PCR-RFLP analysis: a promising technique for host species identification of blood meals from tsetse flies (Diptera: Glossinidae)

A polymerase chain reaction with the restriction fragment length polymorphism (PCR-RFLP) method using universal primers complementary to the conserved region of the cytochrome b gene (cyt b) of the mitochondrion DNA (mtDNA) of vertebrates was applied to the identification of the origin of blood meals in tsetse flies. Blood samples from ten potential tsetse hosts of the family bovidae (cattle, water buffalo, red buffalo, waterbuck, springbok, goat, sheep, sable antelope, oryx and dik-dik) were included in this study. Sites for appropriate restriction endonucleases cuts were chosen by pairwise alignment of the amplified 359 bp fragments. A flow chart of endonucleases digestion using three restriction enzymes (e.g. TaqI, AluI and HindII) for the unequivocal identification of the respective bovid species was developed. A number of additional non-specific DNA fragments attributed to the co-amplification of cytochrome b pseudogenes were observed in some species (e.g. in red buffalo and dik-dik after digestion with AluI) but did not hamper assignment of bovid species. The detection rate of host DNA in tsetse by PCR-RFLP was 100, 80, 60 and 40% at 24, 48, 72 and 96 h after in vitro feeding, respectively. Identification of the last blood meal was possible even when tsetse had previously fed on different hosts.     

Rapid Identification and Typing of Staphylococcus aureus by PCR-Restriction Fragment Length Polymorphism Analysis of the aroA Gene

The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when other Staphylococcus spp. or genera were analyzed by using the same pair of primers. The detection limit for S. aureus cells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 × 10² CFU) when S. aureus cells were suspended in sterilized whole milk. TaqI digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.     

Passive Immunization with Antibodies against Three Distinct Epitopes on Plasmodium yoelii Merozoite Surface Protein 1 Suppresses Parasitemia

We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an ~42-kDa fragment (MSP-1₄₂) that is derived from the C terminus of MSP-1. MSP-1₄₂ is further cleaved to an N-terminal ~33-kDa polypeptide (MSP-1₃₃) and a C-terminal ~19-kDa polypeptide (MSP-1₁₉) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-1₄₂ but not with either of the constituents MSP-1₃₃ and MSP-1₁₉, B4 recognized an epitope within the N terminus of MSP-1₃₃, and B6, B10, F5, and G3 bound to MSP-1₁₉. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes on P. yoelii MSP-1 are recognized by antibodies that suppress parasitemia in vivo.     

Regulatory role of Cdx-2 and Taq I polymorphism of vitamin D receptor gene on chemokine expression in pulmonary tuberculosis

Vitamin D receptor (VDR) gene variants have been shown to be regulating the immune response in tuberculosis. We studied the regulatory role of VDR promoter Cdx-2 and 3′UTR TaqI gene variants on chemokine levels from culture filtrate antigen (CFA) stimulated with or without 1,25(OH)₂D₃ treated peripheral blood mononuclear cells of 50 pulmonary tuberculosis patients (PTB) and 51 normal healthy controls (HCs). In CFA with 1,25(OH)₂D₃ treated cultures, the MIP-1α, MIP-1β, RANTES levels were significantly decreased in Cdx-2 AA genotype compared to GG genotype, while a significantly increased MIG level was observed in Cdx-2 AA genotype (p < 0.05). In TaqI polymorphism, tt genotype significantly decreased MIP-1β and RANTES levels compared to TT genotype. Moreover, a significantly increased level of IP-10 and MIG was observed in TaqI tt genotype compared with TT genotype (p < 0.05). The results suggests that the 1,25(OH)₂D₃ may alter the chemokine response through the VDR polymorphic variants during infection.     

Association between vitamin D receptor polymorphisms and multiple sclerosis: systematic review and meta-analysis of case–control studies

Vitamin D receptor (VDR) polymorphisms have been studied as potential contributors to multiple sclerosis (MS). However, published studies differ with respect to study design and the significance of the effects detected. The aim of this study was to quantify the magnitude of the risk associated with the TaqI, BsmI, ApaI and FokI VDR polymorphisms in MS using a meta-analysis approach. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, we conducted a systematic search and meta-analysis of the literature. Subgroup analyses were performed to detect potential sources of heterogeneity from the selected study characteristics. The stability of the summary risk was evaluated using sensitivity analyses. The meta-analysis included a total of 3300 cases and 3194 controls from 13 case–control studies. There were no significant associations found between TaqI and BsmI polymorphisms and MS risk. The association between the ApaI polymorphism and MS risk was significant in the homozygous and codominant models (P=0.013 and P=0.031, respectively), suggesting that the AA ApaI genotype might be a significant MS risk factor. Publication year and age significantly affected the association between TaqI polymorphisms and MS (P=0.014 and P=0.010, respectively), which indicates a protective effect of the major T allele. The AA ApaI and FF FokI genotypes are significant risk factors for MS. The association between the TaqI polymorphism and MS risk is significantly affected by study characteristics.     

FAS −670 A/G polymorphism may be associated with the depletion of CD4+ T lymphocytes in HIV-1 infection

In this study, the polymorphisms in the FAS and FASL genes was investigated in a sample of 198 HIV-1-seropositive individuals and 191 seronegative controls to evaluate a possible association between polymorphisms and the infection. The identification of the A and G alleles of the FAS −670 polymorphism was accomplished through polymerase chain reaction assays followed by digestion with the restriction enzyme MvaI. The identification of the A and G alleles of the FAS −124 polymorphism and the T and delT alleles of the FAS −169 polymorphism were performed using the amplification-created restriction site method followed by restriction fragment length polymorphism reactions. The comparative analysis of allelic and genotypic frequencies between the groups did not reveal any significant differences. However, the quantitative analysis of CD4⁺ T lymphocytes suggests that the G allele of the FAS −670 A/G polymorphism can be a protective factor against the depletion of these cells in the course of an HIV-1 infection. Polymorphisms in the FAS and FASL genes were not associated with the number of CD8⁺ T lymphocytes or the plasma viral load. Our findings suggest that the FAS −670 polymorphism may be associated with apoptosis of CD4⁺ T lymphocytes after infection by HIV-1.     

Association of vitamin D receptor genotypes with early onset rheumatoid arthritis

The presence of certain vitamin D receptor (VDR) genotypes has been associated with low bone mineral density (BMD) in elderly populations as well as with accelerated bone loss in patients with rheumatoid arthritis (RA). In the present study, VDR genotypes from 120 Spanish patients with RA were investigated. Three VDR gene polymorphisms (BsmI, ApaI and TaqI) were investigated using polymerase chain reaction followed by enzymatic digestion. The distributions of VDR allelic frequencies were similar in patients and controls and therefore no influence of VDR polymorphisms on rheumatoid arthritis susceptibility could be demonstrated. However, in an analysis of the clinical features of the different VDR‐related genetic subgroups, the BB/tt genotype, defined by the BsmI and TaqI restriction site polymorphisms, was identified to be weakly associated with an early onset RA in female patients. This VDR genotype has been associated with a low BMD level in various studies. When patients were stratified according to the presence of the shared HLA epitope SE, it was found that SE + female patients bearing the BB/tt genotype showed the earliest disease onset. The mechanisms by which the VDR polymorphism is associated with RA is unknown, but they could be related to the immunoregulatory properties of vitamin D.     

Major Antigenic Proteins of the Agent of Human Granulocytic Ehrlichiosis Are Encoded by Members of a Multigene Family

Western blot analysis of proteins from a cell culture isolate (USG3) of the human granulocytic ehrlichiosis (HGE) agent has identified a number of immunoreactive proteins, including major antigenic proteins of 43 and 45 kDa. Peptides derived from the 43- and 45-kDa proteins were sequenced, and degenerate PCR primers based on these sequences were used to amplify DNA from USG3. Sequencing of a 550-bp PCR product revealed that it encodes a protein homologous to the MSP-2 proteins of Anaplasma marginale. Concurrently, an expression library made from USG3 genomic DNA was screened with granulocytic Ehrlichia (GE)-positive immune sera. Analysis of two clones showed that they contain one partial and three full-length highly related genes, suggesting that they are part of a multigene family. Amino acid alignment showed conserved amino- and carboxy-terminal regions which flank a variable region. The conserved regions of these proteins are also homologous to the MSP-2 proteins of A. marginale; thus, they were designated GE MSP-2A (45 kDa), MSP-2B (34 kDa), and MSP-2C (38 kDa). The PCR fragment obtained as a result of peptide sequencing was completely contained within the msp-2A clone, and all of the sequenced peptides were found in the GE MSP-2 proteins. Recombinant MSP-2B protein and an MSP-2A fusion protein were expressed in Escherichia coli and reacted with human sera positive for the HGE agent by immunofluorescence assay. These data suggest that the 43- and 45-kDa proteins of the HGE agent are encoded by members of the GE MSP-2 multigene family.     

Conservation of Restriction Sites in Isolates of Streptococcus pneumoniae with Diverse Restriction Fragment Patterns

Separation of large restriction fragments by pulsed-field gel electrophoresis is a commonly used method for epidemiological typing of Streptococcus pneumoniae and many other bacterial species. Information on the genetic changes underlying the restriction fragment polymorphisms that allow discrimination between isolates is scarce. In this study fragments adjacent to ApaI sites in a clinical isolate of S. pneumoniae were cloned and used to probe HindIII and HindIII-plus-ApaI genomic DNA digests from other isolates with very different ApaI fragment patterns. If for a given isolate the HindIII fragment detected by the probe was reduced in size on digestion with ApaI, it was deduced that the ApaI site was conserved in that isolate. The results demonstrate that of six ApaI sites in PN93/908 examined, five were retained in 11 genetically different isolates and one was retained in 2 isolates but lost in 9 others. It was concluded that point mutations at restriction sites are unlikely to account for the restriction fragment length polymorphism observed and that much of the polymorphism may be due to DNA rearrangements, possibly resulting from the insertion or deletion of mobile DNA elements.     

The maxicircle of Trypanosoma brucei kinetoplast DNA hybridizes with a mitochondrial gene encoding cytochrome oxidase subunit II

A restriction endonuclease fragment of the maxicircle of Trypanosoma brucei brucei kinetoplast DNA hybridizes with a cloned mitochondrial DNA sequence which encodes cytochrome oxidase subunit II of Zea mays. A cloned mitochondrial DNA sequence encoding cytochrome oxidase subunit II of Saccharomyces cerevisiae also hybridizes with kDNA, but exhibits less homology with the maxicircle than does the maize gene. The hybridizing maxicircle DNA was localized to a 2.8 kbp segment which is bounded by TaqI restriction endonuclease sites and nearby HindIII and EcoRI restriction sites. The TaqI restriction fragment is conserved between T. brucei brucei, T. brucei rhodesiense and T. brucei gambiense and hybridizes with the Zea mays probe in each case.     

Clinical,Pathological, and Molecular Studies of Two Families with Iodide Organification Defect

Two unrelated families (CA and NA) in which an iodide organification defect (IOD) was present in two siblings of each family were studied. These patients had congenital goiters with hypothyroidism and a positive perchlorate discharge test. Examination of the thyroid tissue revealed no thyroid peroxidase (TPO) activity. Histologic findings were consistent with a microfollicular pattern of hyperplasia. Moderate cellular atypia was present, characterized by nuclear pleomorphism and hyperchromatism. Full length thyroglobulin was purified by gel filtration, but was not iodinated. Immunohistochemical studies using a polyclonal anti-human thyroid peroxidase (hTPO) antibody confirmed the presence of immunoreactive TPO protein in the thyroid tissues. Samples of normal and affected individuals were studied with respect to the presence of various fragments using TPO probes of varying sizes. The two affected siblings from family CA were homozygous for fragments 3.9, 4.6, and 7.0 kb (Bg/II) and 2.3 and 2.9 kb (Taql), whereas the parents were heterozygous. In the other family (NA), theBg/II digestion and TPO-31 hybridization revealed an interesting and informative polymorphism. The parents showed two different polymorphic patterns: the father had a 5.0/4.6 kb pattern and the mother a 4.7/4.5 kb pattern. However, the two affected siblings showed the same heterozygotic allelic pattern at 4.5/4.6 kb. The restriction fragment length polymorphism detected in these two families suggests an association between the TPO gene and an IOD. Results suggest that in these dyshormonogenetic tissues an altered TPO protein molecule is being synthesized, without detectable in vitro activity, but visible by immunostaining techniques in the goitrous tissue. Mutations in the TPO gene sequence are most likely associated with these changes.     

CD4+ T-Lymphocyte and Immunoglobulin G2 Responses in Calves Immunized with Anaplasma marginale Outer Membranes and Protected against Homologous Challenge

Protective immunity against the ehrlichial pathogen Anaplasma marginale has been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain of A. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both whole A. marginale homogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4⁺ T cells, and CD4⁺ T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response to A. marginale and produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinct A. marginale strains, and Anaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes against A. marginale challenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.     

Molecular Genetic Characterization of the Merozoite Surface Protein 1 Gene of Plasmodium vivax from Reemerging Korean Isolates

Plasmodium vivax merozoite surface protein 1 (PvMSP-1) has been considered a major candidate for the development of an antimalaria vaccine, but the molecule exhibits antigenic diversity among isolates. The extent of genetic polymorphism in the region between interspecies conserved blocks 4 and 5 (ICB4 and ICB5) of the PvMSP-1 gene was analyzed for 30 Korean isolates. Two genotypes, SK-A and SK-B, were identified on the basis of amino acid substitution. Almost all the amino acid sequences of the Korean isolates were nearly identical to those of the Solomon Island isolate Solo-83 (97.8 to 99.9% similarity) and Philippine isolates Ph-79, Ph-52-2, and Ph-49 (97.3 to 99.8% similarity). Also, we report two sequences in the isolates that were characterized on the basis of restriction fragment length polymorphism (RFLP). The RFLP profiles following digestion with the DraI restriction enzyme produced two distinguishable patterns. This study might be the first report of the region between ICB4 and ICB5 of the MSP-1 gene of P. vivax in South Korea.Plasmodium vivax malaria represents a major public health problem for many tropical and subtropical countries, which has been exacerbated by the expansion of drug-resistant strains (2, 10, 29, 30). The enormous toll of mortality caused by Plasmodium falciparum has tended to overshadow the public health importance of P. vivax. For this reason and on account of technical difficulties, relatively little investigation has been done toward the development of a vaccine against P. vivax (27). One of the major problems in vaccine development is the antigenic diversity of the vaccine candidates. The critical emerging problem is that the host response to one allele is not very effective against parasites expressing different allelic forms (8, 25). Therefore, genetic variation studies for the antigens of vaccine candidates are very important for P. falciparum and P. vivax. The polymorphism of potential malaria vaccine targets is rather greater for P. vivax than for P. falciparum. Also, the growing resistance of P. vivax strains to chloroquine is spurring the development of a vaccine against P. vivax malaria.The study of polymorphism is important not only for establishing the antigenic repertoire of isolates from regions where malaria is endemic but also for elucidating the mechanisms by which antigenic diversity is generated. The WHO declared in 1979 that malaria had been eradicated in Korea, but in 1993 (4), a case of malaria in a soldier working in the Demilitarized Zone (the border area between North and South Korea) of the Republic of Korea was reported. After 1993, the number of malaria cases expanded exponentially each year, with 3,932 patients diagnosed in 1998 (15). Current epidemiological results suggest that the malaria that has reemerged did not originate from overseas. All indigenous cases of malaria are due to P. vivax, with the occasional imported case of P. falciparum. However, the genetic characteristics of the reemergent Korean strain are not known at present.In previous studies, P. vivax circumsporozoite protein (13), P. vivax Duffy binding protein (12), and P. vivax apical membrane antigen 1 (6) showed genotypes with at least two new phenotypes among Korean isolates. However, the extent of genetic diversity of Korean P. vivax isolates is not accurately known at present, due to the fact that very few polymorphic markers are available for studying P. vivax.P. vivax merozoite surface protein 1 (PvMSP-1) is a well-characterized antigen whose diversity is maintained by host immune selection pressure (20). There is extensive allelic diversity of MSP-1 among isolates (5, 11, 19, 21), and this polymorphism may hamper the development of an effective vaccine against malaria. The polymorphism of PvMSP-1 has been considered to result from interallelic recombination in nature (20). Although the polymorphism of P. falciparum MSP-1 is well characterized, little is known about P. vivax MSP-1. To contribute useful information regarding genetic diversity and to facilitate rational vaccine design, the polymorphism of PvMSP-1 in Korean isolates was investigated in this study. In addition, we also describe the molecular phylogenetic characteristics of Korean P. vivax isolates.     

The Repertoire of Anaplasma marginale Antigens Recognized by CD4+ T-Lymphocyte Clones from Protectively Immunized Cattle Is Diverse and Includes Major Surface Protein 2 (MSP-2) and MSP-3

Major surface proteins of Anaplasma marginale are vaccine candidates. We recently demonstrated that immunization of calves with outer membranes of the Florida strain of A. marginale resulted in protective immunity that correlated with a memory CD4⁺ T-lymphocyte response specific for major surface protein 1 (MSP-1), MSP-2, and MSP-3 (W. C. Brown, V. Shkap, D. Zhu, T. C. McGuire, W. Tuo, T. F. McElwain, and G. H. Palmer, Infect. Immun. 66:5406–5413, 1998). As immunogens, these proteins have been shown to induce complete or partial protection against homologous challenge. To further define the T helper (Th) cell response to these and other A. marginale antigens and to determine conservation of Th cell epitopes among genetically distinct A. marginale strains, Th cell clones obtained prior to challenge from three immunized calves were characterized for antigen-specific responses. Nine distinct antigenic profiles were defined by 11 Th cell clones derived by stimulation with the Florida strain. Several clones responded to MSP-2, MSP-3, or both. All of these MSP-2- or MSP-3-specific clones and the majority of other clones that did not respond to MSPs recognized all bovine blood-passaged strains of A. marginale. These results demonstrate conservation of certain Th cell epitopes between MSP-2 and MSP-3 and show that Th cell epitopes in MSP-2, MSP-3, and undefined antigens are conserved among strains of A. marginale. Of seven clones that responded to the blood-passaged Virginia strain, two did not recognize antigen prepared from this strain cultured in tick cells, suggesting differences in the antigenic composition between these stages. Analysis of the cytokines expressed by the Th cells revealed that all clones expressed gamma interferon and tumor necrosis factor alpha, and most coexpressed interleukin-4. Our results provide a rationale for identifying Th cell epitopes conserved among different strains of A. marginale for inclusion in a nucleic acid or recombinant protein vaccine.     

The genetic affinity of Polynesians: evidence from Y chromosome polymorphisms

Y-linked polymorphisms were studied in a sample of 60 Polynesians, and results were compared with findings from studies on other major population groups. Three previously unreported 49a/TaqI haplotypes were observed, two of which possess a new polymorphic fragment named 12. Frequency data for the 49a/TaqI, XY275, pDP31 and Y Alu polymorphisms indicate that Polynesians have greater affinity to Caucasoids than to African populations. Similar population frequency trends were not observed for the p21A1/TaqI polymorphism, supporting the hypothesis that this polymorphism has arisen more than once.     

Hypervariable polymorphism of autosomal origin detected by the Y-chromosome derived probe,pHY10

A recombinant DNA probe (pHY10) hybridizing specifically to human DNA family DYZ1, 3,000 copies of which are present on the long arm of the Y chromosome, was used for probing human genome DNA digested with various restriction enzymes. To our surprise, the probe detected a hypervariable polymorphism of autosomal origin in human DNA when digested withTaqI. None of other 12 restriction enzymes revealed polymorphic patterns. Codominant segregation of the polymorphism was established in family studies. This probe has been widely used in the detection of the Y chromosome. Its ease of availability as well as highly discriminating polymorphic pattern makes it potentially very useful for forensic and human genetic purposes.     

Differentiation of Candida glabrata, C. nivariensis and C. bracarensis based on fragment length polymorphism of ITS1 and ITS2 and restriction fragment length polymorphism of ITS and D1/D2 regions in rDNA

Different molecular methods for the discrimination of Candida glabrata, C. bracarensis and C. nivariensis were evaluated and the prevalence of these species among Danish blood isolates investigated. Control strains were used to determine fragment length polymorphism in the ITS1, ITS2, ITS1-5.8S-ITS2 regions and in the D1/D2 domain of 26S rDNA using primers designed for this study. A total of 133 blood isolates previously identified as C. glabrata were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the peptide nucleic acid-fluorescent in situ hybridization (PNA-FISH) method. The size of ITS1 allowed differentiation between C. glabrata (483), C. nivariensis (361) and C. bracarensis (385), whereas the ITS2 region was of similar size in C. nivariensis (417) and C. glabrata (418). Sequence analysis of the ITS region suggested that many restriction enzymes were suitable for RFLP differentiation of the species. Enzymatic digestion of the D1/D2 domain with TatI produced unique band sizes for each of the three species. PCR-RFLP and PNA-FISH were in agreement for all of the isolates tested. None of the 133 Danish blood isolates were C. nivariensis or C. bracarensis. Fragment size polymorphism of ITS1 and RFLP of the D1/D2 domain or the ITS region are useful methods for the differentiation of the species within the C. glabrata group. C. bracarensis and C. nivariensis are rare among Danish C. glabrata blood isolates.     

Plasmodium falciparum: a comparative analysis of the genetic diversity in malaria-mesoendemic areas of Brazil and Madagascar

For a better definition of the polymorphic features of Plasmodium falciparum parasite populations, the polymerase chain reaction (PCR) typing technique was used to investigate the genetic diversity and complexity of parasites harbored by acute P. falciparum carriers from three yet unexplored malaria-mesoendemic areas with different transmission levels: two localities in northwestern Brazil (Ariquemes and Porto Velho) and a village in Madagascar (Ankazobe). A total of 89 DNA samples were analyzed by amplification of polymorphic domains from genes encoding merozoite surface antigens 1 and 2 (MSP-1, MSP-2) and thrombospondin-related anonymous protein (TRAP) and by hybridization with allelic-family-specific probes or random-fragment-length polymorphism (RFLP). In all three localities, extensive polymorphism was observed for each marker, but the MSP-2 central repeat was the most diverse one. Similar levels of genetic diversity, allelic frequency, and infection complexity were observed in the two Brazilian localities, although the isolates had been sampled at 2-year intervals, suggesting the stability of the infecting parasite populations presenting in these regions of the Brazilian Amazon. Unexpectedly, although the entomologic inoculation rate was at least 3 times lower in Ankazobe than in the Brazilian areas, Malagasi samples appeared more complex than the Brazilian ones. The implications of these data with regard to parasite population-dynamics studies are discussed. Received: 5 September 1999 / Accepted: 1 October 1999     

The 8-hydroxydeoxyguanosine concentrations according to hormone therapy and S326C polymorphism of OGG1 gene in postmenopausal women

Background

The 8-hydroxydeoxyguanosine (8-OHdG) is widely used for determination of DNA damage since it is excised from oxidative damaged DNA with endonuclease repair enzymes coded by 8-oxoguanine DNA N-glycosylase gene (OGG1). The present study aimed at investigating whether hormone therapy (HT) may influence on the blood/urinary 8-OHdG levels and whether the level of 8-OHdG is different according to OGG1 S326C polymorphism in postmenopausal women receiving HT.

Methods

In 102 postmenopausal women receiving HT, the 8-OHdG levels were measured in the blood and urine using high performance liquid chromatography (HPLC) before HT and 3 months after HT. The genotyping of the S326C polymorphism of the OGG1 was performed by polymerase chain reaction (PCR) and restriction enzyme fragment length polymorphism (RFLP) analysis.

Results

After HT, mean blood 8-OHdG level significantly decreased compared to those before HT (P = 0.003), while urinary 8-OHdG level did not show any difference according to HT. Pre-HT level of 8-OHdG was not different according to OGG1 genotypes and similar finding was demonstrated in post-HT 8-OHdG concentration.

Conclusions

These findings imply that hormone therapy can reduce blood 8-OHdG concentration, one of the markers of oxidative damage. Further study is needed to confirm this association in larger population.     

Lack of association between TaqI A1 allele of dopamine D2 receptor gene and alcohol-use disorders in Atayal natives of Taiwan

Association studies between the A1 allele of the dopamine D2 receptor (DRD2) gene TaqI A polymorphism and alcoholism remain controversial. A recent study from Japan demonstrated that the A1 allele is associated with severe alcoholism in the Japanese population. We were interested in knowing if this association also exists in the Atayals of Taiwan, who were found to have a higher prevalence of alcohol-use disorders than the Han Chinese in Taiwan. Genotype and allele frequencies were determined in alcohol-abusing, alcohol-dependent, and nonalcoholic control Atayal natives in Taiwan. A1 allele frequencies in alcohol-dependent, alcohol-abusing, and normal control Atayals were 0.39, 0.42, and 0.39, respectively. No difference in A1 allele frequency was found among these three groups. Our data do not support the hypothesis that the A1 allele of the TaqI A polymorphism of the DRD2 gene increases susceptibility to alcohol-use disorders in the Atayals of Taiwan. © 1996 Wiley-Liss, Inc.