Analysis of SUMOylated proteins using SUMO-traps

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Sacarificação da polpa cítrica por hidrólise ácida e cultivo pelo processo de batelada simples com reciclo de Trichoderma reesei ou Aspergillus niger com produção de celulases e poligalacturonases

Pós-graduação em Microbiologia - IBILCE

Proceedings of the Sixteenth Annual Biochemical Engineering Symposium

This volume represents the proceedings of the Sixteenth Annual Biochemical Engineering Symposium held at Kansas State University on April 26, 1986. Some of the papers describe the progress of ongoing projects, and others contain the results of completed projects. Only brief summaries are given of many of the papers that will be published in full elsewhere. ContentsEnd-Product Inhibition of the Acetone-Butanol Fermentation—Bob Kuhn, Colorado State University Effect of Multiple Substrates in Ethanal Fermentations from Cheese Whey—C.J. Wang, University of Missouri Extraction and Fermentation of Ensiled Sweet Sorghum—Karl Noah, Colorado State University Removal of Nucleic Acids from Bakers' Yeast—Richard M. Cordes, Iowa State University Modeling the Effects of Plasmid Replication and Product Repression on the Growth Rate of Recombinant Bacteria—William E. Bentley, University of Colorado Indirect Estimates of Cell Concentrations in Mass Cultivation of Bacterial Cells—Andrew Fisher, University of Missouri A Mathematical Model for Liquid Recirculation in Airlift Columns—C.H.Lee, Kansas State University Characterization of Imperfect Mixing of Batch Reactors by Two Compartment Model—Peter Sohn, University of Missouri First Order Breakage Model for the Degradation of Pullalan in the Batch Fermentor—Stephen A. Milligan, Kansas State University Synthesis and Nuclear Magnetic Resonance of 13C-Labeled Amylopectin and Maltooligosaccharides—Bernard Y. Tao, Iowa State University Preparation of Fungal Starter Culture in Gas Fluidized Bed Reactor—Pal Mihaltz, Colorado State University Yeast Flocculation and Sedimentation—David Szlag, University of Colorado Protein Enrichment of Extrusion Cooked Corn by Solid Substrate Fermentation—Lucas Alvarez-Martinez, Colorado State University Optimum Design of a Hollow Fiber Mammalian Cell Reactor—Thomas Chresand, Colorado State University Gas Chromatography and Nuclear Magnetic Resonance of Trifluoroacetylated Carbohydrates—Steven T. Summerfelt, Iowa State University Kinetic and Bioenergetic Considerations for Modeling Photosynthetic Microbial P~ocesses in Producing Biomass and Treating Wastewater—H. Y. Lee, Kansas State University Mathematical Modeling and Simulation of Bicarbonate-Limited Photsynthetic Growth in Continuous Culture—Craig Curless, Kansas State University Data Acquisition and Control of a Rotary Drum Solid State Fermentor—Mnasria A. Habib, Colorado State University Biodegradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D)—Greg Sinton, Kansas State University

Nutritional and functional evaluation of wheat flour cookies supplemented with gram flour

Protein-enriched cookies were prepared by supplementing gram flour into wheat flour at levels of 0%, 10%, 20%, 30%, 40% and 50% and analysed for physicochemical properties. The protein quality of the cookies was assessed by feeding gram flour-supplemented cookies to albino rats for 10 days. The supplementation resulted in a significant increase in protein, fat, crude fibre and ash contents of the cookies. The thickness and spread factor of cookies differ significantly while non-significant effect was observed in the width of the cookies. The protein efficiency ratio, net protein utilization, biological value and true digestibility differed significantly among diets containing cookies with gram flour fed to rats. Cookies with 30% substitution of straight grade flour and gram flour produced acceptable cookies as compared to control. The cookies containing 40-50% gram flour were best regarded as protein bioavailability for rats.

Fresh pasta enrichment with protein concentrate of tilapia: nutritional and sensory characteristics.

With the goal of developing and characterizing the nutritional and sensory aspects of fresh pasta supplemented with tilapia protein concentrate, four types of pasta were prepared, with inclusion of 0, 10, 20, or 30% of tilapia protein concentrate. Linear effects were observed (P < 0.01) in crude protein, total lipids, ash, carbohydrate, and caloric values; these parameters increased with increasing amounts of tilapia protein concentrate in the pasta. The concentration of Na, P, Ca, Mg, and Zn increased linearly (P < 0.01) in correlation with the increase in protein concentrate content, while Fe content decreased linearly (P < 0.01). In the sensory analysis, texture, overall impression, and the acceptance index demonstrated a cubic regression (P < 0.05), with the inclusion of 20% protein concentrate yielding the best scores. Including up to 30% of tilapia protein concentrate in pasta yields an increased nutritional value, but based on the sensory results, 20% of tilapia protein concentrate in pasta is the recommended maximum level.

A functional proteomic method for the enrichment of peripheral membrane proteins reveals the collagen binding protein Hsp47 is exposed on the surface of activated human platelets

Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptor-proximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis.

Enrichment, isolation and characterisation of ruminal bacteria that degrade non-protein amino acids from the tropical legume Acacia angustissima

Acacia angustissima has been proposed as a protein supplement in countries where low quality forages predominate. A number of non-protein amino acids have been identified in the leaves of A. angustissima and these have been linked to toxicity in ruminants. The non-protein amino acid 4-n-acetyl-2,4-diaminobutyric acid (ADAB) has been shown to be the major amino acid in the leaves of A. angustissima. The current study aimed to identify micro-organisms from the rumen environment capable of degrading ADAB by using a defined rumen-simulating media with an amino acid extract from A. angustissima. A mixed enrichment culture was obtained that exhibited substantial ADAB-degrading ability. Attempts to isolate an ADAB-degrading micro-organism were carried out, however no isolates were able to degrade ADAB in pure culture. This enrichment culture was also able to degrade the non-protein amino acids diaminobutyric acid (DABA) and diaminopropionic acid (DAPA) which have structural similarities to ADAB. Two isolates were obtained which could degrade DAPA. One isolate is a novel Grain-positive rod (strain LPLR3) which belongs to the Firmicutes and is not closely related to any previously isolated bacterium. The other isolate is strain LPSR1 which belongs to the Gammaproteobacteria and is closely related (99.93% similar) to Klebsiella pneumoniae subsp. ozaenae. The studies demonstrate that the rumen is a potential rich source of undiscovered micro-organisms which have novel capacities to degrade plant secondary compounds. (c) 2005 Elsevier B.V. All rights reserved.

Daytime pattern of post-exercise protein intake affects whole-body protein turnover in resistance-trained males

Background The pattern of protein intake following exercise may impact whole-body protein turnover and net protein retention. We determined the effects of different protein feeding strategies on protein metabolism in resistance-trained young men. Methods: Participants were randomly assigned to ingest either 80g of whey protein as 8x10g every 1.5h (PULSE; n=8), 4x20g every 3h (intermediate, INT; n=7), or 2x40g every 6h (BOLUS; n=8) after an acute bout of bilateral knee extension exercise (4x10 repetitions at 80% maximal strength). Whole-body protein turnover (Q), synthesis (S), breakdown (B), and net balance (NB) were measured throughout 12h of recovery by a bolus ingestion of [ 15N]glycine with urinary [15N]ammonia enrichment as the collected end-product. Results PULSE Q rates were greater than BOLUS (?19%, P<0.05) with a trend towards being greater than INT (?9%, P=0.08). Rates of S were 32% and 19% greater and rates of B were 51% and 57% greater for PULSE as compared to INT and BOLUS, respectively (P<0.05), with no difference between INT and BOLUS. There were no statistical differences in NB between groups (P=0.23); however, magnitude-based inferential statistics revealed likely small (mean effect90%CI; 0.590.87) and moderate (0.800.91) increases in NB for PULSE and INT compared to BOLUS and possible small increase (0.421.00) for INT vs. PULSE. Conclusion We conclude that the pattern of ingested protein, and not only the total daily amount, can impact whole-body protein metabolism. Individuals aiming to maximize NB would likely benefit from repeated ingestion of moderate amounts of protein (?20g) at regular intervals (?3h) throughout the day.

Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden

Over 80% of women diagnosed with advanced-stage ovarian cancer die as a result of disease recurrence due to failure of chemotherapy treatment. In this study, using two distinct ovarian cancer cell lines (epithelial OVCA 433 and mesenchymal HEY) we demonstrate enrichment in a population of cells with high expression of CSC markers at the protein and mRNA levels in response to cisplatin, paclitaxel and the combination of both. We also demonstrate a significant enhancement in the sphere forming abilities of ovarian cancer cells in response to chemotherapy drugs. The results of these in vitro findings are supported by in vivo mouse xenograft models in which intraperitoneal transplantation of cisplatin or paclitaxel-treated residual HEY cells generated significantly higher tumor burden compared to control untreated cells. Both the treated and untreated cells infiltrated the organs of the abdominal cavity. In addition, immunohistochemical studies on mouse tumors injected with cisplatin or paclitaxel treated residual cells displayed higher staining for the proliferative antigen Ki67, oncogeneic CA125, epithelial E-cadherin as well as cancer stem cell markers such as Oct4 and CD117, compared to mice injected with control untreated cells. These results suggest that a short-term single treatment of chemotherapy leaves residual cells that are enriched in CSC-like traits, resulting in an increased metastatic potential. The novel findings in this study are important in understanding the early molecular mechanisms by which chemoresistance and subsequent relapse may be triggered after the first line of chemotherapy treatment.

Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry

RATIONALE Diseases including cancer and congenital disorders of glycosylation have been associated with changes in the site-specific extent of protein glycosylation. Saliva can be non-invasively sampled and is rich in glycoproteins, giving it the potential to be a useful biofluid for the discovery and detection of disease biomarkers associated with changes in glycosylation. METHODS Saliva was collected from healthy individuals and glycoproteins were enriched using phenylboronic acid based glycoprotein enrichment resin. Proteins were deglycosylated with peptide-N-glycosidase F and digested with AspN or trypsin. Desalted peptides and deglycosylated peptides were separated by reversed-phase liquid chromatography and detected with on-line electrospray ionization quadrupole-time-of-flight mass spectrometry using a 5600 TripleTof instrument. Site-specific glycosylation occupancy was semi-quantitatively determined from the abundance of deglycosylated and nonglycosylated versions of each given peptide. RESULTS Glycoprotein enrichment identified 67 independent glycosylation sites from 24 unique proteins, a 3.9-fold increase in the number of glycosylation sites identified. Enrichment of glycoproteins rather than glycopeptides allowed detection of both deglycosylated and nonglycosylated versions of each peptide, and thereby robust measurement of site-specific occupancy at 21 asparagines. Healthy individuals showed limited biological variability in occupancy, with partially modified sites having characteristics consistent with inefficient glycosylation by oligosaccharyltransferase. Inclusion of negative controls without enzymatic deglycosylation controlled for spontaneous chemical deamidation, and identified asparagines previously incorrectly annotated as glycosylated. CONCLUSIONS We developed a sample preparation and mass spectrometry detection strategy for rapid and efficient measurement of site-specific glycosylation occupancy on diverse salivary glycoproteins suitable for biomarker discovery and detection of changes in glycosylation occupancy in human disease.

Enrichment of circulating interleukin-17-secreting interleukin-23 receptor-positive γ/δ T cells in patients with active ankylosing spondylitis

Objective Ankylosing spondylitis (AS) is a common inflammatory arthritis affecting primarily the axial skeleton. IL23R is genetically associated with AS. This study was undertaken to investigate and characterize the role of interleukin-23 (IL-23) signaling in AS pathogenesis. Methods The study population consisted of patients with active AS (n = 17), patients with psoriatic arthritis (n = 8), patients with rheumatoid arthritis, (n = 9), and healthy subjects (n = 20). IL-23 receptor (IL-23R) expression in T cells was determined in each subject group, and expression levels were compared. Results The proportion of IL-23R-expressing T cells in the periphery was 2-fold higher in AS patients than in healthy controls, specifically driven by a 3-fold increase in IL-23R-positive γ/δ T cells in AS patients. The proportions of CD4+ and CD8+ cells that were positive for IL-17 were unchanged. This increased IL-23R expression on γ/δ T cells was also associated with enhanced IL-17 secretion, with no observable IL-17 production from IL-23R-negative γ/δ T cells in AS patients. Furthermore, γ/δ T cells from AS patients were heavily skewed toward IL-17 production in response to stimulation with IL-23 and/or anti-CD3/CD28. Conclusion Recently, mouse models have shown IL-17-secreting γ/δ T cells to be pathogenic in infection and autoimmunity. Our data provide the first description of a potentially pathogenic role of these cells in a human autoimmune disease. Since IL-23 is a maturation and growth factor for IL-17-producing cells, increased IL-23R expression may regulate the function of this putative pathogenic γ/δ T cell population.

Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays

Aberrant glycosylation of proteins is a hallmark of tumorigenesis, and could provide diagnostic value in cancer detection. Human saliva is an ideal source of glycoproteins due to the relatively high proportion of glycosylated proteins in the salivary proteome. Moreover, saliva collection is non-invasive, technically straightforward and the sample collection and storage is relatively easy. Although, differential glycosylation of proteins can be indicative of disease states, identification of differential glycosylation from clinical samples is not trivial. To facilitate salivary glycoprotein biomarker discovery, we optimised a method for differential glycoprotein enrichment from human saliva based on lectin magnetic bead arrays (saLeMBA). Selected lectins from distinct reactivity groups were used in the saLeMBA platform to enrich salivary glycoproteins from healthy volunteer saliva. The technical reproducibility of saLeMBA was analysed with LC-MS/MS to identify the glycosylated proteins enriched by each lectin. Our saLeMBA platform enabled robust glycoprotein enrichment in a glycoprotein- and lectin-specific manner consistent with known protein-specific glycan profiles. We demonstrated that saLeMBA is a reliable method to enrich and detect glycoproteins present in human saliva.

Comparison of tertiary structures of proteins in protein-protein complexes with unbound forms suggests prevalence of allostery in signalling proteins

Abstract: Background: Most signalling and regulatory proteins participate in transient protein-protein interactions during biological processes. They usually serve as key regulators of various cellular processes and are often stable in both protein-bound and unbound forms. Availability of high-resolution structures of their unbound and bound forms provides an opportunity to understand the molecular mechanisms involved. In this work, we have addressed the question "What is the nature, extent, location and functional significance of structural changes which are associated with formation of protein-protein complexes?" Results: A database of 76 non-redundant sets of high resolution 3-D structures of protein-protein complexes, representing diverse functions, and corresponding unbound forms, has been used in this analysis. Structural changes associated with protein-protein complexation have been investigated using structural measures and Protein Blocks description. Our study highlights that significant structural rearrangement occurs on binding at the interface as well as at regions away from the interface to form a highly specific, stable and functional complex. Notably, predominantly unaltered interfaces interact mainly with interfaces undergoing substantial structural alterations, revealing the presence of at least one structural regulatory component in every complex. Interestingly, about one-half of the number of complexes, comprising largely of signalling proteins, show substantial localized structural change at surfaces away from the interface. Normal mode analysis and available information on functions on some of these complexes suggests that many of these changes are allosteric. This change is largely manifest in the proteins whose interfaces are altered upon binding, implicating structural change as the possible trigger of allosteric effect. Although large-scale studies of allostery induced by small-molecule effectors are available in literature, this is, to our knowledge, the first study indicating the prevalence of allostery induced by protein effectors. Conclusions: The enrichment of allosteric sites in signalling proteins, whose mutations commonly lead to diseases such as cancer, provides support for the usage of allosteric modulators in combating these diseases.

Increased physical activity is not enough to recover astrocytic population from dark-rearing. Synergy with multisensory enrichment is required

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Gene enrichment using antibodies to DNA/RNA hybrids : mapping the ribosomal DNA of slime mold and rat

A novel method for gene enrichment has been developed and applied to mapping the rRNA genes of two eucaryotic organisms. The method makes use of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the synthetic hybrid poly(rA)•poly(dT). Antibodies which cross-react with non-hybrid nucleic acids were removed from the purified IgG fraction by adsorption on columns of DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent purification of the specific DNA/RNA hybrid antibody was carried out on a column of oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these antibodies to CNBr-activated Sepharose produced an affinity resin which specifically binds DNA/RNA hybrids.

In order to map the rDNA of the slime mold Dictyostelium discoideum, R-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs of this organism. This mixture was passed through a column containing the affinity resin, and bound molecules containing R- loops were eluted by high salt. This purified rDN A was observed directly in the electron microscope. Evidence was obtained that there is a physical end to Dictyostelium rDN A molecules approximately 10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding is consistent with reports of other investigators that the rRNA genes exist as inverse repeats on extra-chromosomal molecules of DNA unattached to the remainder of the nuclear DNA in this organism.

The same general procedure was used to map the rRNA genes of the rat. Molecules of DNA which contained R-loops formed with the 188 and 288 rRNAs were enriched approximately 150- fold from total genomal rat DNA by two cycles of purification on the affinity column. Electron microscopic measurements of these molecules enabled the construction of an R-loop map of rat rDNA. Eleven of the observed molecules contained three or four R-loops or else two R-loops separated by a long spacer. These observations indicated that the rat rRNA genes are arranged as tandem repeats. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent repeating units of exactly the same length within the errors of the measurements, although a certain degree of length heterogeneity cannot be ruled out. If significantly shorter or longer repeating units exist, they are probably much less common than the 37.2 kbp unit.

The last section of the thesis describes the production of antibodies to non-histone chromosomal proteins which have been exposed to the ionic detergent sodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did not seem to affect either production of antibodies or their general specificity. Also, a technique is described for the in situ immunofluorescent detection of protein antigens in polyacrylamide gels.