Diabetes and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS): radiolabeled polymerase chain reaction is necessary for accurate detection of low percentages of mutation

A 6-yr-old boy presented with muscle weakness, lactic acidemia, and insulin-dependent diabetes mellitus (IDDM). Using PCR and restriction enzyme analysis, he was found to have the classical A3248G mitochondrial DNA (mtDNA) mutation frequently associated with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). The mutation was confirmed by sequencing muscle mtDNA. The mutation in mtDNA from muscle, lymphoblasts, and blood was clearly demonstrable by standard methods using ethidium bromide staining. His mother also had IDDM, but no A3243G mutation could be detected in her blood or transformed lymphoblasts using the same PCR technique. When PCR was carried out in the presence of [32P]deoxycytidine triphosphate, subsequent autoradiography detected the presence of the mutation at low levels in mtDNA from the mother's lymphoblasts and blood. Study of the mother's muscle showed a mitochondrial myopathy, despite the fact that she was asymptomatic. We emphasize that the increased sensitivity of radiolabeled PCR may be necessary to detect small percentages of heteroplasmic A3243G mtDNA mutation in blood from diabetic subjects. Otherwise the incidence of mtDNA mutations in both IDDM and non-insulin dependent diabetes may be underestimated.     

Repair of aortico-right ventricular tunnel

A short review of the literature on the optimum temperature for the growth of Mycobacterium leprae is followed by a report of an investigation into the correlation of oral surface temperatures with oral leprous lesions. It is concluded that the oral lesions of leprosy occur more frequently in areas of the mouth with a lower surface temperature.     

Genetic databases: online catalogues of inherited disorders

Current information on inherited disorders in domestic animals is available on the internet: Online Mendelian inheritance in animals (OMIA) and Mendelian inheritance in sheep (MIS) are the two major sources of information. OMIA was created (and is maintained) by workers at the University of Sydney. MIS has been compiled by the Committee on Genetic Nomenclature of Sheep and Goats (COGNOSAG), an international group of geneticists. In the future, similar catalogues for other species (starting with goats and cattle) will be made available by COGNOSAG. Electronic access to this information is freely accessible on the world-wide web at http:/(/)www.angis.su.oz.au/Databases/BIRX/om ia (for OMIA), http:/(/)probe.nalusda.gov:8300/animal/omia.h tml (for OMIA in the United States of America) and at http:/(/)www.angis.org.au/Databases/BIRX/mis (for MIS).     

Conjugates of catecholamines with cysteine and GSH in Parkinson's disease: possible mechanisms of formation involving reactive oxygen species

Oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine (DA) to generate semiquinones/quinones, oxygen radicals, and other reactive oxygen species may play a role in neuronal cell death in Parkinson's disease (PD). In particular, semiquinones/quinones can form conjugates with thiol compounds such as GSH and cysteine. Exposure of L-DOPA, DA, and other catecholamines to a system generating O2.- radical led to O2(.-)-dependent depletion of added GSH (or cysteine), accompanied by the formation of thiol-DA or -DOPA adducts as detected by HPLC. Superoxide could additionally cause destruction of these adducts. Iron or copper ions could also promote conjugate formation between GSH or cysteine and DA and L-DOPA, especially if H2O2 was present. We applied HPLC to measure glutathionyl and cysteinyl conjugates of L-DOPA, DA, and 3,4-dihydroxyphenylacetic acid (DOPAC) in postmortem brain samples from PD patients and normal control subjects. Conjugates were detected in most brain areas examined, but levels were highest in the substantia nigra and putamen. In most regions, adduct levels were lower in PD, but there were significant increases in cysteinyl adducts of L-DOPA, DA, and DOPAC in PD substantia nigra, suggesting that acceleration of L-DOPA/DA oxidation occurs in PD, although we cannot say if this is a primary feature of the disease or if it is related to therapy with L-DOPA. In vitro, conjugate formation could be inhibited by the dithiol dihydrolipoate but not by its oxidised form, lipoic acid.     

The effect of the threshold stress on the determination of the Weibull parameters in probabilistic failure analysis

In the analysis of brittle materials and components the probability of failure is commonly modelled using a two-parameter Weibull distribution. Occasionally, a three-parameter model is used when the material shows significant threshold behaviour. In this paper two methods for determining the three-parameter constants are discussed. Two theoretical two- and three-parameter distributions are then analysed to examine the number of samples needed to determine the parameters accurately. The two-parameter models are the best fits of the three-parameter models and their failure distributions are very similar to the three-parameter distributions. It is concluded that far more specimens need to be tested than is usually the case to be confident that the correct distribution has been found.     

Functional expression of three isoforms of human nitric oxide synthase in baculovirus-infected insect cells

Complementary DNAs encoding three human isoforms (neuronal, inducible, and endothelial) of nitric oxide synthase were cloned into the baculovirus expression vector pVL1392/1393. Transfection of Sf-9 insect cells with the recombinant baculovirus resulted in the expression of high levels of nitric oxide synthases. The expressed proteins of neuronal and inducible nitric oxide synthase were predominantly soluble, whereas the endothelial enzyme was for the most part, particulate. Recombinant enzymes were purified with 2',5'-ADP Sepharose affinity chromatography. The effects of reference enzymatic inhibitors (NG-methyl-L-arginine, NG-nitro-L-arginine and N-iminoethyl-L-ornithine) on recombinant expressed proteins were not significantly different from native nitric oxide synthase enzyme preparations. L-aminoguanidine was found to be much less potent in inhibiting recombinant or native human inducible nitric oxide synthase compared to the murine isoform. These findings indicate previously unappreciated interspecies differences in the action of nitric oxide synthase enzymatic inhibitors. The functional expression of human nitric oxide synthase isoforms in a heterologous expression system allowed screening of novel inhibitors. Studies indicated that S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine were potent novel inhibitors of human nitric oxide synthases.     

Is fecundability associated with month of birth? An analysis of 19th and early 20th century family reconstitution data from The Netherlands

The relationship between fecundability and month of birth was investigated in a cohort of 1526 women who married between 1802 and 1929, using only women whose first marriage occurred before the age of 35 years. On the basis of their time to pregnancy (TTP, calculated as time between wedding and first birth minus gestational length), women were categorized into two groups: fecunds (TTP up to 12 months or prenuptial conceptions, n = 1348) and subfecunds (TTP >18 months, n = 118). By use of logistic regression, cosinor functions with a period of 1 year or 6 months and variable shift and amplitude were fitted through the monthly odds of subfecunds versus fecunds. The best fitting curve was unimodal, with a zenith in September (P = 0.13 for H0: no differences). Exclusion of childless women (n = 36, minimum follow-up 5 years) from the subfecunds led to a similar curve (P < 0.01), while childless women, as compared with fecunds, showed a birth distribution that was best represented with a bimodal curve with zeniths in January and July (P = 0.06). This study provides evidence for the existence of differences in fecundability by month of birth. The cause of this relationship is unclear, but may lie in a melatonin-dependent circannual variability of the quality of the oocyte.