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Results for “virtual protein profiling”

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At least 145 records · Page 8Linked to original sources

PubMed1996

Hepatic lipase gene therapy in hepatic lipase-deficient mice. Adenovirus-mediated replacement of a lipolytic enzyme to the vascular endothelium.

Hepatic lipase (HL) is an endothelial-bound lipolytic enzyme which functions as a phospholipase as well as a triacylglycerol hydrolase and is necessary for the metabolism of IDL and HDL. To evaluate the feasibility of replacing an enzyme whose in vivo physiologic function depends on its localization on the vascular endothelium, we have infused recombinant replication-deficient adenovirus vectors expressing either human HL (HL-rAdV; n = 7) or luciferase cDNA (Lucif-rAdV; n = 4) into HL-deficient mice with pretreatment plasma cholesterol, phospholipid, and HDL cholesterol values of 176 +/- 9, 314 +/- 12, and 129 +/- 9, respectively. After infusion of HL-rAdV, HL could be detected in the postheparin plasma of HL-deficient mice by immunoblotting and postheparin plasma HL activities were 25,700 +/- 4,810 and 1,510 +/- 688 nmol/min/ml on days 5 and 15, respectively. Unlike the mouse HL, 97% of the newly synthesized human HL was heparin releasable, indicating that the human enzyme was virtually totally bound to the mouse vascular endothelium. Infusion of HL-rAdV in HL-deficient mice was associated with a 50-80% decrease in total cholesterol, triglyceride, phospholipids, cholesteryl ester, and HDL cholesterol (P < 0.001) as well as normalization of the plasma fast protein liquid chromatography lipoprotein profile by day 8. These studies demonstrate successful expression and delivery of a lipolytic enzyme to the vascular endothelium for ultimate correction of the HL gene defect in HL-deficient mice and indicate that recombinant adenovirus vectors may be useful in the replacement of endothelial-bound lipolytic enzymes in human lipolytic deficiency states.

Adenoviridae↗

PubMed1996

Mitochondria act as a reservoir for the basic amine HIPDM in the lung.

The lungs are a site for the uptake, accumulation, and storage of exogenous basic amines. The compound N-N-N'-trimethyl-N'-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3 propanediamine (HIPDM) is a basic amine that can be labelled with radioactive iodine and detected by external counting. Intravenously injected 123I-HIPDM is extracted by the human lung, where it is retained in a slowly effluxable pool. In the present study, we measured HIPDM lung kinetics and subcellular distribution in rabbits given i.v. 125I-HIPDM. Rabbits were killed from 2 min to 5 h after injection, and the radioactivity retained in their lungs was measured. Subcellular lung fractions (nuclear, mitochondrial, lysosomal, microsomal and postmicrosomal supernatant) were assayed for HIPDM radioactivity, protein content, and distribution of specific marker enzymes. HIPDM lung clearance in rabbits was nearly identical to that of humans. Virtually all the HIPDM radioactivity in lungs (98+/-1%) was associated with subcellular membranous structures. The highest HIPDM specific radioactivity was found in the mitochondrial fraction, and the subcellular distribution profile closely resembled that of the mitochondrial marker enzyme succinate cytochrome c reductase. No redistribution of HIPDM among subcellular compartments was observed over a 5 h period after injection. The data indicate that mitochondria act as reservoir for HIPDM in the lungs and contribute to the pulmonary persistence of this compound. HIPDM can be used to investigate the pulmonary uptake of basic amines in health and in lung disease.

Animals↗

PubMed2001

Differential expression of glucose transporters during chick embryogenesis.

The patterns of Glut1 and Glut3 glucose transporter protein and mRNA expression were assessed during embryogenesis of chicken brain and skeletal muscle, Glut4 protein levels were also evaluated in skeletal muscle and heart, and Glut1 was examined in the developing heart and liver. Glut1 protein expression was detectable throughout brain ontogeny but was highest during early development. Glut1 mRNA levels in the brain remained very high throughout development. Glut3 protein was highest very early and very late and mRNA was highest during the last half of development. In embryonic skeletal muscle, the levels of Glut1 and Glut3 proteins and mRNA were highest very early, and declined severely by mid-development. Glut1 protein and mRNA in the heart also peaked early and then decreased steadily. Although Glut1 mRNA levels were consistently high in the embryonic liver, Glut1 protein expression was not detected. These results suggest that (1) Glut1 is developmentally regulated in chick brain, skeletal muscle, and heart, (2) Glut1 mRNA is present in liver but does not appear to be translated, (3) Glut3 in brain increases developmentally but is virtually absent in muscle, and (4) Glut4 protein and mRNA appear to be absent from chick heart and skeletal muscle.

Animals↗

PubMed1991

Clonal analysis of non-typable Haemophilus influenzae by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of whole cell polypeptides.

A collection of 223 strains of non-typable Haemophilus influenzae was assembled. Most strains were isolated from hospital in-patients in North East Scotland between 1984 and 1989. These isolates were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell polypeptides. Variability was assessed in terms of apparent molecular weight differences between the protein profiles. Isolates were grouped on the basis of Dice coefficients of similarity and assigned to clones. Of the 223 strains, 147 were unique; the remaining strains were assigned to multi-member clones of which 13 clones had two members, one clone had three members, three clones had four members, three clones had five members, two clones had six members and one clone had eight members. The longest time interval between isolation of clonally related strains virtually equalled the limits of the study. Isolates from a childrens' hospital were significantly less diverse than those from patients in adult wards.

Adult↗

PubMed2004

Binding of lithium and boron to human plasma proteins II: results for a bipolar patient not on lithium therapy.

We report further measurements of lithium and boron bound to human plasma proteins using the techniques of gel chromatography, thermal-neutron activation, and high-sensitivity helium isotope mass spectrometry. The plasma sample was donated by a bipolar patient who had never been on lithium therapy. The plasma lithium-binding pattern for the bipolar patient is distinctly different from that previously observed in this laboratory for plasma donated by a normal individual. In the bipolar case, virtually all of the lithium is bound to low-molecular-weight proteins (approx 1000 amu), whereas in the normal case, most of the lithium eluted from the gel column was bound to five high-molecular-weight proteins (approx 50,000 amu to approx 1,000,000 amu). The gel elution profiles for boron were roughly similar for the normal and bipolar cases. The lithium results are in agreement with our previous speculation that lithium-binding plasma proteins are missing or exist in very low concentrations in some individuals suffering from affective disorders.

Bipolar Disorder↗

PubMed2006

Comprehensive protein profiling by multiplexed capillary zone electrophoresis using cross-linked polyacrylamide coated capillaries.

We have recently developed a new process to create cross-linked polyacrylamide (CPA) coatings on capillary walls to suppress protein-wall interactions. Here, we demonstrate CPA-coated capillaries for high-efficiency (>2 x 10(6) plates per meter) protein separations by capillary zone electrophoresis (CZE). Because CPA virtually eliminates electroosmotic flow, positive and negative proteins cannot be analyzed in a single run. A "one-sample-two-separation" approach is developed to achieve a comprehensive protein analysis. High throughput is achieved through a multiplexed CZE system.

Acrylic Resins↗

PubMed1995

The inducible 9, 10-dihydrophenanthrene pathway: characterization and expression of bibenzyl synthase and S-adenosylhomocysteine hydrolase.

Tricyclic 9,10-dihydrophenanthrenes originate from phenylpropane derivatives by chain elongation and cyclization according to the polyacetate rule. Bibenzyls are bicyclic intermediates, and O-methylation is a prerequisite for their conversion into dihydrophenanthrenes. cDNA clones encoding bibenzyl synthases and S-adenosylhomocysteine hydrolase of the orchid Phalaenopsis sp. were isolated from a cDNA library representing the stage of elicitor-induced plants. The deduced amino acid sequences of two clones, pBibSy811 and pBibSy212, indicated that we obtained two full-length sequences of bibenzyl synthases characterized by their homology to stilbene synthases previously investigated. That indeed bibenzyl synthase cDNAs rather than a homologous stilbene synthase cDNA or chalcone synthase cDNA have been isolated was demonstrated by expression of two enzymatically active bibenzyl synthase proteins in Escherichia coli. These proteins showed virtually the same selectivity towards m-hydroxyphenylpropionyl-CoA as substrate as the enzyme isolated from orchid plants. In young sterile Phalaenopsis plants, the formation of both bibenzyl synthase mRNAs and S-adenosylhomocysteine hydrolase mRNAs was increased upon elicitation more than 100-fold. The time courses of gene expression exhibited transient profiles, reaching maximum mRNA levels 20 h after onset of fungal infection followed by a rapid decline to 40 h.

Adenosylhomocysteinase↗

PubMed1994

Immunoreactivity for phosphorylated 200-kDa neurofilament subunit is heterogeneously expressed in human sympathetic and primary sensory neurons.

This study was undertaken to investigate whether human sensory and sympathetic neurons contain phosphorylated neurofilament proteins, and whether they may be classified on the basis of this property, as in other mammalian species. The distribution of the phosphorylated 200-kDa neurofilament protein subunit (p200-NFP) was investigated in lumbar sympathetic and dorsal root ganglia by means of the RT97 monoclonal antibody (against p200-NFP). The intensity of immunostaining, and the size of neuronal body profiles were measured in order to define different neuron subclasses. In dorsal root ganglia, most of the neuronal profiles (96%) were p200-NFP immunoreactive, and the intensity of immunostaining was not related to neuronal perikarya size. In the lumbar paravertebral sympathetic ganglia, virtually all neurons displayed p200-NFP immunoreactivity, and the intensity of immunolabelling was also independent of the size of the neuronal somata. These results demonstrate heterogeneity in the expression of p200-NFP immunoreactivity in human sympathetic and sensory neurons. In contrast to other mammalian species, RT97 immunolabelling cannot be used as a discriminative marker for the two main types of human primary sensory neurons. On the other hand, our findings provide evidence for the occurrence of phosphorylated neurofilaments within peripheral neuron cell bodies.

Adult↗

PubMedDate not supplied

Peptidomics-based discovery of novel neuropeptides.

Modern proteomic methodologies have significantly improved the possibilities of large-scale identification of proteins. However, these methodologies are limited by their inability to reliably detect endogenously expressed peptides. We describe a novel approach of combining sample preparation, comprising focused microwave irradiation and mass spectrometric peptide profiling that has enabled us to simultaneously detect more than 550 endogenous neuropeptides in 1 mg of hypothalamic extracts. Automatic switching tandem mass spectrometry and amino acid sequence determination of the peptides showed that they consist of both novel and previously described neuropeptides. The methodology includes virtual visualization of the peptides as two- and three-dimensional image maps. In addition, several novel and known post-translational modifications of the neuropeptides were identified. The peptidomic approach proved to be a powerful method for investigating endogenous peptides and their post-translational modifications in complex tissues such as the brain. It is anticipated that this approach will complement proteomic methods in the future.

Amino Acid Sequence↗

PubMed2000

Characterization of cuticular proteins in the red flour beetle, Tribolium castaneum.

We are characterizing the cuticular proteins of Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae) to determine their role in the function of the exoskeleton. Based on qualitative analyses of cuticles, we focused on the sodium dodecyl sulfate (SDS)-extractable proteins. A small-scale cuticle "mini-prep" procedure was devised that yields preparations virtually free of contaminating cellular material compared to hand-dissected preparations, as assessed by fluorescent microscopy using DAPI to stain nuclei. Proteins extracted in 1% SDS from various developmental stages (last larval instar, pupal, adult) were analyzed by one-dimensional denaturing polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. The cuticular protein profiles show both similarities and differences among the stages examined. The amino acid composition, glycosylation, and partial amino acid sequence of several abundant cuticular proteins indicate similarity to cuticular proteins of other insects.

Animals↗

PubMed2006

Antibody microarrays: current status and key technological advances.

Antibody-based microarrays are among the novel classes of rapidly evolving proteomic technologies that holds great promise in biomedicine. Miniaturized microarrays (< 1 cm2) can be printed with thousands of individual antibodies carrying the desired specificities, and with biological sample (e.g., an entire proteome) added, virtually any specifically bound analytes can be detected. While consuming only minute amounts (< microL scale) of reagents, ultra- sensitive assays (zeptomol range) can readily be performed in a highly multiplexed manner. The microarray patterns generated can then be transformed into proteomic maps, or detailed molecular fingerprints, revealing the composition of the proteome. Thus, protein expression profiling and global proteome analysis using this tool will offer new opportunities for drug target and biomarker discovery, disease diagnostics, and insights into disease biology. Adopting the antibody microarray technology platform, several biomedical applications, ranging from focused assays to proteome-scale analysis will be rapidly emerging in the coming years. This review will discuss the current status of the antibody microarray technology focusing on recent technological advances and key issues in the process of evolving the methodology into a high-performing proteomic research tool.

Animals↗

PubMedDate not supplied

In vitro release of adriamycin from drug-loaded albumin and haemoglobin microspheres.

Release of adriamycin from albumin and haemoglobin microspheres has been determined in vitro using a flow through system. Release from cross-linked albumin microspheres is controlled by the percentage of glutaraldehyde used but release profiles of spheres of 23, 41 and 60 microns diameter were virtually superimposable. Not all of the adriamycin is released from the microspheres over 20 h; as biodegradation occurs in vivo after 24 h the amount of drug remaining in the system at 20 h is likely to be principally released by degradation of the protein matrix in vivo. Estimates of the retained adriamycin vary from 16-26 per cent for albumin (n = 3) and 30 per cent for haemoglobin (n = 1).

Albumins↗

PubMed2001

Neuron-specific expression of therapeutic proteins: evaluation of different cellular promoters in recombinant adenoviral vectors.

In order to achieve neuron-restricted expression of antiapoptotic proteins, cellular promoters were investigated for their expression profiles in the context of adenoviral vectors. Both the synapsin 1 gene and the tubulin alpha1 gene promoters were strictly neuron specific in cocultures of primary neurons with their essential feeder cells. The neuron-specific enolase gene promoter exhibited only weak activity in cultured hippocampal neurons and was not neuron specific in preparations of cerebellar granule cells. By attaining virtually 100% transduction efficiency we were able to generate "quasi-transgenic" primary neuron cultures using both differentiated and completely undifferentiated hippocampal neurons. In a functional assay, we used the synapsin promoter to evaluate the effect of Bcl-X(L) overexpression on potassium-withdrawal-induced apoptosis of cerebellar granule neurons. We found nearly complete inhibition of caspase-9 and -3 activation and apoptosis, indicating a major role for mitochondrial pathways in this paradigm of neuronal cell death. The excellent suitability of the synapsin promoter as a strong panneuronal promoter was further demonstrated by its restricted neuronal activity in various brain regions of adult rats in vivo.

Adenoviridae↗

PubMed2003

In silico analysis of the EPS8 gene family: genomic organization, expression profile, and protein structure.

EPS8 codes for a protein essential in Ras to Rac signaling leading to actin remodeling. Three genes highly homologous to EPS8 were discovered, thereby defining a novel gene family. Here, we report the genomic structure of EPS8 and the EPS8-related genes in human and mouse. We performed BLASTN searches against the Celera Human Genome and Mouse Fragments Database. The mouse fragments were manually assembled, and the organization of both human and mouse genes was reconstructed. The gene structures in Celera annotations of the human and mouse genomes were compared to outline correspondences and divergences. We also compared the EPS8 family gene structures predicted by Celera with those predicted by NCBI. Moreover, we performed a virtual analysis of the expression of the EPS8 gene family members by using the SAGEmap Database in NCBI. Finally, we analyzed the domain organization of the gene products and their evolutionary conservation to define novel putative domains, thereby helping to predict novel modality of action for the members of this gene family. The data obtained will be instrumental in directing further experimental functional characterization of these genes.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Genetic diversity and relationships among Streptococcus pyogenes strains expressing serotype M1 protein: recent intercontinental spread of a subclone causing episodes of invasive disease.

Chromosomal diversity and relationships among 126 Streptococcus pyogenes strains expressing M1 protein from 13 countries on five continents were analyzed by multilocus enzyme electrophoresis and restriction fragment profiling by pulsed-field gel electrophoresis. All isolates were studied for the presence of the gene encoding streptococcal pyrogenic exotoxin A by PCR. Strain subsets were also examined by automated DNA sequencing for allelic polymorphism in genes encoding M protein (emm), streptococcal pyrogenic exotoxin A (speA), streptokinase (ska), pyrogenic exotoxin B (interleukin-1 beta convertase) (speB), and C5a peptidase (scp). Seven distinct emm1 alleles that encode M proteins differing at one or more amino acids in the N-terminal variable region were identified. Although substantial levels of genetic diversity exist among M1-expressing organisms, most invasive disease episodes are caused by two subclones marked by distinctive multilocus enzyme electrophoretic profiles and pulsed-field gel electrophoresis restriction fragment length polymorphism (RFLP) types. One of these subclones (ET 1/RFLP pattern 1a) has the speA gene and was recovered worldwide. Identity of speA, emm1, speB, and ska alleles in virtually all isolates of ET 1/RFLP type 1a means that these organisms share a common ancestor and that global dispersion of this M1-expressing subclone has occurred very recently. The occurrence of the same emm and ska alleles in strains that are well differentiated in overall chromosomal character demonstrates that horizontal transfer and recombination play a fundamental role in diversifying natural populations of S. pyogenes.

Amino Acid Sequence↗

PubMed1992

Characterization of the snake venoms from seven Brazilian species of Bothrops by FPLC anion-exchange chromatography.

1. The elution profiles and the caseinolytic, myotoxic, coagulant and hemorrhagic activities of the venoms of seven Bothrops species fractionated on a Mono-Q FPLC column were analyzed. 2. Each venom separated into 16-20 peaks, with good reproducibility and the activities were concentrated in virtually discrete regions of the chromatogram. 3. There is a considerable overlap of active proteins in the different species venoms and our results indicate that a venom pool with the species B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. atrox venoms would contain the major active proteins determined in the seven species.

Animals↗

PubMed1991

Antibodies to non-beta regions of the beta-amyloid precursor protein detect a subset of senile plaques.

A central unresolved issue in Alzheimer's disease is the origin of the extracellular amyloid beta protein (A beta P) found in senile plaques and its relationship to the dystrophic neurites that intimately surround it. Here the presence and distribution within senile plaques of various epitopes of the beta-amyloid precursor protein (APP) are compared with the distribution of A beta P itself and markers for plaque neurites. Several principal findings emerge: 1) antibodies to regions of APP outside of A beta P ('APP antibodies') recognize only a subgroup of senile plaques; 2) within these plaques, APP antibodies label discrete globular and granular structures morphologically resembling neurites; 3) virtually all of the plaques labeled by APP antibodies also contain neurites reactive with antibodies to tau; 4) double labeling with anti-tau and an APP antibody shows that the neuritelike profiles stained by the APP antibody are always closely associated with tau-positive neurites within the same plaque and that a minority of profiles appear to be labeled by both antibodies; and 5) antibodies to different regions throughout APP label the same profiles within plaques, suggesting the presence of the full-length precursor. The authors conclude that only a subgroup of senile plaques contain APP epitopes and that the immunostained structures are neurites. Because many A beta P-containing plaques in neocortex, cerebellum, and striatum were found to be devoid of any APP labeling, as were vascular A beta P deposits, it is unlikely that the extracellular A beta P is principally derived from the APP found within dystrophic neurites. The immunodetection of apparently full-length APP, an axonally transported protein, in selected plaque neurites provides yet another protein marker of neuritic dystrophy, possibly indicative of an aberrant regenerative response.

Alzheimer Disease↗

PubMed1993

Subcellular and gel chromatographic distribution of manganese in the mouse brain: relation to the chemical form of chronically-ingested manganese.

The subcellular distribution of manganese and the binding characteristics of manganese to protein in the mouse brain were examined on G-75 Sephadex gel columns. Four manganese compounds were included at 2 g/kg in each food eaten by ddY mice for 12 months. The cerebral cortex manganese concentrations in the virtually insoluble manganese compounds were significantly higher than those in the control group. The brain striatal subcellular distribution and gel chromatographic profiles of manganese were similar among the divalent manganese compounds. On the contrary, the behaviour of MnO2 was little different from the divalent manganese compounds. There was more manganese associated with fast-migrating ligands in the striatal cytosol of the manganese-exposed group than in the control groups.

Acetates↗