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Biomedical subjects

A Hu

Publications and source records attributed to A Hu.

At least 37 records · Page 2Linked to original sources
PubMed1996

Selective expression of a subset of measles virus receptor-competent CD46 isoforms in human brain.

The human cell surface protein CD46 is the main measles virus (MV) receptor. We analyzed the CD46 isoforms expressed in the brain of three patients who died with persistent MV infections and in an unaffected brain. Complete CD46 cDNAs were produced and found to code exclusively for CD46 isoforms with cytoplasmic tail 2. Selective expression of tail 2 isoforms was shown in a second control brain by Western blots with antibodies specific for each of the cytoplasmic tails. Binding of purified MV particles and virus-dependent cell fusion were tested after transient expression of brain-derived CD46 proteins in mouse cells. All the brain-derived proteins mediated MV binding and virus-dependent fusion. Isoforms containing both serine/threonine/proline (STP)-rich domains were more active in virus binding, whereas isoforms with only one STP domain were more efficient in mediating fusion.

Amino Acid Sequence↗
PubMed1996

Construction of a 750-kb bacterial clone contig and restriction map in the region of human chromosome 21 containing the progressive myoclonus epilepsy gene.

The gene responsible for progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is located on human chromosome 21q22.3 in a region defined by recombination breakpoints and linkage disequilibrium. As part of an effort to clone the EPM1 gene on the basis of its chromosomal location, we have constructed a 753-kb bacterial clone contig that encompasses the region containing the gene. Because DNA markers from the region did not identify intact yeast artificial chromosome (YAC) clones after screening several libraries, we built the contig from cosmid clones and used bacterial artificial chromosome (BAC) and bacteriophage P1 clones to fill gaps. In addition to constructing the clone contig, we determined the locations of the EcoRI, SacII, EagI, and NotI restriction sites in the clones, resulting in a high-resolution restriction map of the region. Most of the contig is represented by a level of redundancy that allows the orders of most restriction sites to be determined, provides multiple data points supporting the clone orders and orientations, and allows a set of clones with a minimum degree of overlap to be chosen for efficient additional analysis. The clone and restriction maps are in excellent agreement with maps generated of the region by other methods. These ordered bacterial clones and the mapping information obtained from them provide valuable reagents for isolating candidate genes for EPM1, as well as for determining the nucleotide sequence of a 750 kb region of the human genome.

Base Sequence↗
PubMed1996

[Investigation of soluble tumor necrosis factor receptor in patients with active tuberculosis].

It is assumed that soluble tumor necrosis factor receptor I and II (sTNF-RI, II) play important roles in the regulation of tumor necrosis factor alpha (TNF-alpha) activity. We measured the levels of circulating sTNF-R in patients with active pulmonary tuberculosis (n = 31) and the correlation between TNF-alpha and sTNF-R in serum level was investigated. We also compared sTNF-R levels before and after the treatment in 7 cases. Significant increase of circulating sTNF-R were found in patients with tuberculosis compared with the normal controls (n = 28) (p < 0.01). Moreover, significant positive correlations were found between TNF-alpha and sTNF-R I and II (r = 0.520, r = 0.553) in serum comparing sTNF-R levels before and after the treatment for patients with tuberculosis, significant fall was found in sTNF-R I, but not in sTNF-R II. As a result, it is suggested that sTNF-R regulates TNF-alpha activity in patients with tuberculosis, and that sTNF-R I levels could be used as one of the indices to evaluate the clinical activity of tuberculosis.

Adult↗
PubMed1996

Field evaluation of the QBC technique for rapid diagnosis of vivax malaria.

The QBC (quantitative buffy coat) technique was compared with that of the Giemsa-stained thick blood film (GTF) under field conditions in Junlian and Mingshan counties, Sichuan, China, for rapid diagnosis of vivax malaria. Blood samples were collected from 364 volunteer villagers, and each sample was examined with both the QBC and GTF techniques. For each GTF sample (10 microliters of blood), as many as 300 oil-immersion fields were examined; each QBC tube was inspected for up to 5 minutes. The GTF technique resulted in 86 positive blood samples and 278 negative; the QBC technique indicated 89 positive and 275 negative samples. Relative to the results obtained with GTF, the QBC technique had a sensitivity and specificity of 87.2% and 95.0%, respectively; concordance between the tests was 93.1%. The median time-to-positive diagnosis with the QBC technique (1.12 min) was 11% of that with GTF. The distribution of different developmental stages of Plasmodium vivax parasites was also examined in the centrifuged QBC tubes: all stages except schizonts could be found in the lower part of the platelet zone (the interphase between the monocyte and platelet layers), especially ring forms.

Azure Stains↗
PubMed1995

Neutrophils from human immunodeficiency virus (HIV)-seronegative donors induce HIV replication from HIV-infected patients' mononuclear cells and cell lines: an in vitro model of HIV transmission facilitated by Chlamydia trachomatis.

Infection with a sexually transmitted disease (STD) increases the risk for human immunodeficiency virus (HIV) infection. Polymorphonuclear leukocytes (PMNs) are recruited into the genital tract by STD pathogens, such as Chlamydia trachomatis. Semen of HIV-infected men contains HIV associated with mononuclear cells. This study investigated the interaction among PMNs from HIV-uninfected persons, C. trachomatis, and HIV-infected cells and examined the mechanisms for enhanced HIV replication. We demonstrated that PMNs from HIV-seronegative donors induced HIV replication in mononuclear cells from 17 HIV-infected patients in medium without exogenous IL-2. HIV in the cell-free supernatants from cocultures of PMNs and patients' peripheral blood mononuclear cells (PBMCs) was replication competent, as indicated by their capacity to propagate HIV in a second round of culture using PBMCs from HIV-seronegative individuals and by the fact that proviral DNA was found in these cells. PMNs from HIV-seronegative donors increased HIV replication over 100-fold in chronically HIV-infected cell lines of the monocytic, T, and B cell lineages. Moreover, PMNs increased U1 cells' production of p24 antigen by as much as ninefold when compared with U1 cells cocultured with PBMCs. The addition of C. trachomatis to PMN and U1 coculture increased HIV replication by an additional ninefold at 24 h, whereas C. trachomatis alone had no effect on p24 antigen production by U1 cells. Thus, C. trachomatis serves not only to recruit PMNs, but also to interact with PMNs to increase HIV replication. HIV replication is triggered by contact of HIV-infected cells with PMNs, by the generation of reactive oxygen intermediates (ROIs), and by soluble factors such as TNF-alpha and IL-6. This is based on the findings that production of p24 antigen, IL-6, and TNF-alpha induced by PMNs is abrogated by disrupting or partitioning PMNs from HIV-infected cells; is inhibited by superoxide dismutase and catalase, enzymes that destroy ROIs; is enhanced by differentiated HL60 cells capable of producing ROIs; and is induced by PMNs tested negative for CMV. Furthermore, the production of ROIs is independent of HIV infection of mononuclear cells, since PMNs cocultured with HIV-uninfected parental monocytic and T cell lines generated ROIs. Therefore, the increased risk for acquiring HIV infection associated with chlamydia cervicitis may be related to the local recruitment of PMNs by C. trachomatis and the induction of infectious virus from mononuclear cells present in semen. These observations provide a rationale for strategies to reduce HIV transmission by control of STD.

B-Lymphocytes↗
PubMed1995

Synergistic effects on ganglionic herpes simplex virus infections by mutations or drugs that inhibit the viral polymerase and thymidine kinase.

Herpes simplex virus encodes proteins, such as DNA polymerase, that are essential for its replication and proteins, such as thymidine kinase, that are not essential for replication in cell culture, but are important for pathogenesis in animal models. However, certain mutations affecting these proteins exert little or no effect on replication or pathogenesis. We tested the effects of combining two such mutations--one that alters DNA polymerase and one that decreases but does not abolish thymidine kinase activity--on replication in cultured cells and on acute and latent infections in mice. The double mutant replicated similarly to the single mutants and wild-type virus both in cell culture and acutely in the mouse eye. However, it was severely impaired for acute replication in trigeminal ganglia and for reactivatable latent infections. This impairment depended upon the polymerase mutation. Similarly, although Ro 31-5140, a thymidine kinase inhibitor, did not potentiate the antiviral effects of phosphonoacetic acid, a polymerase inhibitor, in cell culture, the two drugs in combination substantially inhibited viral reactivation from latency at concentrations that had little or no effect when used singly. These synergistic effects may have implications for viral functions during pathogenesis and for antiviral chemotherapy.

Animals↗
PubMed1995

Influence of N-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein.

The fusion (F) glycoprotein of measles virus, a structural component of the virion envelope, contains four potential sites for attachment of N-linked oligosaccharides. Three are located in the F2 subunit of the protein and one in the signal peptide. Four mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Ser/Thr to Ser-X-Ser/Thr. The wild-type and altered forms of the F protein were expressed in BHK-21 and HeLa T4 cells by use of the recombinant vaccinia virus-encoding T7 polymerase system. Analysis of these proteins revealed that three (residues 29, 61 and 67) potential sites for addition of N-linked glycans in the F2 subunit are actually utilized. The functional glycosylation sites were systematically removed in all possible combinations from the F protein to form a panel of mutants from which the role of carbohydrates, singly or in various combinations, could be evaluated. One single-site mutant protein lacking the glycosylation site of Asn-67 was processed, transported to the cell surface and could induce cell fusion. However, the other two single-site mutant proteins with deletions of glycosylation sites Asn-29 or Asn-61 exhibited a defect in processing, were not transported to cell surface and thus induced no cell fusion. The absence of any two of the three or of all three glycosylation sites resulted in protein retention in the endoplasmic reticulum. Therefore, it appears that glycosylation of sites Asn-29 and Asn-61 has important roles in maintaining the native structure of the F protein.

Amino Acid Sequence↗
PubMed1994

Intracellular processing and antigenic maturation of measles virus hemagglutinin protein.

The intracellular processing and antigenic maturation of the measles virus (MV) hemagglutinin (H) protein in virus infected cells were probed with murine monoclonal antibodies (Mabs) that reacted with continuous and discontinuous epitopes. The antibodies distinguished between the immature, cotranslational monomeric form of the protein and the mature, dimeric hemagglutinin structure. This was evidenced by testing of immunoreactivity of the Mabs with synthetic peptides, by in vitro synthesized H protein analysis, and by pulse-chase analysis of gel separated monomeric and dimeric forms of the H protein. Time kinetics analysis showed that the protein was synthesized as monomers and most of them were converted into dimers with t1/2 about 30 min. The H protein remained endoglycosidase H (Endo H) sensitive up to 30 min and started to acquire partial resistance to Endo H between 30 and 60 min (t1/2 about 60 min) after synthesis. Oligomerization of the H protein was unaffected in virus infected cells treated with a compound (carbonylcyanide m-chlorophenylhydrazone, CCCP) that blocks transport from the endoplasmic reticulum (ER) to the Golgi complex. These results suggest that the H protein dimerization takes place in the ER before its transport to the medial Golgi complex. The Mabs specific for discontinuous epitopes reacted with the H protein in cells treated with CCCP. Thus conformational antigenic epitope formation appears to take place in the ER.

Amino Acid Sequence↗
PubMed1994

Role of N-linked oligosaccharide chains in the processing and antigenicity of measles virus haemagglutinin protein.

The effects of N-linked oligosaccharides on the haemagglutinin (H) protein of measles virus (MV) were assessed with respect to the processing and antigenicity of the molecule. The functional glycosylation sites on the H protein were determined by eliminating each of the five potential positions, Asn-168, Asn-187, Asn-200, Asn-215 and Asn-238, for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant H proteins in BHK-21 cells by a recombinant vaccinia virus encoding T7 polymerase indicated that the first four sites were used in the H glycoprotein for the addition of N-linked oligosaccharide chains. Heterogeneity of oligosaccharide processing was demonstrated. One of the four glycosylation sites had a different carbohydrate structure from those of the other three glycosylation sites and this varied glycosylation was responsible for the appearance of two forms of the H protein. The functional glycosylation sites were systematically removed in various combinations from the H protein to form a panel of mutants in which the role of carbohydrate chains, singly or in different combinations, could be evaluated. Investigations of these glycosylation mutants indicated that (i) two of the four individual carbohydrate side-chains have a large influence on the antigenicity of the molecule; (ii) individual carbohydrate side-chains have little effect on the folding and oligomerization of the molecule, and are not sufficient or necessary alone to facilitate the transport of the molecule to the plasma membrane; (iii) at least two carbohydrate side-chains are required for the H protein to move along the exocytic pathway to the plasma membrane and various combinations of oligosaccharide side-chains, irrespective of the carbohydrate localizations, influence equally the processing of the molecule.

Amino Acid Sequence↗
PubMed1994

Role of individual cysteine residues in the processing and antigenicity of the measles virus haemagglutinin protein.

The haemagglutinin (H) protein is the dominant envelope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide bonds impaired the capacity of the protein to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of individual cysteine residues to the folding of H protein into its native conformation. Site-directed oligonucleotide mutagenesis was used to create 13 mutants, each with a serine replacing a cysteine. The mutated genes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic structure and intracellular processing properties of the mutant proteins reveal the following outcome. (i) Replacements of cysteine residues 139, 154, 188, 386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a mutant with a replaced cysteine residue 154 displayed modified migration properties. (ii) Alterations of cysteine residues 381 or 494 displayed a moderate effect on H protein properties. The two mutants expressed discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex and failed to be transported to the cell surface. (iii) Substitutions of cysteine residues 287, 300, 394, 579 or 583 resulted in a complete loss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epitope. No dimeric form of the proteins was observed and only high mannose oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not oligomerize and were retained in the endoplasmic reticulum. In conclusion, cysteine residues 287, 300, 381, 394, 494, 579 and 583 appear to play a particularly critical role in the antigenic structure and processing of the H molecules and they probably participate in the inter- or intramolecular disulphide bonding.

Animals↗
PubMed1993

Antibody responses of children to the C-terminal peptide of the SH protein of respiratory syncytial virus and the immunological characterization of this protein.

The SH protein of RSV, a small integrated hydrophobic membrane protein, consists of 64 amino acid residues in the polypeptide of subgroup A and 65 amino acid residues in the polypeptide of subgroup B. We synthesized five peptides, representing the SH protein of each RSV subgroup comprised of the following amino acid residues: 2-16, 12-26, 35-49, 45-60, and for subgroup A, 51-64 and for subgroup B, 51-65. Peptides 2-16 and 51-64/65 represented the N-terminal and C-terminal ends of the protein, respectively. In RIPA, under reducing conditions with mercaptoethanol, hyperimmune guinea pig (GP) serum against C-terminal peptide of the two subgroups precipitated the homologous 7.5 kDa and 21-30 kDa SH proteins. Under nonreducing conditions, the GP antipeptide sera precipitated all three SH proteins, suggesting that the 13-15 kDa protein exists as a dimer. The subgroup A 7.5 and 13-15 kDa proteins had apparent molecular weights about 1-2 kDa higher than the corresponding subgroup B proteins. The C-terminal peptides of subgroups A and B were used to characterize the immune response of 11 children, age 1 month to 1 year, with presumed primary RSV infection. Three of 4 children with subgroup A infection and 4 of 7 children with subgroup B infection developed homologous 4-fold rises in antibody to C-terminal peptide (aa 51-64/65) during convalescence. Except for one child with subgroup A and one child with subgroup B infection, the other 5 children developed heterologous rises also.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Molecular characterization of epitopes on the measles virus hemagglutinin protein.

The measles virus (MV) hemagglutinin (H) gene nucleotide sequences of the LEC-WI strain and 11 branched sequential neutralization escape variants of the strain derived by selection with five monoclonal antibodies (Mabs) were determined by direct analysis of amplified polymerase chain reaction products. The parental LEC-WI strain isolated from a patient with subacute sclerosing panencephalitis exhibited H gene sequence characteristics similar to other persistent virus strains derived from brain materials. Mostly single-point H gene mutations, coding for single amino acid substitutions in the H protein, were found to provide explanations for the resistance to the individual Mabs. Resistance to Mabs 16-CD11 and I-41 resulted from changes of Gly-491 to Asp (or Val) and Phe-552 to Val, respectively. One variant (B89) selected by Mab 16-CD11 had a mutation introduced by a single nucleotide deletion and subsequent nucleotide insertion, which caused a shift in the open reading frame. The epitope of Mab I-29 was assigned to Ser-313 or Gly-314, which were changed to Leu and Arg, respectively. The variants subjected to the Mab I-44 selection exhibited change of Ser-189 to Pro. Radioimmunoprecipitation assay and endoglycosidase H (Endo H) treatment revealed that this change destroyed a potential N-linked glycosylation site, indicating that the carbohydrate chain participates in formation of the epitope or indirectly influences its properties. Resistance to Mab 16-DE6 involved three specific amino acid changes in three different places, Gly-211 to Ser, Gly-388 to Asp, and Ser-532 to Phe or Arg-533 to Gly, reflecting the occurrence of a conformational epitope. In conclusion, this study identifies the precise positions of several critical sites on the MV H protein which react with neutralizing antibodies.

Amino Acid Sequence↗