Genomic organization and chromosomal localization of three members of the UDP-N-acetylgalactosamine: polypeptide N- acetylgalactosaminyltransferase family

A homologous family of UDP- N -acetylgalactosamine: polypeptide N - acetylgalactosaminyltransferases (GalNAc-transferases) initiate O- glycosylation. These transferases share overall amino acid sequence similarities of approximately 45-50%, but segments with higher similarities of approximately 80% are found in the putative catalytic domain. Here we have characterized the genomic organization of the coding regions of three GalNAc-transferase genes and determined their chromosomal localization. The coding regions of GALNT1 , -T2 , and -T3 were found to span 11, 16, and 10 exons, respectively. Several intron/exon boundaries were conserved within the three genes. One conserved boundary was shared in a homologous C. elegans GalNAc- transferase gene. Fluorescence in situ hybridization showed that GALNT1 , -T2 , and -T3 are localized at chromosomes 18q12-q21, 1q41-q42, and 2q24-q31, respectively. These results suggest that the members of the polypeptide GalNAc-transferase family diverged early in evolution from a common ancestral gene through gene duplication.      

Improved microfluorometric DNA determination in biological material using 33258 Hoechst.

A simple and rapid microfluorometric method is described for the determination of DNA in submicrogram quantities using 33258 Hoechst fluorochrome. A high degree of reproducibility was obtained using calf thymus and phage DNA, mouse liver chromatin, and HeLa cells homogenate preparations. None or very little interference by the routinely used preparation reagents or by the cellular components was found. Compared to other commonly used procedures this innovative and versatile technique can be conveniently applied to DNA microdetermination for the high sensibility/reproducibility ratio and can also be used without the need of previous purification steps.     

Thymidylate synthetase catalyzed dehalogenation of 5-bromo-and 5-iodo-2′-deoxyuridylate

Thymidylate synthetase catalyzes a facile dehalogenation of 5-bromo-and 5-iodo-2′-deoxyuridylate in the presence of dithiothreitol. The chloro and fluoro nucleotides are not dehalogenated under similar conditions. A mechanism for this reaction is proposed which is in complete accord with previously ascertained features of the catalytic mechanism as well as model chemical reactions. This reaction is likely an important pathway in the biological dehalogenation of 5-halogenated uracil derivatives.     

Heterologous expression of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major

The bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) of Leishmania major has been cloned and expressed in Escherichia coli and Saccharomyces cerevisiae. The strategy involved placing the entire 1560-bp coding sequence into a parent cloning plasmid that was designed to permit introduction of unique restriction sites at the 5'- and 3'-ends. In this manner, the entire coding sequence could be easily subcloned into a variety of expression vectors. High levels of TS-DHFR gene expression were driven by tac, pL and T7 RNA pol promoters in E. coli, and the GAPDH-ADH-2 promoter in S. cerevisiae. L. major TS-DHFR also complemented TS deficiency in E. coli. In E. coli, the protein accumulated to very high levels, but most was present as inactive inclusion bodies. Nevertheless, substantial amounts were soluble; up to 2% of the soluble protein was catalytically active TS-DHFR. In the yeast systems, essentially all of the bifunctional protein was soluble and catalytically active, and crude extracts contained about 100-fold more enzyme than do extracts from wild-type L. major. The expressed TS-DHFR from yeast and E. coli was purified to homogeneity by methotrexate-Sepharose affinity chromatography. About 8.5 mg of homogeneous, catalytically active protein is obtained from a 1-L culture of yeast, and 1.5 mg was obtained from 1 L of E. coli culture. A 200-L fermentation of the yeast expression system yielded a crude extract containing over 4 g of TS-DHFR.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.     

Prevalence of scoliosis in school-children from Mostar, Bosnia and Herzegovina

In the school-year 2002/2003 a prospective epidemiological study was performed with the aim of evaluating the prevalence and distribution of scoliosis in the population of schoolchildren from Mostar, Bosnia and Herzegovina. The general check-up of primary-school children covered a total of 2,517 children aged 7-14. The children in which at least one positive symptom of scoliosis was found were directed to undergo orthopedic examination and--if indicated--radiography. Incorrect posture was noted in 33.4% of children, and 11.8% of children were sent to orthopedic specialist examination. The prevalence of structural scoliosis amounted to 3.1%, with the spine curvature threshold being 10 degrees. In eight children (0.32%; 1 boy and 7 girls) a curvature of 20 degrees or more was diagnosed. The most common type of curvature was the thoracal (39%) and the thoraco-lumbar (39%) while 14 children had a double curvature (17.8%). A scoliosis was detected due to here performed check-up in 83.5% of children with scoliosis. No case of serious spine deformity (45 degree or more) was recorded, due to regular general check-ups taking place biannually in this population.     

Plasma nociceptin/orphanin FQ levels rise after spontaneous episodes of angina, but not during induced myocardial ischemia

The aim of our study was to evaluate the effects of repeated episodes of angina and induced myocardial ischemia on plasma nociceptin/orphanin FQ (N/OFQ) levels. Patients with unstable angina (23 with new onset severe angina or accelerated angina and 18 with subacute angina at rest) who had had repeated spontaneous episodes of chest pain in the last week before the study underwent myocardial perfusion single-photon emission computed tomography using adenosine infusion. Twenty subjects without clinical symptoms of angina matched for age, sex and cardiac risk factors served as a control group. N/OFQ levels were significantly (P < 0.01) higher in the patients (15.2 ± 2.1 pg/ml) than in the control group (8.5 ± 2.6 pg/ml). Blood pressure and heart rate did not significantly differ. All patients showed transient adenosine infusion myocardial ischemia that did not induce chest pain or significantly modify plasma N/OFQ levels or hemodynamic parameters. Our findings show that unstable angina is associated with a significant increase in circulating N/OFQ levels unrelated to intervening transient myocardial ischemia or hemodynamic changes. This increase is probably related to the chest pain repeatedly occurring in the course of coronary artery disease, but absent during transient adenosine-induced myocardial ischemia.