PubMed Health⌕ Search

Biomedical subjects

M Mach

Publications and source records attributed to M Mach.

At least 55 records · Page 3Linked to original sources
PubMed1990

The gp116 of the gp58/116 complex of human cytomegalovirus represents the amino-terminal part of the precursor molecule and contains a neutralizing epitope.

The glycoprotein complex gp58/116 of human cytomegalovirus (HCMV) represents a dominant antigen for the humoral immune response. We have used the human monoclonal antibody C23, which is capable of neutralizing HCMV in tissue culture without the addition of complement, to study the origin of gp116 as well as the amino acid sequence recognized by the antibody. Our results show that gp116 is derived from the same open reading frame as gp58 and that it represents the amino-terminal portion of the precursor protein. Using prokaryote-expressed beta-galactosidase-gp116 fusion proteins, the binding site of C23 was located to between amino acids 27 to 84 of the amino-terminal portion of gp116. Analyses of HCMV-positive human sera revealed that this portion of the molecule is immunogenic during natural infection.

Amino Acid Sequence↗
PubMed1990

Large-scale screening of human sera with cytomegalovirus recombinant antigens.

One of the major problems regarding cytomegalovirus (CMV) serodiagnosis is the use of poorly defined viral antigens. Individual CMV proteins expressed via recombinant DNA procedures are a promising approach to solving this problem. In this work, 10 different fusion proteins containing antigenic epitopes of the major CMV structural proteins of 150, 71, 65, 38, and 28 kilodaltons and of the nonstructural protein of 52 kilodaltons were subjected to Western (immuno-) blotting to assay their reactivities with immunoglobulins G and M in 395 CMV-seropositive and 100 CMV-seronegative unselected human serum samples. As a whole, the results obtained indicate that CMV can be replaced by recombinant viral proteins in the serological evaluation of anti-CMV antibodies.

Antigens, Viral↗
PubMed1989

Identification of a neutralizing epitope on glycoprotein gp58 of human cytomegalovirus.

Human cytomegalovirus contains an envelope glycoprotein of 58 kilodaltons (gp58). The protein, which is derived from a glycosylated precursor molecule of 160 kilodaltons via proteolytic cleavage, is capable of inducing neutralizing antibodies. We have mapped the epitopes recognized by the neutralizing monoclonal antibody 7-17 and a second antibody (27-287) which is not neutralizing. Overlapping fragments of the carboxy-terminal part of the open reading frame coding for gp58 were expressed in Escherichia coli as beta-galactosidase fusion proteins. The reactivities of antibodies 7-17 and 27-287 were determined by Western blot (immunoblot) analysis. Both antibodies recognized sequences between amino acids 608 and 625 of the primary gp58 translation product. The antibodies almost completely inhibited one another in a competitive binding assay with intact virus as antigen. Moreover, antibody 27-287 was able to inhibit the complement-independent neutralizing activity of antibody 7-17.

Antibodies, Monoclonal↗
PubMed1989

Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses.

A rabbit antiserum was raised against envelope material from purified human cytomegalovirus strain AD169. The serum recognized polypeptides 200, 170, 160, 75, 58, and 45 kilodaltons in size. It was used to screen a cDNA library constructed from poly(A)+ RNA from human cytomegalovirus-infected cells in the expression vector lambda gt11. A recombinant bacteriophage expressing cytomegalovirus-specific sequences was identified, and the corresponding gene was mapped to the HindIII R fragment. The gene is transcribed into a late 1.5-kilobase RNA. The nucleotide sequence of the coding region was determined. Computer analysis of the gene product revealed a polypeptide containing multiple potential membrane-spanning domains, representing a type of protein not identified in the envelope of herpesviruses before. The protein shows homology on the amino acid level to hypothetical proteins from reading frames BBRF3 of Epstein-Barr virus, UL10 of herpes simplex virus type 1, and ORF50 of varicella-zoster virus. By using an antiserum raised against procaryote-expressed parts of the cytomegalovirus membrane protein, a 45-kilodalton structural component of the virus was identified as the gene product.

Amino Acid Sequence↗
PubMed1988

Identification and procaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp28) of human cytomegalovirus.

Human cytomegalovirus contains a structural polypeptide that is 28 kilodaltons in apparent molecular size and is reactive in Western blot (immunoblot) analysis with the majority of human sera. The gene coding for this polypeptide was mapped on the genome of human cytomegalovirus strain AD169. A monoclonal antibody specific for the 28-kilodalton polypeptide was used to screen a cDNA library constructed from poly(A)+ RNA of human cytomegalovirus-infected cells in the procaryotic expression vector lambda gt11. Hybridization of cDNA with cosmid and plasmid clones mapped the gene to the HindIII R fragment. The gene was transcribed into a late 1.3-kilobase RNA. The nucleotide sequence of the coding region was determined. Parts of the 28-kilodalton polypeptide were expressed in Escherichia coli as hybrid proteins fused to beta-galactosidase. In Western blots these proteins were recognized by human sera. Antibodies raised against the hybrid proteins reacted specifically with the viral antigen in immunoprecipitations and Western blots. In vitro phosphorylation of HCMV virions and immunoprecipitation showed that the 28-kilodalton polypeptide was phosphorylated.

Amino Acid Sequence↗
PubMed1987

Map position and nucleotide sequence of the gene for the large structural phosphoprotein of human cytomegalovirus.

Human cytomegalovirus particles contain a phosphoprotein of 150,000 (pp150) apparent molecular weight in their matrix; the protein appears particularly reactive in Western blot analyses with human antisera. The gene for pp150 was mapped by screening a bacteriophage lambda gt11 cDNA expression library with monospecific rabbit antisera. Subsequent hybridization of cDNA with cosmid and plasmid clones containing the human cytomegalovirus strain AD169 genome mapped the gene to HindIII fragments J and N. The gene is transcribed into a late 6.2-kilobase RNA. The nucleotide sequence of this region was determined, and a transcription initiation site and two polyadenylation sites of an abundant transcript were located by primer extension and nuclease protection experiments. The reading frame for pp150, deduced from computer analyses, gives rise to a polypeptide of 1,048 amino acids in length; protein secondary structure analysis revealed multiple beta-pleated sheets in hydrophilic clusters, providing a possible explanation for the immunogenic properties of the polypeptide.

Amino Acid Sequence↗
PubMed1987

X-linked hypogammaglobulinaemia with a late secretion defect. A family study.

Three boy cousins suffering from x-linked hypogammaglobulinaemia have been described. Their disease is quite different from the classical x-linked hypogammaglobulinaemia. We present data from 15 family members of three families. The three mothers are sisters and 3 boys from 5 are suffering from immunodeficiency. HLA A, B and DR typing and in vitro diagnostics of immune functions of all family members were carried out. In the patients we could demonstrate a well functioning T cell system which seems to be regular for T cell help on PMW driven B cell maturation into cytoplasmic immunoglobulin positive (cIg+) cells of all immunoglobulin classes but we had no evidence for IgG secreting cells in a plaque forming cell (PFC) assay. The registration of spontaneous suppressor phenomena should be taken into account. If patients undergo an gamma-globulin therapy the changed suppressor effects come back to normal values but the secretion defect is unchanged. The three healthy mothers express one identical haplotype (A2 B8 DR3) which we could demonstrate in the three patients but also in one healthy boy.

Agammaglobulinemia↗
PubMed1986

Isolation of a cDNA clone encoding S-adenosylmethionine decarboxylase. Expression of the gene in mitogen-activated lymphocytes.

S-Adenosylmethionine decarboxylase was purified from bovine liver and digested with endopeptidase Lys-C; the resulting peptides were chromatographically separated. Peptides containing either methionine or tryptophan were subjected to sequence analysis. An oligonucleotide mixture of 48 sequences, which was 17 nucleotides in length, was synthesized based on one of these peptide sequences. This synthetic oligonucleotide mixture was labeled and used to screen a bovine cDNA library in phage lambda gt11. A clone was identified which contained a 1350-nucleotide insert. This insert contained nucleotide sequences coding for amino acid sequences of two of the peptides that were analyzed, thus proving that this cDNA clone codes for S-adenosylmethionine decarboxylase. A subcloned fragment from the coding region of the cDNA was used as a probe to analyze the expression of this gene in mitogen-activated lymphocytes. Northern blots revealed two message species of 2.4 and 3.6 kilobases in length. Both mRNAs were coordinately expressed and were present in polysomes. The levels of these mRNAs increased approximately 4-fold by 9 h after activation of the cells. The magnitude of the increase in these messages is to be compared with an 8- to 10-fold increase in the rate of synthesis of the protein. The apparent increase in translational efficiency of this message upon lymphocyte activation was confirmed by analyzing polysomes from these cells. In resting lymphocytes, the average size of polysomes containing mRNA coding for S-adenosylmethionine decarboxylase was 1.4 ribosomes per mRNA, and this value increased to 2.7 in stimulated cells. Thus, it appears that the increase in translational efficiency of this mRNA arises from an elevated rate of translational initiation, leading to more ribosomes per polysome encoding this particular message. This is not a general effect on the expression of all proteins, since there is no change in the translational efficiency of cytoplasmic actin upon activation of lymphocytes.

Adenosylmethionine Decarboxylase↗
PubMed1986

Mapping of the major glycoprotein gene of human cytomegalovirus.

The gene coding for the most abundant glycoprotein (gp58) of human cytomegalovirus (HCMV), strain AD169, was physically mapped on the viral genome. A monospecific rabbit antiserum against gp58 was used to screen a cDNA library that was constructed from poly(A)+ RNA of HCMV-infected cells in the prokaryotic expression vector lambda gt11. A cDNA clone was identified which synthesized part of the glycoprotein. It allowed localization of the coding region within the right terminal sequence of the HindIII-F fragment between map coordinates 0.344 and 0.380 of HCMV virion DNA.

Antibodies, Monoclonal↗
PubMed1984

Polyamine requirement for microbial protein synthesis: structural specificity in cell-free systems of Escherichia coli.

Cell-free protein synthesis was performed with synthetic or natural mRNA in an E. coli system containing physiological concentrations of Ca2, Mg2 and either one or both of the two natural polyamines of E. coli, spermidine and putrescine, or corresponding homologues. Putrescine does not permit poly(U)-dependent poly(Phe) synthesis unless spermidine or nor-spermidine is added. Spermidine supports homopeptide synthesis sufficiently well, its effect being stimulated by putrescine or homologous diamines with increasing chain length from 4 to 7 carbon atoms. Diaminopropane completely inhibits the spermidine-activated system in a competitive manner. Translation of MS2 phage RNA is supported by putrescine, the rate and quality (read through to the termination signal) of translation is optimized by spermidine or triamine homologues. MS2 phage RNA translation is supported by spermidine, putrescine has no further stimulatory effect but diaminoheptane enhances the rate of translation. In this case, however, premature chain termination does occur. The results indicate that spermidine is necessary for optimal poly(U) and MS2 phage RNA translation, that the aminopropyl moiety is important for its function and that the remaining side chain can be extended from C4 to C8. Putrescine may cooperate with spermidine but its chain length is rather critical, it cannot substitute for spermidine. The results indicate that the polyamines facilitate mRNA/tRNA/ribosome interactions in a specific manner.

Amino Acids↗
PubMed1983

Excretion of polyamines by children with leukemia during chemotherapy.

Evidence is presented showing a relation between polyamine concentration and the methylation of tRNA in vitro and in vivo. Polyamine excretion in urine of children with ALL and AML is slightly elevated before the commencement of chemotherapy. Immediately thereafter, excretion of acetylputrescine increases drastically over a period of at least 30-60 days. The elevation seems to be caused by different factors, e.g., destruction of tumor cells, induction of ornithindecarboxylase, and cell recovery after termination of chemotherapy. Acetylspermidine excretion also increases 3-4 days after the beginning of chemotherapy. A positive correlation exists between leukocyte counts and excretion of acetylspermidine. The ratio of acetylspermidine excretion at days 3-4 of the therapy to that before commencement of chemotherapy could be an indicator of response to the therapy.

Child↗