Molecular genetic analysis of the GJB1 gene: a study of six mutations

Charcot-Marie-Tooth type X1 disease (CMTX1) is an X-dominant peripheral neuropathy caused by mutations in the GJB1 gene. Molecular genetic analysis of the GJB1 gene is crucial for CMTX1 diagnosis and for genetic counselling. To date, molecular genetic analysis of the GJB1 gene revealed 264 mutations in the GJB1 gene. In spite of the rising number of GJB1 gene mutations, family history was documented in only a few CMTX1 cases. The aim of this study was a molecular genetic analysis of the GJB1 gene in 7 families, performed in 19 CMTX1-affected patients and 13 healthy family members. Moreover, we attempted to report evidence of effects of 6 amino-acid substitutions described in this study. To the best of our knowledge, the G110D, V152D and K167E mutations are novel substitutions, which have not been reported so far.     

Interplay of DNA supercoiling and catenation during the segregation of sister duplexes

The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in vivo.     

Disruption of Growth Hormone Receptor Prevents Calorie Restriction from Improving Insulin Action and Longevity

Most mutations that delay aging and prolong lifespan in the mouse are related to somatotropic and/or insulin signaling. Calorie restriction (CR) is the only intervention that reliably increases mouse longevity. There is considerable phenotypic overlap between long-lived mutant mice and normal mice on chronic CR. Therefore, we investigated the interactive effects of CR and targeted disruption or knock out of the growth hormone receptor (GHRKO) in mice on longevity and the insulin signaling cascade. Every other day feeding corresponds to a mild (i.e. 15%) CR which increased median lifespan in normal mice but not in GHRKO mice corroborating our previous findings on the effects of moderate (30%) CR on the longevity of these animals. To determine why insulin sensitivity improves in normal but not GHRKO mice in response to 30% CR, we conducted insulin stimulation experiments after one year of CR. In normal mice, CR increased the insulin stimulated activation of the insulin signaling cascade (IR/IRS/PI3K/AKT) in liver and muscle. Livers of GHRKO mice responded to insulin by increased activation of the early steps of insulin signaling, which was dissipated by altered PI3K subunit abundance which putatively inhibited AKT activation. In the muscle of GHRKO mice, there was elevated downstream activation of the insulin signaling cascade (IRS/PI3K/AKT) in the absence of elevated IR activation. Further, we found a major reduction of inhibitory Ser phosphorylation of IRS-1 seen exclusively in GHRKO muscle which may underpin their elevated insulin sensitivity. Chronic CR failed to further modify the alterations in insulin signaling in GHRKO mice as compared to normal mice, likely explaining or contributing to the absence of CR effects on insulin sensitivity and longevity in these long-lived mice.     

Selenium levels in blood of Upper Silesian population

The selenium status and the relationship of whole-blood selenium and plasma homocysteine are reported for healthy human subjects living in Upper Silesia. A total of 1063 individuals (627 male and 436 female) examined for whole-blood selenium were subdivided into six groups according to age; the youngest included adolescents (n=143) aged 10–15 yr, and the oldest were centenarians (n=132). The mean Se content was relatively low (62.5±18.4 μg/L), and it tended to be higher in men (65.9±17.2 μg/L) than in women (57.5±18.9 μg/L). Selenium levels appeared to be age dependent, as the highest values were observed in young and middle-age adults (21–40 yr), whereas they were significantly lower in adolescents and in the elderly. In more than 40% of apparently healthy adults (aged 21–69 yr), the Se concentration was within the range 60–80 μg/L (i.e., below the lower limit of the nutritional adequacy range [80 μg/L]). A significant inverse correlation between whole-blood selenium and plasma total homocysteine was detected in a smaller population sample of middle-aged and elderly persons (n=204).     

Purification of Low Molecular Weight Metal-Binding Proteins by Preparative Polyacrylamide Gel Electrophoresis: Properties of Electrophoretically Purified Rat Liver (Cd,Zn)-Metallothioneins

A method is proposed for purification of metallothionein by preparative polyacrylamide gel electrophoresis. The method enables purification of 100–700 mg of a preparation containing (Cd, Zn] - metallothionein yielding preperations of considerably higher purity as compared with those obtained by ion-exchange chromatography.     

In vitro regeneration of <Emphasis Type="Italic">Carlina acaulis</Emphasis> subsp. <Emphasis Type="Italic">simplex</Emphasis> from seedling explants

The aim of the study was to obtain an efficient system for Carlina acaulis subsp. simplex propagation. The experimental materials were shoot tips, fragments of hipocotyls, cotyledons and roots isolated from 10-day-old seedlings. The explants were transferred to the proliferation medium supplemented with different types of cytokinin: BA (13.3 μM), kinetin (13.9 μM) and zeatin (13.7 μM) in combination with NAA (0.54 μM). The best morphogenetic response was observed when explants were cultured on the BA supplemented medium. The maximum shoot organogenesis frequency was observed for shoot tip (nearly 94%). On average 8.6 axillary shoots were induced per explant. Multiplication rate increased during the first three subcultures. The shoots revealed a wide range of morphogenetic responses. Differences were observed in the presence or absence of hair on the surface of lamina. These changes had epigenetic character and were the effect of changes in DNA methylation, which is shown by differences in methylation pattern between 18S rRNA and 25S rRNA genes in the analyzed regenerated plants. Nearly 94% of plantlets were rooted on auxin lacking medium. Addition of auxin (NAA or IAA) increased both the rooting percentage (100%) and the number of roots per shoot, but their growth was inhibited. Shortening of the auxin exposition time reduced the number of roots. Moreover, high efficiency (90%) was observed for ex vitro rooting. Plantlets with a large number of roots survived better than the ones with only a few roots. Plants were able to flower and gave viable seeds.     

Serum from rats fed red or yellow tomatoes induces Connexin43 expression independently from lycopene in a prostate cancer cell line

Epidemiologic studies suggested a protective effect of tomatoes against prostate cancer brought by lycopene, a carotenoid conferring the red colour of tomatoes. However, intervention studies on patients have shown that the preventive effect of tomato was more potent than that of lycopene. The aim of this study was to compare the effects of red tomato, yellow tomato (devoid of lycopene) and lycopene on Connexin43 (Cx43) expression, a protein regulating cell growth, on a prostate cancer cell line expressing the androgen receptor. Cells were incubated with serum from rats fed a control diet (CS) or control diet supplemented with red tomato (RTS), yellow tomato (YTS) or lycopene beadlets (LBS). After exposure of the cells to RTS or YTS for 48 h, the expression of Cx43 was significantly increased compared to cells exposed to CS. Whereas LBS effect was not significantly different. The cells incubated with RTS and LBS had similar levels of lycopene, while those incubated with YTS contained no lycopene. These data first show that serum nutritionally enriched with red and yellow tomatoes could up-regulate Cx43 turn-over in PC3AR cells independently from lycopene level. Within the physiological approach used in the present study, it can be concluded that compounds other than lycopene contribute to the preventive effect of tomatoes.     

Oral health status and oral hygiene practices of patients with peptic ulcer and how these affect Helicobacter pylori eradication from the stomach

Background: Helicobacter pylori eradication from the oral cavity is more difficult than from the stomach. Thus, if the bacterium survives the antibacterial therapy in the oral cavity, it would be able to re‐infect the stomach within a few weeks. Since oral health status could correspond to oral infection with H. pylori, the aim of the study was to determine whether oral health and oral hygiene practices affect the efficacy of H. pylori eradication from the stomach. Material and Methods: The study was performed in 137 patients with peptic ulcer who had undergone a 7‐day course of eradication treatment with one of two sets of drugs: 1, omeprazole, amoxicillin, and tinidazole or 2, omeprazole, clarithromycin, and tinidazole. The efficacy of H. pylori eradication from the stomach was evaluated at the second gastroscopy 4 weeks after cessation of eradication therapy by means of two methods: rapid urease test and histology. The examination of natural dentition and prosthetic restorations as well as the assessment of hygienic procedures referring to natural dentition and dentures accompanied the second gastroscopy. Results: No association was found between the efficacy of H. pylori eradication from the stomach and the number of natural teeth, decayed teeth, use of dentures, debris index, or periodontal index. However, an association between eradication success and some oral hygiene procedures were noted. Unexpectedly, in patients treated with omeprazole, amoxicillin and tinidazole, the removal of dental prosthesis for the night and brushing the natural teeth twice a day or more reduced the efficacy of H. pylori eradication from the stomach. Conclusions: Oral health and oral hygiene practices seem unlikely to increase the efficacy of H. pylori eradication from the stomach.