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Results for “virtual protein profiling”

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PubMed1999

Amino acid neurochemistry of the vertebrate retina.

The dominant neurochemicals involved in encoding sensory information are the amino acid neurotransmitters, glutamate, gamma-aminobutyrate (GABA) and glycine, which mediate fast point-to-point synaptic transmission in the retina and other parts of the central nervous system. The relative abundance of these neurochemicals and the existence of neuronal and glial uptake mechanisms as well as a plethora of receptors support the key role these neurochemicals play in shaping neural information. However, in addition to subserving neurotransmitter roles, amino acids subserve normal metabolic,cellular functions, may be precursors for other amino acids, and may also be associated with protein synthesis. Post-embedding immunocytochemistry of small molecules has allowed the characterization of multiple amino acid profiles within subpopulations of neurons in the vertebrate retina. The general theme emerging from these studies is that the retinal through pathway uses glutamate as its neurotransmitter, and the lateral elements, GABA and/or glycine. Co-localization studies using quantitative immunocytochemistry have shown that virtually all neuronal space can be accounted for by the three dominant amino acids. In addition, co-localization studies have demonstrated that there are no purely aspartate, glutamine, alanine. leucine or ornithine immunoreactive neurons and thus these amino acids are likely to act as metabolites and may sustain glutamate production through a multitude of enzymatic pathways. The mapping of multiple cellular metabolic profiles during development or in degenerating retinas has shown that amino acid neurochemistry is a sensitive marker for metabolic activity. In the degenerating retina, (RCS retina), neurochemical anomalies were evident early in development (from birth), even before photoreceptors mature at PND6-8 implying a generalized metabolic dysfunction. Identification of metabolic anomalies within subpopulation of neurons is now possible and can be used to investigate a multitude of retinal functions including amino acid metabolic and neurochemical changes secondary to external insult as well as to expand our understanding of the intricate interrelationship between neurons and glia.

Amino Acids↗

PubMed2000

Human protein tyrosine phosphatase-like gene: expression profile, genomic structure, and mutation analysis in families with ARVD.

The mouse protein tyrosine phosphatase-like gene (Ptpla) was recently cloned and data suggested that it plays a role in myogenesis and cardiogenesis. The human homologue (PTPLA) was mapped to chromosome 10p13-14, a region where we have mapped a locus responsible for arrhythmogenic right ventricular dysplasia (ARVD). As a positional candidate gene, we characterized PTPLA by determining its tissue expression, its genomic structure, and we also screened for mutations in the ARVD patients. Northern analysis demonstrated PTPLA is preferentially expressed in both adult and fetal heart. A much lower expression was detected in skeletal and smooth muscle tissues. Virtually no expression was observed in other tissues. The protein-encoding sequences of PTPLA consist of seven exons. A sequence variation (Lys64Gln) was found in all the affecteds in a large ARVD family. However, the same variant was also detected in normal control subjects (three alleles/100 chromosomes). Thus, the variant (Lys64Gln) is not responsible for ARVD in our family and is a benign polymorphism. Nevertheless, its tissue-specific expression in the developing and adult heart suggest PTPLA has a role in regulating cardiac development, differentiation, or other cellular events. The genomic structure and intragenic polymorphism of PTPLA should be useful for further clinical and genetic studies such as gene targeting of PTPLA.

Arrhythmogenic Right Ventricular Dysplasia↗

PubMed2004

Virtual screening of human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase against the NCI diversity set by use of AutoDock to identify novel nonfolate inhibitors.

AICAR transformylase (5-aminoimidazole-4-carboxamide ribonucleotide transformylase) is a folate-dependent activity of the bifunctional protein ATIC (AICAR transformylase and IMP cyclohydrolase) and is responsible for catalyzing the penultimate step of the de novo purine biosynthetic pathway. As such, AICAR transformylase has been proposed as a potential target for antineoplastic drug design. Virtual screening of the human AICAR transformylase active site by use of AutoDock against the NCI diversity set, a library of compounds with nonredundant pharmacophore profiles, has revealed 44 potential inhibitor candidates. In vitro inhibition assay of 16 soluble compounds from this list revealed that eight compounds with novel scaffolds, relative to the general folate template, had micromolar inhibition. Subsequent extension of docking trials on compounds with similar scaffolds from the entire NCI-3D database has unveiled 11 additional inhibitors that were confirmed by the in vitro inhibition assay. In particular, one compound, NSC30171, had nanomolar inhibition (K(i) = 154 nM, IC(50) = 600 nM) against AICAR transformylase. These 19 inhibitors serve as novel templates/scaffolds for development of more potent and specific non-folate-based AICAR transformylase inhibitors.

Binding Sites↗

PubMed2006

Sex differences in atherosclerosis in mice with elevated phospholipid transfer protein activity are related to decreased plasma high density lipoproteins and not to increased production of triglycerides.

Plasma phospholipid transfer protein (PLTP) has atherogenic properties in genetically modified mice. PLTP stimulates hepatic triglyceride secretion and reduces plasma levels of high density lipoproteins (HDL). The present study was performed to relate the increased atherosclerosis in PLTP transgenic mice to one of these atherogenic effects. A humanized mouse model was used which had decreased LDL receptor expression and was transgenic for human cholesterylester transfer protein (CETP) in order to obtain a better resemblance to the plasma lipoprotein profile present in humans. It is well known that female mice are more susceptible to atherosclerosis than male mice. Therefore, we compared male and female mice expressing human PLTP. The animals were fed an atherogenic diet and the effects on plasma lipids and lipoproteins, triglyceride secretion and the development of atherosclerosis were measured. The development of atherosclerosis was sex-dependent. This effect was stronger in PLTP transgenic mice, while PLTP activity levels were virtually identical. Also, the rates of hepatic secretion of triglycerides were similar. In contrast, plasma levels of HDL were about 2-fold lower in female mice than in male mice after feeding an atherogenic diet. We conclude that increased atherosclerosis caused by overexpression of PLTP is related to a decrease in HDL, rather than to elevated hepatic secretion of triglycerides.

Animals↗

PubMed1999

Phytoene synthase-2 enzyme activity in tomato does not contribute to carotenoid synthesis in ripening fruit.

The characteristic yellow fruit phenotype of the r,r mutant and Psy-1 (phytoene synthase-1) antisense tomatoes is due to a mutated or down-regulated phytoene synthase protein, respectively, resulting in the virtual absence of carotenoids. Based on detailed carotenoid determinations Psy-1 appeared to barely contribute to the formation of carotenoids in chloroplast-containing tissues. Despite the virtual absence of carotenoids in ripe fruit the formation of phytoene in vitro was detected in fruit of both mutants. When [14C]isopentenyl pyrophosphate (IPP) was used as the substrate for phytoene synthase a reduction (e.g. r,r mutant, 5-fold) in the formation of phytoene was observed with an accumulation (e.g. r,r mutant, 2-fold) of the immediate precursor geranylgeranyl pyrophosphate (GGPP). Contrastingly, reduced phytoene synthase activity was not detected when [3H]GGPP was used as the substrate. The profile of phytoene formation during ripening was also different in the down-regulated mutants compared to the wild-type. Using specific primers, RT-PCR analysis detected the presence of Psy-2 transcripts in the down-regulated mutants and wild-type throughout fruit development and ripening. These data were supported by the detection of phytoene synthase protein on western blots. Both GGPP formation and phytoene desaturation were elevated in these mutants. Therefore, it appears that despite the absence of carotenoids in ripe fruit, both the mutants have the enzymic capability to synthesize carotenoids in this tissue. Implications of the data with respect to the regulation of carotenoid formation and the channelling of prenyl lipid precursors in tomato (and its potential manipulation) are discussed.

Alkyl and Aryl Transferases↗

PubMed1989

Immunological identification of five troponin T isoforms reveals an elaborate maturational troponin T profile in rabbit myocardium.

Myocardium is generally thought to express no more than two isoforms of troponin T (TnT). We have recently reported that TnT purified from rabbit myocardium is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into five proteins (TnT1, TnT2, TnT3, TnT4, and TnT5). In this study, these proteins are characterized immunologically and a novel elaborate maturational profile is described. Myocardium was obtained from 23 days of gestation fetal rabbits and 2-day, 6-week, 3-month, and 6-month postnatal rabbits. The major species in the adult myocardium, TnT4, was identified on sodium dodecyl sulfate-polyacrylamide gels and excised. The protein was electroeluted and purified. An amino acid microsequence of a cleaved fragment of this protein was found to be virtually identical to residues 86-99 from adult rabbit cardiac TnT. The protein, TnT4, was used to raise a polyclonal antibody. This antibody recognized all five isoforms from purified cardiac TnT, but none of the TnT isoforms from fast skeletal muscle. A monoclonal antibody, Mab JLT-12, raised against a highly conserved epitope of rabbit fast skeletal muscle, recognized all five cardiac as well as five skeletal muscle isoforms. Western blots performed on intact myocardial preparations demonstrated that TnT1, the cardiac isoform with the slowest electrophoretic mobility, was expressed prominently in the immature hearts, in addition to TnT2, TnT3, and TnT4, but TnT1 was not evident in the 3-month and 6-month postnatal hearts. The expression of TnT2 also decreased with maturation. Thus, the number of TnT isoforms expressed in the rabbit decreases with maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

Evidence for high-affinity binding sites for the adenosine A2A receptor agonist [3H] CGS 21680 in the rat hippocampus and cerebral cortex that are different from striatal A2A receptors.

The binding of the adenosine A2A receptor selective agonist 2-[4-(2-p-carboxyethyl)phenylamino] -5'-N-ethylcarboxamidoadenosine (CGS 21680) to the rat hippocampal and cerebral cortical membranes was studied and compared with that to striatal membranes. [3H] CGS 21680, in the concentration range tested (0.2-200 nM), bound to a single site with a Kd of 58 nM and a Bmax of 353 fmol/mg protein in the hippocampus, and with a Kd of 58 nM and a Bmax of 264 fmol/mg protein in the cortex; in the striatum, the single high-affinity [3H] CGS 21680 binding site had a Kd of 17 nM and a Bmax of 419 fmol/mg protein. Both guanylylimidodiphosphate (100 microM) and Na+ (100 mM) reduced the affinity of [3H] CGS 21680 binding in the striatum by half and virtually abolished [3H] CGS 21680 binding in the hippocampus and cortex. The displacement curves of [3H] CGS 21680 binding with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), N6-cyclohexyladenosine (CHA), 5'-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) were biphasic in the hippocampus and cortex as well as in the striatum. The predominant [3H]CGS 21680 binding site in the striatum (80%) had a pharmacological profile compatible with A2A receptors and was also present in the hippocampus and cortex, representing 10-25% of [3H]CGS 21680 binding. The predominant [3H]CGS 21680 binding site in the hippocampus and cortex had a pharmacological profile distinct from A2A receptors: the relative potency order of adenosine antagonists DPCPX, 1,3-dipropyl- 8-¿4-[(2-aminoethyl)amino]carbonylmethyl- oxyphenyl¿ xanthine (XAC), 8-(3-chlorostyryl)caffeine (CSC), and (E)-1,3-dipropyl-8-(3,4-dimethoxystyryl)- methylxanthine (KF 17,837) as displacers of [3H] CGS 21680 (5 nM) binding in the hippocampus and cerebral cortex was DPCPX > XAC >> CSC approximately KF 17,837, and the relative potency order of adenosine agonists CHA, NECA, CADO, 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5'-N- ethylcarboxamidoadenosine (APEC), and 2-phenylaminoadenosine (CV 1808) was CHA approximately NECA > or = CADO > APEC approximately CV1808 > CGS 21680. In the presence of DPCPX (20 nM), [3H] CGS 21680 (0.2-200 nM) bound to a site (A2A-like) with a Kd of 20 nM and a Bmax of 56fmol/mg protein in the hippocampus and with a Kd of 22 nM and a Bmax of 63fmol/mg protein in the cortex. In the presence of CSC (200 nM), [3H]CGS 21680(0.2-200 nM) bound to a second high-affinity site with a Kd of 97 nM and a Bmax of 255 fmol/mg protein in the hippocampus and with a Kd of 112 nM and a Bmax of 221 fmol/mg protein in the cortex. Two pharmacologically distinct [3H]CGS 21680 binding sites were found in synaptosomal membranes of the hippocampus and cortex and in the striatum, one corresponding to A2A receptors and the other to the second high-affinity [3H]CGS 21680 binding site. In contrast, the pharmacology of [3H]CHA binding was similar in synaptosomal membranes of the three brain areas. The present results establish the existence of at least two high-affinity [3H]CGS 21680 binding sites in the CNS and demonstrate that the [3H]CGS 21680 binding site predominant in the hippocampus and cerebral cortex has different binding characteristics from the classic A2A adenosine receptor, which predominates in the striatum.

2-Chloroadenosine↗

PubMed1988

Characterization of equine herpesvirus type 1 immediate early proteins.

EHV-1 immediate early (IE) gene expression in lytic infection results in the production of four high mol wt immediate early polypeptides (IEPs), designated IE1, IE2, IE2, and IE4; however, IE transcription is limited to the synthesis of a single 6-kb mRNA. Together, these findings raised questions as to whether the four IEPs were related products of the same gene. In the present study the IEPs were characterized with respect to their structural similarities, antigenic relatedness, and postsynthetic modifications. IE1 was the most abundant IEP, in that it accounted for approximately 80% of the IEP-incorporated radiolabel in infected rabbit kidney cells labeled under IE conditions with [35S]methionine or 14C-labeled amino acid mixtures. IE1 also was the major phosphorylated species. Limited proteolytic digestion of isolated radiolabeled IEP bands with Staph V8 protease yielded virtually identical fragment profiles in SDS-PAGE, as did digestions with chymotrypsin and N-chlorosuccinimide. Monospecific rabbit antisera raised against each of the four isolated IEPs reacted with all the IEP species in immunoblotting assays. Pulse-chase experiments indicated that all the IEPs were detectable immediately after a 15-min pulse and that several alterations in the IEP profile occurred during subsequent chase periods. Thus, the EHV-1 IEPs are closely related structurally and antigenically and appeared to be either produced simultaneously or processed to yield the individual forms immediately.

Antibodies, Viral↗

PubMed2007

Agrococcus casei sp. nov., isolated from the surfaces of smear-ripened cheeses.

Seven Gram-positive, coryneform bacteria with virtually identical whole-organism protein patterns were isolated from the surface of smear-ripened cheeses. Representatives of these strains were the subject of a polyphasic study designed to establish their taxonomic status. The organisms formed a distinct branch in the Microbacteriaceae 16S rRNA gene tree and were most closely related to members of the genus Agrococcus, sharing sequence similarities of 95.4-98.7 %. The chemotaxonomic profiles of the strains were consistent with their classification in the genus Agrococcus. The combined genotypic and phenotypic data show that the isolates should be classified in the genus Agrococcus as representatives of a novel species. The name Agrococcus casei sp. nov. is proposed for this taxon. Isolate R-17892t2(T) (=DSM 18061(T)=LMG 22410(T)) is the type strain of Agrococcus casei sp. nov.

Actinomycetales↗

PubMed1993

Safety of combined oligosaccharide conjugate Haemophilus influenzae type b (HbOC) and whole cell diphtheria-tetanus toxoids-pertussis vaccine in infancy. The Kaiser Permanente Pediatric Vaccine Study Group.

The safety of the combined oligosaccharide conjugate Haemophilus influenzae (Hib) type b (HbOC) and whole cell diphtheria-tetanus toxoids-pertussis (DTP) vaccine (Tetramune, HbOC-DTP; Lederle) in infancy was evaluated in 6644 recipients of this vaccine and compared with 3914 recipients of separate injections of whole cell DTP and HbOC vaccines when given as a three dose regimen to infants at 2, 4 and 6 months of age in each group. Of the total number of infants in the study, a subset of 1435 were enrolled into the study and then randomly assigned to receive either the Hib-DPT combined vaccine or the separate components. This subset was used to assess local and systemic side effects which were evaluated utilizing telephone interviews 48 to 72 hours after vaccine. The remaining children in the study population were enrolled in a nonrandomized manner. For these children parents were offered the experimental Hib-DPT vaccine and refusers were given HbOC and DTP. Both of these groups of children as well as the randomized subset described above were used to assess rates of episodes of hospitalization, emergency room utilization and sudden infant death syndrome in HbOC-DTP recipients and children who received HbOC and DTP separately. Immunogenicity was evaluated in 123 children by collection of a single serum sample 30 days after the third dose of HbOC-DTP. The observed immunogenicity was comparable to that observed in other recent studies for HbOC and DTP component antigens. The profile of local and systemic side effects observed was virtually identical to that observed after DTP plus HbOC given separately.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed2006

Biochemical profiling in silico--predicting substrate specificities of large enzyme families.

A general high-throughput method for in silico biochemical profiling of enzyme families has been developed based on covalent docking of potential substrates into the binding sites of target enzymes. The method has been tested by systematically docking transition state--analogous intermediates of 12 substrates into the binding sites of 20 alpha/beta hydrolases from 15 homologous families. To evaluate the effect of side chain orientations to the docking results, 137 crystal structures were included in the analysis. A good substrate must fulfil two criteria: it must bind in a productive geometry with four hydrogen bonds between the substrate and the catalytic histidine and the oxyanion hole, and a high affinity of the enzyme-substrate complex as predicted by a high docking score. The modelling results in general reproduce experimental data on substrate specificity and stereoselectivity: the differences in substrate specificity of cholinesterases toward acetyl- and butyrylcholine, the changes of activity of lipases and esterases upon the size of the acid moieties, activity of lipases and esterases toward tertiary alcohols, and the stereopreference of lipases and esterases toward chiral secondary alcohols. Rigidity of the docking procedure was the major reason for false positive and false negative predictions, as the geometry of the complex and docking score may sensitively depend on the orientation of individual side chains. Therefore, appropriate structures have to be identified. In silico biochemical profiling provides a time efficient and cost saving protocol for virtual screening to identify the potential substrates of the members of large enzyme family from a library of molecules.

Binding Sites↗

PubMed2004

Transformation potency of ErbB heterodimer signaling is determined by B-Raf kinase.

Cellular transformation occurs only in cells that express both ErbB1 and ErbB4 receptors, but not in cells expressing only one or the other of these receptors. However, when both receptors are coexpressed and ligand-stimulated, they interact with virtually the same adaptor/effector proteins as when expressed singly. To reveal the underlying regulatory mechanism of the kinase/phosphatase network in ErbB homo- and heterodimer receptor signaling, extracellular signal-regulated kinase (ERK) and Akt activities were evaluated in the presence of several enzyme inhibitors in ligand-induced cells expressing ErbB1 (E1), ErbB4 (E4), and ErbB1/ErbB4 (E1/4) receptor. The PP2A inhibitor okadaic acid showed receptor-specific inhibitory profiles for ERK and Akt activities. Moreover, B-Raf isolated only from E1/4 cells could induce in vitro phosphorylation for MEK; this B-Raf kinase activity was abolished by pretreatment of the cells with okadaic acid. Our study further showed that the E1/4 cell-specific B-Raf activity was stimulated by PLC gamma and subsequent Rap1 activation. The present study suggests that B-Raf kinase, which was specifically activated in the cells coexpressing ErbB1 and ErbB4 receptors, elevates total ERK activity within the cell and, therefore, can induce cellular transformation.

Animals↗

PubMed1997

Identification of a novel gene encoding the yeast mitochondrial dicarboxylate transport protein via overexpression, purification, and characterization of its protein product.

A gene encoding the mitochondrial dicarboxylate transport protein (DTP) has been identified for the first time from any organism. Our strategy involved overexpression of putative mitochondrial transporter genes, selected based on analysis of the yeast genome, followed by purification and functional reconstitution of the resulting protein products. The DTP gene from the yeast Saccharomyces cerevisiae encodes a 298-residue basic protein which, in common with other mitochondrial anion transporters of known sequence and function, displays the mitochondrial transporter signature motif, three homologous 100-amino acid sequence domains, and six predicted membrane-spanning regions. The product of this gene has been abundantly expressed in Escherichia coli where it accumulates in inclusion bodies. Upon solubilization of the overexpressed DTP from isolated inclusion bodies with Sarkosyl, 28 mg of DTP was obtained per liter of E. coli culture at a purity of 75%. The purified, overexpressed DTP was then reconstituted in phospholipid vesicles where both its kinetic properties (i.e. Km = 1. 55 mM and Vmax = 3.0 micro;mol/min/mg protein) and its substrate specificity were determined. The intraliposomal substrates malonate, malate, succinate, and phosphate effectively supported [14C]malonate uptake, whereas other anions tested did not. External substrate competition studies revealed a similar specificity profile. Inhibitor studies indicated that the reconstituted transporter was sensitive to inhibition by n-butylmalonate, p-chloromercuribenzoate, mersalyl, and to a lesser extent pyridoxal 5'-phosphate but was insensitive to N-ethylmaleimide and selective inhibitors of other mitochondrial anion transporters. In combination, the above findings indicate that the identified gene encodes a mitochondrial transport protein which upon overexpression and reconstitution displays functional properties that are virtually identical to those of the native mitochondrial dicarboxylate transport system. In conclusion, the present investigation has resulted in identification of a gene encoding the mitochondrial DTP and thus eliminates a major impediment to molecular studies with this metabolically important transporter. Based on both structural and functional considerations, the yeast DTP is assignable to the mitochondrial carrier family. Additionally, the development of a procedure that enables the expression and isolation of large quantities of functional DTP provides the foundation for comprehensive investigations into the structure/function relationships within this transporter via site-directed mutagenesis, as well as for the initiation of crystallization trials.

Amino Acid Sequence↗

PubMed1985

Cell surface features associated with differentiation-induction of methylcholanthrene-transformed AKR-2B fibroblasts by N,N-dimethylformamide.

Methylcholanthrene-transformed AKR-2B mouse embryonal fibroblasts (AKR-MCA cells) were examined for cell surface alterations after growth in culture medium containing N,N-dimethylformamide (DMF) using the lactoperoxidase-glucose oxidase radioiodination procedure with subsequent electrophoresis. DMF has been shown to induce maturational changes in a variety of transformed cells in vitro and has been reported to produce a more normal phenotype when applied to cultured AKR-MCA cells. The electrophoretic profile of 125I-labeled surface proteins from AKR-MCA cells exhibited a prominent peak of labeled material with a molecular weight of approximately 85,000. After growth of AKR-MCA cells in medium containing DMF, the Mr 85,000 peak was substantially reduced, while there was a large increase in Mr 200,000 to 250,000 radioiodinated surface material. This cell surface labeling pattern was virtually identical to that of the nontransformed AKR-2B fibroblasts from which AKR-MCA cells were derived. The cell surface alterations observed upon exposure of AKR-MCA cells to DMF occurred as a function of time of growth in DMF and DMF concentration. Growth of AKR-MCA cells in DMF resulted in a steady increase in cell surface 125I incorporation up to the fourth day of exposure to DMF. At this time, the incorporation level was 22.9-fold greater than that for untreated AKR-MCA cells. Incorporation of radiolabel was decreased after the fifth and sixth days of AKR-MCA exposure to DMF. This trend was also manifested by AKR-2B fibroblasts grown in the presence of DMF. The data suggest that there was increased expression of the Mr 200,000 to 250,000 surface protein on both AKR-2B and AKR-MCA cells when grown in DMF. DMF also inhibited morphological transformation and the cell surface changes associated with transformation of AKR-2B cells by exogenous transforming growth factors.

Animals↗

PubMed2001

Cycle-regulated genes and cell cycle regulation.

The transcriptional profile of the entire Caulobacter crescentus genome over a synchronous cell cycle was recently described. The analysis reveals a stunning 553 cell-cycle-regulated genes or orfs, nearly 19% of the genome, including putative functions in virtually all biological activities. Over a quarter of these genes/orfs respond to the Caulobacter master regulator, CtrA, most of them apparently indirectly. The analysis confirms and extends earlier observations showing that many proteins involved in cell cycle functions are expressed at the cell age when they are needed. Conversely, the data suggest that proteins specifically expressed at a particular age may be involved in a process taking place then.

Bacterial Proteins↗

PubMed1993

Characterization of "plasma proteins" secreted by cultured rat macroglial cells.

The brain is isolated behind a blood-tissue barrier that restricts the access of circulating proteins to neural cells. There is evidence that some of these proteins are synthesized within the central nervous system. The present study examines the synthesis and secretion of such proteins by cultured macroglial cells. Primary glial cultures were derived from cortical and subcortical regions of neonatal rat brains, and subsequent secondary cultures were enriched in type-1 astrocytes, type-2 astrocytes, or oligodendrocytes. Newly synthesized proteins were immunoprecipitated from the culture media using antisera directed against whole rat serum. All three types of glial cells secreted a range of plasma proteins. In general, type-1 astrocytes secreted more of these proteins than did type-2 astrocytes or oligodendrocytes, although the one-dimensional polyacrylamide gel electrophoresis (PAGE) profiles were specific for each cell type. Anti-sera directed against specific plasma proteins identified three of the most abundant proteins secreted by type-1 astrocytes as transferrin, alpha-2-macroglobulin, and ceruloplasmin. Northern blot analysis of cellular RNA confirmed that type-1 astrocytes contained transferrin mRNA, and that it was more abundant in cultures derived from subcortical regions than from cortical regions. In situ hybridization studies revealed that virtually all type-1 and type-2 astrocytes contained transferrin mRNA. Since the proteins identified in this study have been proposed to have a variety of neurotrophic roles in the central nervous system, these data further extend the range of possible functions that glial cells may serve in the CNS.

Animals↗

PubMed2006

Intensity-based hierarchical Bayes method improves testing for differentially expressed genes in microarray experiments.

BACKGROUND: The small sample sizes often used for microarray experiments result in poor estimates of variance if each gene is considered independently. Yet accurately estimating variability of gene expression measurements in microarray experiments is essential for correctly identifying differentially expressed genes. Several recently developed methods for testing differential expression of genes utilize hierarchical Bayesian models to "pool" information from multiple genes. We have developed a statistical testing procedure that further improves upon current methods by incorporating the well-documented relationship between the absolute gene expression level and the variance of gene expression measurements into the general empirical Bayes framework. RESULTS: We present a novel Bayesian moderated-T, which we show to perform favorably in simulations, with two real, dual-channel microarray experiments and in two controlled single-channel experiments. In simulations, the new method achieved greater power while correctly estimating the true proportion of false positives, and in the analysis of two publicly-available "spike-in" experiments, the new method performed favorably compared to all tested alternatives. We also applied our method to two experimental datasets and discuss the additional biological insights as revealed by our method in contrast to the others. The R-source code for implementing our algorithm is freely available at http://eh3.uc.edu/ibmt. CONCLUSION: We use a Bayesian hierarchical normal model to define a novel Intensity-Based Moderated T-statistic (IBMT). The method is completely data-dependent using empirical Bayes philosophy to estimate hyperparameters, and thus does not require specification of any free parameters. IBMT has the strength of balancing two important factors in the analysis of microarray data: the degree of independence of variances relative to the degree of identity (i.e. t-tests vs. equal variance assumption), and the relationship between variance and signal intensity. When this variance-intensity relationship is weak or does not exist, IBMT reduces to a previously described moderated t-statistic. Furthermore, our method may be directly applied to any array platform and experimental design. Together, these properties show IBMT to be a valuable option in the analysis of virtually any microarray experiment.

Animals↗

PubMed2006

Discovery of protein phosphatase 2C inhibitors by virtual screening.

Protein phosphatase 2C (PP2C) is an archetype of the PPM Ser/Thr phosphatases, characterized by dependence on divalent magnesium or manganese cofactors, absence of known regulatory proteins, and resistance to all known Ser/Thr phosphatase inhibitors. We have used virtual ligand screening with the AutoDock method and the National Cancer Institute Diversity Set to identify small-molecule inhibitors of PP2Calpha activity at a protein substrate. These inhibitors are active in the micromolar range and represent the first non-phosphate-based molecules found to inhibit a type 2C phosphatase. The compounds docked to three recurrent binding sites near the PP2Calpha active site and displayed novel Ser/Thr phosphatase selectivity profiles. Common chemical features of these compounds may form the basis for development of a PP2C inhibitor pharmacophore and may facilitate investigation of PP2C control and cellular function.

Binding Sites↗