the 48-residue polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I). Spin systems were first identified by using two-dimensional relayed or multiple quantum filtered correlation spectroscopy, double quantum spectroscopy, and spin lock experiments. Specific resonance assignments were then obtained from nuclear Overhauser enhancement (NOE) connectivities between protons from residues adjacent in the amino acid sequence. Of a total of 265 potentially observable resonances, 248 (i.e., 94%) were assigned, arising from 39 completely and 9 partially assigned amino acid spin systems. The secondary structure ofmore » Sh I was defined on the basis of the pattern of sequential NOE connectivities. NOEs between protons on separate strands of the polypeptide backbone, and backbone amide exchange rates. Sh I contains a four-stranded antiparallel {beta}-sheet encompassing residues 1-5, 16-24, 30-33, and 40-46, with a {beta}-bulge at residues 17 and 18 and a reverse turn, probably a type II {beta}-turn, involving residues 27-30. No evidence of {alpha}-helical structure was found.« less

Structures and functions of proteins and nucleic acids in protein biosynthesis

NASA Astrophysics Data System (ADS)

Miyazawa, Tatsuo; Yokoyama, Shigeyuki

Infrared and Raman spectroscopy is useful for studying helical conformations of polypeptides, which are determined by molecular structure parameters. Nuclear magnetic resonance spectroscopy, as well as X-ray analysis, is now established to be important for conformation studies of proteins and nucleic acids in solution. This article is mainly concerned with the conformational aspect and function regulation in protein biosynthesis. The strict recognition of transfer ribonucleic acid (tRNA) by aminoacyl-tRNA synthetase (ARS) is achieved by multi-step mutual adaptation. The conformations of ARS-bound amino acids have been elucidated by transferred nuclear Overhauser effect analysis. Aminoacyl-tRNA takes the 3‧-isomeric form in the polypeptide chain elongation cycle. The regulation of codon recognition by post-transcriptional modification is achieved by conversion of the conformational characteristic of the anticodon of tRNA. The cytidine → lysidine modification of the anticodon of minor isoleucine tRNA concurrently converts the amino acid specificity and the codon specificity. As novel protein engineering, a basic strategy has been established for in vivo biosynthesis of proteins that are substituted with unnatural amino acids (alloproteins).

Classical late infantile neuronal ceroid lipofuscinosis fibroblasts are deficient in lysosomal tripeptidyl peptidase I.

PubMed

Vines, D J; Warburton, M J

1999-01-25

Tripeptidyl peptidase I (TPP-I) is a lysosomal enzyme that cleaves tripeptides from the N-terminus of polypeptides. A comparison of TPP-I amino acid sequences with sequences derived from an EST database suggested that TPP-I is identical to a pepstatin-insensitive carboxyl proteinase of unknown specificity which is mutated in classical late infantile neuronal ceroid lipofuscinosis (LINCL), a lysosomal storage disease. Both TPP-I and the carboxyl proteinase have an M(r) of about 46 kDa and are, or are predicted to be, resistant to inhibitors of the four major classes of proteinases. Fibroblasts from LINCL patients have less than 5% of the normal TPP-I activity. The activities of other lysosomal enzymes, including proteinases, are in the normal range. LINCL fibroblasts are also defective at degrading short polypeptides and this defect can be induced in normal fibroblasts by treatment with a specific inhibitor or TPP-I. These results suggest that the cell damage, especially neuronal, observed in LINCL results from the defective degradation and consequent lysosomal storage of small peptides.

Transcriptional control in Alicyclobacillus acidocaldarius and associated genes, proteins, and methods

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

Lee, Brady D.; Thompson, David N.; Apel, William A.

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of modulating transcription or transcription or transcriptional control using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Enhanced processive cellulases

DOEpatents

Adney, William S.; Beckham, Gregg T.; Jarvis, Eric; Himmel, Michael E.; Decker, Stephen R.; Linger, Jeffrey G.; Podkaminer, Kara; Baker, John O.; Taylor, II, Larry; Xu, Qi; Singh, Arjun

2017-06-20

Nucleic acid sequences encoding chimeric polypeptides that exhibit enhanced cellulase activities are disclosed herein. These nucleic acids may be expressed in hosts such as fungi, which in turn may be cultured to produce chimeric polypeptides. Also disclosed are chimeric polypeptides and their use in the degradation of cellulosic materials.

Transcriptional control in Alicyclobacillus acidocaldarius and associated genes, proteins, and methods

DOEpatents

Lee, Brady Deneys; Thompson, David N; Apel, William A.; Thompson, Vicki Slavchev; Reed, David W; Lacey, Jeffrey A

2014-05-06

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of modulating transcription or transcription or transcriptional control using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Transcriptional control in Alicyclobacillus acidocaldarius and associated genes, proteins, and methods

DOEpatents

Lee, Brady D.; Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

2015-11-17

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of modulating transcription or transcription or transcriptional control using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Transcriptional control in alicyclobacillus acidocaldarius and associated genes, proteins, and methods

DOEpatents

Lee, Brady D; Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A

2016-11-22

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of modulating transcription or transcription or transcriptional control using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Caffeine-water-polypeptide interaction in aqueous solution

NASA Astrophysics Data System (ADS)

Ghabi, Habib; Dhahbi, Mahmoud

1999-04-01

The interaction of caffeine monomer with the synthetic polypeptides polyasparagine (pAg) and polyaspartic acid (pAsp) was studied by UV spectrophotometry. The results show that different types of interactions are possible depending on the nature of polypeptide. The form of the complex was discussed.