Comparative susceptibility to anthelmintics of Brugia pahangi in jirds infected by different methods

It is possible to infect jirds with Brugia pahangi by three methods. Infective larvae (L3) can be injected either intraperitoneally (ip), when adults develop in the peritoneal cavity, or sub-cutaneously (sc), when they develop in the lymphatics or the heart and blood vessels associated with the lungs. Alternatively adult worms which have been grown in the peritoneal cavities of jirds can be implanted into the peritoneal cavities of other jirds. This latter system has been widely used for screening for new filaricides. We have compared the activity of 9 macrofilaricidal compounds against these 3 types of infection. Mebendazole and albendazole were more active against implanted adults than against L3 induced adults in the peritoneal cavity. Oxibendazole, flubendazole, CGP24588A and oxfendazole were equally active against both types of worm. CGP20376, Mel Ga and Mel Ni were more active against adult worms derived from inoculated L3 than implanted worms. When comparing intra-lymphatic and ip adults (both derived from L3 infections and in the same jirds) albendazole and CGP20376 were active at the same levels against both types of infection. Mebendazole, flubendazole, oxfendazole, CGP24588A, Mel Ga and Mel Ni were more active against ip adults than intra-lymphatic adults. No drug was more active against intra-lymphatic adults than against adults.     

Detection of carriers of hemophilia A by testing for HindIII polymorphism in the factor VIII gene by PCR

Representatives of 62 families from Moscow and Leningrad with haemophilia A observed in the pedigree were tested for HindIII polymorphism in the factor VIII gene. The proposed scheme of investigation was based on intron 19 of the FVIII gene amplification by the PCR technique followed by restriction analysis with the inner control of hydrolysis. 207 unrelated X-chromosomes were analysed, the frequency of the incidence of the polymorphic HindIII site in the given population found to be 0.29. The frequency of incidence of the HindIII heterozygotes calculated according to Hardy-Weinberg equation was 0.41. This value evidences for relatively high informativity of this polymorphism for carrier detection and prenatal diagnosis of haemophilia A. 23 families (37%) out of 62 examined in the study were informative for this criteria. The new scheme proved to be effective in testing HindIII polymorphism for haemophilia A carrier detection and prenatal diagnosis. The whole procedure takes one day, the radiolabelled probes are not used. The scheme described was inculcated in the All-Union Research Center for Haematology, Ministry of Health, USSR, Moscow, Research Institute for Obstetrics and Gynecology, Leningrad, Institute of Medical Genetics, Greifswald, DDR.     

Molecular nature of beta-thalassemia in Tajikistan: a four base pair deletion in codons 41-42 of the beta-globin gene]

Thirty tajiks, whose relatives had beta-thalassemia traits (revealed in previous investigations by determination of the HbA-2 and HbF levels) were selected to screen beta-thalassemia mutations. DNA samples from each individual were subjected to the PCR (polymerase chain reaction) to amplify the 635 bp beta-globin gene fragment. One additional band was detected in three samples after the amplified fragment underwent electrophoresis in 2% agarose gel and the EtBr was stained, and two additional ones were revealed by 6% PAAGE and staining of the EtBr. All additional bands migrated more slowly than appropriate 635 bp fragment. It is supposed that additional bands are heteroduplexes formed from the wild type chains and mutated chains carrying a deletion or insertion. The 4 bp deletion of the 41-42 (-tctt) was detected after the direct sequencing of the amplified fragments. This mutation is common among Chinese but it was not revealed in the Middle Asia populations. The mutation can be easily screened using the PCR and electrophoresis in 2% agarose gel or PAAG of the amplified beta-globin gene fragments.     

Disruption of functional activity of mitochondria during MTT assay of viability of cultured neurons

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨ_m), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨ_m-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨ_m decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.     

Incorporation of yeast tRNA into mouse L 1210 lympholeukemic cells

[32P]tRNA from baker's yeast is incorporated without degradation into lympholeukotic cells of L1210 mice. The tRNA incorporation determined after tRNA hydrolysis on cell surface by RNAase increases linearly with a rise in the initial concentration from 0.5 to 500 micrograms per ml. According to gel electrophoresis of intracellular nucleic acids, after a 3 hour incubation the [32P]tRNA incorporated into the cells by 50% to form tRNA fragments without any conspicuous reutilization. The kinetic curve of tRNA incorporation during the first 60 min demonstrates a severalfold decrease in the initial maximal incorporation of [32P]tRNA into the cells (2 min), with a subsequent restoration of the incorporation within 2-3 hours.     

Genomic carrier detection and prenatal diagnosis of haemophilia A in families at risk using the polymerase chain reaction (PCR).

The polymerase chain reaction (PCR) was applied in genomic analysis of families at risk for haemophilia A using the intragenic Bel I and Hind III polymorphism in introns 18 and 19, respectively, of factor VIII gene. For the latter the primers derived from exon 19 and 20 sequences allowed to amplify the whole intron 19 resulting in a 730 bp fragment. Hind III restriction of this fragment provides polymorphic fragments of 250 bp or 160 bp and 90 bp respectively. An also occurring 480 bp fragment can be used as internal control to circumvent misdiagnosis due to incomplete or failure of restriction. The Hind III polymorphism was successfully used in prenatal diagnosis of an affected male in the first trimenon of pregnancy. Fetal sexing was also performed by PCR technique using Y specific primers.     

Three Novel Mutations in Porphobilinogen Deaminase Gene Identified in Russian Patients with Acute Intermittent Porphyria

Porphobilinogen deaminase (PBGD) is a key enzyme of the heme biosynthetic pathway. Defects in the PBGD gene lead to an autosomal dominant disease, acute intermittent porphyria (AIP). Almost all AIP patients with rare exceptions are heterozygous for the defective gene. To date, at least 160 different mutations causing AIP are identified. Extensive investigations along this line are conducted in many countries of the world. In Russia these studies had not been hitherto performed. Here we report the results of molecular genetic examination of four Russian patients with AIP diagnosed from clinical symptoms. By direct sequencing of the PBGD gene or the corresponding cDNA, we have detected four mutations, three of which were not previously encountered in the world population. These are TAAG deletion in intron 7 between positions +2 and +5 (IVS7 2–5 delTAAG); T deletion in the initiation codon ATG of exon 3, and the G for C replacement at position –1 of intron 5 (IVS5 as –1 G–C), which disrupts splicing. In addition, in one female patient, a known deletion CT in codon 68 was revealed. In two patients, expression of PBGD gene alleles was significantly disproportional, so that normal mRNA prevailed in one case and mutant mRNA of nonerythroid type in the other. Deletion in intron 7 was easily detectable due to the formation of a heteroduplex fragment with abnormal electrophoretic mobility directly in PCR. This simple heteroduplex analysis allowed us to exclude AIP carriage in son and daughter of a female patient with the genetic defect.