Y chromosome--a tool in infertility studies of Latvian population.

UNLABELLED: Human Y chromosome is used as a tool in male infertility and population genetic studies. The aims of this research were to analyse the prevalence of Y chromosome microdeletions among infertile Latvian men, and to identify possible lineages of Y chromosome that may be at increased risk of developing infertility. A study encompassed 105 infertile men with different spermatogenic disturbances. Deletions on Y chromosome were detected in 5 out of 105 (approximately 5%) cases analysed in this study. Three of them carried deletion in AZFc region and two individuals had AZFa + b + c deletion. Study of Y chromosome haplogroups showed that N3a1 and R1a1 lineages were found less frequently in the infertile male group compared to ethnic Latvian group, however K* cluster was predominantly found in infertile male Y chromosomes. CONCLUSIONS: 1) Our study advocates running Y chromosome microdeletion analyses only in cases of severe form of infertility; 2) Y chromosome haplogroup analysis showed statistically significant tendencies that some haplogroups are more common in ethnic male group, but others are more common in infertile males.

Recombinant RNA phage Q beta capsid particles synthesized and self-assembled in Escherichia coli.

The Escherichia coli RNA phage Q beta coat protein-encoding gene (C) was amplified from native Q beta RNA using a reverse transcription-PCR technique. Gene C contains sequences coding for both the 133-amino acid (aa) Q beta coat protein (CP) and the 329-aa read-through protein (A1) consisting of CP and an additional 196-aa C-terminal sequence, separated from CP within the C gene by an opal (UGA) stop codon. Primers ensuring the natural environment for gene C, especially within the ribosome-binding site, and supplying C with unique restriction sites at both ends have been prepared. An amplified 1062-bp PCR fragment was positioned under the control of the strong E. coli trp promoter (Ptrp) within a pGEM-derived plasmid. The synthesis of gene C products was confirmed electrophoretically and immunologically. An immunodiffusion test with anti-Q beta phage antibodies and electron microscopy evaluation of the purified recombinant products showed that when expressed, the Q beta C gene was responsible for high-level synthesis and correct self-assembly of Q beta CP monomers into capsids indistinguishable morphologically and immunologically from Q beta phage particles, which we plan to use as surface display vectors.

Synthesis of recombinant atrial natriuretic peptide (rANP) using hybrid fusion protein-phage fr coat/ANP (CP/ANP).

Recombinant atrial natriuretic peptide (rANP) was expressed in and isolated from E. coli. rANP was purified using HPLC. Amino acid analysis, partial sequencing, and molecular mass were determined. Fused protein was used to rise polyclonal antibodies and to develop of immunoenzymatic assays of rANP and CP/ANP. Experiments were designed to study rANP effects on isolated rabbit aortic strips and to examine hypotensive, diuretic, and natriuretic activity, as well as renal creatinine clearance, in an in vivo rat model. Identity of recombinant and commercial ANP has been confirmed. Physiological activity of CP/ANP has allowed the investigators to predict the conformation of CP/ANP, pro-ANP processing, and the method by which fusion protein interacts with ANP receptors.

Prevalence of Beijing genotype in Latvian multidrug-resistant Mycobacterium tuberculosis isolates.

SETTING: Predominant genotypes of Mycobacterium tuberculosis include the Beijing family, which has caused large tuberculosis outbreaks and has been associated with increased virulence and multidrug resistance (MDR). OBJECTIVE: To search for the Beijing genotype among Latvian MDR patients to characterise their DNA isolates at the molecular level. DESIGN: MDR isolates were spoligotyped and tested for gene mutations by automatic nucleotide sequencing. RESULTS: Of 109 isolates examined, 95 were located in six clusters of 2 to 63 isolates each. The 63 isolates in the largest cluster had an identical pattern corresponding to the Beijing genotype. The remaining isolates were of a non-Beijing genotype and formed another large group whose similarity ranged from 72% to 100%. Mutations in the rpoB and katG genes were compared in the Beijing and non-Beijing strains. In both groups, the rpoB gene mutations predominated in codons S531L (52.2%) and D516V (14.7%). Double mutations in the rpoB gene were observed in 8.2% of the isolates, most of them located among Beijing-type isolates. The katG gene mutation S315T (98.4%) was prevalent among all isolates. CONCLUSION: Molecular analysis of MDR isolates of M. tuberculosis demonstrates that the Beijing genotype, most likely due to recent transmission, is prevalent in Latvia among MDR patients and that this genotype can be associated with double mutations.

High level expression of alpha-human atrial natriuretic factor as a fusion polypeptide with phage fr coat protein in Escherichia coli.

A synthetic DNA sequence coding for the 28 amino acid residues of alpha-human atrial natriuretic factor (alpha-hANF) and the N-terminal linker tripeptide Ile-Asp-Lys was inserted in the 3'-terminal part of the RNA bacteriophage fr coat protein gene. The cloned hybrid gene was isolated and placed into an expression vector under the control of the inducible E. coli tryptophan promoter and phage fr coat protein translation initiation region (TIR) sequence. In an appropriate host strain the expressed fusion protein accounts for at least 10% of the total cellular protein. In order to achieve high-cell density in a bioreactor while maintaining efficiency of alpha-hANF expression, improved cultivation conditions were selected using modified Shielach-Bauer's culture media containing glucose, yeast extract and bacto tryptone at an initial concentration of 2 g l-1 of each, adding concentrate of medium throughout the microbial growth and maintaining the dissolved oxygen in a range of 25-30%. At 13-14 h cultivation, the cell density reached 40 g cell dry weight per liter and the yield of fusion protein exceeded 45 mg g-1 cell dry weight. Fusion protein from solubilized E. coli cells was purified to homogeneity by ion exchange chromatography on DEAE-, CM-cellulose, QAE Sephadex A25 columns and selective precipitation.

Recent nosocomial transmission and genotypes of multidrug-resistant Mycobacterium tuberculosis.

SETTING: Multidrug-resistant tuberculosis (MDR-TB) is a serious health problem in Eastern European countries, including Latvia. OBJECTIVE: To investigate the proportion of tuberculosis, including MDR-TB cases, attributable to recent transmission and risk factors associated with clustering. DESIGN: Retrospective nested case-control study. The data set incorporated a wide spectrum of social features, as well as genotypes of Mycobacterium tuberculosis isolates determined by insertion sequence 6110 restriction fragment length polymorphism analysis of PvuII cleaved genomic DNA and spoligotyping. RESULTS: In comparison with non-clustered M. tuberculosis, the Beijing genotype (OR 12.15) and multidrug resistance (OR 5.61, P < 0.01) were associated with clustering. In comparison with clustered drug-susceptible M. tuberculosis, clustering of MDR M. tuberculosis was associated with Beijing genotype (OR 41.67), previous hospitalisation (OR 18.33) and previous TB treatment (OR 17.68, P < 0.05). Direct epidemiological links in hospitals were found for almost one third (32%) of MDR Beijing cases. CONCLUSIONS: MDR cases were more likely to be found in clusters than drug-susceptible cases (74.0% vs. 33.6%). Recent nosocomial transmission of MDR-TB is an important risk factor for the spread of multiresistance, and is associated with the Beijing genotype. Special attention should be paid to infection control measures in hospitals and ambulatory treatment should be enforced.

Mitochondrial DNA portrait of Latvians: towards the understanding of the genetic structure of Baltic-speaking populations.

Mitochondrial DNA (mtDNA) variation was investigated in a sample of 299 Latvians, a Baltic-speaking population from Eastern Europe. Sequencing of the first hypervariable segment (HVS-I) in combination with analysis of informative coding region markers revealed that the vast majority of observed mtDNAs belong to haplogroups (hgs) common to most European populations. Analysis of the spatial distribution of mtDNA haplotypes found in Latvians, as well as in Baltic-speaking populations in general, revealed that they share haplotypes with all neighbouring populations irrespective of their linguistic affiliation. Hence, the results of our mtDNA analysis show that the previously described sharp difference between the Y-chromosomal hg N3 distribution in the paternally inherited gene pool of Baltic-speaking populations and of other European Indo-European speakers does not have a corresponding maternal counterpart.

Mutations in the rpoB and katG genes leading to drug resistance in Mycobacterium tuberculosis in Latvia.

To characterize the genetic basis of drug resistance in Mycobacterium tuberculosis in Latvia, mutations involved in rifampin (rpoB gene) and isoniazid (katG gene) resistance in DNA from 19 drug-susceptible and 51 multidrug-resistant M. tuberculosis complex isolates were analyzed. The most frequent rpoB gene mutations found by the Line Probe assay were the S531L (14 of 34 isolates), D516V (7 of 34), H526D (4 of 34), and D516Y plus P535S (4 of 34) mutations. Direct sequencing of seven isolates with unclear results from Line Probe assay showed the presence of the L533P mutation and the Q510H plus H526Y (1 of 34) and D516V plus P535S (4 of 34) double mutations, neither of which has been described previously. Single-strand conformation polymorphism analysis showed strand mobility differences between the rifampin-susceptible and -resistant samples for the D516V, H526D, and D516Y plus P535S mutations but not for the S531L mutation. Nucleotide substitution at codon 315 (AGC-->ACC) of the katG gene was found in 48 of 51 multidrug-resistant samples by sequencing. Furthermore, katG gene restriction fragment length polymorphism analysis with endonuclease AciI confirmed the nucleotide change in codon 315.

Development of immunoenzymatic reactions of peptides using recombinant hybrid proteins.

A recombinant hybrid protein comprising the bacteriophage fr coat protein, a short linker, and atrial natriuretic factor (ANF) as well as native and recombinant phage coat proteins, ANF, and variants of the hybrid recombinant protein were used in the development of various immunological reactions including immunization, preparation of affinity columns, purification of antibodies, synthesis of conjugates, and immunoenzyme assay of ANF and the recombinant protein. The hybrid protein is effective in competitive assay of ANF and other constructs that include the phage protein.