Critical nuclear DNA size and distribution associated with S phase initiation

Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology and autoradiography grain patterns between middle G₁ and middle S phases: Our results show two distinct [³H]-thymidine labeling patterns. The first “peripheral” labeling pattern has a characteristic nuclear size distribution, in contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of very early S phase. The sizes of the second labeling pattern—homogeneous or inhomogeneous grain distribution throughout the nucleus—are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size (reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel experimentation with rat liver cells—synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography—confirms the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near the nuclear periphery.     

High-resolution separation of disaccharide and oligosaccharide alditols from chondroitin sulphate, dermatan sulphate and hyaluronan using CarboPac PA1 chromatography

Recent literature indicates that specific glycosaminoglycanstructures are involved in various biological processes, suchas anticoagulation, growth factor activation and viral infection.The initial step in the structural analysis of glycosaminoglycansis a definitive compositional analysis of its characteristicdisaccharide repeat structures. Current chromatographic or electrophoreticprocedures may have limitations in analysing glycosaminoglycansamples that are in low abundance, contain novel structuresthat need to be further characterized, or are metabolicallylabelled from radioactive precursors as a result of biosyntheticexperiments. This study presents a new methodology for analysingdisaccharides and oligosaccharides derived from chondroitinsulphate, dermatan sulphate and hyaluronan that fulfils theabove criteria. The procedure involves the separation of reducedforms of these glycoconjugates on a CarboPac PA1 column usingalkaline eluants. This study adopted a strategy which uses specificenzymes to release these disaccharides from their glycosaminoglycanforms. A borohydride reduction reaction was modified to be compatiblewith the buffer conditions commonly used with these enzymesin order to quantitatively reduce the disaccharides to theiralditol forms (thereby stabilizing them to alkaline pH). Chromatographyconditions were established which separated all known disaccharidealditol structures from chondroitin sulphate, dermatan sulphateand hyaluronan with extremely high resolution in a single run.Integrated pulsed amperometry was compared to UV absorbancemeasurement at 232 nm as two sensitive methods for detectingthese reduced disaccharides; most of them could be routinelydetected in the range of 50–500 ng. Data are presentedapplying this method to quantify hyaluronan in a biologicalsample which contains {small tilde}5000 cells and only {smalltilde}10 ng of hyaluronan. Additional data are presented todemonstrate that this procedure will also separate oligosaccharidealditols derived from hyaluronan. borohydride reduction glycosaminoglycans integrated pulsed amperometry     

Heritability of adult body height: a comparative study of twin cohorts in eight countries.

A major component of variation in body height is due to genetic differences, but environmental factors have a substantial contributory effect. In this study we aimed to analyse whether the genetic architecture of body height varies between affluent western societies. We analysed twin data from eight countries comprising 30,111 complete twin pairs by using the univariate genetic model of the Mx statistical package. Body height and zygosity were self-reported in seven populations and measured directly in one population. We found that there was substantial variation in mean body height between countries; body height was least in Italy (177 cm in men and 163 cm in women) and greatest in the Netherlands (184 cm and 171 cm, respectively). In men there was no corresponding variation in heritability of body height, heritability estimates ranging from 0.87 to 0.93 in populations under an additive genes/unique environment (AE) model. Among women the heritability estimates were generally lower than among men with greater variation between countries, ranging from 0.68 to 0.84 when an additive genes/shared environment/unique environment (ACE) model was used. In four populations where an AE model fit equally well or better, heritability ranged from 0.89 to 0.93. This difference between the sexes was mainly due to the effect of the shared environmental component of variance, which appears to be more important among women than among men in our study populations. Our results indicate that, in general, there are only minor differences in the genetic architecture of height between affluent Caucasian populations, especially among men.     

Electron paramagnetic resonance studies and effects of vanadium in Saccharomyces cerevisiae

Vanadium uptake by whole cells and isolated cell walls of the yeast Saccharomyces cerevisiae was studied. When orthovanadate was added to wild-type S. cerevisiae cells growing in rich medium, growth was inhibited as a function of the VO₄ ^3- concentration and the growth was completely arrested at a concentration of 20 mM of VO₄ ^3- in YEPD. Electron paramagnetic resonance (EPR) spectroscopy was used to obtain structural and dynamic information about the cell-associated paramagnetic vanadyl ion. The presence of EPR signals indicated that vanadate was reduced by whole cells to the vanadyl ion. On the contrary, no EPR signals were detected after interaction of vanadate with isolated cell walls. A mobile and an immobile species associated in cells with small chelates and with macromolecular sites, respectively, were identified. The value of rotational correlation time _r indicated the relative motional freedom at the macromolecular site. A strongly immobilized vanadyl species bound to polar sites mainly through coulombic attractions was detected after interaction of VO²⁺ ions with isolated cell walls.     

Update on genotoxicity and carcinogenicity testing of 472 marketed pharmaceuticals

This survey is a compendium of genotoxicity and carcinogenicity information of 838 marketed drugs, whose expected clinical use is continuous for at least 6 months or intermittent over an extended period of time. Of these 838 drugs, 366 (43.7%) do not have retrievable genotoxicity or carcinogenicity data. The remaining 472 (56.3%) have at least one genotoxicity or carcinogenicity test result. Of the 449 drugs with at least one genotoxicity test result, 183 (40.8%) have at least one positive finding. Of the 338 drugs with at least one carcinogenicity test result, 160 (47.3%) have at least one positive result. Concerning the predictivity of genetic toxicology findings for long-term carcinogenesis assays, of the 315 drugs which have both genotoxicity and carcinogenicity data 116 (36.8%) are neither genotoxic nor carcinogenic, 50 (15.9%) are non-carcinogens which test positive in at least one genotoxicity assay, 75 (23.8%) are carcinogenic in at least one sex of mice or rats but test negative in genotoxicity assays, and 74 (23.5%) are both genotoxic and carcinogenic. Only 208 (24.8%) of the 838 drugs considered have all data required by current guidelines for testing of pharmaceuticals. However, it should be noted that a large fraction of the drugs considered were developed and marketed prior to the present regulatory climate. Although the laws do not require re-testing based on revised standards, in the absence of epidemiological studies excluding a carcinogenic risk to humans, a re-evalutation would be appropriate.     

The plant invertase inhibitor shares structural properties and disulfide bridges arrangement with the pectin methylesterase inhibitor

Attempts to purify the inhibitor of pectin methylesterase (PMEI) from the soluble extract of ripe apricot (Prunus armeniaca) fruit led to isolation of a protein (Pa-INH) similar to PMEI, but having invertase inhibitory activity against vacuolar invertase from tomato. The molecular charge, the native and SDS-PAGE molecular weights were similar to those of PMEI. Partial amino acid sequence indicated a high level of identity with invertase inhibitors and a significant identity with PMEI. Circular dichroism analysis showed a mainly -helix secondary structure for both the inhibitors and a higher thermostability of Pa-INH. Four Cys residues forming disulfide bridges in PMEI were conserved in Pa-INH. Similarly to PMEI, these residues were linked by disulfide bridges (first to second and third to fourth). The free Cys139 of PMEI is substituted by Ala in Pa-INH. The results reported in this study suggest a common structural arrangement of the two inhibitors.     

Prolonged lifespan with enhanced exploratory behavior in mice overexpressing the oxidized nucleoside triphosphatase hMTH1

The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1‐Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1‐Tg mice express high levels of the hMTH1 hydrolase that degrades 8‐oxodGTP and 8‐oxoGTP and excludes 8‐oxoguanine from both DNA and RNA. Compared to wild‐type animals, hMTH1‐overexpressing mice have significantly lower steady‐state levels of 8‐oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age‐dependent accumulation of DNA 8‐oxoguanine that occurs in wild‐type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1‐Tg animals live significantly longer than their wild‐type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1‐Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1‐Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1‐Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids.     

Bisphenol-A Impairs Insulin Action and Up-Regulates Inflammatory Pathways in Human Subcutaneous Adipocytes and 3T3-L1 Cells

Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA) is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPARγ and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-γ, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function.     

Association of Genetic Variation in Adaptor Protein APPL1/APPL2 Loci with Non-Alcoholic Fatty Liver Disease

The importance of genetics and epigenetic changes in the pathogenesis of non alcoholic fatty liver disease (NAFLD) has been increasingly recognized. Adiponectin has a central role in regulating glucose and lipid metabolism and controlling inflammation in insulin-sensitive tissues and low adiponectin levels have been linked to NAFLD. APPL1 and APPL2 are adaptor proteins that interact with the intracellular region of adiponectin receptors and mediate adiponectin signaling and its effects on metabolism. The aim of our study was the evaluation of a potential association between variants at APPL1 and APPL2 loci and NAFLD occurrence. The impact on liver damage and hepatic steatosis severity has been also evaluated. To this aim allele frequency and genotype distribution of APPL1- rs3806622 and -rs4640525 and APPL2-rs 11112412 variants were evaluated in 223 subjects with clinical diagnosis of NAFLD and compared with 231 healthy subjects. The impact of APPL1 and APPL2 SNPs on liver damage and hepatic steatosis severity has been also evaluated. The minor-allele combination APPL1-C/APPL2-A was associated with an increased risk of NAFLD (OR = 2.50 95% CI 1.45–4.32; p<0.001) even after adjustment for age, sex, body mass index, insulin resistance (HOMA-IR), triglycerides and adiponectin levels. This allele combination carrier had higher plasma alanine aminotransferase levels (Diff = 15.08 [7.60–22.57] p = 0.001) and an increased frequency of severe steatosis compared to the reference allele combination (OR = 3.88; 95% CI 1.582–9.531; p<0.001). In conclusion, C-APPL1/A-APPL2 allele combination is associated with NAFLD occurrence, with a more severe hepatic steatosis grade and with a reduced adiponectin cytoprotective effect on liver.