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F D Rumjanek

Publications and source records attributed to F D Rumjanek.

At least 19 recordsLinked to original sources
PubMed2002

Dolichol phosphate mannose synthase is differentially expressed in male and female worms of Schistosoma mansoni.

The cDNA encoding the Schistosoma mansoni dolichol phosphate mannose synthase was completely sequenced, displaying the highest homology with Cricetulus griseus and Saccharomyces pombe genes. The Schistosome enzyme had a K(m) of 0.127 microM, a value that is within the range of those reported for several other species. Thin-layer chromatography of the radiolabelled schistosome lipid intermediate showed it was identical to dolichol-phosphate (C80-C105). Expression of dolichol phosphate mannose synthase of S. mansoni (SmDPMS) was analysed by Northern blot and quantified by semi-quantitative RT-PCR with cDNA from mature and immature male and female worms. Northern blot analysis revealed a single 1-kb band. Both approaches confirmed a higher level of expression in mature female worms, as compared to immature and male worms.

Amino Acid Sequence↗
PubMed2001

Lack of DNA methylation in Schistosoma mansoni.

Schistosoma mansoni genomic DNA from male and female adult worms was subjected to restriction by the isoschizomeric endonucleases HpaII and MspI, which display different sensitivities with respect to cytosine methylation. The digested DNA was hybridized with 13 S. mansoni probes. Southern blot analysis showed that there were no observable differences in the restriction patterns of the two isoschizomers and that the patterns were identical in male and female parasites. Adenine methylation was also ruled out since no differences were observed with DpnI, Sau3A1, or MboI restriction enzymes. The methylation-dependent restriction endonuclease McrBC, which cleaves DNA containing methylcytosine and will not cleave unmethylated DNA, did not digest S. mansoni genomic DNA. These results demonstrate that the genome of adult S. mansoni is not methylated.

Animals↗
PubMed2000

Immunological cytokine correlates of protective immunity and pathogenesis in leprosy.

The in vitro production of interferon (IFN)-gamma, interleukin (IL)-5, tumour necrosis factor (TNF)-alpha and IL-10 by blood mononuclear cells in response to whole Mycobacterium leprae and polyclonal stimulii of 23 individuals, representing a variety of conditions in relation to exposure/susceptibility to M. leprae, was assayed. In most cases, healthy household contacts of newly diagnosed multibacillary leprosy patients, designated exposed household contacts (EC), showed low-to-undetectable in vitro IFN-gamma production in addition to substantial TNF-alpha production in response to M. leprae. In contrast, peripheral blood mononuclear cells from previously exposed contacts (R) regarded as resistant-to-leprosy released low-to-moderate levels of IFN-gamma together with a mixed cytokine profile resembling a T helper (Th)0-type response. TNF-alpha/IL-10 ratios in response to M. leprae and Concanavalin A were significantly higher in EC than in R contacts suggesting a role for the TNF-alpha/IL-10 ratio in restraining mycobacteria proliferation and spreading early in infection. The cytokine profiles of leprosy patients were taken as reference points. Post-treatment lepromatous leprosy patients secreted relatively high levels of IL-10 in response to M. leprae, whereas one self-cured tuberculoid leprosy patient produced simultaneously high levels of IFN-gamma and TNF-alpha. In addition, the quantitative changes in the cytokines released by peripheral blood mononuclear cells in EC contacts after Bacille Calmette-Guérin (BCG) vaccination were investigated. Vaccination induced amplification of IFN-gamma production with a concomitant decrease in TNF-alpha/IL-10 ratios that resembled the cytokine pattern observed in R contacts. IFN-gamma production was observed in response to both a cross-reactive antigen (Ag 85) and a M. leprae-specific protein (MMP-I), which attests to a BCG nonspecific stimulation of the immune system, thereby casting these antigens as likely candidates for inclusion in a subunit vaccine against leprosy. Finally, a model for protective x pathologic response to mycobacteria is presented.

Adolescent↗
PubMed1999

Comparison between site-specific DNA binding proteins of male and female Schistosoma mansoni.

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.

Animals↗
PubMed1999

Dolichol phosphate is a rate-limiting factor in mannosyl transferase activity of adult male worms of Schistosoma mansoni.

The formation of mannolipid through catalysis by mannosyl transferase of adult females of Schistosoma mansoni was found to be 2-3-fold higher than male worms. In contrast, mannosyl transferase in immature females generated approximately the same amount of mannolipid as male worms, immature or not. Exogenous dolichol phosphate added to homogenates of male worms produced a stoichiometric increase in mannolipid formation. Saturating amounts of dolichol phosphate generated similar mannosyl transferase activities in male and female homogenates, showing that in S. mansoni, dolichol phosphate is the lipid intermediate in the glycosylation reaction and that this mannolipid is a rate-limiting substrate. Thin layer chromatography revealed that the mannolipid was identical in male and female worms. Adult males incubated with 14C-acetate synthesised several apolar compounds, one of which displayed a Rf identical to that of the mannolipid. When exposed to 14C-acetate treated males in vitro, untreated females were able to incorporate a compound, which partitioned in the same way as the mannolipid. The increased mannosyl transferase-dependent mannolipid formation in adult females may reflect a higher energy demand by these parasites, which is probably associated with oogenesis.

Animals↗
PubMed1997

The interaction of human LDL with the tegument of adult Schistosoma mansoni.

The occurrence of a receptor for human LDL was investigated in the tegument of adult Schistosoma mansoni employing several approaches. Binding of LDL to SDS-PAGE fractionated tegument proteins was measured directly on nitro-cellulose membranes and visualised by an anti-human LDL antibody. Proteins with an Mr of 60, 35 and 14 kDa were evidenced. Affinity chromatography of 125I-labelled tegument proteins on a LDL-Sepharose column, revealed the same pattern of proteins observed in the immunoblot experiments. Finally, the binding of human LDL to the intact tegument was measured by microcalorimetry. Binding was shown to be an exothermic reaction, releasing approximately 2500 kcal/mol, it was saturable, and reproducibly displayed a biphasic curve suggesting that binding of LDL to S. mansoni might occur through a two step process, initiated by a nonspecific hydrophobic interaction followed by a specific high affinity ligand-receptor reaction. Pre-treatment of the tegument with trypsin reduced the binding of LDL to the tegument. Furthermore, albumin, which is an abundant lipid carrier protein in the serum and thus a potential ligand, failed to release any measurable heat when incubated with the tegument.

Animals↗
PubMed1995

Cloning and characterization of a putative calcium-transporting ATPase gene from Schistosoma mansoni.

Complementary DNA was isolated, encoding a putative Ca(2+)-transport ATPase (SMA1) of the human parasitic trematode Schistosoma mansoni. The cDNA was isolated by a nested polymerase chain reaction based strategy. The oligonucleotides used were designed on the basis of conserved amino-acid regions found in P-type ATPases. The complete nucleotide sequence was determined. The primary structure and topology of the enzyme were deduced. SMA1 has 1022 amino acids and a predicted molecular mass of 113 kDa. This protein is 67% identical and phylogenetically related to several sarco/endoplasmic reticulum Ca(2+)-ATPases but lacks the phospholamban-binding domain that exists in the SERCA isoforms 1 and 2. The membrane topology predicted for SMA1 is characteristic of the P-type ATPases, showing two major cytoplasmic loops and ten conserved hydrophobic segments. Sequences and residues that are important for the function of the SER Ca(2+)-ATPase, such as the high-affinity Ca(2+)-binding sites, the putative fluorescein isothiocyanate binding site, the 5'-(p-fluorosulfonyl)benzoyladenosine binding site and the aspartyl phosphorylation site, are conserved in SMA1, suggesting that the cloned gene is a Ca(2+)-transport ATPase of the SERCA family. In addition, three PCR products were cloned which share homology with another SER Ca(2+)-ATPase, with the yeast secretory pathway Ca(2+)-ATPase PMR1 and its mammalian homologue, and with the alpha subunit of a Na+,K(+)-ATPase.

Adenosine Triphosphatases↗
PubMed1995

F-10 nuclear binding proteins of Schistosoma mansoni: structural and functional features.

By incubating total protein extracts of Schistosoma mansoni with 3H-17-beta-estradiol and 20-hydroxyecdysone, steroid binding proteins were detected in both male and female worms. The interaction of nuclear proteins with a restriction fragment of the gender and stage-specific gene F-10 was investigated using the 'band-shift' technique. Male and female nuclear proteins bound in a distinct way to the fragment of this gene containing putative regulatory consensus motifs. Among the nuclear proteins, only those rich in cysteine residues bound to DNA. In vitro incubation of live worms with the oestrogen antagonist Tamoxifen, altered the pattern of the DNA binding proteins, producing in females a profile similar to that obtained with male worm protein extracts. This effect of Tamoxifen could not be correlated to inhibition of protein biosynthesis. These results suggest that the regulation of transcription of the F-10 gene might involve steroid receptors.

Animals↗
PubMed1994

Characterization of a HMG2-like protein from Schistosoma mansoni.

An HMG2-like protein was purified from nuclear extracts of adult Schistosoma mansoni. Investigation of the amino acid composition of the schistosome HMG2-like protein showed that glutamic acid, glycine, aspartic acid and lysine were the most abundant. Carbohydrate analysis showed that the HMG2-like protein presented a low degree of glycosylation, galactose or glucose being the major monosaccharide constituent. Incubation of live schistosomes with 32P followed by isolation of nuclear proteins showed that the HMG-2 like protein could be phosphorylated. Partial sequence analysis of cyanogen bromide peptides revealed the occurrence of a phosphorylation consensus motif. The schistosome HMG2-like protein was found to bind preferentially to single-stranded DNA. The results suggest that the major non-histone S. mansoni nuclear protein belongs to the HMG family.

Amino Acid Sequence↗
PubMed1993

Protein interactions with a gender-specific gene of Schistosoma mansoni: characterization by DNase I footprinting, band shift and UV cross-linking.

A schistosome gender specific gene (F-10), was used as a probe to characterize DNA binding proteins from adult male and female Schistoma mansoni. Using the band-shift and DNase I footprinting methods, it was found that proteins from male and female worms bound to the intact F-10 gene and to restriction fragments corresponding to different domains of the gene, generating relatively long protected sites. Clear differences between male and female proteins were only observed when nuclear proteins were tested. Thus, gender-specific binding was detected in fragments corresponding to the 5' and 3' ends. UV-induced cross-linking between schistosome proteins and a synthetic oligonucleotide bearing a steroid response element present in the 3' untranslated end of the F-10 gene, revealed a major DNA binding protein with a molecular mass of 30 kDa, in both male and female worms. These results suggested that the activation of transcription of the F-10 gene may depend essentially on nuclear proteins.

Animals↗
PubMed1992

Extraction and partial characterization of non-histone nuclear proteins of Schistosoma mansoni.

A pool of nuclear proteins from adult worms of Schistosoma mansoni was analyzed for amino acid composition and found to be compatible with high mobility group (HMG) proteins. One of the schistosome HMG proteins was identified as HMG 2 by one-dimensional and two-dimensional PAGE. Stage-specific differences in the HMG-like protein composition were encountered when adult worms were compared to schistosomula, the larval form. Immobilization of the adult male and female nuclear proteins onto nitrocellulose, followed by hybridization against 32P-F-10, a schistosome sex specific gene encoding a major egg shell protein, revealed distinct banding patterns. On the other hand, a synthetic oligonucleotide, derived from the 3' untranslated end of the F-10 gene and possibly containing one regulatory element of the gene, bound mainly to male low MW proteins.

Aging↗
PubMed1992

Protein-DNA associations in a gender-specific gene of Schistosoma mansoni: characterization by UV cross-linking, DNase I footprinting and band shift assays.

Protein extracts obtained from male and female schistosomes were incubated with a gender-specific gene, F-10, transcribed only in adult females and encoding a major egg-shell protein. The protein/DNA interaction was measured using the band shift, DNase-I-footprinting and UV cross-linking techniques. The results showed a clear band shift when a 302 bp restriction fragment containing the 3' end of the gene was incubated with either female or male proteins. This fragment also contained a putative steroid hormone regulatory element (HRE). In contrast, only the male proteins produced a shift with the 495 bp fragment corresponding to the middle region of the gene. DNase I footprinting showed that proteins from males and females interacted with the F-10 gene by binding to multiple adjacent sites along the DNA, thus generating relatively long protected fragments of approximately 100 bp. This result suggested that the adjacent binding of several moles of protein occurred at the 5' end of the gene. UV cross-linking between schistosome proteins and a 21 bp synthetic oligonucleotide containing the F-10 HRE, evidenced proteins having MWS of 30, 45 and 65 kDa. These proteins are presumably involved in the regulation of transcription of the F-10 gene.

Animals↗
PubMed1991

Serology and leprosy: immunoassays comparing immunoglobulin G antibody responses to 28- and 30-kilodalton proteins purified from Mycobacterium bovis BCG.

Two major proteins from Mycobacterium bovis BCG culture filtrates with molecular masses of 28 kDa (P28) and 30 kDa (P30), identified as components of the BCG 85 complex, were purified and used in enzyme-linked immunosorbent assays (ELISAs) for the determination of specific immunoglobulin G (IgG) levels in patients with leprosy or tuberculosis or with exposure to these diseases. High reactivity to both antigens was observed with sera from lepromatous leprosy patients, whereas antibody levels in sera from paucibacillary leprosy patients were not significantly different from those in sera from healthy individuals from an area in which leprosy is endemic. High IgG responses were also found in some contacts of lepromatous leprosy patients. A comparison of the levels of anti-P28 and anti-P30 within the multibacillary leprosy patient group showed much higher IgG reactivity to P28 than to P30, suggesting that the antibody response of lepromatous patients is directed predominantly against the 28-kDa protein. A high degree of correlation in values of ELISAs based on P28 and on the phenolic glycolipid of Mycobacterium leprae was observed in all groups analyzed. The potential use of an assay based on the 28-kDa protein to selectively distinguish individuals destined to develop multibacillary leprosy is discussed, as also is the likelihood that the 28-kDa-30-kDa complex, part of the fibronectin-binding family, is an important component of M. leprae.

Antibodies, Bacterial↗