Intranuclear DNA density affects chromosome condensation in metazoans

Chromosome condensation is critical for accurate inheritance of genetic information. The degree of condensation, which is reflected in the size of the condensed chromosomes during mitosis, is not constant. It is differentially regulated in embryonic and somatic cells. In addition to the developmentally programmed regulation of chromosome condensation, there may be adaptive regulation based on spatial parameters such as genomic length or cell size. We propose that chromosome condensation is affected by a spatial parameter called the chromosome amount per nuclear space, or “intranuclear DNA density.” Using Caenorhabditis elegans embryos, we show that condensed chromosome sizes vary during early embryogenesis. Of importance, changing DNA content to haploid or polyploid changes the condensed chromosome size, even at the same developmental stage. Condensed chromosome size correlates with interphase nuclear size. Finally, a reduction in nuclear size in a cell-free system from Xenopus laevis eggs resulted in reduced condensed chromosome sizes. These data support the hypothesis that intranuclear DNA density regulates chromosome condensation. This suggests an adaptive mode of chromosome condensation regulation in metazoans.     

Lack of tissue-specificity of calcium-activated neutral proteases from skeletal muscle and lung of rabbit

Calcium-activated neutral proteases (CANPs) with high sensitivity (microCANP) and low sensitivity (mCANP) to calcium ions were purified individually from rabbit skeletal muscle and rabbit lung and compared as to their electrophoretic properties, calcium requirements and peptide mapping of fragments produced by S. aureus V8 protease digestion of separated subunits. All of the results suggested that there is no difference between the microCANPs as well as between the mCANPs obtained from the two tissues, with respect to the chemical and enzymatic properties. However, the contents of CANPs in these tissues were different.     

Chloride transport of yeast vacuolar membrane vesicles: a study of in vitro vacuolar acidification.

Effects of various solutes on acidification inside the vacuolar membrane vesicles of the yeast Saccharomyces cerevisiae were examined. ATP-dependent acidification was stimulated by the presence of chloride salts. There was essentially no difference in the stimulatory effects of NaCl, KCl, LiCl, and choline chloride. The membrane potential across the vacuolar membrane was reduced by the presence of Cl- salts. Transport of 36Cl- is driven by the protonmotive force across the vacuolar membrane. Kinetic analyses have revealed that the stimulatory effect of Cl- on internal acidification depends on two distinct components. One shows linear dependency on chloride concentration and is inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid (DIDS). The other exhibits saturable kinetics with an apparent Km for chloride of 15-20 mM. We conclude that the vacuolar membrane of yeast is equipped with Cl- transport systems contributing to the formation of a chemical gradient of protons across the vacuolar membrane by shunting the membrane potential generated by proton translocation.     

Partitioning, Movement, and Positioning of Nucleoids in Mycoplasma capricolum

The nucleoids in Mycoplasma capricolum cells were visualized by phase-combined fluorescence microscopy of DAPI (4', 6-diamidino-2-phenylindole)-stained cells. Most growing cells in a rich medium had one or two nucleoids in a cell, and no anucleate cells were found. The nucleoids were positioned in the center in mononucleoid cells and at one-quarter and three-quarters of the cell length in binucleoid cells. These formations may have the purpose of ensuring delivery of replicated DNA to daughter cells. Internucleoid distances in binucleoid cells correlated with the cell lengths, and the relationship of DNA content to cell length showed that cell length depended on DNA content in binucleoid cells but not in mononucleoid cells. These observations suggest that cell elongation takes place in combination with nucleoid movement. Lipid synthesis was inhibited by transfer of cells to a medium lacking supplementation for lipid synthesis. The transferred cells immediately stopped dividing and elongated while regular spaces were maintained between the nucleoids for 1 h. After 1 h, the cells changed their shapes from rod-like to round, but the proportion of multinucleoid cells increased. Inhibition of protein synthesis by chloramphenicol induced nucleoid condensation and abnormal positioning, although partitioning was not inhibited. These results suggest that nucleoid partitioning does not require lipid or protein synthesis, while regular positioning requires both. When DNA replication was inhibited, the cells formed branches, and the nucleoids were positioned at the branching points. A model for the reproduction process of M. capricolum, including nucleoid migration and cell division, is discussed.     

Risk-sensitive decisions during nesting may increase maternal provisioning capacity in the subsocial shield bug Parastrachia japonensis

Abstract 1. Females of the monophagous shield bug Parastrachia japonensis Scott provision their nymph-containing nests with high-quality drupes of the single host tree, Schoepfia jasminodora , a resource that is poor and unstable, under an array of variable environmental constraints.
2. Although the number of drupes provisioned is correlated positively with enhanced nymphal development and survival, there is great variability in the number of drupes that females provide.
3. The ability to adjust behaviour and/or physiology, i.e. make risk-sensitive decisions, should be adaptive for organisms such as P. japonensis that are confronted with extreme variability in food availability and in the conditions under which they must forage. To assess whether females of this species use risk-sensitivity to improve their provisioning success, data on the parameters of provisioning activity obtained during a long-term field study were analysed. The relationships between variation in three female-controlled factors during nesting (nest site, active stage during the provisioning season, duration of provisioning activity) and the conditions of three environmental factors (drupe availability, intraspecific competition, weather) were examined. The relationships between all of these factors and provisioning capacity of females were investigated.
5. Nest site, intraspecific competition, and timing of provisioning activity only affected provisioning capacity when drupe availability was extremely good or bad or weather was particularly bad.
6. The findings suggest that females use risk-sensitive decisions to increase provisioning capacity under extreme conditions of low and high drupe availability and inclement weather.     

Role of JC virus agnoprotein in virion formation

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T' antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein-negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus-like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S-transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1-pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.     

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy

ABSTRACT

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.

Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2     

Increased susceptibility to tumor necrosis factor-alpha in butyric acid-induced apoptosis is caused by downregulation of cFLIP expression in Jurkat T cells

Butyric acid is one of the major extracellular metabolites of periodontopathic Gram-negative bacteria. We previously demonstrated that butyric acid induced apoptosis in human T cells. In the present study, we examined the interaction between butyric acid and TNF-alpha in Jurkat T-cell apoptosis. Simultaneous treatment with TNF-alpha enhanced butyric acid-induced apoptosis by promoting caspase activity more than was achieved by either reagent alone. We examined which genes were associated with the increased susceptibility to TNF-alpha caused by butyric acid, and revealed that expression of cFLIP decreased with increased concentrations of butyric acid. Furthermore, exogenous expression of cFLIP protein suppressed the enhancing effect by TNF-alpha in the apoptosis. These results suggest that butyric acid downregulates cFLIP expression and increases the susceptibility to TNF-alpha by activating caspases via the death receptor signal.