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Objective Patients with light chain–predominant multiple myeloma have been shown to exhibit shorter survival. Retrospective comparison of clinical and laboratory data was undertaken to ascertain the likely cause(s) of this observation. Methods Records of patients with multiple myeloma seen at 1 institution revealed 316 patients with conventional and 71 patients with light chain–predominant multiple myelomas with secretion of intact immunoglobulins. Laboratory and clinical findings in the 2 groups were compared. Results Patients with light chain–predominant multiple myeloma had a significantly higher death rate, a higher rate of chronic dialysis, a lower estimated glomerular filtration rate and serum albumin, a significantly higher urine protein concentration, and a significantly higher prevalence of hypertension and blood transfusion requirements. Other clinical and laboratory parameters surveyed were not significantly different between the 2 groups. Conclusion The shorter survival of patients with light chain–predominant multiple myeloma is clearly associated with renal damage caused by excess free immunoglobulin light chains. Renal damage may be ameliorated by early aggressive treatment with chemotherapy, plasmapheresis, and dialysis; a multi-institutional prospective controlled trial would be needed to test this hypothesis. [ABSTRACT FROM AUTHOR]

The polygenic variety of chylomicronemia occurs in adults in whom factors such as obesity, diabetes, alcoholism, renal disease, and certain drugs can precipitate chylomicronemia. A rare cause of polygenic chylomicronemia is hyperlipoproteinemia type 3 (HLP3). We report on a 54-year-old male who presented with chylomicronemia with triglycerides (TG) >2000 mg/dL. From admission, the ratio of total cholesterol to total triglycerides was not below 0.2 but was closer to 0.5, suggesting that his condition was not classic chylomicronemia. We confirmed that the patient had HLP3 based on his very-low-density lipoprotein cholesterol (VLDL-C)/TG ratio, which was ≥0.3, and lipoprotein electrophoresis showing a broad beta band. Because he was not responsive to initial therapy, we considered an interferent impairing lipolysis and TG reduction. The interferent was an M-protein that may also have falsely elevated both apolipoprotein-B and direct-LDL-C levels. In this case study, we report on a patient with chylomicronemia resulting from HLP3 complicated by a circulating M-protein. [ABSTRACT FROM AUTHOR]

The absence of clinical tools to evaluate individual variation in the pace of aging represents a major impediment to understanding aging and maximizing health throughout life. The human lens is an ideal tissue for quantitative assessment of molecular aging in vivo. Long-lived proteins in lens fiber cells are expressed during fetal life, do not undergo turnover, accumulate molecular alterations throughout life, and are optically accessible in vivo. We used quasi-elastic light scattering (QLS) to measure age-dependent signals in lenses of healthy human subjects. Age-dependent QLS signal changes detected in vivo recapitulated time-dependent changes in hydrodynamic radius, protein polydispersity, and supramolecular order of human lens proteins during long-term incubation (~1 year) and in response to sustained oxidation (~2.5 months) in vitro. Our findings demonstrate that QLS analysis of human lens proteins provides a practical technique for noninvasive assessment of molecular aging in vivo. [ABSTRACT FROM AUTHOR]

Background Concentration of serum free light chains has been promoted as an assay in the evaluation of monoclonal gammopathic manifestations. A high false-positive rate in patients without monoclonal gammopathic manifestations and a high false-negative rate in patients with those manifestations have been reported. The false-negative rate for lambda chain–associated lesions is higher than that for kappa chain–associated lesions. Objective To assess the serum free light chains in light-chain myelomas. Methods Concentrations of involved and uninvolved serum free light chains were retrospectively reviewed in patients with light-chain myelomas. Results The highest recorded levels of involved light chains in kappa-chain myelomas and lambda-chain myelomas were comparable. The levels of uninvolved light chains were higher, although not statistically significantly, in lambda-chain lesions. Conclusions The results of serum free light chains in light-chain myelomas support the clinical usefulness of the assay in monitoring patients with light chain myeloma. [ABSTRACT FROM AUTHOR]

Objectives: This study identified and characterized mcr-1-positive Enterobacteriaceae (MCRPE) and carbapenemase-producing Enterobacteriaceae (CPE) in hospital sewage water.Methods: Influent and effluent sewage samples were collected from five tertiary hospitals in Beijing in December 2016. Samples were screened for MCRPE and CPE using antibiotic selection media. Results were confirmed by PCR amplification of β-lactamase and colistin resistance (mcr-1 and mcr-2) genes and by sequencing. Antimicrobial susceptibility testing, MLST, conjugation and plasmid typing and S1-nuclease-PFGE/Southern blotting were performed for all MCRPE and CPE isolates.Results: Nine MCRPE and 12 CPE isolates were obtained. All mcr-1-positive isolates (n = 9) were Escherichia coli and belonged to eight different STs. The blaKPC-2-positive Enterobacteriaceae included Klebsiella pneumoniae (n = 4), Enterobacter cloacae (n = 4) and Citrobacter freundii (n = 1) isolates. Two C. freundii isolates and one E. cloacae isolate harboured the blaNDM-1 gene. MLST analysis revealed distinct genetic relatedness among all ST11 K. pneumoniae but not among any other carbapenemase-producing isolates. Conjugation and plasmid typing confirmed that three MCRPE isolates harboured mcr-1 on the self-transmissible IncX4 plasmid and the blaNDM-1 gene on the IncX3 plasmid. The sizes of the plasmids harbouring mcr-1, blaNDM-1 and blaKPC-2 were ∼33 to ∼240, ∼40 to ∼75 and ∼30 to ∼90 kb, respectively.Conclusions: To the best of our knowledge, this is the first report of mcr-1-positive E. coli and blaNDM-1-carrying E. cloacae and C. freundii in hospital sewage water. These findings, especially the diversity of MCRPE and K. pneumoniae ST11 that harbour the blaKPC-2 gene, suggest that monitoring and management of hospital sewage water should be enhanced. [ABSTRACT FROM AUTHOR]

Objectives: Vancomycin-intermediate Staphylococcus aureus (VISA) strains have spread globally. We previously isolated an ST239 VISA (XN108) with a vancomycin MIC of 12 mg/L. The mechanism for XN108 resistance to vancomycin was investigated in this study.Methods: Genome comparison was performed to characterize mutations that might contribute to the XN108 resistance phenotype. The novel mutation WalK(S221P) was identified and investigated using allelic replacement experiments. Vancomycin susceptibilities, autolytic activities and morphologies of the strains were examined. Autophosphorylation activities of WalK and the WalK(S221P) mutant were determined in vitro with [λ- 32 P]ATP, and binding activity of WalK(S221P)-activated WalR to the promoter region of its target gene lytM was determined by electrophoretic mobility shift assay.Results: Genome comparison revealed three mutations, GraS(T136I), RpoB(H481N) and WalK(S221P), which might be responsible for vancomycin resistance in XN108. The introduction of WalK(S221P) to the vancomycin-susceptible strain N315 increased its vancomycin MIC from 1.5 to 8 mg/L, whereas the allelic replacement of WalK(S221P) with the native N315 WalK allele in XN108 decreased its vancomycin MIC from 12 to 4 mg/L. The VISA strains have thickened cell walls and decreased autolysis, consistent with observed changes in the expression of genes involved in cell wall metabolism and virulence regulation. WalK(S221P) exhibited reduced autophosphorylation, which may lead to reduced phosphorylation of WalR. WalK(S221P)-phosphorylated WalR also exhibited a reduced capacity to bind to the lytM promoter.Conclusions: The naturally occurring WalK(S221P) mutation plays a key role in vancomycin resistance in XN108. [ABSTRACT FROM AUTHOR]

Background: A genome-wide association study (GWAS) suggested inherited genetic single-nucleotide polymorphisms (SNPs) affecting overall survival (OS) in advanced pancreatic cancer. To identify robust clinical biomarkers, we tested the strongest reported candidate loci in an independent patient cohort, assessed cellular drug sensitivity, and evaluated molecular effects.Methods: This study comprised 381 patients with histologically verified pancreatic ductal adenocarcinoma treated with gemcitabine-based chemotherapy. The primary outcome was the relationship between germline polymorphisms and OS. Functional assays addressed pharmacological dose-response effects in lymphoblastoid cell lines (LCLs) and pancreatic cancer cell lines (including upon RNAi), gene expression analyses, and allele-specific transcription factor binding. All statistical tests were two-sided.Results: The A allele (26% in Caucasians) at SNP rs11644322 in the putative tumor suppressor gene WWOX conferred worse prognosis. Median OS was 14 months (95% confidence interval [CI] = 12 to 15 months), 13 months (95% CI = 11 to 15 months), and nine months (95% CI = 7 to 12 months) for the GG, GA, and AA genotypes, respectively (P trend < .001 for trend in univariate log-rank assuming a codominant mode of inheritance; advanced disease subgroup P trend < .001). Mean OS was 25 months (95% CI = 21 to 29 months), 19 months (95% CI = 15 to 22 months), and 13 months (95% CI = 10 to 16 months), respectively. This effect held true after adjustment for age, performance status according to Eastern Cooperative Oncology Group classification, TNM, grading, and resection status and was comparable with the strongest established prognostic factors in multivariable analysis. Consistently, reduced responsiveness to gemcitabine, but not 5-fluorouracil, along with lower WWOX expression was demonstrated in LCLs harboring the AA genotype. Likewise, RNAi-mediated WWOX knockdown in pancreatic cancer cells confirmed differential cytostatic drug sensitivity. In electrophoretic mobility shift assays, the A allele exhibited weaker binding of Sp family members Sp1/Sp3.Conclusions: WWOX rs11644322 represents a major predictive factor in gemcitabine-treated pancreatic cancer. Decreased WWOX expression may interfere with gemcitabine sensitivity, and allele-specific binding at rs11644332 might be a causative molecular mechanism behind the observed clinical associations. [ABSTRACT FROM AUTHOR]

The article presents information on proteins (polymers of amino acids joined by peptide bonds) in the human body, with a focus on the use of the protein albumin in disease detection and management. Topics include the analysis of protein structures, the biochemical properties of proteins, and methods for the measurement of proteins.

The purpose of this study was to isolate the coding sequence of the p24 protein (encoded by the gag gene) from sera collected from Iranian patients, to clone and express this sequence in Escherichia coli (E coli), and to assess the immunoreactivity of the expressed recombinant protein. The complete coding sequence for the Gag protein was isolated by nested real-time polymerase chain reaction {RT-PCR) from the serum of an infected individual and then cloned into a PTZ57R (T) vector. The resulting vector, PTZ-gag53-IR, was sequenced. Subsequently, the coding sequence of p24 protein was isolated by PCR from the PTZ-gag53-IR vector and inserted into a pET102/D-TOPO vector using TOPO directional cloning and then expressed in E coli in fusion with His-Patch (HP)-thioredoxin. The immunoreactivity of the expressed protein was tested by immunoblotting and enzyme-linked immunosorbent assay (ELISA). RT-PCR revealed a sequence belonging to the CRF35_AD subtype of the HIV-1 virus, which is highly prevalent in Iran and Afghanistan. Recombinant p24 protein from this isolate showed 100% sensitivity and 100% specificity in an antibody assay. [ABSTRACT FROM AUTHOR]

The article discusses a study which investigated the production of reactive oxygen species (ROS), as well as its apoptosis induction through Gts1p-mediated protein aggregate formation. The study used agarose gel electrophoresis to examine the degrees of Gts1p-mediated protein aggregation. Results suggested that protein aggregates mediated by Gts1p influence the mitochondrial regulation of ROS apoptosis and production.

Background. Soft-laser therapy has been used to treat rheumatic diseases for decades. The major effects of laser treatment may be dependent not on thermal mechanisms but rather on cellular, photochemical mechanisms. However, the exact cellular and molecular mechanisms of action have not been elucidated. Objective. The aim of this study was to investigate the ex vivo effects of low-level laser treatment (with physical parameters similar to those applied previously) on protein expression in the synovial membrane in rheumatoid arthritis (RA). Design. Synovial tissues were laser irradiated, and protein expression was analyzed. Methods. Synovial membrane samples obtained from 5 people who had RA and were undergoing knee surgery were irradiated with a near-infrared diode laser at a dose of 25 J/cm2 (a dose used in clinical practice). Untreated synovial membrane samples obtained from the same people served as controls. Synovial protein expression was assessed with 2-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry. Results. The expression of 12 proteins after laser irradiation was different from that in untreated controls. Laser treatment resulted in the decreased expression of α-enolase in 2 samples and of vimentin and precursors of haptoglobin and complement component 3 in 4 samples. The expression of other proteins, including 70-kDa heat shock protein, 96-kDa heat shock protein, lumican, osteoglycin, and ferritin, increased after laser therapy. Limitations. The relatively small sample size was a limitation of the study. Conclusions. Laser irradiation (with physical parameters similar to those used previously) resulted in decreases in both α-enolase and vimentin expression in the synovial membrane in RA. Both proteins have been considered to be important autoantigens that are readily citrullinated and drive autoimmunity in RA. Other proteins that are expressed differently also may be implicated in the pathogenesis of RA. Our results raise the possibility that low-level laser treatment of joints affected with RA may be effective, at least in part, by suppressing the expression of autoantigens. Further studies are needed. [ABSTRACT FROM AUTHOR]

Electrophoresis in a chromatographic column packed with porous gel has been successfully established to preparatively separate proteins and DNA. This method shows good separative performance. Since separation of small polar nucleotides and oligonucleotides is challenging to conventional reversed phase high-performance liquid chromatography (RP-HPLC), it is of significance to establish preparative electrophoresis for laboratory-scale purification of them. In this paper, milligram of model polar samples, cytidine 5'-monophosphate (CMP) and uridine 5'-monophosphate (UMP), could be completely separated by a column (0.6 cm i.d.) packed with Sepharose 6B at pH 3.6 in 120 V/cm electric field, but never be separated without electric field. The orientation and strength of electric field could affect retention, resolution, and recovery of mononucleotides. With cathode at the column outlet and anode at the column inlet, the electric field force on UMP was strong enough to counteract the buffer flow, retain UMP at the column inlet, and markedly enhance the resolution between UMP and CMP. With this electrical retention mode, the maximum sample capacity was 7.5 mg on a column of 40 × 0.6 cm i.d. (bed volume 11.4 mL), which is much more than that of a RP-HPLC column. This electrokinetic method is a potential system for the separation of small polar compounds from natural extracts. [ABSTRACT FROM AUTHOR]

This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The PII proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of PII might play a role in the control of nitrogen metabolism in H. seropedicae. [ABSTRACT FROM AUTHOR]

Seed storage proteins are of great importance in nutrition and in industrial transformation because of their functional properties. Brachypodium distachyon has been proposed as a new model plant to study temperate cereals. The protein composition of Brachypodium grain was investigated by separating the proteins on the basis of their solubility combined with a proteomic approach. Salt-soluble proteins as well as salt-insoluble proteins separated by two-dimensional gel electrophoresis revealed 284 and 120 spots, respectively. Proteins from the major spots were sequenced by mass spectrometry and identified by searching against a Brachypodium putative protein database. Our analysis detected globulins and prolamins but no albumins. Globulins were represented mainly by the 11S type and their solubility properties corresponded to the glutelin found in rice. An in silico search for storage proteins returned more translated genes than expressed products identified by mass spectrometry, particularly in the case of prolamin type proteins, reflecting a strong expression of globulins at the expense of prolamins. Microscopic examination of endosperm cells revealed scarce small-size starch granules surrounded by protein bodies containing 11S globulins. The presence of protein bodies containing glutelins makes B. distachyon closer to rice or oat than to wheat endosperm. [ABSTRACT FROM PUBLISHER]

Vanillin dehydrogenases (VDHs) were purified and characterized from two bacterial strains that have different pH dependencies for growth. The alkaliphile Micrococcus sp. TA1, isolated from an alkaline spa, can grow on several aromatic compounds such as ferulic acid, vanillin, vanillic acid, and protocatechuic acid under alkaline conditions. The neutrophile Burkholderia cepacia TM1, which was isolated previously, also grew on the above-mentioned compounds because they functioned as the sole carbon source under neutral conditions. Purified VDHs showed activities toward some aromatic aldehydes. These enzymes have the same subunit molecular mass of about 57 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differed in some of their observed properties. Native molecular masses also differed between the purified enzymes. These were 250 kDa for the enzyme from alkaliphilic strain TA1 and 110 kDa for that from neutrophilic strain TM1, as determined by gel filtration. The enzyme from strain TA1 required NADP+ as a coenzyme for its activity, but that from strain TM1 required NAD+. These results are important because this is the first report of an alkaliphilic bacterium consuming lignin monomers. [ABSTRACT FROM AUTHOR]

Aims: Although wine yeast gene expression has been thoroughly investigated only few data are available on the evolution the proteome during alcoholic fermentation. This work aimed at specifying the change in proteome during fermentation and to assess its connection with transcriptome. Methods and Results: The proteome of a wine yeast was monitored by 2-D gel electrophoresis throughout alcoholic fermentation. Proteome was analysed in exponential growth and stationary phase. Among 744 spots, detected we observed significant changes in abundance with 89 spots displaying an increase in intensity and 124 a decrease. We identified 59 proteins among the most regulated and/or the most expressed. Glycolysis and ethanol production, amino acid and sulfur metabolism were the most represented functional categories. We found only a weak correlation between changes in mRNA and protein abundance, which is strongly dependent on the functional category. Conclusions: There are substantial changes in protein abundance during alcoholic fermentation, but they are not directly associated with changes at transcript level suggesting that mRNA is selectively processed and/or translated in stationary phase. Significance and Impact of the Study: These data show that proteome is a relevant level of analysis to gain insight into wine yeast adaptation to alcoholic fermentation. [ABSTRACT FROM AUTHOR]

The ferric-uptake regulator (Fur) protein is an Fe2+-dependent transcriptional repressor. To clarify the global regulation of Helicobacter pylori proteins by Fur according to the growth phase, we compared the proteome profiles of H. pylori 26695 and its isogenic fur mutant, harvested during in vitro culture. Clustering analysis of the proteome profiles of the two strains revealed that the growth-phase-dependent protein regulation in the wild-type strain was largely altered in the fur mutant. Reverse transcriptase-PCR analysis of several H. pylori genes showed that a major switch in transcription occurred 12 h earlier than in the wild type, indicating that the fur mutation induced an earlier transcriptional switch from log to stationary phase. Several H. pylori proteins also showed changes in their patterns of protein post-translational modification (PTM). In particular, the HydB protein, which was detected as four spots on 2-dimensional electrophoresis gels, underwent two types of PTM, which were under different kinds of regulation. These data demonstrate that a fur mutation affects the growth-phase-dependent regulation of proteins and mRNAs, suggesting a role for Fur in controlling the global regulation of cellular processes in response to changing growth environments. [ABSTRACT FROM AUTHOR]

MS/MS techniques are well customized now for proteomic analysis, even for non-sequenced organisms, since peptide sequences obtained by these methods can be matched with those found in databases from closely related sequenced organisms. We used this approach to characterize the protein content of the “Rovabio™ Excel”, an enzymatic cocktail produced by Penicillium funiculosum that is used as feed additive in animal nutrition. Protein separation by bi-dimensional electrophoresis yielded more than 100 spots, from which 37 proteins were unambiguously assigned from peptide sequences. By one-dimensional SDS-gel electrophoresis, 34 proteins were identified among which 8 were not found in the 2-DE analysis. A third method, termed ‘peptidic shotgun’, which consists in a direct treatment of the cocktail by trypsin followed by separation of the peptides on two-dimensional liquid chromatography, resulted in the identification of two additional proteins not found by the two other methods. Altogether, more than 50 proteins, among which several glycosylhydrolytic, hemicellulolytic and proteolytic enzymes, were identified by combining three separation methods in this enzymatic cocktail. This work confirmed the power of proteome analysis to explore the genome expression of a non-sequenced fungus by taking advantage of sequences from phylogenetically related filamentous fungi and pave the way for further functional analysis of P. funiculosum. [ABSTRACT FROM AUTHOR]

A proteomic approach combining two-dimensional electrophoresis, Western blot and matrix-assisted laser desorption tandem time-of-flight mass spectrometry has been used to map the extracellular proteins of Streptococcus equi ssp. zooepidemicus ( S. zooepidemicus) strain ATCC 35246. These bioinformatic technologies facilitated the identification of novel S. zooepidemicus vaccine candidate antigens and therapeutic agents. Despite the limitations posed by the unavailability of complete genome and proteome data for S. zooepidemicus, seven of 15 chosen immunogenic spots were successfully identified as streptococcal proteins (AE1 and AE4 c. 10) from homologous Streptococcus species. Among these, AE6 and AE7 were identified as S. zooepidemicus UDP- N-acetyl-glucosamine pyrophosphorylase and UDP-glucose pyrophosphorylase proteins. In addition, AE4 was determined to be glyceraldehyde-3-phosphate dehydrogenase from Enterococcus faecalis. Followingsignalip 3.0 ( ) prediction, data suggested that AE5, AE7 and AE9 contained signal peptides.blast ( ) results found that nucleotide sequences of all identified proteins shared high homology (≥65%) with S. zooepidemicus. The majority of proteins identified in our study remain formally unreported in S. zooepidemicus. However, these proteins serve a vital role in the immune system and reproduction of host species. Therefore, we further evaluated the proteins as vaccine candidates in this study. [ABSTRACT FROM AUTHOR]

We describe the proteomic identification of two pathogenesis-related group 5 (PR-5) proteins, an acidic thaumatin-like protein (TLP) and a basic TLP isolated from the pollination drop of hybrid yew (Taxus × media Rehder). The basic TLP (TxmTLPb) was the most abundant protein in the yew pollination drop based on protein spot size after two-dimensional electrophoresis. The acidic TLP (TxmTLPa) is also a major protein component of the yew ovular secretion and appears to be encoded by a number of mRNAs transcribed from a TLP gene family that has undergone limited sequence divergence. We have sequenced five acidic TLP-encoding cDNAs (TxmTLPa-1,2,3,4 and 5) isolated from the yew ovule that vary from each other by no more than five out of 233 amino acid residues in their predicted protein sequences. All of the cDNA variants encode TLPs possessing the 16 conserved cysteine residues and five charged amino acid side chains associated with antifungal activity. Amplification of genomic DNA with TxmTLPa primers indicated that at least 11 acidic TLPs with highly similar amino acid sequences may be expressed in yew tissues. Antibodies against TLPs confirmed the identity of TxmTLPa and TxmTLPb in the yew pollination drop and detected TLPs in the ovular secretions of four other species from three other conifer families. Our results suggest that TLPs are a conserved component of conifer ovular secretions and are involved in broad spectrum pathogen defence of ovules. [ABSTRACT FROM PUBLISHER]

Serratia entomophila and Serratia proteamaculans cause amber disease of the grass grub Costelytra zealandica ( Coleoptera: Scarabaeidae). Three genes required for virulence, sepABC, are located on a large plasmid, pADAP. The translated products of the sep genes are members of the toxin complex (Tc) family of insecticidal toxins that reside in the genomes of some Enterobacteriaceae. Each of the sep genes was placed either singly or as various combinations under the control of an inducible arabinose promoter, allowing their inductive expression. Western Immunoblot confirmed that each of the Sep proteins migrated at their predicted size on sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. Bioassays of sonicated filtrates derived from the various arabinose-induced para-SEP constructs showed that only when sepA, sepB and sepC were coexpressed were amber disease symptoms observed in grass grub larvae. Fourteen days after ingestion of the Sep protein filtrate, ∼64% of the larvae reverted from a diseased to a healthy phenotype. Redosing the revertents with a fresh Sep protein filtrate reinitiated the amber pathotype, indicating that the Sep proteins are needed to be continuously present to exert an effect. [ABSTRACT FROM AUTHOR]

Nonomuraea sp. ATCC 39727 is an aerobic actinomycete, industrially important as a producer of the glycopeptide A40926, which is used as a precursor of the semi-synthetic antibiotic dalbavancin. Previous studies showed that the production of A40926 is depressed by calcium, but promoted byl-glutamine orl-asparagine. In this study, the protein expression changes of Nonomuraea sp. ATCC 39727 in these two different growth and antibiotic-production conditions have been investigated by two-dimensional electrophoresis and mass spectrometry (MS) analysis. Few protein spots show statistically significant expression changes, and, among this group of proteins, malate dehydrogenase (MDH) shows a significant decrease in the overproduction condition. The decrease of MDH is of particular interest because it is the first described significant change in the expression levels of enzymes of the central metabolism related with A40926 overproduction. [ABSTRACT FROM AUTHOR]

Protein extraction is the most important step to reveal a proteome by Two-Dimensional Gel Electrophoresis. Usually, the urea/thiourea based standard protein extraction buffer (SB) is combined with detergents with the aim of achieving better resolution and solubilization of different classes of proteins. In order to produce better gels and achieve the greatest spot resolution of Human Brain Proteins, comparisons using 2-DE of extracted proteins from Human Brain Frontal Cortex with SB constituents (7M Urea, 2M Thiourea and 100mM DTT) were made, using different detergent compositions in the buffer. SB preparations in combination with CHAPS and ASB-14 as well as with ASB- 16 (reported for the first time in 2-DE experiments) have been tested. Our results confirm that the most efficient solubilizing solution for 2-DE analysis of cytosolic and membrane Human Brain Proteins is SB combined with 4% CHAPS and 2% ASB-14. [ABSTRACT FROM AUTHOR]

Brassica napus (cultivar Reston) seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering in biological quadruplicate using two-dimensional gel electrophoresis. Developmental expression profiles for 794 protein spot groups were established and hierarchical cluster analysis revealed 12 different expression trends. Tryptic peptides from each spot group were analyzed in duplicate using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry. The identity of 517 spot groups was determined, representing 289 nonredundant proteins. These proteins were classified into 14 functional categories based upon the Arabidopsis (Arabidopsis thaliana) genome classification scheme. Energy and metabolism related proteins were highly represented in developing seed, accounting for 24.3% and 16.8% of the total proteins, respectively. Analysis of subclasses within the metabolism group revealed coordinated expression during seed filling. The influence of prominently expressed seed storage proteins on relative quantification data is discussed and an in silico subtraction method is presented. The preponderance of energy and metabolic proteins detected in this study provides an in-depth proteomic view on carbon assimilation in B. napus seed. These data suggest that sugar mobilization from glucose to coenzyme A and its acyl derivative is a collaboration between the cytosol and plastids and that temporal control of enzymes and pathways extends beyond transcription. This study provides a systematic analysis of metabolic processes operating in developing B. napus seed from the perspective of protein expression. Data generated from this study have been deposited into a web database (http://oilseedproteomics.missouri.edu) that is accessible to the public domain. [ABSTRACT FROM AUTHOR]

Small heat-shock proteins (sHsps) are widespread molecular chaperones for which a peroxisomal localization has not yet been reported. The Arabidopsis (Arabidopsis thaliana) genome encodes two sHsps with putative peroxisomal targeting signals type 1 or 2 (PTS1 or PTS2). As demonstrated by double-labeling experiments using full-length fusion proteins with enhanced yellow fluorescent protein and deletion constructs lacking the putative targeting domains, AtHsp15.7 (At5g37670) and AtAcd31.2 (At1g06460) are targeted to the peroxisome matrix by a functional PTS1 (SKL>) and a functional PTS2 (RLx5HF), respectively. The peroxisomal localization of AtAcd31.2 was further confirmed by isolation of leaf peroxisomes from Arabidopsis by two successive sucrose density gradients, protein separation by one- and two-dimensional gel electrophoresis, and mass spectrometric protein identification. When AtHsp15.7 and AtAcd31.2 were heterologously expressed in yeast (Saccharomyces cerevisiae) and directed to the cytosol by deletion of the PTSs, both sHsps were able to complement the morphological phenotype of yeast mutants deficient in the cytosolic homologs ScHsp42 or ScHsp26. According to expression studies by reverse transcription-PCR, AtAcd31.2 is constitutively expressed, whereas AtHsp15.7 is hardly expressed under normal conditions but strongly induced by heat and oxidative stress, the latter of which was triggered by the catalase inhibitor 3-aminotriazole or the herbicide methyl viologen applied by watering of whole plants or infiltration of rosette leaves. Thus, plants are exceptional among eukaryotes in employing sHsps in the peroxisome matrix to prevent unspecific aggregation of partially denatured proteins under both physiological and stress conditions. [ABSTRACT FROM AUTHOR]

Two-dimensional gel electrophoresis coupled to mass spectrometry analysis was used to examine for the first time the effect of a herbicide (flumioxazin) on a crop species (Vitis vinifera L.) at the proteome level. Examination of 2-D maps derived from chemically stressed tissues revealed the presence of 33 spots displaying a differential expression pattern. The presence of stress responsive proteins in the different plant organs analysed suggests that flumioxazin could act systemically. Among the responsive proteins, some photosynthesis-related proteins, including several fragments of the enzyme Rubisco, were identified. This effect suggests that photosynthesis could be impaired by the herbicide. The induction of several enzymatic antioxidant systems was also observed, probably as a result of an oxidative stress. Moreover, the photorespiration pathway was stimulated, as suggested by the induction of some key enzymes involved in this process. Changes in carbon metabolism-associated proteins presumably reflect altered patterns of carbon flux in response to impaired photosynthesis and an increased need for osmotic adjustment in affected tissues. Finally, plant defences were stimulated as revealed by the induction of a set of proteins belonging to the pathogenesis-related 10 class, suggesting that they could play an essential role in cell defence mechanisms against flumioxazin. [ABSTRACT FROM PUBLISHER]

Sample complexity frequently interferes with the analysis of low-abundance proteins by two-dimensional gel electrophoresis (2DGE). Ideally, high abundance proteins should be removed, allowing low-abundance proteins to be applied at much higher concentrations than is possible with the unfractionated sample. One approach is to partition the sample in a manner that segregates the bulk of extraneous proteins from the protein(s) of interest. Solution phase isoelectric focusing in the multi-compartment electrolyser generates fractions of discrete isoelectric point (pl) intervals allowing isolated narrow segments of a proteome to be analysed individually by 2DGE. It is particularly useful for the isolation of low-abundance proteins of extremely basic or acidic pl. [ABSTRACT FROM AUTHOR]

The comprehensive analysis of gene expression in complex biological systems has demanded the development of new technologies to study the cell transcriptome and the cell proteome. Each approach has advantages and disadvantages from both the conceptual and the methodological viewpoints. Differential proteomics, the comparison of distinct proteomes (eg normal versus diseased cells, diseased versus treated cells etc) is of paramount importance. Several approaches can be used and these typically involve electrophoresis and/or chromatography combined with chemical or metabolic labelling and mass spectrometry. These approaches aim to identify molecular targets, namely proteins, involved in different physiopathological states. Incorporating this knowledge with knowledge from other technologies lays the foundations of active principles at the molecular level. Here, the various gel- and non-gel-based approaches that are used in a wide range of biological systems for the study of differentially expressed proteins will be reviewed. [ABSTRACT FROM AUTHOR]

Compares the use of wet and dry two-dimensional electrophoresis gels for in-gel tryptic digestion and subsequent analysis by mass spectrometry. Utility of dry electrophoretic gels for proteomics of insect extracts; Means of identifying various proteins; Relationship between the absorbance and the amount of peptide.

Plant roots have important roles not only in absorption of water and nutrients, but also in stress tolerance such as desiccation, salt, and low temperature. We have investigated stress-response proteins from rice roots using 2-dimensional polyacrylamide-gel electrophoresis and found a rice protein, RO-292, which was induced specifically in roots when 2-week-old rice seedlings were subjected to salt and drought stress. The full-length RO-292 cDNA was cloned, and was determined to encode a protein of 160 amino acid residues (16.9 kDa, pI 4.74). The deduced amino acid sequence showed high similarity to known rice PR10 proteins, OsPR10a/PBZ1 and OsPR10b. RO-292 mRNA accumulated rapidly upon drought, NaCl, jasmonic acid and probenazole, but not by exposure to low temperature or by abscisic acid and salicylic acid. The RO-292 gene was also up-regulated by infection with rice blast fungus. Interestingly, induction was observed almost exclusively in roots, thus we named the gene RSOsPR10 (root specific rice PR10). The present results indicate that RSOsPR10 is a novel rice PR10 protein, which is rapidly induced in roots by salt, drought stresses and blast fungus infection possibly through activation of the jasmonic acid signaling pathway, but not the abscisic acid and salicylic acid signaling pathway. [ABSTRACT FROM AUTHOR]

To provide information on virulence expression of Escherichia coli in healthy hosts, stool, periurethral, and urine samples were collected weekly from healthy 3-6-year-old girls who lived in a small rural community. Dominant and nondominant clones were defined in stool specimens, and the expression of virulence factors was determined. We found that healthy girls commonly shared dominant clones. P adhesin, hemolysin, and type I adhesin were commonly found in clones in the stool and in clones in the urinary tract. In addition, expression of virulence factors, among both dominant and nondominant clones in the stool, changed from week to week. The presence of P adhesin was a marker for the persistence of a dominant clone in the stool and was associated with an increased likelihood that a nondominant clone would be detected in the urinary tract. Type I adhesin was ubiquitous among stool strains, with orientation of the fimbrial switch being both in the "off" position and in both the "on" and "off" positions. In summary, the intestinal flora of healthy girls is complex, with frequent changes in virulence expression. [ABSTRACT FROM AUTHOR]

Antifreeze proteins (AFPs) are known as thermal hysteresis proteins, which can depress the freezing points of the solution by noncolligative effects, but do not affect the melting points. Although some AFPs have been found in some plants, the identity of most proteins remains unclear, owing to insufficient quantity and quality to characterize them. In this report, we describe the purification of an AFP from the winter leaves of Ammopiptanthus mongolicus using a combination of column chromatography and gel electrophoresis. After homogenization in ascorbate-acid-containing Tris buffers (pH 7.4) the soluble proteins are captured by (diethylamino)ethyl-cellulose 52 material. An elution with 0.1-0.3M KCl leads to a crude active fraction. The crude fraction is further purified on a Superdex 75 prep-grade column and finally a Poros 20HP2 column. A complex, consisting of two proteins with relative molecular masses of 34,700 and 37,100, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, is obtained by this protein purification protocol. The recovery of two proteins from the gel is carried out by electrophoresis. The purified protein, with a molecular mass of 37,100, shows thermal hysteresis activity (THA) and can modify the normal growth of ice crystals. The THA of this purified antifreeze protein is 0.24°C at the concentration of 5 mg/mL. [ABSTRACT FROM AUTHOR]

Examines the proteins present at the peribacteroid membrane (PBM) by proteome analysis. Purification of PBM from root nodules by an aqueous polymer two-phase system; Separation of peptides by nanoscale liquid chromatography; Analysis of the electrophoresis of total PBM.

For the process of aging in epidermal cells to be characterized, the status of oxidized and damaged protein accumulation and removal by the proteasome has been investigated. Modified protein content and proteasome activity were assayed in lysates of epidermal cells from donors of different ages. Increased levels of oxidized proteins, glycated proteins, and proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were observed in cells from old donors. At the same time, a decline of chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities of the proteasome was found in aging keratinocytes. This age-related decline of the proteasome peptidase activities can be explained, at least in part, by a decreased proteasome content as observed by immunoblotting and enzyme-linked immunosorbent assay. In keratinocyte cultures, a decrease of proteasome activity and content was observed upon serial passaging. In cultures, as well as in skin, an inverse relationship was found between the aging marker 1-galactosidase and the proteasome content. These results suggest that proteasome is downregulated during replicative senescence as well as in aged cells in vivo, possibly resulting in the accumulation of modified proteins. [ABSTRACT FROM AUTHOR]

Background: Previous studies have suggested that male hormones (androgens) and certain forms of oxygen (reactive oxygen species) are linked to the development of prostate cancer. We hypothesized that androgens contribute to prostate carcinogenesis by increasing oxidative stress. We further hypothesized that antioxidants reduce prostate cancer risk by modulating androgen effects on cellular processes.Methods: To test these hypotheses, we looked for 1) a change in the level of reactive oxygen species in the presence of androgens, 2) androgen-induced binding activity of transcriptional activators AP-1 and NF-kappaB, whose activities are known to be altered during cell proliferation, and 3) the effect of antioxidants on androgen-induced transcription factor binding.Results: Physiologic concentrations (1 nM) of 5alpha-dihydrotestosterone or 1-10 nM R1881, a synthetic androgen, produced sustained elevation of AP-1 and NF-kappaB DNA-binding activity in LNCaP cells, an androgen-responsive human prostate carcinoma cell line. Androgen-independent DU145 cells (another human prostate carcinoma cell line) were unaffected by R1881 treatment. AP-1-binding activity increased 5 hours after 1 nM R1881 treatment; NF-kappaB DNA-binding activity increased after 36 hours. Both activities remained elevated for at least 120 hours. Nuclear AP-1 and NF-kappaB protein levels were not elevated. Antioxidant vitamins C plus E blocked both androgen-induced DNA-binding activity and production of reactive oxygen species.Conclusion: Physiologic concentrations of androgens induce production of reactive oxygen species and cause prolonged AP-1 and NF-kappaB DNA-binding activities, which are diminished by vitamins C and E. [ABSTRACT FROM AUTHOR]

Amyloid obtained from the myocardium of a patient (Han) with familial amyloid cardiomyopathy of Danish origin was studied. Gel filtration and electrophoresis of purified and denatured amyloid fibrils Han revealed various fractions ranging in mol. wt from 40,000 to 8.000 daltons. Amyloid Han and fractions reacted with an antiserum against amyloid Han showing a reaction of identity with each other: partial identity between Han and human pre-albumin was observed. while no reaction was seen with AA or AL proteins. Cardiac tissue sections from Han showed reactivity with antisera to amyloid Han, pre-albumin and protein AP. but not with anti-AA or anti-AL in indirect immunofluorescence. Amino acid composition and sequence studies of a protein fraction of amyloid Han with mol. wt 15,000 daltons confirmed the structural relationship with pre-albumin. [ABSTRACT FROM AUTHOR]

Detergent extracts of human trophoblast plasma membanes (TrPM) and of other fetal tissues from the same conceptus were prepared and the proteins separated by use of isoelectric focusing. A major band of isoelectric point (pI) 4.1 was present in TrPM together with minor bands of (pI) 49, 59, 73, 78 and 7.9. These bands were absent from non-trophoblastic fetal tissues. The pI 4.1 protein reacted with an 125I-labelled IgG fraction of a heterologous antiserum raised against TrPM and was found to have an apparent molecular weight of 100,000. This protein did not appear to be placental alkaline phosphatase, SP1, transferrin or human chorionic gonadotrophin. Many neoplastic cell lines tested also revealed a major protein band of pI 4.1 but it has not been established that this was the same protein as found in the TrPM preparations. [ABSTRACT FROM AUTHOR]

Polyacrylamide gel electrophoresis of water-soluble proteins from sunflower (Helianthus annuus L.) cotyledons, followed by specific staining for superoxide dismutase activity, discriminated, according to their electrophoretic mobility, two distinct achromatic bands for Cu/Zn superoxide dismutase. Zymograms of proteins from isolated chloroplasts showed that the chloroplast-located Cu/Zn superoxide dismutase (Cu/ZnSODchl) migrated faster in the SOD activity-stained gels. An electrophoretic variant pattern, whose mobility is lower than the control pattern, was identified in the ABA-deficient mutant w-1. The variant is coded by a nuclear gene with two codominant alleles. [ABSTRACT FROM PUBLISHER]

Investigates the protein-DNA complex composed of NtrC protein and its target promoter DNA. Analysis of the protein-DNA interaction using capillary narrow-tube electrophoresis; Behavior of the complex under high-pressure conditions; Role of the complex in pressure-regulated transcription process.

The article presents abstracts of several studies about milk protein and enzymes. "Composition of the Air Interface in Ice Cream as Affected by Protein and Emulsifier Content," by Z. Zhang and D. Goff; "Evidence that the Major Novel Disulfide Bond in Heated Cows Milk is Between β-lactoglobulin Cys160 and K-Casein Cys88," by E. K. Lowe, S. G. Anema, M. J. Boland, R. Jiménez-Flores, and L. K. Creamer; "Application of Novel Gel Electrophoresis to Analyze Native, Heat-, and Pressure-Treated Milk Protein Systems," by H. A. Patel, H. Singh, and L. K. Creamer; "Effects of Genetic Modification, Pressure, and Heat on the Binding of Various Probes to β-lactoglobulin," by K. A. N. S. Ariyaratne, G. B. Jameson, T. S. Loo, G. E. Norris, H. A. Patel, T. Considine, H. Singh, L. K. Creamer, and R. Jiménez-Flores.

Evaluates a new technique from Molecular Dyamics, high-resolution dynamic imaging (HRDI), enabling researchers to observe protein bands during electrophoretic separations as samples move through gel. Similarities between HRDI and phase contrast imaging used in microscopy; Use of helium-neon laser light in identifying protein bands in gel.