The Golgi Protein ACBD3, an Interactor for Poliovirus Protein 3A,Modulates Poliovirus Replication

We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence.     

Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells.

The cellular content of 170kD and 180kD topoisomerase II was studied as a function of the proliferation state and cell cycle position in NIH-3T3 cells. When the cells were synchronized by serum starvation and then stimulated to enter the cell cycle by addition of fresh growth medium, the amount of 170kD topoisomerase II present was undetectable until the cells reached late S phase, peaked in G2-M phase cells, and decreased as the cells completed mitosis. The amount of 180kD topoisomerase II was constant once the cells entered the cell cycle. When exponentially growing cells were induced to enter G0 by serum starvation, the amount of 170kD topoisomerase II decreased in parallel with the loss of cells from the S and G2-M phases of the cell cycle and was undetectable once all of the cells reached G0. In contrast, the 180kD enzyme was still present after all of the cells had entered G0. The tightness of association of the two enzymes with chromatin was measured by determining the concentration of salt required to extract them from isolated nuclei. The 180kD enzyme required a higher concentration of NaCl for extraction than did the 170kD enzyme. The different patterns of expression of the two forms of topoisomerase II suggest that they perform different functions in cells.     

Long-term mortality in a cohort of severely obese persons in Italy

Few large studies on Northern European or US populations reported on mortality of severely obese individuals (BMI > or = 40 kg/m(2)). We studied a historical cohort in Italy to compare its mortality with previous findings, to investigate its relationship with BMI in the >40 range, and to provide evidence useful for clinical decision-making on treatment. The cohort comprised 4,837 persons with a BMI > or =40 kg/m(2) and aged > or =18 at first consultation, referred to six centers for obesity treatment between 1975 and 1996. After exclusion of persons with missing personal identification data or those untraceable, 4,498 (972 men, 3,526 women) remained for analyses. We calculated standardized mortality ratios (SMRs) and carried out Cox proportional hazards modeling. General mortality (484 deaths: 153 men, 331 women) was in excess, with SMRs (95% confidence intervals) of 2.78 (2.36-3.26) for men and 2.10 (1.88-2.34) for women. Excess mortality (i) was observed in all BMI categories, except among women weighing 40-42.4 kg/m(2); (ii) increased with increasing BMI; (iii) increased less among persons recruited in recent calendar periods; (iv) was inversely related to age attained at follow-up; and (v) was due to cardiovascular and respiratory diseases and violent deaths but not malignant neoplasms. Excess mortality was similar to that observed in Northern European and US cohorts. Its steady increase with BMI levels > or =40 suggests that benefits proportional to weight reduction are expected and that even limited control may be beneficial. The smaller excess among persons recruited most recently might reflect better treatment.     

ATP-dependent Ca2+ transport in vesicles isolated from the bile canalicular region of the hepatocyte plasma membrane

Three plasma membrane subfractions have been isolated and characterized from rat liver cells. The high affinity Ca2+-stimulated ATPase is highly enriched in the bile canalicular subfraction. Taking into account cross-contamination by the blood sinusoidal and lateral membranes it is suggested that the high-affinity Ca2+-ATPase is located exclusively in this fraction. The high-affinity Ca2+-ATPase is coupled to Ca2+ transport, is calmodulin-insensitive, sensitive to vanadate under appropriate experimental conditions and is strongly inhibited by La3+. In the presence of Ca2+ and ATP the ATPase forms a phosphorylated intermediate of molecular mass about 200 kDa.     

An Overview of the Genetic Structure within the Italian Population from Genome-Wide Data

In spite of the common belief of Europe as reasonably homogeneous at genetic level, advances in high-throughput genotyping technology have resolved several gradients which define different geographical areas with good precision. When Northern and Southern European groups were considered separately, there were clear genetic distinctions. Intra-country genetic differences were also evident, especially in Finland and, to a lesser extent, within other European populations. Here, we present the first analysis using the 125,799 genome-wide Single Nucleotide Polymorphisms (SNPs) data of 1,014 Italians with wide geographical coverage. We showed by using Principal Component analysis and model-based individual ancestry analysis, that the current population of Sardinia can be clearly differentiated genetically from mainland Italy and Sicily, and that a certain degree of genetic differentiation is detectable within the current Italian peninsula population. Pair-wise F_ST statistics Northern and Southern Italy amounts approximately to 0.001 between, and around 0.002 between Northern Italy and Utah residents with Northern and Western European ancestry (CEU). The Italian population also revealed a fine genetic substructure underscoring by the genomic inflation (Sardinia vs. Northern Italy = 3.040 and Northern Italy vs. CEU = 1.427), warning against confounding effects of hidden relatedness and population substructure in association studies.     

Inhibition of hepatocyte plasma membrane Ca2+-ATPase activity by menadione metabolism and its restoration by thiols

Incubation of isolated rat hepatocytes with cytotoxic concentrations of menadione resulted in inhibition of plasma membrane Ca2+-ATPase activity. This could be restored by subsequent treatment with either dithiothreitol or reduced glutathione, suggesting that the inhibition by menadione was due to oxidation of sulfhydryl groups critical for Ca2+-ATPase activity.     

Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II

The p170 and p180 forms of topoisomerase II have been compared. The concentration dependence of ATP for catalytic activity of the two forms of the enzyme was identical, and each was equally sensitive to novobiocin. Orthovanadate was found to be a potent inhibitor of catalytic activity of both p170 and p180, with an IC50 value of about 2 microM for each. Under standard reaction conditions, relaxation of supercoiled pBR322 by p180 was highly processive, while p170 performed the same reaction in a distributive manner. The optimal concentration of KCl for catalytic activity of p180 was 20-30 mM higher than that for p170. Comparison of their thermal stability showed that p180 was inactivated at twice the rate of p170. Teniposide and merbarone selectively inhibited catalytic activity of p170, requiring concentrations 3-fold and 8-fold lower, respectively, than those required for equivalent inhibition of p180. Similar selectivity for p170 was seen for teniposide-stimulated DNA cleavage or its inhibition by merbarone. Analysis of sites of DNA cleavage indicated a subset of sites that were either preferred or unique for each of the enzymes. A synthetic oligonucleotide representative of p170 sites selectively inhibited the p170 enzyme. Immunoblotting of p170 and p180 from U937 cells at different stages of proliferation showed that p170 levels declined as the cells reached the plateau phase of growth, while p180 levels were low during rapid proliferation and increased as the growth rate slowed. The data indicate that the p170 and p180 forms of topoisomerase II can be distinguished biochemically, pharmacologically, and by differential cellular regulation.     

Metallothionein turnover in mammalian cells. Implications in metal toxicity

Metallothioneins are low molecular weight, cysteine-rich proteins believed to participate in metal detoxification. Turnover of Cd-, Zn-, and Au-induced metallothionein was studied in a Chinese hamster ovary cell line which was resistant to Cd and the Au-containing drug auranofin. Cd, Zn, and Au were potent inducers of metallothionein mRNA and resulted in accumulation of approximately equal amounts of mRNA. Pulse-chase studies with [35S]cysteine revealed that the half-life of Au-, Zn-, and Cd-induced metallothionein was 0.75, 10, and 24 h, respectively. The differences in the half-life of metallothionein may be related to the tertiary structure of metal-metallothionein complexes. These results have implications in the mechanism of resistance to gold compounds.     

Antimicrobial and anticancer activity of tetrahedral, chelated, diphosphine silver(I) complexes: comparison with copper and gold

Tetrahedral, bischelated Ag(I) diphosphine complexes [Ag(P-P)2]NO3, where P-P is Ph2P(CH2)2PPh2 (dppe), Et2P(CH2)2PPh2 (depe), and cis-Ph2P(CH = CH)PPh2 (dppey), are potently cytotoxic to B16 melanoma cells in vitro (IC50 4 microM) and exhibit good activity against ip P388 leukemia in mice. The complex [Ag(dppe)2]NO3 is active against M5076 reticulum cell sarcoma. The antibacterial and antifungal activities of Ag(I) diphosphine and related Cu(I) and Au(I) complexes were assessed. The complexes [Au(dppey)2]Cl, [Au(dppp)2]Cl and (CuCl)2(dppe)3 show modest activity against three of the 12 bacterial strains tested, but all complexes exhibit antifungal activity against three strains of C. albicans in a "defined" medium, [Ag(depe)2]NO3 and [Au(dppp)2]Cl having comparable activity to fungizone. Antifungal activity of the complexes is reduced in Sabouraud's broth medium, and lost altogether for the Ag(I) complexes. Reactions of some of the Ag(I) complexes with glutathione and blood plasma were studied by 31P NMR.