Biomedical subjects

N Agabian

Publications and source records attributed to N Agabian.

At least 73 records · Page 4Linked to original sources

PubMed1988

Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase.

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.

Amino Acid Sequence↗

PubMed1988

Schistosome heat-shock proteins are immunologically distinct host-like antigens.

Constitutively expressed schistosome homologues of heat-shock protein Hsp70 elicit a dominant antibody response in humans infected with either Schistosoma japonicum or Schistosoma mansoni; in each case the parasite antigens are immunologically distinct and noncrossreactive. The antigenic site of the homologues is located near their carboxyl terminus where phylogenetic divergence between Hsp70 proteins is greatest. Nucleotide sequence comparison between these regions predicts very few amino acid differences between the schistosome protein and that of their human host. Thus strikingly limited diversity is sufficient to elicit a discriminatory antibody response to these parasite host-like antigens.

Amino Acid Sequence↗

PubMed1988

Parasite heat-shock proteins.

Many parasites, including most of those of medical or veterinary importance, experience a major increase in ambient temperature at some stage during their life cycle. This occurs when a cyst or free-living larval form is ingested by a warm-blooded host, when a poikilotherm-infecting parasite is transmitted to a homeotherm, or when a transiently free-living invasive larva penetrates the skin of a mammal. This sudden change in temperature could be expected to stress the intruder, as it should dramatically alter rates of metabolic reactions and of denaturation of proteins. This would especially affect the function of near-equilibrium, regulatory, and membrane-bound enzymes (changes in temperature affect membrane fluidity). In this article George Newport, Janice Culpepper and Nina Agabian consider how parasites cope with this problem, emphasizing the possible role of heat-shock proteins (HSPs), how the expression of these molecules is regulate, and how HSPs interact with the host immune system.

Journal Article↗

PubMed1988

Transcriptional analysis of the major surface array gene of Caulobacter crescentus.

The major component of the paracrystalline surface array of Caulobacter crescentus CB15 and one of the most abundant cellular proteins is a protein designated 130K. We have determined the DNA sequence of the 5' portion of the 130K gene, including the N-terminal one-third of the protein coding region, and analyzed the transcription of the gene. The site of transcription initiation was determined by S1 mapping of Caulobacter RNA. Although the DNA sequence upstream from the transcription start site showed significant homology to the consensus promoter sequences of Escherichia coli, S1 analysis of RNA from E. coli carrying the 130K gene on a plasmid indicated that the 130K promoter was not transcribed by E. coli RNA polymerase in vivo. Quantitative S1 analysis of RNA isolated from synchronously growing Caulobacter cells suggested that this promoter was not under developmental regulation; the amount of 130K transcript varied no more than 1.5-fold during the cell cycle. The length of the 130K mRNA was determined to be 3.3 kilobases by Northern (RNA blot) analysis, indicating that the 130K mRNA is not part of a polycistron. The amino acid sequence predicted from the DNA sequence agreed well with the N-terminal amino acid sequence determined by sequencing of the 130K protein. The 130K protein appears to be synthesized without an N-terminal leader sequence, but the N-terminal 20 amino acids are relatively hydrophobic and may function like a signal sequence during transmembrane translocation.

Amino Acid Sequence↗

PubMed1988

Identification, molecular cloning, and expression of a schistosome antigen displaying diagnostic potential.

Recombinant DNA techniques were employed to address the problem of specificity in a serodiagnostic test for schistosomiasis. Immunoprecipitation of in vitro translation products of adult Schistosoma mansoni RNA with sera from Egyptian donors revealed that the human IgG response to schistosome proteins is highly heterogeneous, does not correlate with clinical status, and remains essentially unaltered 6 months after chemotherapeutic cure; peptides of 38 and 70 kDa are recognized by sera from patients infected with S. mansoni or S. haematobium but not by sera from individuals harboring other helminth infections. Using serum from C57BL/6J mice acutely infected with S. mansoni, which strongly reacts with these peptides and very weakly with other worm proteins, portions of the 70 kDa peptide were cloned from an expression cDNA library. The value and limitations of using recombinant schistosome antigens in serodiagnostic assays is discussed.

Animals↗

PubMed1987

A major immunogen in Schistosoma mansoni infections is homologous to the heat-shock protein Hsp70.

A 70,000 mol wt protein of Schistosoma mansoni was shown to be a major immunogen that invariably elicited an antibody response in infected humans. The universality of the response to this abundant antigen was confirmed in experimental animals and included the antibody response associated with the protective irradiated cercarial vaccine. We identified the 70,000 mol wt antigen as an S. mansoni homologue of the major eukaryotic heat-shock protein hsp70 by DNA sequence analysis of a cDNA insert from a lambda gt11 clone expressing the antigen and located the immunodominant epitope near the COOH-terminus of the molecule. The antigenic relationship of hsp70 to schistosome infections suggested an important role for this protein in parasite development and pathogenesis.

Amino Acid Sequence↗

PubMed1987

Molecular cloning of Schistosoma mansoni myosin.

A cDNA library representative of adult Schistosoma mansoni mRNA populations was screened with serum from infected rats (refractory hosts), positive plaques being rescreened with serum from infected mice and humans. Based on general reactivity, one clone was selected for further study. As judged by immunofluorescence data, size of corresponding mRNA, and nucleotide sequence analysis, the recombinant expresses approximately 625 amino acids of a schistosome muscle myosin rod. Antibodies evoked by the protein do not cross-react with human cardiac or skeletal muscle, are not invariably stimulated in naturally infected human beings, and rise in titer after chemotherapeutic cure, findings which suggest that the antigen is not a causative agent of Katayama fever, and is probably presented by degenerating worms. The schistosome sarcomeric myosin gene, the most primitive examined to date, appears to be unique inasmuch as it may not be a member of a multigene family and encodes a single mRNA transcript; nonetheless, predicted higher order structure of its translation product is consistent with expected function.

Amino Acid Sequence↗

PubMed1987

Trypanosome mRNAs have unusual "cap 4" structures acquired by addition of a spliced leader.

A single capped oligonucleotide is released from Trypanosoma brucei poly(A)+ RNA upon digestion with RNase T2. This observation supports the hypothesis that all T. brucei mRNAs share a common leader sequence. Digestion of the T2-resistant species with nucleotide pyrophosphatase shows that the capping nucleotide is 7-methylguanosine 5'-monophosphate (pm7G). Additional characterization of the T2-resistant fragment indicates that modifications are present on the first four transcribed nucleotides; the 5' termini of T. brucei mRNAs can, therefore, be described as "cap 4" structures. Identical 5'-cap structures are found on the T. brucei spliced leader (SL) RNA; an observation compatible with the hypothesis that the small SL RNA acts as a donor of the SL for the mRNA. However, we find that within a population of purified SL RNAs are species that are capped but incompletely modified. The presence of these unmodified and partially modified species allowed us to analyze the 5' sequence of the SL RNA transcript. The results indicate that transcription begins four nucleotides upstream of the reported 5' end. Therefore, the T. brucei SL transcript is actually 39 rather than 35 nucleotides long. We have also analyzed the capped oligonucleotide of a distantly related Trypanosomatid, Leptomonas collosoma and find it to be identical to that of T. brucei. The potential significance of these results is discussed in light of observations of trypanosome gene expression.

Animals↗

PubMed1987

Genomic and cDNA actin sequences from a virulent strain of Entamoeba histolytica.

Invasiveness of Entamoeba histolytica strains that cause acute amoebiasis is characterized by aggressive behavior associated with cell motility and actin function. Analysis of actin genes from E. histolytica was initiated by devising methods for the isolation of biologically active nucleic acids, which allowed the preparation of cDNA and genomic DNA libraries. E. histolytica actin-encoding cDNAs and genomic clones have been isolated from libraries prepared from the virulent HM1:IMSS strain using a heterologous actin probe. Nucleotide sequence analysis of three independent cDNA clones and one genomic clone reveals a highly unusual codon bias and the absence of intervening sequences in E. histolytica actin. The coding sequence of the genomic clone is identical to that of two of the three cDNA clones. These represent at least two distinct mRNAs differing only by five silent changes in the protein coding sequence. Multiple genomic copies of the actin gene can be detected by Southern hybridization. E. histolytica actin exhibits a higher degree of homology to cytoplasmic than to muscle actin. Although the protein has been shown not to bind DNase I, the inferred amino acid sequence indicates conservation of all residues implied to participate in this binding.

Actins↗

PubMed1987

Successful treatment with aerosolized pentamidine of Pneumocystis carinii pneumonia in rats.

We examined both the therapeutic efficacy and tissue distribution of aerosolized pentamidine in immunosuppressed rats with Pneumocystis carinii pneumonia. In rats immunosuppressed by 5 weeks of pretreatment with dexamethasone, a 2-week course of 5 mg of aerosolized pentamidine per kg per day, administered free or encapsulated in the drug carrier system (liposomes), eradicated P. carinii pneumonia in 75% of treated animals. At this dose, extrapulmonary drug uptake as measured by a sensitive high-pressure liquid chromatography assay was negligible. No significant differences in tissue distribution were noted between aerosolized free and liposome-encapsulated pentamidine. In rats receiving dexamethasone for 6 weeks prior to treatment with pentamidine, both lung uptake and therapeutic efficacy of aerosolized pentamidine (5 mg/kg per day) were substantially reduced. Aerosolized pentamidine appears to be an effective therapy for P. carinii pneumonia in rats and produces significantly lower extrapulmonary drug deposition than parenteral administration. The severity of P. carinii involvement at the time of treatment influences both the level of drug delivery to the lung and the response to aerosolized pentamidine therapy.

Aerosols↗

PubMed1987

Schistosome elastases: biological importance, structure, function and stage-specific expression.

Larval schistosomes (Digenea: Trematoda) invade their definitive host by directly penetrating the skin. During the process they secrete a number of macromolecules, ostensibly to facilitate their entry. Among these we have identified and characterized a dominant proteolytic species: a serine protease capable of fragmenting keratin, types IV and VIII collagen, proteoglycan, fibronectin, laminin, and elastin. The enzyme exhibits the specificity characteristic of elastases, has a molecular mass of 30,000 Da and pI of 7.8, and is potently immunogenic in its native form. Specificity of the active site has been analysed, tetrapeptides having large hydrophobic or aromatic amino acids at size P1 serving as best substrates. The amino terminal 20 amino acids of the mature enzyme have been sequenced and the information derived has been used to construct an oligonucleotide (22-mer) complement of its corresponding mRNA. The latter has been used to establish, by Northern analysis, that expression of the enzyme is stage specific (differing in this respect from most schistosome immunogens), and under transcriptional control. Transcripts are encoded by a multigene family. Several cDNAs hybridizing to the oligonucleotide have been isolated, subcloned into bacteriophage M-13, and sequenced by the di-deoxy method. It is our expectation that this line of investigation will lead towards: (i) an anti-infection vaccine; (ii) a means for chemically preventing infection (using enzyme inhibitors), and/or (iii) a rapid diagnostic assay of prepatent infection.

Amino Acid Sequence↗

PubMed1986

Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing.

We present evidence that addition of the 35 nucleotide spliced leader (SL) to the 5' end of T. brucei mRNAs occurs via trans RNA splicing. A 100 nucleotide fragment of the 135 base SL RNA (100-mer) is revealed by S1 nuclease analysis of total and poly(A)+ RNA. This 100-mer is not detected by Northern hybridization analysis, indicating that it does not exist free in the cell. The 5' end of the 100-mer maps precisely to the conserved splice junction sequence of the SL RNA. Purified debranching enzyme releases this 100-mer RNA as a free, 100 nucleotide species. This indicates that the 100-mer is covalently linked to poly(A)+ RNA by a 2'-5' phosphodiester bond, that the branched intermediate has a discontinuous intron or Y structure (rather than a lariat), which is expected of a trans-spliced mRNA, and that the SL RNA is indeed the donor of the SL sequence to trypanosome mRNAs.

Animals↗

PubMed1986

The trypanosome spliced leader small RNA gene family: stage-specific modification of one of several similar dispersed genes.

Diverse mRNAs of Trypanosoma brucei possess the same 5' terminal 35 nucleotides, termed the spliced leader (SL), which appears to be derived from a separate 135 nucleotide transcript. This small SL RNA is encoded within a 1.4 kb unit of DNA which is tandemly reiterated in the genome. In addition, there are at least 4 orphon elements containing SL sequences dispersed from the tandem array. Here we show that during the trypanosome life cycle one of the SL orphons undergoes a stage-specific modification that prevents cleavage of an EcoRV site and we further demonstrate that although only one orphon is modified, three of the SL orphons are flanked by very similar sequences. Each of these contains SL reiteration units including the non-transcribed spacer DNA, suggesting that they did not originate through an RNA intermediate. In addition no evidence of direct repeats at the junction of 1.4 kb and non-1.4 kb DNA was observed. Finally, a phylogenetic survey indicates that while many trypanosomatid species possess similarly organized SL-like sequences, only the SL orphons of closely related subspecies of the T. brucei - T. evansi complex share similar flanking regions.

Animals↗

PubMed1986

Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2.

The structural gene for the major outer membrane protein (MOMP) from Chlamydia trachomatis was cloned and sequenced. A lambda gt11 recombinant (lambda gt11/L2/33) that contains a portion of the MOMP coding sequence was used to probe a lambda 1059 library constructed from DNA obtained from C. trachomatis serovar L2. Selected lambda 1059 recombinants were mapped with endonuclease restriction enzymes. The MOMP gene was mapped to the 5' end of a BamHI fragment of approximately 9 kilobases. Contiguous endonuclease restriction fragments identified within this region permitted the selection of specific fragments for subcloning and DNA sequencing. The MOMP gene consisted of a 1,182-base-pair open reading frame that encoded 394 amino acids and ended with three stop codons. The known amino-terminal amino acid was preceded by 22 amino acids whose sequence was compatible with a leader or signal sequence. The primary structure of MOMP determined from the translated DNA sequence demonstrated nine cysteine residues and a remarkably homogeneous distribution of charged and hydrophobic residues.

Amino Acid Sequence↗

PubMed1985

Preferential activation of telomeric variant surface glycoprotein genes in Trypanosoma brucei.

During an infection, Trypanosoma brucei expresses diverse variant surface glycoprotein (VSG) genes in a quasi-sequential order. Numerous VSG genes have intrachromosomal locations but many are located adjacent to telomeres. We have tested whether telomeric VSG genes are preferentially activated compared to intrachromosomal VSG genes during an antigenic switch. The frequency with which the IsTat 11 VSG gene is expressed in first relapse populations has been compared for variant antigenic types (VATs) A3 and A11. These VATs express the same A VSG gene from the same chromosome but VAT A11 contains an inactive telomeric 11 VSG gene which is absent in VAT A3. The 11 gene is activated at a much higher frequency in first relapse populations from VAT A11 than from VAT A3. A resultant VAT 11 clone was examined in detail and shown to have reactivated the telomeric 11 VSG gene. These results suggest that a telomeric location can result in a greater frequency of activation of a VSG gene. This preferential activation may explain, in part, the order of expression of VSG genes.

Animals↗

PubMed1985

A 5' spliced leader is added in trans to both alpha- and beta-tubulin transcripts in Trypanosoma brucei.

The approximately 15 alpha- and 15 beta-tubulin genes of Trypanosoma brucei are arranged in a tandem array of alternating alpha- and beta-tubulin genes. We have examined the structure of mRNA transcripts from the tubulin gene family and have found at the 5' ends of both alpha- and beta-tubulin messages a 35-nucleotide spliced leader (SL) identical to that identified previously on the 5' ends of variant surface glycoprotein (VSG) mRNAs. No 35-nucleotide SL sequences were encoded in the tubulin intergenic regions; instead, the SL sequence apparently originates from small RNA transcripts encoded at sites that are unlinked to the tubulin genes. The tandem arrangement of the tubulin genes and the presence of the SL at the 5' end of both alpha- and beta-tubulin transcripts establishes that RNA joining between the SL and tubulin mRNA occurs in trans.

Animals↗

PubMed1985

Antigenic variation in clones of Trypanosoma brucei grown in immune-deficient mice.

We have produced monoclonal antibodies against six variant surface glycoproteins from early variant antigen types (VATs) of the IsTaR 1 serodeme of Trypanosoma brucei brucei. We have used these in fixed cell immunofluorescence assays to follow the VAT composition of populations of each early VAT when passaged through irradiated mice. The IsTat 1.A and 1.7a populations were stable for more than 30 days (approximately 150 generations), but 1.1a, 1.3a, 1.5a, and 1.11a all changed to 1.A within this time. The time and rate of this antigenic switch were characteristic for each VAT. Growth rates of the VATs were determined when they were both grown separately and grown with 1.A. It appeared that the order of growth rates was 1.7a greater than 1.A = 1.1a greater than 1.11a greater than 1.5a greater than 1.3a. We have generated theoretical curves for the replacement of one VAT by another based on differences in their growth rates and the rate at which one VAT switches to another (switch frequency). These curves closely match those derived experimentally. We postulate that the differences in growth rates between VATs and the different switch frequencies for VATs may be sufficient to generate the loosely defined sequence of VATs seen in chronic infections.

Animals↗

PubMed1985

Molecular cloning and expression of Chlamydia trachomatis major outer membrane protein antigens in Escherichia coli.

DNA obtained from Chlamydia trachomatis (serovar L2) was partially digested with DNase I and inserted into the beta-galactosidase gene of bacteriophage lambda gt11. Seven recombinants were selected that produced immunoreactive fusion proteins which were detected with anti-C. trachomatis rabbit serum. One recombinant, designated lambda gt11/L2/33, reacted with various monoclonal antibodies that recognize species-, subspecies-, and type-specific determinants on the chlamydial major outer membrane protein (MOMP). Immunoblot analysis of a lambda gt11/L2/33 lysogen revealed a fusion protein that expressed a approximately 15,000-dalton carboxyl-terminal peptide of the chlamydial MOMP. This moiety of the MOMP possesses epitopes responsible for each of the unique reactivities demonstrated by anti-MOMP monoclonal antibodies. The lambda gt11/L2/33 recombinant contained a 1.1-kilobase DNA insert which hybridized to DNA isolated from each of the 15 C. trachomatis serovars.

Antibodies, Monoclonal↗