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At least 1,711 records · Page 95Linked to original sources
PubMed1992

Serine protein kinase associated with varicella-zoster virus ORF 47.

Varicella-zoster virus (VZV) ORF 47 lies in the unique long region of the VZV genome. Sequence homology studies have demonstrated that gene 47 possessed conserved protein kinase motifs. In this study, we investigated the properties of the ORF 47 product. First, a rabbit antiserum was raised against a protein generated from the fusion of the most antigenic ORF 47 domain with Escherichia coli beta-galactosidase. The high-titer antiserum reacted specifically with ORF 47 polypeptides translated in vitro. When incubated with VZV-infected cell lysate, the antiserum immunoprecipitated a phosphoprotein of M(r) 54,000, a size comparable with the predicted molecular mass. The precipitated viral protein was phosphorylated in a protein kinase assay; subsequent phosphoamino acid analysis indicated that the phosphotransferase associated with the ORF 47 protein was a serine protein kinase. Synthesis of the ORF 47 product in VZV-infected cell culture increased in the first and second days and plateaued after the third day of infection. The protein kinase activity associated with VZV ORF 47 had several distinctive biochemical properties: (i) its phosphotransferase activity was enhanced more by manganese than by magnesium, (ii) it utilized both ATP and GTP as donors of phosphate, and (iii) it phosphorylated both acidic and basic substrates. In summary, this report lends support to the computer homology data which predicted that VZV ORF 47 would encode a serine protein kinase.

Antibodies, Viral↗
PubMed1996

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.

Animals↗
PubMed2003

Generating data for databases--the peptide repertoire of HLA molecules.

During the past few years, a huge amount of information about HLA-presented peptides has been compiled: several thousand naturally processed ligands of such cell surface receptors are already known. Nevertheless, our knowledge covers only a minute proportion of the total peptide repertoire. The overall amount of different peptides presented by one given HLA class I molecule lies between 1000 and 10000 individual sequences per cell. There is, however, no HLA molecule of which more than 100 ligands have been published so far. The situation is further complicated by the fact that different cells present different sets of peptides by the same HLA molecules, a feature that provides great hope for immunotherapy. We have been analysing HLA-presented peptides for many years for three reasons. First, the basic rules of peptide presentation (the 'peptide motifs') had to be established. Second, the listing of individual peptides presented by HLA molecules is steadily continuing, although a comprehensive catalogue of all possible HLA-presented peptides is utopical in our days. Third, quantitative differences in the presentation of individual HLA ligands provide information about the dynamic state of the host cells. Comprehensive information about HLA-presented peptides enables accurate epitope prediction and provides a basis for diagnostic assessment and therapeutic intervention.

Allergy and Immunology↗
PubMed1996

A third interferon-gamma-induced subunit exchange in the 20S proteasome.

The 20S proteasome is a protease complex of functional importance for antigen processing. Two of the 14 proteasome subunits, delta and MB1, can be replaced by the major histocompatibility complex (MHC)-encoded and interferon-gamma (IFN-gamma)-inducible subunits LMP2 and LMP7, respectively. LMP2 and LMP7 alter the cleavage site specificity of the 20S proteasome and are required for the efficient generation of T cell epitopes from a number of viral proteins and for optimal MHC class I cell surface expression. We compared the 20S proteasome subunit pattern from IFN-gamma-induced and non-induced mouse fibroblasts on two-dimensional gels and identified a third subunit exchange by microsequencing: the non-MHC-encoded subunit MECL-1 is induced by IFN-gamma and replaces a sofar barely characterized beta subunit designated 'MC14'. In analogy to LMP2 and LMP7, MECL-1 may be functional in MHC class I-restricted antigen presentation.

Amino Acid Sequence↗
PubMed2004

Guanylate cyclase-activating proteins: structure, function, and diversity.

The guanylate cyclase-activating proteins (GCAPs), Ca2+-binding proteins of the calmodulin gene superfamily, function as regulators of photoreceptor guanylate cyclases. In contrast to calmodulin, which is active in the Ca2+-bound form, GCAPs stimulate GCs in the [Ca2+]-free form and inhibit GCs upon Ca2+ binding. In vertebrate retinas, at least two GCAP1 and two GCs are present, a third GCAP3 is expressed in humans and fish, and at least five additional GCAP4-8 genes have been identified or are predicted in zebrafish and pufferfish. Missense mutations in GCAP1 (Y99C, I143NT, E155G, and P50L) have been associated with autosomal dominant cone dystrophy. Absence of GCAP1/2 in mice delays recovery of the photoresponse, a phenotype consistent with delay in cGMP synthesis. In the absence of GCAP2, GCAP1 supports the generation of wild-type flash responses in both rod and cone cells. Recent progress revealed an unexpected complexity of the GC-GCAP system, pointing, out a number of unsolved questions.

Amino Acid Sequence↗
PubMed2004

Electrochemical determination of triple helices: electrocatalytic oxidation of guanine in an intramolecular triplex.

Electrocatalytic oxidation of the oligonucleotide 5'- GAA GAG GTT TTT CCT CTT CTT TTT CTT CTC C (TS) by Ru(bpy)(3)(2+) was studied by cyclic voltammetry. This oligonucleotide forms either an intramolecular triplex, hairpin, or single strand, depending on the pH (Plum, G. E.; Breslauer, K. J. J. Mol. Biol. 1995, 248, 679-695). In the triplex form, the guanine doublet in TS is buried inside the folded structure, and as such is less susceptible to oxidation by electrogenerated Ru(bpy)(3)(3+). Digital simulations of the catalytic voltammograms gave a rate constant of 3.5 +/- 0.2 x 10(2) M(-1) s(-1) for oxidation of the triplex form, while oxidation of the duplex and single-stranded forms occurred with much faster rate constants of (3.5-9.1) x 10(4) M(-1) s(-1). Experiments using a truncated form of TS that lacked the third strand of the triplex were consistent with these measurements. The Ru(bpy)(3)(3+) complex was also generated by photolyzing Ru(bpy)(3)(2+) in the presence of Fe(CN)(6)(3-). This reaction produced strand scission following piperidine treatment, which was visualized using high-resolution gel electrophoresis. These experiments showed decreased reactivity for the triplex form, and also gave an unusual reversal of a common selectivity for the 5'-G of GG doublets generally seen in B-form DNA. This reversal was ascribed to strain caused by the location of the GG doublet adjacent to the hairpin loop.

Base Sequence↗
PubMed1998

TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae.

The production of several virulence factors in Vibrio cholerae O1, including cholera toxin and the pilus colonization factor TCP (toxin-coregulated pilus), is strongly influenced by environmental conditions. To specifically identify membrane proteins involved in these signal transduction events, we examined a transposon library of V. cholerae generated by Tnbla mutagenesis for cells that produce TCP when grown under various nonpermissive conditions. To select for TCP-producing cells we used the recently described bacteriophage CTX phi-Kan, which uses TCP as its receptor and carries a gene encoding resistance to kanamycin. Among the isolated mutants was a transposon insertion in a gene homologous to nqrB from Vibrio alginolyticus, which encodes a subunit of a Na(+)-translocating NADH:ubiquinone oxidoreductase, and tcpI, encoding a chemo-receptor previously implicated in the negative regulation of TCP production. A third transposon mutant had an insertion in tcpP, which is in an operon with tcpH, a known positive regulator of TCP production. However, TcpP was shown to be essential for TCP production in V. cholerae, as a tcpP-deletion strain was deficient in pili production. The amino-terminal region of TcpP shows sequence homology to the DNA-binding domains of several regulatory proteins, including ToxR from V. cholerae and PsaE from Yersinia pestis. Like ToxR, TcpP activates transcription of the toxT gene, an essential activator of tcp operon transcription. Furthermore, TcpH, with its large periplasmic domain and inner membrane anchor, has a structure similar to that of ToxS and was shown to enhance the activity of TcpP. We propose that TcpP/TcpH constitute a pair of regulatory proteins functionally similar to ToxR/ToxS and PsaE/PsaF that are required for toxT transcription in V. cholerae.

Amino Acid Sequence↗
PubMed1985

Structure and surface exposure of protein IIs of Neisseria gonorrhoeae JS3.

Colonies of Neisseria gonorrhoeae JS3, each bearing a predominate protein II (PII) type, were derived from a progenitor transparent colony. Five distinct PIIs were identified and isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The PII bands were excised from gels of unlabeled whole cells and from gels containing lysates of surface-radioiodinated bacteria. These were subjected to alpha-chymotrypsin digestion and two-dimensional peptide mapping, which allowed for a comparison of both the primary structures of the PIIs and the identification of surface-exposed regions of the molecules. The results demonstrated that PIIs are unrelated to either Protein I or Protein III in structure but are closely related to one another, sharing about two-thirds of the peptides generated by alpha-chymotrypsin. The remaining third of the peptides varied with each PII, resulting in unique portions of the molecule being exposed on the bacterial surface. However, the variable peptides were not always among the exposed peptides, suggesting that the structural differences in the PIIs occur at a discrete site (or sites) of the PII molecule and not randomly throughout the protein. Such alterations can result in the exposure of distant, nonvariant portions of the molecule to the surface, perhaps by conformational changes. These bacteria can thus present a variety of new immunodeterminant sites to the host during the course of disease.

Amino Acid Sequence↗
PubMed1983

Deletion mutants of Moloney murine leukemia virus which lack glycosylated gag protein are replication competent.

A series of deletion mutations localized near the 5' end of the Moloney murine leukemia virus genome was generated by site-specific mutagenesis of cloned viral DNA. The mutants recovered from such deleted DNAs failed to synthesize the normal glycosylated gag protein gPr80gag. Two of the mutants made no detectable protein, and a third mutant, containing a 66-base pair deletion, synthesized an altered gag protein which was not glycosylated. All the mutants made normal amounts of the internal Pr65gag protein. The viruses were XC positive and replicated normally in NIH/3T3 cells as well as in lymphoid cell lines. These results indicate that the additional peptides of the glycosylated gag protein are encoded near the 5' end, that the glycosylated and internal gag proteins are synthesized independently, and that the glycosylated gag protein is not required during the normal replication cycle. In addition, the region deleted in these mutants apparently encodes no cis-acting function needed for replication. Thus, all essential sequences, including those for packaging viral RNA, must lie outside this area.

Animals↗
PubMed2003

Episomal persistence of recombinant adenoviral vector genomes during the cell cycle in vivo.

Previously we showed that recombinant adenoviral helper-dependent (HD) vectors result in long-term transgene expression levels in vivo which slowly declined by 95% over a period of 1 year. In this study, we further establish that this was not predominantly immune mediated. To determine if cell turnover was responsible for the loss of transgene expression, we induced rapid hepatocyte cell cycling in mouse liver, by performing a surgical two-thirds partial hepatectomy. We observed a 55 and 65% reduction in transgene expression levels and a 50 and 71% loss of vector genomes for the HD vector and the first-generation adenoviral vector. In sharp contrast, in nonviral, episomal plasmid DNA-injected mice, transgene expression levels and DNA copy numbers decreased by 95 and 99%, respectively. These findings suggest that cell division alone was not the primary reason for the slow decrease in transgene expression levels and that recombinant adenoviral vectors have a more robust mechanism for maintaining persistence during cell cycling. Several potential mechanisms are proposed.

Adenoviridae↗
PubMed2007

Physiologically based controller for generating overground locomotion using functional electrical stimulation.

The physiological control of stepping is governed both by signals descending from supraspinal systems and by circuitry residing within the lumbosacral spinal cord. The goal of this study was to evaluate the capacity of physiologically based controllers to restore functional overground locomotion after neurological damage, such as spinal cord injury when used in conjunction with functional electrical stimulation. For this purpose we implemented and tested two controllers: 1) an intrinsically timed system that generated a predetermined rhythmic output and 2) a sensory-based system that used feedback signals to make appropriate transitions between the unloaded (flexion) and loaded (extension) phases of the gait cycle. A third controller, a combination of the intrinsically timed and sensory-driven controllers, was implemented and two sessions were conducted to demonstrate the functional advantages of this approach. The controllers were tested in anesthetized cats, implanted with intramuscular electrodes in six major extensor and flexor muscles of the hindlimbs. The cats were partially supported on a sliding trolley that was propelled by the hindlimbs along a 2.5-m instrumented walkway. Ground reaction forces and limb positions were measured by force plates in the walkway and by accelerometers secured to the legs of the cat, respectively. The controllers were used to generate patterns of stimulation that would elicit alternating flexor (swing) and extensor (stance) movements in the hindlimbs. Using either the intrinsically timed or sensory-driven controllers, the cats were able to travel a distance of 2.5 m, taking five to 12 steps. Functional stepping sequences were more easily achieved using the intrinsically timed controller as the result of a lower sensitivity to the selection of initial stimulation parameters. However, unlike the sensory-driven controller, the intrinsically timed controller was unable to adjust to overcome walkway resistance and muscle fatigue. Neither system was consistently able to ensure load-bearing stepping. Therefore we propose the use of a "combined controller" that relies heavily on intrinsic timing but that can be reset based on sensory signals. A combined controller such as this one may provide the best solution for restoring robust overground locomotion after spinal cord injury.

Animals↗
PubMed1995

Beta-Amyloid peptide and amyloid precursor proteins in olfactory mucosa of patients with Alzheimer's disease, Parkinson's disease, and Down syndrome.

Dystrophic neurites are present in olfactory epithelium (OE) of patients with Alzheimer's disease (AD), Parkinson's disease (PD), and Down syndrome (DS) and occasionally in normal individuals. Cultured olfactory neuroblasts from AD patients generate carboxy terminal amyloid precursor protein (APP) fragments that contain beta-amyloid (A beta), but it is not known if deposits of A beta and/or APP fragments occur in the OE of individuals with or without AD, PD, or DS. To determine if A beta accumulates in the OE in situ, we probed postmortem samples of olfactory mucosa from patients with AD, PD, and AD (PD/AD), and DS and AD (DS/AD), as well as from controls, using polyclonal and monoclonal antibodies to A beta and flanking sequences in APPs. Samples of OE also were examined by thioflavin-S and electron microscopy. Labeling of A beta was observed in 10 of 12 AD cases, 2 of 3 PD/AD cases, 3 of 4 DS/AD cases, 3 of 10 adult controls, and 4 of 6 fetal cases. The A beta staining was seen in the basal third of the OE, in axons projecting through the lamina propria, and in metaplastic respiratory epithelium within the OE. Antibodies to other APP domains stained the OE of patients and controls. Thioflavin-S staining was present in the basal third of the OE of 8 of 9 AD patients and several PD/AD and DS/AD patients, but only in rare cells of 3 controls. Electron microscopy did not reveal amyloid fibrils in the OE.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed2005

[Oxaliplatin].

Oxaliplatin (L-OHP) is a new third generation 1,2-DACH-platinum derivative. Pre-clinical studies from human cell lines suggested that L-OHP was efficacious in treating advanced or recurrent colorectal cancer. Furthermore, L-OHP and fluorouracil combination was shown to be synergistic both in experimental studies and in clinical trials. Toxicity profiles also differ from other platinum derivatives. Neurotoxicity is much more frequent, but renal toxicity, alopecia, and ototoxicity are less. Combination chemotherapy with L-OHP, infusional 5-fluorouracil, and leucovorin-a treatment regimen known as FOLFOX, has been shown clinical benefit in response rate, time to progression, and overall survival as compared to those achieved with 5-fluorouracil+leucovorin. Thus, introduction of irinotecan and L-OHP into clinical use has been a major advances for patients with metastatic colorectal cancer. Additional research must be required to define optimal dose schedule and sequence of these agents. Inducing remission with first-line treatment has been shown a significant correlation between tumor response and survival (progression free and overall).

Animals↗
PubMed1994

Measuring CDR3 length variability in individuals during ontogeny.

The portion of the antibody sequence that contributes most to the conformation of the antigen-combining site is the third complementarity-determining region (CDR3). The CDR3 in antibody heavy chains are somatically produced by the rearrangement of three different immunoglobulin (Ig) gene segments, VH, D and JH, and are therefore highly variable in sequence and length. We have devised a technique for rapid analysis of CDR3 length variability from a large number of cDNA samples. This technique allows comparative evaluation of IgH heterogeneity in individuals without recourse to DNA cloning and sequencing. We also present data on Ig sequences from different stages of development in the amphibian, Xenopus laevis. Most of the larval and post-metamorphic Ig samples contained CDR3 of 3-10 codons, two codons shorter than those of the adult CDR3, which were 5-12 codons. Antibodies may by immunized tadpoles have been shown to be much less diverse than those produced by adults to the same antigen. The shorter tadpole CDR3 perhaps generates a more limited spectrum of antigen-combining site structures and, in this way, makes for a more restricted antibody repertoire.

Animals↗
PubMed2005

Identification of a functional envelope protein from the HERV-K family of human endogenous retroviruses.

Genome-wide screening of sequence databases for human endogenous retroviruses (HERVs) has led to the identification of 18 coding env genes, among which two-the syncytin genes-encode fusogenic ENV proteins possibly involved in placenta physiology. Here we show that a third ENV, originating from the most "recent" HERV-K(HML2) family, is functional. Immunofluorescence analysis of env-transduced cells demonstrates expression of the protein at the cell surface, and we show that the protein confers infectivity to simian immunodeficiency virus pseudotypes. Western blot analysis of the pseudotyped virions further discloses the expected specific cleavage of the ENV precursor protein. This functional ENV could play a role in the amplification--via infection of the germ line--of the HERV-K genomic copies, all the more as coding HERV-K gag and pol genes can similarly be found in the human genome, which could therefore generate infectious virions of a fully endogenous origin.

Endogenous Retroviruses↗
PubMed2005

Amino acid residues that are important for Hyal2 function as a receptor for jaagsiekte sheep retrovirus.

BACKGROUND: Infection by jaagsiekte sheep retrovirus (JSRV) and by enzootic nasal tumor virus (ENTV) depends on cell-surface expression of the virus entry receptor, hyaluronidase 2 (Hyal2). Human Hyal2 binds the envelope (Env) proteins of these viruses and is functional as a receptor, but Hyal2 from mice does not bind Env nor does it mediate entry of either virus. Here we have explored the amino acid determinants that account for the difference in receptor function. RESULTS: Analysis of human-mouse Hyal2 chimeric proteins showed that amino acid differences responsible for the difference in Hyal2 receptor activity were localized to the central third of Hyal2. Human Hyal2 mutants containing single or double amino acid replacements with the respective mouse amino acids were generated across this region and were assayed for activity. None of the single or double mutation reduced the receptor activity of human Hyal2 by more than 10-fold, whereas mouse Hyal2 activity is reduced 1,000-fold from that of human Hyal2. While the 3-dimensional structures of mammalian Hyal2 proteins are unknown, bee venom hyaluronidase shows significant amino acid similarity to human and mouse Hyal2 and its structure has been determined. Many mutations having the largest negative effects on human Hyal2 function mapped to a small region of the bee venom hyaluronidase close to but not overlapping the active site of the enzyme, suggesting that this site represents the binding site for Env. Analysis of synonymous and non-synonymous nucleotide substitutions in the coding sequences of multiple mammalian Hyal2 proteins shows that the proteins are undergoing strong selection for amino acid conservation. We found no evidence for positive selection of amino acid changes that might reflect evolution of mammalian hosts to resist JSRV or ENTV infection. CONCLUSION: These results show that the greatly reduced receptor activity of mouse Hyal2 in comparison to that of human Hyal2 is determined by multiple amino acid changes acting in concert. In particular, no one amino acid change blocks infection. However, the most important amino acids map to a small patch on a predicted 3-dimensional Hyal2 structure, which may represent the binding site for Env.

Animals↗
PubMed1990

Alternative splicing generates two forms of mRNA coding for human heparin-binding growth factor 1.

Human class 1 heparin-binding growth factor (HBGF-1), also known as acidic fibroblast growth factor, is a mitogen for a variety of mesoderm- and neutroectoderm-derived cells in vitro as well as an angiogenic factor in vivo. Several oncogenes and growth factors have been shown to be homologous to HBGF-1. Four cDNA clones coding for HBGF-1 have been isolated from a human brain stem cDNA library. Nucleotide sequence analysis revealed that alternative splicing generated at least two different forms of HBGF-1 mRNA. Because the difference occurs in the 5'-untranslated regions, these transcripts may result from the usage of alternative promoters. One of the cDNA clones contains the polyadenylation signal, AATAAA, and a poly(A) tail, representing the 3'-end of an HBGF-1 mRNA. RNAase protection assays suggested this cDNA clone corresponds to a minor transcript, and the majority of the HBGF-1 mRNA terminates at 3.1 kbp downstream from the translation termination codon. The biological significance of this unusually long 3'-untranslated sequence is not known. To study the HBGF-1 gene structure, we have isolated 50 kbp of contiguous genomic DNA coding for the HBGF-1 protein. Both restriction enzyme mapping and nucleotide sequencing established that the distance between the first and second protein-coding exons is 13.6 kbp while that between the second and third is 5.3 kbp. By using the HBGF-1 cDNA as a probe, we showed that human fetal heart expresses high levels of HBGF-1 mRNA. Thus, HBGF-1 may be involved in mediating processes such as embryonic development and vascular growth in the heart.

Amino Acid Sequence↗
PubMed2002

Extracellular domains of the neurokinin-1 receptor: structural characterization and interactions with substance P.

The technical difficulties associated with the structure determination of membrane proteins have limited the structural information available for the ligand binding to G-protein coupled receptors (GPCRs). Here, we describe a reductionist approach to GPCR structure determination in which the extracellular domains of the receptor are examined by high-resolution NMR in the presence of a membrane mimetic. The resulting structural features are then incorporated into a molecular model of the receptor, utilizing the x-ray structure of rhodopsin to generate the topological orientation of the transmembrane helices. The results of our study of the neurokinin-1 receptor (NK-1R) and its interactions with substance P (SP) are detailed here. The structure of the N-terminus, NK-1R(1-39), and of the third extracellular loop, NK-1R(264-290), in the presence of dodecylphosphocholine micelles is described. Our findings provide a structural basis for the interpretation of the results from other methods including mutagenesis, fluorescence, and photoaffinity labeling experiments, resulting in an experimentally based, high-resolution model of SP binding to NK-1R.

Amino Acid Sequence↗