Serum concentrations of myostatin and myostatin-interacting proteins do not differ between young and sarcopenic elderly men.

Sarcopenia is the loss of muscle size and function during ageing. The aim of this study was to test whether serum concentrations of myostatin and interacting proteins (GASP-1, FLRG, and follistatin) differed between young and elderly sarcopenic men. Isometric knee extensor maximal voluntary contraction and quadriceps cross-sectional area (magnetic resonance imaging measurement) were significantly higher in young (22 ± 2 years; 266 ± 54 N/m; 8,686 ± 1,154 mm(2)) than in mildly sarcopenic (69 ± 3 years; 183 ± 17 N/m; 6,621±718 mm(2)) and severely sarcopenic men (76 ± 6 years; 127 ± 23 N/m; 5,846 ± 591 mm(2)), respectively (p ≤ .01 for all comparisons). There was a trend (p = .06) toward higher FLRG in young (20 ± 8 ng/mL) than in mildly (15 ± 6 ng/mL) and severely sarcopenic men (17 ± 8 ng/mL). Myostatin, follistatin, GASP-1, tumor necrosis factor α, and interleukin-6 did not differ significantly. Insulin-like growth factor-1 and free testosterone were both significantly lower in sarcopenic men (p < .001). This suggests that altered serum concentrations of myostatin and myostatin-interacting proteins are not contributing to sarcopenia with the possible exception of FLRG.

Intrahepatic cholestasis of pregnancy: three novel MDR3 gene mutations.

BACKGROUND: The aetiology of intrahepatic cholestasis of pregnancy is unknown, but more than 10 different MDR3 gene mutations have recently been identified. AIM: To evaluate the genetic contribution of the MDR3 gene in the pathogenesis of intrahepatic cholestasis of pregnancy in Italian subjects. METHODS: We performed a multicentre prospective case-control study, enrolling 80 women with intrahepatic cholestasis of pregnancy at the third trimester of pregnancy and 80 pregnant women without intrahepatic cholestasis of pregnancy. Genomic DNA was extracted from peripheral venous blood leucocytes using standard procedures. The polymerase chain reaction was used to amplify exon 14 of the MDR3 gene and the polymerase chain reaction products were sequenced using a Big Dye Terminator Cycle Sequencing kit. RESULTS: Three novel non-synonymous heterozygous mutations in exon 14 were found (4%; E528D, R549H, G536R) among the 80 intrahepatic cholestasis of pregnancy patients, whereas the pregnant controls were all negative for exon 14 polymorphisms. The three patients involved had normal GGT and bilirubin, but high levels of both ALT and serum bile acids. One had cholesterol bile stones. The outcome of pregnancy was normal for two (with vaginal delivery), while foetal distress was recorded in the third. CONCLUSIONS: These three novel mutations add further information on the involvement of the MDR3 gene in intrahepatic cholestasis of pregnancy. As in other studies, we found only heterozygous mutations that could cause an impaired transport protein function, not its absence (which is responsible for more severe liver disease). Different genetic backgrounds might justify the presence of novel MDR3 gene mutations.

Evaluation and characterization of micronuclei in early spermatids of mice exposed to 1,3-butadiene.

The frequency of micronuclei induced in mouse meiotic cells after exposure to 1,3-butadiene has been evaluated in early spermatids. Germ cells were isolated from mice exposed to three butadiene concentrations (130, 250 and 500 ppm), at time intervals allowing to evaluate effects induced in late spermatocytes or at the stage of prelepotene/differentiating spermatogonia. The characterization of the origin of micronuclei, by simultaneous detection of centromeric and telomeric sequences, was also done on spermatid preparations from the 250 ppm concentration. The same analysis was carried out on a group of mice treated with the major butadiene metabolite, 1,2,3,4-diepoxybutane. The results obtained indicate a weak clastogenic effect of butadiene to premeiotic germ cells in the mouse.

Micronucleus induction in germ and somatic cells of the mouse after exposure to the butadiene metabolites diepoxybutane and epoxybutene.

The genotoxicity of diepoxibutane (DEB) and epoxybutene (EB), two of the main metabolites of 1,3-butadiene, was tested in the germ and somatic cells of the mouse by applying an MN assay in early spermatids, and in peripheral blood reticulocytes of a subgroup of the same animals. DEB (0.17 and 0.35 mmol/kg) and EB (0.35, 0.70 and 1.04 mmol/kg) were administered i.p. In the germ cell assay, significant increases of MN were observed after treatment of premeiotic S-phase cells with both butadiene metabolites, but DEB was shown to be more powerful than EB in the induction of chromosomal damage. A weak effect of the same compounds was also found after treatment of late spermatocytes, approaching the meiotic divisions. From the MN assay in peripheral blood reticulocytes, a statistically significant increase of the frequency of MN was detected at each dose tested for both chemicals. However, the results have again shown that DEB is much more efficient than EB in inducing chromosome damage.

PRINS localization of centromeres and telomeres in micronuclei indicates that in mouse splenocytes chromatid non-disjunction is a major mechanism of aneuploidy.

Primed In Situ DNA Synthesis (PRINS) of telomeric and centromeric (minor satellite DNA) sequences has been applied together with the cytokinesis block micronucleus (MN) assay in mouse splenocytes, with the aim of understanding the mechanism of origin of spontaneous and induced MN. Splenocyte cultures were treated in vitro either with the clastogenic agent mitomycin C or with the aneugenic compound colcemid. The relative proportions of MN carrying 1 to 4 telomeric signals were in agreement with the known mechanism of action of the chemicals tested, i.e., an higher number of MN with less than 4 telomeres were found in MMC-than in colcemid-treated cultures. No MN lacking the telomeric sequences (0 spot) were found, indicating that the observed distributions should not be affected by false-negative data. Furthermore, all MN carrying a single telomere were negative for the centromere, thus indicating that this class represents true chromosome acentric fragments. Finally, MN with 4 telomeric spots always carried the centromeric sequence, as expected on the hypothesis that these MN correspond to whole chromosomes. With respect to centromere-positive MN, more than one half carried 4 telomeric signals (whole chromosomes), and only 1/4 or less showed 2 telomeric signals (probably corresponding to a single chromatid). This difference was statistically significant, either in untreated cultures or in cultures exposed to mitomycin C or colcemid. On the whole, these data indicate that non-disjunction followed by whole chromosome loss (with the production of two daughter monosomic nuclei) may be the main mechanism of malsegregation leading to MN formation.

Detection of minor and major satellite DNA in cytokinesis-blocked mouse splenocytes by a PRINS tandem labelling approach.

A protocol for the simultaneous visualization of minor and major satellite DNA by primed in situ DNA synthesis (PRINS) was developed in cytokinesis-blocked murine splenocytes. After individuation of optimal experimental conditions, a micronucleus (MN) test was carried out by treating splenocytes in vitro with the clastogenic agent mitomycin C and the aneugenic compound Colcemid. It was found that PRINS gives highly reproducible results, also comparable with the literature on MN results obtained by fluorescent in situ hybridization (FISH). Therefore the PRINS methodology may be proposed as a fast alternative to FISH for the characterization of induced MN.