Alterations of housekeeping proteins in human aged and diseased hearts.

Pathological remodeling includes alterations of ion channel function and calcium homeostasis and ultimately cardiac maladaptive function during the process of disease development. Biochemical assays are important approaches for assessing protein abundance and post-translational modification of ion channels. Several housekeeping proteins are commonly used as internal controls to minimize loading variabilities in immunoblotting protein assays. Yet, emerging evidence suggests that some housekeeping proteins may be abnormally altered under certain pathological conditions. However, alterations of housekeeping proteins in aged and diseased human hearts remain unclear. In the current study, immunoblotting was applied to measure three commonly used housekeeping proteins (ß-actin, calsequestrin, and GAPDH) in well-procured human right atria (RA) and left ventricles (LV) from diabetic, heart failure, and aged human organ donors. Linear regression analysis suggested that the amounts of linearly loaded total proteins and quantified intensity of total proteins from either Ponceau S (PS) blot-stained or Coomassie Blue (CB) gel-stained images were highly correlated. Thus, all immunoblotting data were normalized with quantitative CB or PS data to calibrate potential loading variabilities. In the human heart, ß-actin was reduced in diabetic RA and LV, while GAPDH was altered in aged and diabetic RA but not LV. Calsequestrin, an important Ca2+ regulatory protein, was significantly changed in aged, diabetic, and ischemic failing hearts. Intriguingly, expression levels of all three proteins were unchanged in non-ischemic failing human LV. Overall, alterations of human housekeeping proteins are heart chamber specific and disease context dependent. The choice of immunoblotting loading controls should be carefully evaluated. Usage of CB or PS total protein analysis could be a viable alternative approach for some complicated pathological specimens.

Expression of a heptelidic acid-insensitive recombinant GAPDH from Trichoderma virens, and its biochemical and biophysical characterization.

Trichoderma virens genome harbors two isoforms of GAPDH, one (gGPD) involved in glycolysis and the other one (vGPD) in secondary metabolism. vGPD is expressed as part of the "vir" cluster responsible for the biosynthesis of volatile sesquiterpenes. The secondary metabolism-associated GAPDH is tolerant to the anti-cancer metabolite heptelidic acid (HA), produced by T. virens. Characterizing the HA-tolerant form of GAPDH, thus has implications in cancer therapy. In order to get insight into the mechanism of HA-tolerance of vGPD, we have purified recombinant form of this protein. The protein displays biochemical and biophysical characteristics analogous to the gGPD isoform. It exists as a tetramer with Tm of about 56.5 °C, and displays phosphorylation enzyme activity with Km and Kcat of 0.38 mM and 2.55 sec-1, respectively. The protein weakly binds to the sequence upstream of the vir4 gene that codes for the core enzyme (a terpene cyclase) of the "vir" cluster. The EMSA analysis indicates that vGPD may not act as a transcription factor driving the "vir" cluster, at least not by directly binding to the promoter region. We also succeeded in obtaining small crystals of this protein. We have constructed structural models of vGPD and gGPD of T. virens. In silico constrained docking analysis reveals weaker binding of heptelidic acid in vGPD, compared to gGPD protein.

Effects of mutation and selection on plasticity of a promoter activity in Saccharomyces cerevisiae.

Phenotypic plasticity is an evolvable property of biological systems that can arise from environment-specific regulation of gene expression. To better understand the evolutionary and molecular mechanisms that give rise to plasticity in gene expression, we quantified the effects of 235 single-nucleotide mutations in the Saccharomyces cerevisiae TDH3 promoter (PTDH3 ) on the activity of this promoter in media containing glucose, galactose, or glycerol as a carbon source. We found that the distributions of mutational effects differed among environments because many mutations altered the plastic response exhibited by the wild-type allele. Comparing the effects of these mutations with the effects of 30 PTDH3 polymorphisms on expression plasticity in the same environments provided evidence of natural selection acting to prevent the plastic response in PTDH3 activity between glucose and galactose from becoming larger. The largest changes in expression plasticity were observed between fermentable (glucose or galactose) and nonfermentable (glycerol) carbon sources and were caused by mutations located in the RAP1 and GCR1 transcription factor binding sites. Mutations altered expression plasticity most frequently between the two fermentable environments, with mutations causing significant changes in plasticity between glucose and galactose distributed throughout the promoter, suggesting they might affect chromatin structure. Taken together, these results provide insight into the molecular mechanisms underlying gene-by-environment interactions affecting gene expression as well as the evolutionary dynamics affecting natural variation in plasticity of gene expression.

GAPDH--a recruits a plant virus movement protein to cortical virus replication complexes to facilitate viral cell-to-cell movement.

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.

Effects of copper overload in P19 neurons: impairment of glutathione redox homeostasis and crosstalk between caspase and calpain protease systems in ROS-induced apoptosis.

Copper, a transition metal with essential biological functions, exerts neurotoxic effects when present in excess. The aim of the present study was to better elucidate cellular and molecular mechanisms of CuSO4 toxicity in differentiated P19 neurons. Exposure to 0.5 mM CuSO4 for 24 h provoked moderate decrease in viability, accompanied with barely increased generation of reactive oxygen species (ROS) and caspase-3/7 activity. Glutathione (GSH) and ATP contents were depleted, lactate dehydrogenase inactivated, and glyceraldehyde-3-phosphate dehydrogenase overexpressed. In severely damaged neurons exposed to only two times higher concentration, classical caspase-dependent apoptosis was triggered as evidenced by marked caspase-3/7 activation and chromatin condensation. Multifold increase in ROS, together with very pronounced ATP and GSH loss, strongly suggests impairment of redox homeostasis. At higher copper concentration protease calpains were also activated, and neuronal injury was prevented in the presence of calpain inhibitor leupeptin through the mechanism that affects caspase activation. MK-801 and nifedipine, inhibitors of calcium entry, and H-89 and UO126, inhibitors of PKA and ERK signaling respectively, exacerbated neuronal death only in severely damaged neurons, while ROS-scavenger quercetin and calcium chelator BAPTA attenuated toxicity only at lower concentration. In a dose-dependent manner copper also provoked transcriptional changes of genes involved in intracellular signaling and induction of apoptosis (p53, c-fos, Bcl-2 and Bax). The obtained results emphasize differences in triggered neuronal-death processes in a very narrow range of concentrations and give further insight into the molecular mechanisms of copper toxicity with the potential to improve current therapeutic approaches in curing copper-related neurodegenerative diseases.

Promotion of glycerol utilization using ethanol and 1-propanol in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe does not grow in media containing glycerol as a sole carbon source but uses glycerol in the presence of ethanol. Ethanol, but not glycerol, triggered upregulation of gld1+ and fbp1+ during glucose starvation even though gld1+ and fbp1+ are essential for growth on glycerol. This upregulation occurred at a very low concentration of ethanol. The transcriptional regulation of gld1+ was tested in the presence of various alcohols, and both ethanol and 1-propanol were found to induce gld1+ and to support growth in glycerol-containing media. We suggest that S. pombe has a novel ethanol and/or 1-propanol recognition mechanism that upregulates glycerol utilization during glucose starvation.

Proteomic profiling of human bone marrow mesenchymal stem cells under shear stress.

Mesenchymal stem cells (MSCs) are promising seed cells for tissue engineering of blood vessels. As seed cells, MSCs must endure blood fluid shear stress after transplantation. It has been shown that fluid shear stress can regulate the proliferation and differentiation of MSCs. However, the effects of fluid shear stress on MSCs including the types of proteins modulated are still not well understood. In this study, we exposed human mesenchymal stem cells (HMSCs) to 3 dyn/cm(2) shear stress for 6 h and compared them to a control group using proteomic analysis. Thirteen specific proteins were affected by shear stress, 10 of which were up-regulated. Shear stress especially induced sustained increases in the expression of Annexin A2 and GAPDH, which have been specifically shown to affect HMSCs function. We present here the first comparative proteome analysis of effect of shear stress on HMSCs.

Satellite cell activity in muscle regeneration after contusion in rats.

1. The role of satellite cells in muscle growth during development is well documented, but the involvement of these cells in muscle repair after contusion is less well known. In the present study, we investigated the time-course of satellite cell activity (from 3h to 7days) after contusion of rat gastrocnemius muscle using specific molecular markers for immunofluorescence and real-time polymerase chain reaction (PCR). 2. Inflammation of the injured muscle occurred within 6h, followed by disintegration of the damaged myofibres within 12h. Newly formed myofibres appeared by Day 7. 3. The number of MyoD-positive nuclei (activated satellite cells) in the injured muscle was significantly increased by 6h, reaching a maximum by 12h after contusion. However, the number of MyoD-positive nuclei decreased towards control levels by Day 7. Changes in the number of bromodeoxyuridine-labelled nuclei (proliferating satellite cells) paralleled the changes seen in the number of MyoD-positive nuclei. Conversely, expression of myogenin protein was not apparent until Day 3 and increased further by Day 7. Colabelling of MyoD and myogenin was seen in only a few cells. 4. The time-course of MyoD mRNA expression corresponded with MyoD protein expression. However, there were two peaks in myogenin mRNA expression: 6h and Day 7 after contusion. The second peak coincided with upregulation of myostatin mRNA levels. 5. The results of the present study suggest that contusion activates a homogeneous population of satellite cells to proliferate within 3days, followed by differentiation to form new myofibres. The latter may be regulated, in part, by myostatin.

Expression profile and synthesis of different collagen types I, II, III, and V of human gingival fibroblasts, osteoblasts, and SaOS-2 cells after bisphosphonate treatment.

Bisphosphonates (BP) are used in the treatment of malignant osteolytic processes and postmenopausal osteoporosis. There have been a number of incidents in patients treated with BP. The incidents are described as an osteonecrosis of the jaw (ONJ). The main medications associated with these reports are zoledronic acid (ZOL) and pamidronate (PAM). The clinical presentations describe a deterioration of the jaw bones and surrounding tissues. The purpose of this study was to investigate expression of collagen types I, II, III, and V in human gingival fibroblasts, osteoblasts, and osteosarcoma cells (SaOS-2 cells) by ELISA and reverse transcription PCR (RT-PCR) with constant exposure (28 days) to ZOL and PAM. The real-time PCR indicates that ZOL inhibited gene expression below 16% at any concentration used. Hence, an amplification of extracellular matrix was only possible for PAM at concentrations of 1 microM. The following expression levels were for fibroblasts at a maximum of 31%, exceptionally high for the osteoblasts at 56%, and for SaOS-2 cells the peak was 14%. Principally, a decreased production of collagen was measured. With this in vitro study, we demonstrated how negatively influencing a long exposure to ZOL and PAM can be. Therefore, a reduction in extracellular matrix production of these cell lines under BP exposure could be a possible clinical indication as to why patients experience ONJ and have wound healing problems. However, it remains uncertain as to why an osteonecrosis is mainly found in the jaws and not other bones. As there are many influencing factors, further investigation needs to be pursued.

Selection and validation of reference genes by RT-qPCR for murine cementoblasts in mechanical loading experiments simulating orthodontic forces in vitro.

Different structures and cell types of the periodontium respond to orthodontic tooth movement (OTM) individually. Cementoblasts (OC/CM) located in the immediate vicinity of the fibroblasts on the cement have found way to the centre of actual research. Here, we identify and validate possible reference genes for OC/CM cells by RT-qPCR with and without static compressive loading. We investigated the suitability of 3 reference genes in an in vitro model of cementoblast cells using four different algorithms (Normfinder, geNorm, comparative delta-Ct method and BestKeeper) under different confluences and time. Comparable to our previous publications about reference genes in OTM in rats and human periodontal ligament fibroblasts (hPDLF), Rpl22 in murine OC/CM cells appears as the least regulated gene so that it represents the most appropriate reference gene. Furthermore, unlike to the expression of our recommended reference genes, the expression of additionally investigated target genes changes with confluence and under loading compression. Based on our findings for future RT-qPCR analyses in OC/CM cells, Rpl22 or the combination Rpl22/Tbp should be favored as reference gene. According to our results, although many publications propose the use of Gapdh, it does not seem to be the most suitable approach.

Inhibitory Traits of Dendrosome Curcumin (DNC) on Breast Cancer Compared to Curcumin Single Compound.

PURPOSE: Vast therapeutic traits and very low toxicity of curcumin compound have made it and related formulations promising for treatment purposes. The aim of this study was the assessment of dendrosome curcumin (DNC) inhibitory effects on breast cancer therapy compared to single curcumin compound. METHODS: DNC was synthesized and MCF-7 cells were prepared. The cultured cells were treated with 20 µg/ml and 25 µg/ml of DNC. Real-time quantitative PCR (RT-qPCR) was performed to measure the expression of FOXCUT and MEG3 genes. Additionally, flow cytometry was applied to measure cell death rate and apoptosis. RESULTS: The results outlined that DNC enhanced the MEG3 gene expression significantly higher than the control. Furthermore, DNC was associated with a significant decrease in the expression of FOXCUT gene as compared to the control. The application of DNC in the MCF7 cell line enhanced cell death and reduced necrosis. CONCLUSIONS: The rate of apoptosis (programmed cell death) was enhanced, but necrosis was decreased in treated cancer cells compared to those treated with single curcumin. Accordingly, the DNC can be applied to hinder the growth and dissemination of cancer cells as a preferred approach to reduce the complications of other strategies such as chemotherapy and radiation therapy. Indeed, we concluded that DNC enhanced the expression of MEG3, a tumor suppressor, at 25 µm dose, but reduced the expression of the FOXCUT gene, possibly via the methylation of the gene. Thereby, DNC exerted a promising tumor inhibitory growth potential for the eradication of cancer cells.

siRNA-mediated gene silencing in the salivary gland using in vivo microbubble-enhanced sonoporation.

OBJECTIVES: siRNA-induced gene silencing in the salivary gland using microbubble-enhanced sonoporation was used to develop an in vivo gene knockdown technique. METHODS: siRNA targeting rat glyceraldehyde-3-phosphate dehydrogenas (GAPDH) was mixed with echo-enhanced microbubbles and reverse-injected into rat parotid glands using transdermal ultrasound. To compare direct and transdermal ultrasound efficiencies, an incision was made on the lateral neck to expose the parotid glands for direct application. The efficiency of gene suppression was determined using quantitative reverse transcription-polymerase chain reaction 24-72 h after siRNA delivery. Cytotoxicity was assessed using histological analysis. RESULTS: Expression of rat GAPDH in the parotid glands was silenced 48 h after siRNA was delivered by ultrasound (frequency: 1 MHz; intensity: 2 W cm(-2); exposure time: 2 min). High-intensity ultrasound induced tissue damage and apoptotic change. Echo-enhanced microbubbles significantly improved siRNA-induced gene silencing by 10-50%. Compared with transdermal application, direct-exposure ultrasound was only slightly effective, and no significant difference in gene expression was observed. CONCLUSION: The results indicate that microbubble-enhanced sonoporation can yield in vivo siRNA gene silencing in the rat parotid gland. This technique could be applied to provide gene knockdown organs for functional genomic analyses and to develop siRNA-based gene therapy.

AMPK represses TOP mRNA translation but not global protein synthesis in liver.

The AMP-activated protein kinase (AMPK) represses signaling through the mammalian target of rapamycin complex 1 (mTORC1). In muscle, repression of mTORC1 leads to a reduction in global protein synthesis. In contrast, repression of mTORC1 in the liver has no immediate effect on global protein synthesis. In the present study, signaling through mTORC1 and translation of specific mRNAs such as those bearing a 5'-terminal oligopyrimidine (TOP) tract and were examined in rat liver following activation of AMPK after treadmill running. Activation of AMPK repressed translation of the TOP mRNAs encoding rpS6, rpS8, and eEF1alpha. In contrast, neither global protein synthesis nor translation of mRNAs encoding GAPDH or beta-actin was changed. Basal phosphorylation of the mTORC1 target 4E-BP1, but not S6K1 or rpS6, was reduced following activation of AMPK. Thus, in liver, AMPK activation repressed translation of TOP mRNAs through a mechanism distinct from downregulated phosphorylation of S6K1 or rpS6.

GAPDH, 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restriction.

Comparative gene expression studies in the placenta may provide insights into molecular mechanisms of important genomic alterations in pregnancy disorders. Endogenous reference genes often referred to as housekeeping genes, are routinely used to normalise gene expression levels. For this reason, it is important that these genes be empirically evaluated for stability between placental samples including samples from complicated pregnancies. To address this issue, six candidate housekeeping genes including several commonly used ones (ACTB, GAPDH, 18S rRNA, TBP, SDHA and YWHAZ) were investigated for their expression stability in placentae obtained from pregnancies complicated by idiopathic FGR (n=25) and gestation-matched control pregnancies (n=25). Real-time PCR was performed using pre-validated gene expression assay kits. The geNorm program was used for gene stability measure (M) for the entire housekeeping genes in all control and FGR-affected placental samples. Results showed that GAPDH and 18S rRNA were most stable, with an average expression stability of M=0.441 and 0.443, respectively, followed by YWHAZ (M=0.472). SDHA, ACTB and TBP were the least stable housekeeping genes (M=0.495, 0.548 and 1.737, respectively). We recommend geometric averaging of two or more housekeeping genes to determine relative gene expression levels between FGR-affected and control placentae.

Quantitative detection of micrometastases in pelvic lymph nodes in patients with clinically localized prostate cancer by real-time reverse transcriptase-PCR.

PURPOSE: Routine pathologic examination can miss micrometastatic tumor foci in the lymph nodes of patients with prostate cancer, resulting in confusion during tumor staging and clinical decision-making. The objective of this study was to clarify the significance of micrometastases in pelvic lymph nodes in patients who underwent radical prostatectomy for prostate cancer. EXPERIMENTAL DESIGN: The expression of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) in 2,215 lymph nodes isolated from 120 patients with clinically localized prostate cancer was assessed by a fully quantitative real-time reverse transcriptase-PCR. We regarded specimens in which either PSA or PSMA mRNAs were positive as proof of the "presence of micrometastasis." Immunohistochemical staining of lymph node specimens with an antibody against PSA was also done. RESULTS: Pathologic examinations detected tumor cells in 29 lymph nodes from 11 patients, and real-time reverse transcriptase-PCR further identified micrometastasis in 143 lymph nodes from 32 patients with no pathologic evidence of lymph node involvement. The presence of micrometastatic cancer cells was confirmed by immunohistochemical staining in 61 lymph nodes from 17 patients with pathologically negative lymph nodes. The presence of micrometastases was significantly associated with other conventional prognostic variables, including serum PSA value, pathologic stage, Gleason score, and tumor volume. Biochemical recurrence was detected in 32 patients, 17 of whom were negative for lymph node metastasis by pathologic examination (including 4 patients with pathologically organ-confined disease), but were diagnosed as having micrometastasis. Biochemical recurrence-free survival rate in patients without micrometastasis was significantly higher than in those with micrometastasis irrespective of the presence of pathologically positive nodes. Furthermore, only the presence of micrometastasis was independently associated with biochemical recurrence regardless of other factors examined. CONCLUSIONS: These findings suggest that approximately 30% of clinically localized prostate cancers shed cancer cells to the pelvic lymph nodes, and that biochemical recurrence after radical prostatectomy could be explained, at least in part, by micrometastases in pelvic lymph nodes.

Accumulation and nuclear import of HIF1 alpha during high and low oxygen concentration in skeletal muscle cells in primary culture.

The hypoxia-inducible-factor-1 (HIF1) mediates the transcriptional upregulation of several target genes during hypoxia. HIF1 itself is known to be regulated essentially by ubiquitinylation and proteolytic degradation of the subunit HIF1alpha of the dimeric transcription factor HIF1. In contrast to other tissues, skeletal muscle expresses high amounts of HIF1alpha in normoxia as well as in hypoxia. In view of this, we aimed to investigate HIF1alpha accumulation and subcellular localization as well as the transcriptional activity of the HIF1alpha-regulated gene of glyceraldehyde dehydrogenase (GAPDH) in skeletal muscle cells exposed to low oxygen concentration (3% O2), normoxia (20% O2) or high oxygen concentration (42% O2). Immunofluorescence analysis reveals that under normoxic and high oxygen conditions, significant amounts of HIF1alpha can be found exclusively in the cytoplasm of the myotubes. Muscle cells treated with CoCl2, a known inhibitor of HIF1alpha degradation, show even higher levels of HIF1alpha, again exclusively in the cytoplasm. Under conditions of low oxygen, HIF1alpha in controls as well as in CoCl2-treated cells is found in the nuclei. CdCl2 inhibits nuclear import of HIF1alpha at low oxygen concentration and leads to a transcriptional downregulation of the marker enzyme of anaerobic glycolysis GAPDH. Immunoprecipitation with anti-HIF1alpha antibody co-precipitates HSP90 in an oxygen-dependent manner, more at high pO2 than at low pO2. Cadmium-treated samples also show high amounts of co-immunoprecipitated HSP90, independent of oxygen concentration. We conclude that in skeletal muscle cells, HIF1alpha, in contrast to other tissues, may, in addition to its regulation by degradation, also be regulated by binding to HSP90 and subsequent inhibition of its import into the nuclei.

Age-related changes in relative expression of real-time PCR housekeeping genes in human skeletal muscle.

The purpose of this investigation was to examine the expression of three commonly used housekeeping genes -- glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta(2)-microglobulin (beta(2)M), and RNA polymerase 2a (polR2a) -- in elderly (E) compared to young (Y) subjects. Nine young subjects (22.7 +/- 3.4 yrs) and 11 elderly subjects (73.0 +/- 9.5 yrs) underwent a percutaneous skeletal muscle biopsy of the vastus lateralis. Equal concentrations of isolated mRNA from these samples were used to perform real-time polymerase chain reaction with primer/probe combinations specific to each gene of interest. The expression of GAPDH, beta(2)M, and polR2a was obtained as the value of cycle threshold (C(T)). An independent t-test with a level of significance at p < or = 0.05 was used to determine differences between groups. There was no difference in average C(T) of GAPDH between groups (p=0.869) (Y = 16.92 +/- 2.25 vs. E = 17.08 +/- 2.09) and polR2a (p = 0.089) (Y = 28.00 +/- 0.89 vs. E = 26.73 +/- 1.91). However, there was a significant difference (p < or = 0.05) in the average C(T) of beta(2)M (Y =21.79 +/- 0.44 vs. E = 21.05 +/- 0.51). The results indicate that special consideration needs to be made when selecting housekeeping genes for comparisons in real-time reverse-transcriptase polymerase chain reaction, depending upon the age of the populations of interest.

Cloning, expression and characterization of a mucin-binding GAPDH from Lactobacillus acidophilus.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis. It is also referred to as a moonlighting protein as it has many diverse functions like regulation of apoptosis, iron homeostasis, cell-matrix interactions, adherence to human colon etc. apart from its principal role in glycolysis. Lactobacilli are lactic acid bacteria which colonize the human gut and confer various health benefits to humans. In the present study, we have cloned, expressed and purified the GAPDH from Lactobacillus acidophilus to get a recombinant product (r-LaGAPDH) and characterized it. Size exclusion chromatography shows that r-LaGAPDH exists as a tetramer in solution and have a mucin binding and hemagglutination activity indicating carbohydrate like binding adhesion mechanism. Fluorescence spectroscopy studies showed an interaction of r-LaGAPDH with mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine with a Kd of 3.6±0.7×10(-3)M, 4.34±0.09×10(-3)M, 4±0.87×10(-3)M and 3.7±0.28×10(-3)M respectively. We hope that this preliminary data will generate more interest in further elucidation of the roles of GAPDH in the adhesion processes of the bacteria.

CDK9 inhibitors define elongation checkpoints at both ends of RNA polymerase II-transcribed genes.

Transcription through early-elongation checkpoints requires phosphorylation of negative transcription elongation factors (NTEFs) by the cyclin-dependent kinase (CDK) 9. Using CDK9 inhibitors and global run-on sequencing (GRO-seq), we have mapped CDK9 inhibitor-sensitive checkpoints genome wide in human cells. Our data indicate that early-elongation checkpoints are a general feature of RNA polymerase (pol) II-transcribed human genes and occur independently of polymerase stalling. Pol II that has negotiated the early-elongation checkpoint can elongate in the presence of inhibitors but, remarkably, terminates transcription prematurely close to the terminal polyadenylation (poly(A)) site. Our analysis has revealed an unexpected poly(A)-associated elongation checkpoint, which has major implications for the regulation of gene expression. Interestingly, the pattern of modification of the C-terminal domain of pol II terminated at this new checkpoint largely mirrors the pattern normally found downstream of the poly(A) site, thus suggesting common mechanisms of termination.

Differential abundance of sarcoplasmic proteome explains animal effect on beef Longissimus lumborum color stability.

The sarcoplasmic proteome of beef Longissimus lumborum demonstrating animal-to-animal variation in color stability was examined to correlate proteome profile with color. Longissimus lumborum (36 h post-mortem) muscles were obtained from 73 beef carcasses, aged for 13 days, and fabricated to 2.5-cm steaks. One steak was allotted to retail display, and another was immediately vacuum packaged and frozen at -80°C. Aerobically packaged steaks were stored under display, and color was evaluated on days 0 and 11. The steaks were ranked based on redness and color stability on day 11, and ten color-stable and ten color-labile carcasses were identified. Sarcoplasmic proteome of frozen steaks from the selected carcasses was analyzed. Nine proteins were differentially abundant in color-stable and color-labile steaks. Three glycolytic enzymes (phosphoglucomutase-1, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase M2) were over-abundant in color-stable steaks and positively correlated (P<0.05) to redness and color stability. These results indicated that animal variations in proteome contribute to differences in beef color.