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Biomedical subjects

A Landa

Publications and source records attributed to A Landa.

At least 19 recordsLinked to original sources
PubMed2001

Timing of SOX9 downregulation and female sex determination in gonads of the sea turtle Lepidochelys olivacea.

The SRY-related gene SOX9 is involved in the differentiation of Sertoli cells in male gonads of vertebrates with different kinds of sex determination. In the olive ridley Lepidochelys olivacea, a species with temperature sex determination (TSD), the SOX9 protein is expressed at stages 21-24 in medullary cells in gonads of embryos incubated at both male-(MPT) or female-promoting temperatures (FPT). However, at FPT the expression of SOX9 protein decreases at stage 25 and disappears at stage 26, suggesting this as the critical period for SOX9 regulation by temperature. Here, we used reverse transcriptase polymerase chain reaction (RT-PCR) to detect SOX9 transcripts in gonads of embryos switched from MPT to FPT at stage 23 and sampled at days 6-14. Simultaneously, groups of embryos were switched back to MPT and gonadal sex was established. SOX9 transcripts were detected at days 6-12 of switching, when embryos reached stage 25 and were no longer detected at day 14, when the embryos were at stage 26. Embryos switched back to MPT at days 6 or 8 formed testes, whereas embryos switched at days 10 or 14 developed ovaries. Results suggest that at MPT the male sex-determining pathway that maintains SOX9 expression in male gonads is established at stage 24. In contrast, at FPT, the female sex-determining pathway involved in downregulation of SOX9 in female gonads occurs within two days at stage 25. J. Exp. Zool. 290:498-503, 2001.

Amino Acid Sequence↗
PubMed2001

A beta-lactam-based stereoselective access to beta,gamma-dihydroxy alpha-amino acid-derived peptides with either alpha,beta-like or unlike configurations.

A concise access to alpha,beta-dihydroxy alpha-amino acid-derived N-carboxy anhydrides (NCAs) with either like or unlike relative configuration is described. The key steps of the synthetic route are the preparation of the nonracemic 4-alkenyl beta-lactams, through either Horner-type olefination of a common 4-formyl beta-lactam or the Corey-Winter alkene synthesis applied to 4-dihydroxyalkyl beta-lactams, followed by the Sharpless AD reaction, and a subsequent ring expansion of the corresponding 4-substituted 3-hydroxy beta-lactams promoted by TEMPO. The opening of thus-prepared NCAs upon treatment with different O- and N-nucleophiles, including alpha-amino esters which lead to peptides, has also been studied under various reaction conditions.

Amino Acids↗
PubMed2001

Genetic variation and population structure in Scandinavian wolverine (Gulo gulo) populations.

Wolverine (Gulo gulo) numbers in Scandinavia were significantly reduced during the early part of the century as a result of predator removal programmes and hunting. Protective legislation in both Sweden and Norway in the 1960s and 1970s has now resulted in increased wolverine densities in Scandinavia. We report here the development of 15 polymorphic microsatellite markers in wolverine and their use to examine the population sub-structure and genetic variability in free-ranging Scandinavian wolverine populations as well as in a sample of individuals collected before 1970. Significant subdivision between extant populations was discovered, in particular for the small and isolated population of southern Norway, which represents a recent recolonization. Overall genetic variability was found to be lower than previously reported for other mustelids, with only two to five alleles per locus and observed heterozygosities (H(O)) ranging from 0.269 to 0.376 across the examined populations, being lowest in southern Norway. Analysis of the mitochondrial DNA control region revealed no variation throughout the surveyed populations. As the historical sample did not show higher levels of genetic variability, our results are consistent with a reduction in the genetic variation in Scandinavian wolverines that pre-dates the demographic bottleneck observed during the last century. The observed subdivision between populations calls for management caution when issuing harvest quotas, especially for the geographically isolated south Norwegian population.

Animals↗
PubMed2001

Comparison of sexual dysfunction in male schizophrenic patients maintained on treatment with classical antipsychotics versus clozapine.

BACKGROUND: Antipsychotic treatment is frequently associated with sexual dysfunction. The objective of the present study was to evaluate and compare sexual function and behavior in male schizophrenic patients who regularly take either classical neuroleptic drugs or the prototypical atypical antipsychotic agent, clozapine. METHOD: Participants included 60 schizophrenic male patients (DSM-IV criteria); 30 maintained on treatment with classical antipsychotics and 30 on treatment with clozapine. The patients were evaluated with a detailed 18-item sexual function questionnaire. RESULTS: Both groups reported sexual dysfunction, although scores were significantly higher, indicating better functioning, in the clozapine-treated group in the domains of orgasmic function (number of orgasms per month, p = .037; frequency of orgasm during sex, p = .046), enjoyment of sex (p = .013), and sexual satisfaction (p = .0004). Equivocal results were obtained for the desire parameters. CONCLUSION: Maintenance therapy with the atypical neuroleptic clozapine may be associated with a lesser degree of sexual dysfunction than the classical antipsychotics in male outpatients with chronic schizophrenia.

Adult↗
PubMed2000

Cloning, characterization and functional expression of a cyclophilin of Entamoeba histolytica.

Full-length Entamoeba histolytica cyclophilin gene (EhCyp) was isolated, characterized and recombinantly expressed in bacterial cells. The deduced amino acid sequence of EhCyp shows 60-70% identity with cyclophilins from other organisms and has conserved the cyclophilin signature motifs and residues involved in cyclosporin A binding. Upstream of the 501 bp open reading frame of EhCyp, sequences resembling the putative consensus E. histolytica CE1, CE2 and CE3 regulatory elements were found. Northern blot assays revealed a single transcript of 0.63 kb. The transcription start was determined by primer extension at position -13 relative to the initial ATG codon. Cyclosporin A binding and peptidyl-proplyl cis-trans isomerase activities characteristic of cyclophilin were detected in soluble extracts of E. histolytica trophozoites and in the recombinant protein. In both cases, the isomerase activity was inhibited by nanomolar concentrations of cyclosporin A. Treatment of cultured trophozoites with cyclosporin A decreased their proliferation with a 50% inhibition value of 1 microg/ml and was lethal in doses over 50 microg/ml.

Amino Acid Sequence↗
PubMed2000

A strategy for the asymmetric aminohomologation of alpha, beta-dihydroxy aldehydes: application to the synthesis of the southwest tripeptide segment of echinocandin B.

The synthesis of the (2S,3S,4S)-3,4-dihydroxyhomotyrosine amino acid segment, present in echinocandin B, in its activated form ready for peptide coupling is described. The key steps of the approach are the enantioselective AD reaction of 4-methoxycinnamic acid methyl ester, a completely diastereoselective [2 + 2] hydroxyketene-imine cycloaddition, and the TEMPO-assisted cycloexpansion of the resulting 3-hydroxy beta-lactam to the corresponding alpha-amino acid N-carboxy anhydride (NCA). The smooth opening of the latter upon treatment with L-Thr(OSi(t)BuPh(2))OMe and further acylation with the N-Cbz protected L-4-tert-butyldiphenylsilyloxy proline rendered the southwest portion of echinocandin B.

Aldehydes↗
PubMed2000

Cloning, expression and characterisation of a recombinant triosephosphate isomerase from Taenia solium.

We isolated and characterised the cDNA that encodes the glycolytic enzyme, triosephosphate isomerase from Taenia solium. A 450 bp DNA fragment was obtained by the polymerase chain reaction using a cDNA from larval stage as template and degenerate oligonucleotides designed from conserved polypeptide sequences from TPIs of several organisms. The fragment was used to screen a T. solium larval stage cDNA library. The isolated cDNA, encoding a protein of 250 amino acids shares 44.8-59.6% positional identity with other known TPIs, in which the catalytic enzyme residues were conserved. The complete coding sequence of the T. solium TPI cDNA was cloned into the expression vector pRSET and expressed as a fusion protein with an N-terminal tail of six histidine residues. The catalytic activity of the purified protein was similar to other TPI enzymes. Northern and Southern blot analysis suggest that in T. solium, single gene exists for triosephosphate isomerase and that the gene is expressed in all stages of the parasite.

Amino Acid Sequence↗
PubMed1999

Characterization of glutathione S-transferase of Taenia solium.

A Taenia solium glutathione-S-transferase fraction (SGSTF) was isolated from a metacestode crude extract by affinity chromatography on reduced glutathione (GSH)-sepharose. The purified fraction displayed a specific glutathione S-transferase (GST) activity of 2.8 micromol/min/mg and glutathione peroxidase selenium-independent activity of 0.22 micromol/min/mg. Enzymatic characterization of the fraction suggested that the activity was closer to the mammalian mu-class GSTs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and enzyme activity analysis showed that the fraction was composed of a major band of Mr = 26 kd and that the active enzyme was dimeric. Immunohistochemical studies using specific antibodies against the major 26-kd band of the SGSTF indicated that GST protein was present in the tegument, parenchyma, protonephridial, and tegumentary cytons of the T. solium metacestode. Antibodies generated against the SGSTF tested in western blot showed cross-reactivity against GSTs purified from Taenia saginata, T. taeniaeformis, and T. crassiceps, but did not react with GSTs from Schistosoma mansoni, or mice, rabbit, and pig liver tissue. Furthermore, immunization of mice with SGSTF reduced the metacestode burden up to 74.2%. Our findings argue in favor of GST having an important role in the survival of T. solium in its hosts.

Animals↗
PubMed1998

Molecular and functional characterization and tissue localization of 2 glucose transporter homologues (TGTP1 and TGTP2) from the tapeworm Taenia solium.

Tapeworms absorb and consume large quantities of glucose through their syncytial tegument, storing the excess as glycogen. Although some studies on the metabolism of glucose in several tapeworms are available, the proteins that mediate its uptake and distribution in their tissue have not been identified. We describe the isolation and characterization of cDNA clones encoding 2 facilitated diffusion glucose transporters (TGTP1 and TGTP2) from Taenia solium, the causal agent of human and porcine cysticercosis. Radio-isotope labelled hexose uptake mediated by TGTP1 expressed in Xenopus oocytes is inhibited by the natural stereoisomers D-glucose and D-mannose but not by L-glucose. Transport by TGTP1 is sensitive to classical inhibitors of facilitated diffusion such as phloretin and cytochalasin B, and insensitive to ouabain. TGTP2 did not function in Xenopus oocytes. Localization studies using specific anti-TGTP1 and anti-TGTP2 antibodies show that TGTP1 is abundant in a number of structures underlying the tegument in adult parasites and larvae, whereas TGTP2 appears to be localized only on the tegumentary surface of the larvae and is not detected in adults.

Amino Acid Sequence↗
PubMed1997

Sequencing, expression and properties of triosephosphate isomerase from Entamoeba histolytica.

We have isolated a cDNA clone of the glycolytic enzyme, triosephosphate isomerase (TPI) from Entamoeba histolytica. Degenerate oligonucleotides obtained by reverse translation of conserved polypeptide sequences, derived from TPIs of other organisms, were used to amplify a 450-bp fragment using E. histolytica cDNA as a template. The fragment was used to screen a cDNA library. The isolated cDNA, encoding a protein of 261 amino acids, shares 43-52.6% positional identity with other known protozoan TPIs. The catalytic residues were conserved; nevertheless, several indels occurred at other regions in the protein sequence. The complete coding sequence of the E. histolytica TPI gene was cloned into the expression vector pRSET and expressed as a wild-type TPI enzyme (E. histolytica TPI) and as a fusion protein with an N-terminal tail of six histidine residues E. histolytica TPI-His6); both recombinant proteins were purified. Molecular modeling of E. histolytica TPI showed an identical topology to the known structures of other TPI molecules, but with a remarkable feature; more than 10 inserted residues are located in the same region of the molecular surface. Studies were performed to detect possible changes that might be caused by the inserted amino acids. The catalytic activity and oligomeric state of the purified protein were similar to that reported for TPI from other sources. In contrast, stability towards dilution, as well as thermal inactivation and unfolding assays, showed that E. histolytica TPI is significantly more stable towards denaturation than Trypanosoma brucei TPI.

Amino Acid Sequence↗
PubMed1997

Cloning, expression, purification and characterization of triosephosphate isomerase from Trypanosoma cruzi.

The gene that encodes for triosephosphate isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for triosephosphate isomerase. The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that triosephosphate isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L. mexicana. Its circular dichroism spectrum was almost identical to that of triosephosphate isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent. The disposition of the cysteine in triosephosphate isomerase from T. cruzi appears to make it unique for inhibition by modification of its cysteine.

Amino Acid Sequence↗
PubMed1997

Mammary gland type I iodothyronine deiodinase is encoded by a short messenger ribonucleic acid.

Lactating rat mammary gland expresses a deiodinating activity that, on the basis of kinetic characteristics, corresponds to the so-called 5'-deiodinase type I (D1). In the present study we amplified and sequenced several D1 complementary DNA (cDNA) fragments from rat lactating mammary gland. The mammary cDNA was found to be identical to the previously reported rat liver cDNA in the coding region, but 465 nucleotides shorter on its 3'-untranslated region, suggesting that the D1 is the same in both tissues. D1 messenger RNA (mRNA) was also detected by reverse transcriptase-PCR in mammary glands from puberal and late pregnant rats, but not in virgin animals. Densitometric analysis showed a close and direct correlation between mRNA content and enzyme specific activity in mammary gland. Our results also show that rat liver contains both D1 mRNA forms and that the large form may respond to the thyroid status. These data suggest a differential and organ-specific expression of these mRNA forms, which could play a role in the functional regulation of D1 activity.

Animals↗
PubMed1994

Purification and ultrastructural localization of surface glycoproteins of Taenia solium (Cestoda) cysticerci.

A glycoprotein-enriched fraction was obtained by Concanavalin A-Sepharose 4B affinity chromatography from a crude extract of T. solium cysticerci. The six most prominent glycoproteins with molecular sizes of 180, 103, 96, 68, 55 and 45 kDa were purified by electro-elution from polyacrylamide gel slices. Ultrastructural localization assays using hyperimmune rabbit sera to each glycoprotein, demonstrated their presence on the tegumentary surface of the bladder wall of T. solium cysticerci. Similar studies showed that the 180 kDa glycoprotein is also present on the surface of the T. solium and T. saginata adult worms, as well as in T. saginata, T. pisiformis and T. crassiceps cysticerci. The 55 kDa glycoprotein, which is one of the most abundant on the cyst surface, was found to correspond to the heavy chain of pig IgG by Western blotting.

Animals↗
PubMed1994

Protein uptake by cysticerci of Taenia crassiceps.

The internalization of host macromolecules to the vesicular fluid of T. crassiceps cysticerci was studied in vitro. Uptake of purified class G immunoglobulin was not significantly affected by the specificity of its antigen-recognition site and bovine serum albumin was internalized at a similar rate. Internalization was inhibited at low temperature, being optimal at 37 degrees C and saturation was accomplished only at a protein concentration in the culture medium over 12 mg/ml which is close to the physiological concentration of serum proteins in the host. Morphological studies using markers for adsorptive endocytosis allowed visualization of endocytic vesicles and tracking of their movement across the bladder wall tissue. Degradation of internalized proteins was observed at longer times of incubation, suggesting that proteins are later processed and that degraded host macromolecules can be nutrients for cysticerci. Quantification of this capability of internalization suggests that it might play a role in the in vivo removal of potentially damaging host macromolecules, such as antibodies or complement factors, from the host-parasite interface.

Animals↗
PubMed1992

Alternative splicing of the Schistosoma mansoni gene encoding a homologue of epidermal growth factor receptor.

The complete coding DNA for a Schistosoma mansoni homologue of the epidermal growth factor receptor (SER) was characterized from cDNA clones obtained by homology to the tyrosine kinase domain of erbB. The DNA sequence predicts a 200-kDa translation product that contains a secretory leader, a cysteine-rich extracellular domain, a hydrophobic transmembrane sequence, and an intracellular tyrosine kinase domain. The SER transcript is present in cercariae and adult schistosomes. In addition to SER transcripts, schistosomes produce at least 3 variant transcripts encoding truncated SER products that include the secretory leader and a small portion of the extracellular domain followed by short sequences of unrelated, C-terminal amino acids. Based on these sequences, 2 of the variant mRNAs (class 2 and 5) appear to encode soluble, secreted proteins while one (class 4) encodes an SER variant protein with a hydrophobic C-terminus that may serve as a membrane anchor. Class 2 SER variant transcripts are present at levels comparable to SER transcripts in adult worms but are not detected in cercariae. Class 4 and 5 SER variant transcripts are also found within adult worms but at lower levels. Genomic cloning and characterization demonstrate that the variant SER transcripts arise through alternative splicing of the SER gene.

Amino Acid Sequence↗