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R D Horstmann

Publications and source records attributed to R D Horstmann.

At least 55 records · Page 3Linked to original sources
PubMed1992

Lysis of pathogenic and nonpathogenic Entamoeba histolytica by human complement: methodological analysis.

The effect of nonimmune human serum on Entamoeba histolytica trophozoites was studied: (a) using whole serum in the presence of Ca and Mg ions allowing complement activation via both the alternative and classical pathways or in the presence of MgEGTA permitting alternative pathway activation only; (b) using different E. histolytica isolates; (c) varying serum and trophozoite concentrations and the time of incubation; and (d) using three different methods to quantify lysis, i.e., microscopic inspection, flow cytometry and 111In release. All three methods yielded similar results, with flow cytometry being most sensitive in identifying membrane damage and 111In release being most valid in determining cell death. Microscopic analysis was reliable only when a chamber was used to calculate the number of complement treated cells in relation to the initial cell count. E. histolytica isolates were classified into three groups according to their susceptibility to lysis by complement: (i) pathogenic isolates after long term cultivation in vitro were susceptible; (ii) pathogenic isolates after recent in vivo passage were less susceptible; and (iii) nonpathogenic isolates were nearly unaffected by exposure to the alternative pathway alone. The extent of lysis of the various isolates correlated with the degree of complement consumption in the serum samples, suggesting that unlysed isolates did not activate complement under the conditions employed. In general, lysis of susceptible trophozoites increased with the serum concentration and with the time of incubation. However, when the trophozoite concentration was 10(6)/ml or higher, lysis no longer reflected complement susceptibility because of exhaustion of the complement supply.

Animals↗
PubMed1992

Role of fibrinogen in complement inhibition by streptococcal M protein.

M protein, the major virulence factor of group A streptococci, has antiopsonic activity in that it inhibits activation of the alternative complement pathway on the streptococcal surface. Two properties of M protein have been claimed to account for the inhibitory activity, namely, (i) its binding affinity for complement factor H, which is an inhibitor of alternative pathway activation, and (ii) its high binding affinity for fibrinogen. We have recently shown that fibrinogen, like M protein, inhibits alternative pathway activation by possessing binding affinity for factor H. Here we report that fibrinogen effectively competes with factor H for binding to M protein but retains its own binding affinity for factor H. The presence of fibrinogen did not significantly affect alternative pathway inhibition on the streptococcal surface.

Antigens, Bacterial↗
PubMed1992

Host tissue destruction by Entamoeba histolytica: molecules mediating adhesion, cytolysis, and proteolysis.

Entamoeba histolytica, the protozoan parasite causing human amoebiasis, has recently been found to comprise two genetically distinct forms, potentially pathogenic and constitutively nonpathogenic ones. Host tissue destruction by pathogenic forms is believed to result from cell functions mediated by a lectin-type adherence receptor, a pore-forming peptide involved in host cell lysis, and abundant expression of cysteine proteinase(s). Isolation and molecular cloning of these amoeba products have provided the tools for structural analyses and manipulations of cell functions including comparisons between pathogenic and nonpathogenic forms.

Animals↗
PubMed1992

Recent progress in the molecular biology of Entamoeba histolytica.

Entamoeba histolytica, a protozoan parasite causing human amoebiasis, has recently been found to comprise two genetically distinct forms, potentially pathogenic and constitutively nonpathogenic ones. Host tissue destruction by pathogenic forms is believed to result from cell functions mediated by a lectin-type adherence receptor, a pore-forming peptide involved in host cell lysis, and abundant expression of cysteine proteinases. Comparisons of these products from pathogenic and nonpathogenic E. histolytica suggest that they have evolved to serve functions in free-living or commensal behaviour. Isolation of the corresponding genes have provided the tools for detailed structural studies and manipulations of amoeba cell functions.

Animals↗
PubMed1992

[Recent findings on the pathogenicity of Entamoeba histolytica].

Entamoeba histolytica is the causative agent of human amoebiasis. During recent years, research in amoebiasis has concentrated on two subjects: 1. the dual manifestation of the infection as harmless colonization of the intestinal cavity or pathogenic tissue invasion and 2. the molecular analysis of functions of E. histolytica that are considered essential for pathogenicity. Besides epidemiological studies and isoenzyme analyses, molecular genetics have revealed additional evidence that two genetically distinct forms of E. histolytica do exist, named "pathogenic" and "nonpathogenic" forms, respectively. Both can infect humans but only the "pathogenic" form is able to invade the tissue and cause disease whereas the "nonpathogenic" is not. Questions remain open about the mechanism that triggers "pathogenic" E. histolytica to become invasive and about the molecules that are involved. Current data indicate that at least three functions of the amoebae are considered essential for pathogenic tissue invasion. Pathogenicity is viewed as a result of 1. adherence of the amoeba to host cells, predominantly mediated by a galactose- and N-acetylgalactosamine-inhibitable lectin, 2. killing of host cells by a pore-forming peptide known as amoebapore, and 3. proteolysis of the host's extracellular matrix mediated by cysteine proteinases. Structural detailed molecular analysis including cloning of the corresponding genes have led to a better understanding of the function of these proteins.

Animals↗
PubMed1991

Direct sequencing of variable HLA gene segments after in vitro amplification and allele separation by temperature-gradient gel electrophoresis.

Previously unrecognized variants of human leukocyte antigens (HLA) are currently being analyzed by in vitro amplification and sequencing of the variable gene segments. In heterozygous individuals, molecular cloning is required to separate the two concomitantly amplified haplotypic gene segments. A method is presented which facilitates the procedure of separating the two haplotypic gene segments by using a temperature-gradient gel electrophoresis (TGGE). The procedure comprises PCR amplification of the variable HLA gene segments, allele separation by TGGE, re-amplification of each of the separated allelic segments, and direct DNA sequencing using the PCR primers.

Amino Acid Sequence↗
PubMed1991

Pore-forming peptide of pathogenic Entamoeba histolytica.

A polypeptide that causes pore formation in target-cell membranes is implicated in the potent cytolytic activity of pathogenic Entamoeba histolytica. Pore-forming material was purified to apparent homogeneity by a multistep procedure, and its analysis by NaDodSO4/PAGE revealed one peptide of 4-5 kDa under nonreducing or under reducing conditions. Pore-forming activity was measured by depolarization of liposome membrane potential and was found to be optimally expressed at low pH. Active material preferentially inserted into negatively charged lipid vesicles. Treatment of purified amoeba peptide in solution or bound to liposomes with glutaraldehyde revealed oligomers upon NaDodSO4/PAGE, suggesting functionally relevant peptide-peptide interactions. The NH2-terminal amino acid sequence of the amoeba peptide was determined by protein sequencing and revealed a structural similarity to melittin, the membranolytic peptide of bee venom.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of a human serum protein structurally related to complement factor H.

Two cDNA clones termed H36-1 and H36-2 were isolated from a human liver cDNA library. Clone H36-1 appears to represent the recently isolated human serum proteins h37 and h42, which are two differently glycosylated forms of a protein antigenically related to human complement factor H. The H36-1 deduced protein sequence is 327 amino acid long and possesses a leader sequence. The secreted part of the protein is comprised of five tandem repeating units, termed short consensus repeats (SCRs). SCR 1 and 2 display high homology to the corresponding region of the recently isolated murine factor H-related cDNA clone 13G1. In contrast, the 3'-end of the H36-1 clone shows sequence homology to the 3'-end of human complement factor H. The second clone, H36-2, is nearly identical to H36-1. Within 1148 base pairs, where the two clones overlap, their nucleotide sequences differed at nine positions. One nucleotide exchange in the sequence of H36-2 which was located within SCR 1 creats a stop codon (TAA). Consequently, the corresponding mRNA cannot code for a functional protein, suggesting that this clone is a transcribed pseudogene. These two clones represent new human members of the family of proteins structurally related to complement factor H.

Amino Acid Sequence↗
PubMed1991

Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytica. Differences in structure and expression.

A cDNA clone derived from the gene encoding a cysteine proteinase of pathogenic Entamoeba histolytica was isolated using an antiserum to the purified enzyme. This clone was used to identify the homologous clone in a cDNA library from nonpathogenic E. histolytica. Sequence analysis and comparison of the predicted amino acid sequences revealed a sequence divergence of 16%. Southern blot analyses indicated that (i) pathogenic isolates may contain more genes coding for these or related enzymes than nonpathogenic isolates, (ii) the structure and organization of these genes are conserved within each group of amoebae, and (iii) none of the genes is found in both pathogenic and nonpathogenic E. histolytica, underlining the notion that the two groups are genetically distinct. Northern blot analyses suggested that the cysteine proteinase is expressed by pathogenic isolates in substantially higher amounts than by nonpathogenic isolates. Overexpression of this enzyme may be an important factor in the pathogenicity of E. histolytica.

Amino Acid Sequence↗
PubMed1991

Primary structure of the 170-kDa surface lectin of pathogenic Entamoeba histolytica.

The adherence of Entamoeba histolytica to colonic mucins and to host cells appears to be predominantly mediated by a 170-kDa surface lectin of the amoebae. By using an antiserum to the purified lectin, the corresponding cDNA was isolated from an expression library of the pathogenic E. histolytica isolate HM-1:IMSS. Northern blot analysis indicated a transcript of approximately 4 kilobases, and Southern blot analyses suggested that multiple genes may encode the lectin or closely related proteins in HM-1:IMSS trophozoites. The cDNA-deduced amino acid sequence revealed an N-terminal signal peptide and a mature protein of 1270 amino acids corresponding to a molecular mass of 143 kDa, which comprises a short C-terminal cytoplasmic domain with potential phosphorylation sites, a transmembrane region, and a large extracellular portion with nine potential asparagine-linked glycosylation sites. The extracellular portion may be separated into a cysteine-poor domain and a cysteine-rich domain, the latter of which shows in part repetitive structural elements with a low degree of sequence homology to wheat germ agglutinin and to pDd63, a developmentally expressed protein of Dictyostelium discoideum.

Amino Acid Sequence↗
PubMed1991

Two major serum components antigenically related to complement factor H are different glycosylation forms of a single protein with no factor H-like complement regulatory functions.

Factor H is a 150-kDa serum glycoprotein with key regulatory functions in the alternative pathway of complement activation. Two glycoproteins with a molecular mass of approximately 42 and 37 kDa that react with an antiserum against factor H were purified from human plasma. The two glycoproteins have identical N-terminal amino acid sequences but differ in glycosylation. Sequence comparisons indicated that they both correspond to a 1.4-kb mRNA recently cloned from human liver cDNA. The serum concentration of the two glycoproteins together was estimated to be approximately 40 mg/liter. They were found not to exert factor H-like regulatory functions in the alternative pathway. Thus, the 42-kDa glycoprotein described here appears to be distinct from the previously characterized factor H-related protein of similar size, suggesting that human serum contains two factor-H related molecules which both have a molecular mass of 41 to 43 kDa but which differ largely in structure.

Amino Acid Sequence↗
PubMed1991

Pathogenic and nonpathogenic Entamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase.

Superoxide dismutase (SOD) activity was determined in the cell lysate of the axenically cultured Entamoeba histolytica isolate HM-1:IMSS. Under anaerobic culture conditions, 18.7 (+/- 4.9) units SOD activity (mg protein)-1 were found. By inhibition studies the activity was attributed to an iron-containing type of SOD (FeSOD). Using degenerate oligonucleotide primers derived from regions highly conserved in prokaryotic FeSOD sequences, a genomic DNA fragment was amplified by the polymerase chain reaction. The fragment was used to isolate FeSOD specific cDNA clones from a pathogenic and a nonpathogenic E. histolytica isolate. A comparison of the 2 sequences revealed 5% nucleotide differences resulting in a single amino acid exchange. The primary structure showed the characteristics of an iron-containing type of SOD with a homology of approximately 55% with other FeSOD sequences. The enzyme was found to be encoded by single copy genes in both the pathogenic and the nonpathogenic E. histolytica, but restriction fragment lengths differed between the 2 groups. In 5 isolates studied, no correlation was found between pathogenic behavior of the amebae and the expression of FeSOD-related mRNA.

Amino Acid Sequence↗