Plasmodium falciparum proteinases: cloning of the putative gene coding for the merozoite proteinase for erythrocyte invasion (MPEI) and determination of hydrolysis sites of spectrin by Pf37 proteinase

Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI), involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.

Trypanosoma cruzi ribosomal protein S4: characterization of its coding locus, analysis of transcripts, and antigenicity of the protein

Two allelic genomic fragments containing ribosomal protein S4 encoding genes (rpS4) from Trypanosoma cruzi (CL-Brener strain) were isolated and characterized. One allele comprises two complete tandem repeats of a sequence encoding an rpS4 gene. In the other, only one rpS4 gene is found. Sequence comparison to the accessed data in the genome project database reveals that our two-copy allele corresponds to a variant haplotype. However, the deduced aminoacid sequence of all the gene copies is identical. The rpS4 transcripts processing sites were determined by comparison of genomic sequences with published cDNA data. The obtained sequence data demonstrates that rpS4 genes are expressed in epimastigotes, amastigotes, and trypomastigotes. A recombinant version of rpS4 was found to be an antigenic: it was recognized by 62.5% of the individuals with positive serology for T. cruzi and by 93.3% of patients with proven chronic chagasic disease.

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

A 37-kb restriction map of the human immunoglobulin lambda variable locus, VB cluster, harboring four functional genes and two non-coding Vl sequences

The human immunoglobulin lambda variable locus (IGLV) is mapped at chromosome 22 band q11.1-q11.2. The 30 functional germline v-lambda genes sequenced untill now have been subgrouped into 10 families (Vl1 to Vl10). The number of Vl genes has been estimated at approximately 70. This locus is formed by three gene clusters (VA, VB and VC) that encompass the variable coding genes (V) responsible for the synthesis of lambda-type Ig light chains, and the Jl-Cl cluster with the joining segments and the constant genes. Recently the entire variable lambda gene locus was mapped by contig methodology and its one- megabase DNA totally sequenced. All the known functional V-lambda genes and pseudogenes were located. We screened a human genomic DNA cosmid library and isolated a clone with an insert of 37 kb (cosmid 8.3) encompassing four functional genes (IGLV7S1, IGLV1S1, IGLV1S2 and IGLV5a), a pseudogene (VlA) and a vestigial sequence (vg1) to study in detail the positions of the restriction sites surrounding the Vl genes. We generated a high resolution restriction map, locating 31 restriction sites in 37 kb of the VB cluster, a region rich in functional Vl genes. This mapping information opens the perspective for further RFLP studies and sequencing

A novel polymorphism in the coding region of the vasopressin type 2 receptor gene

Nephrogenic diabetes insipidus (NDI) is a rare disease characterized by renal inability to respond properly to arginine vasopressin due to mutations in the vasopressin type 2 receptor (V2(R)) gene in affected kindreds. In most kindreds thus far reported, the mode of inheritance follows an X chromosome-linked recessive pattern although autosomal-dominant and autosomal-recessive modes of inheritance have also been described. Studies demonstrating mutations in the V2(R) gene in affected kindreds that modify the receptor structure, resulting in a dys- or nonfunctional receptor have been described, but phenotypically indistinguishable NDI patients with a structurally normal V2(R) gene have also been reported. In the present study, we analyzed exon 3 of the V2(R) gene in 20 unrelated individuals by direct sequencing. A C®T alteration in the third position of codon 331 (AGC®AGT), which did not alter the encoded amino acid, was found in nine individuals, including two unrelated patients with NDI. Taken together, these observations emphasize the molecular heterogeneity of a phenotypically homogeneous syndrome

Comparative sequence analysis of the P-, M- and L-coding region of the measles virus CAM-70 live attenuated vaccine strain

Measles virus is a highly contagious agent which causes a major health problem in developing countries. The viral genomic RNA is single-stranded, nonsegmented and of negative polarity. Many live attenuated vaccines for measles virus have been developed using either the prototype Edmonston strain or other locally isolated measles strains. Despite the diverse geographic origins of the vaccine viruses and the different attenuation methods used, there was remarkable sequence similarity of H, F and N genes among all vaccine strains. CAM-70 is a Japanese measles attenuated vaccine strain widely used in Brazilian children and produced by Bio-Manguinhos since 1982. Previous studies have characterized this vaccine biologically and genomically. Nevertheless, only the F, H and N genes have been sequenced. In the present study we have sequenced the remaining P, M and L genes (approximately 1.6, 1.4 and 6.5 kb, respectively) to complete the genomic characterization of CAM-70 and to assess the extent of genetic relationship between CAM-70 and other current vaccines. These genes were amplified using long-range or standard RT-PCR techniques, and the cDNA was cloned and automatically sequenced using the dideoxy chain-termination method. The sequence analysis comparing previously sequenced genotype A strains with the CAM-70 Bio-Manguinhos strain showed a low divergence among them. However, the CAM-70 strains (CAM-70 Bio-Manguinhos and a recently sequenced CAM-70 submaster seed strain) were assigned to a specific group by phylogenetic analysis using the neighbor-joining method. Information about our product at the genomic level is important for monitoring vaccination campaigns and for future studies of measles virus attenuation.

Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%), an extended strand (17.13%), a ß turn (5.61%), and a random coil (43.30%). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

Comparative evaluation of underlying causes of death processed by the Automated Classification of Medical Entities and the Underlying Cause of Death Selection Systems

INTRODUCTION: The correct identification of the underlying cause of death and its precise assignment to a code from the International Classification of Diseases are important issues to achieve accurate and universally comparable mortality statistics These factors, among other ones, led to the development of computer software programs in order to automatically identify the underlying cause of death. OBJECTIVE: This work was conceived to compare the underlying causes of death processed respectively by the Automated Classification of Medical Entities (ACME) and the "Sistema de Seleção de Causa Básica de Morte" (SCB) programs. MATERIAL AND METHOD: The comparative evaluation of the underlying causes of death processed respectively by ACME and SCB systems was performed using the input data file for the ACME system that included deaths which occurred in the State of S. Paulo from June to December 1993, totalling 129,104 records of the corresponding death certificates. The differences between underlying causes selected by ACME and SCB systems verified in the month of June, when considered as SCB errors, were used to correct and improve SCB processing logic and its decision tables. RESULTS: The processing of the underlying causes of death by the ACME and SCB systems resulted in 3,278 differences, that were analysed and ascribed to lack of answer to dialogue boxes during processing, to deaths due to human immunodeficiency virus [HIV] disease for which there was no specific provision in any of the systems, to coding and/or keying errors and to actual problems. The detailed analysis of these latter disclosed that the majority of the underlying causes of death processed by the SCB system were correct and that different interpretations were given to the mortality coding rules by each system, that some particular problems could not be explained with the available documentation and that a smaller proportion of problems were identified as SCB errors. CONCLUSION: These results, disclosing a very low and insignificant number of actual problems, guarantees the use of the version of the SCB system for the Ninth Revision of the International Classification of Diseases and assures the continuity of the work which is being undertaken for the Tenth Revision version.

Central nervous system involvement in Chagas' disease: an updating

A review was made of the available literature on central nervous system (CNS) involvement in Chagas' disease. Thirty-one works concerning the acute nervous form and 17 others dealing with the chronic nervous form, all presenting neuropathologic studies, were critically analysed. Based on this analysis, an attempt was made to establish the possible natural history of CNS involvement in Chagas' disease. Among others, the following facts stand out: 1) the initial, acute phase of Trypanosoma cruzi infection is usually asymptomatic and subclinical; 2) only a small percentage of cases develop encephalitis in the acute phase of Chagas' disease; 3) the symptomatic acute forms accompanied by chagasic encephalitis are grave, with death ensuing in virtually all cases as a result of the brain lesions per se or of acute chagasic myocarditis, this being usually intense and always present; 4) individuals with the asymptomatic acute form and with the mild symptomatic acute form probably have no CNS infection or, in some cases, they may have discrete encephalitis in sparse foci. In the latter case, regression of the lesions may be total, or residual inflammatory nodules of relative insignificance may persist. Thus, no anatomical basis exists that might characterize the existence of a chronic nervous form of Chagas' disease; 5) reactivation of the CNS infection in the chronic form of Chagas' disease is uncommon and occurs only in immunosuppressed patients.

Immune responses induced by a Leishmania (Leishmania) amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-g, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasis

Genotype distribution of the GB virus C in citizens of São Paulo City, Brazil

There has been several studies worldwide on phylogenetics and genotype distribution of the GB-virus C / Hepatitis G virus (GBV-C/HGV). However, in their great majority, those investigations were based on some epidemiologically linked group, rather than on a representative sampling of the general population. The present is a continuation of the first study in Brazil with such a population; it addresses the GBV-C/HGV phylogenetics and genotype distribution based on samples identified among more than 1,000 individuals of the city of São Paulo. For this purpose, a 728 bp fragment of the 5´non-coding region (5´NCR) of the viral genome, from 24 isolates, was sequenced and subjected to phylogenetic analysis. Genotypes 1, 2a and 2b were found at 8.3% (2/24), 50% (12/24) and 41.7% (10/24), respectively. In conclusion São Paulo displays a genotype distribution similar to the published data for other States and Regions of Brazil, endorsing the notion that types 1 and 2 would have entered the country with African and European people, respectively, since its earliest formation.

Virulence factors of uropathogenic Escherichia coli from a University Hospital in Ribeirão Preto, São Paulo, Brazil

The aim of the study was to determine the occurrence of virulence genes expressing fimbriae, production of hemolysin, colicin and aerobactin among a hundred Escherichia coli isolates obtained from in-and outpatients of a tertiary-care teaching hospital, between July and August 2000, showing clinical and laboratory signs of urinary tract infection (UTI). The presence of genes (pap, afa, sfa) for fimbriae expression was assayed using specific primers in a polymerase chain reaction. Among the isolates studied, the prevalence of the virulence factors was 96.0%, 76.0%, 24.0%, for hemolysin, aerobactin and colicin, respectively; the prevalence of genes coding for fimbrial adhesive systems was 32.0%, 19.0% and 11.0% for pap, sfa and afa respectively. The strains isolated from the outpatients displayed a greater number of virulence factors compared to those from hospitalized subjects, emphasizing the difference between these two kinds of patients.

Candida esophagitis: species distribution and risk factors for infection

Although Candida albicans is the main cause of fungal esophagitis, other species such as C. tropicalis, C. krusei and C. stellatoidea have also been implicated. Several studies have identified risk factors for C. albicans esophagitis. However, data for non-C. albicans species is still sparse. The aim of this study was to determine the etiology of Candida esophagitis in our medical centre over an 18-month period. Additionally, we aimed to investigate predisposing conditions for esophageal candidosis caused by different Candida species. A total of 21,248 upper gastroscopies were performed in Santa Casa Complexo Hospitalar between January 2005 and July 2006. The prevalence of Candida esophagitis was 0.74% (n = 158). C. albicans caused the vast majority of infections (96.2%), followed by C. tropicalis (2.5%), C. lusitaniae (0.6%) and C. glabrata (0.6%). There were 81 women (51.3%) and 77 men (48.7%). No case of mixed infection occurred. Concomitant oral candidosis was documented for 10.8% (n = 17). Most of cases (55.1%) involved outpatients. Around one fifth of patients in our cohort had no identifiable risk factors for esophageal candidosis (20.8%). Since nearly all infections were caused by C. albicans we were not able to determine risk factors for esophagitis caused by other Candida species.

Molecular characterization of Cryptosporidium spp. from HIV infected patients from an urban area of Brazil

Cryptosporidium spp. are important cause of enteric disease in humans, but may also infect animals. This study describes the relative frequency of several Cryptosporidium species found in human specimens from HIV infected patients in the São Paulo municipality obtained from January to July 2007. Sequence analysis of the products of nested-PCR based on small subunit rRNA and Cryptosporidium oocyst wall protein coding genes revealed 17 (63.0%) isolates of C. hominis, four (14.8%) C. parvum, five (18.5%) C. felis and one (3.7%) C. canis. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis whereas the anthroponotic transmission of cryptosporidiosis caused by C. hominis seems to predominate.