Changes in the level of poly ADP-ribosylation during a cell cycle

In order to analyze the fluctuation of the poly ADP-ribosylation level during the cell cycle of synchronously growing He La S3 cells, we have developed three different assay systems; intact and disrupted nuclear systems, and poly(ADP-ribose) polymerase in vitro system. The optimum conditions for poly ADP-ribosylation in each assay system were similar except the pH optimum. Under the conditions favoring poly ADP-ribosylation, little radioactivity incorporated into poly(ADP-ribose) was lost after termination of the poly ADP-ribosylation by addition of nicotinamide which inhibits the reactions by more than 90% in any system. In the intact nuclear system, the level of poly ADP-ribosylation increased slightly subsequent to late G2 phase with a peak at M phase. The high level of poly ADP-ribosylation in M phase was also confirmed by using selectively collected mitotic cells which were arrested in M phase by Colcemid. The level in mitotic chromosomes was 5.1-fold higher than that in the nuclei from logarithmically growing cells. Colcemid has no effect on the poly ADP-ribosylation. In the disrupted nuclear system, a relatively high level of poly ADP-ribosylation was observed during mid S-G2 phase. When poly(ADP-ribose) polymerase was extracted from the nuclei with a buffer solution containing 0.3 M KCl, more than 90% of the enzyme activity was recovered. The poly(ADP-ribose) polymerase in vitro system was dependent on both DNA and histone—10 μg each. In the enzyme system, enzyme activity was detected throughout the cell cycle and was observed to be highest in G2 phase. The high level at M phase observed in the intact nuclear system was not seen in the other two systems. Under the assay conditions, little influence of poly(ADP-ribose) degrading enzymes was noted on the level of poly ADP-ribosylation in any of the three systems. This was confirmed at various stages during the cell cycle through pulse-labeling and “chasing” by adding nicotinamide.     

Binding specificity of a HeLa DNA-binding protein to DNA and homopolymers

A protein which has affinity for single-stranded DNA but not for double-stranded DNA has been isolated from HeLa cells by DNA-cellulose chromatography. This protein having a molecular weight of 34,000 was accounted for approximately 3% of total soluble proteins. Its binding specificity to DNA and nucleotide homopolymers has been investigated by Sephadex G-200 column chromatography. Specific binding to single-stranded DNA has been confirmed also by this method and furthermore strong binding to poly U has been found.     

Colliding fragment islands transport independent lineages of endemic rock-crawlers (Grylloblattodea: Grylloblattidae) in the Japanese archipelago

Fragment islands, viewed from the paradigm of island biogeographic theory, depend on continual immigration from continental sources to maintain levels of species diversity, or otherwise undergo a period of relaxation where species diversity declines to a lower equilibrium. Japan is a recently derived fragment island with a rich endemic flora and fauna. These endemic species have been described as paleoendemics, and conversely as recently derived Pleistocene colonists. Geological events in the Miocene period, notably the fragmentation and collision of islands, and the subsequent uplift of mountains in central Japan, provided opportunities for genetic isolation. More recently, cyclical climatic change during the Pliocene and Pleistocene periods led to intermittent land bridge connections to continental Asia. Here we investigate the pattern and timing of diversification in a diverse endemic lineage in order to test whether ongoing migration has sustained species diversity, whether there is evidence of relaxation, and how geological and climatic events are associated with lineage diversification. Using multi-locus genetic data, we test these hypotheses in a poorly dispersing, cold-adapted terrestrial insect lineage (Grylloblattodea: Grylloblattidae) sampled from Japan, Korea, and Russia. In phylogenetic analyses of concatenated data and a species tree approach, we find evidence of three deeply divergent lineages of rock-crawlers in Japan consistent with the pattern of island fragmentation from continental Asia. Tests of lineage diversification rates suggest that relaxation has not occurred and instead endemism has increased in the Japanese Grylloblattidae following mountain-building events in the Miocene. Although the importance of climate change in generating species diversity is a commonly held paradigm in Japanese biogeography, our analyses, including analyses of demographic change and phylogeographic range shifts in putative species, suggests that Pleistocene climatic change has had a limited effect on the diversification of rock-crawlers.     

srGAP1 regulates lamellipodial dynamics and cell migratory behavior by modulating Rac1 activity

The distinct levels of Rac activity differentially regulate the pattern of intrinsic cell migration. However, it remains unknown how Rac activity is modulated and how the level of Rac activity controls cell migratory behavior. Here we show that Slit-Robo GAP 1 (srGAP1) is a modulator of Rac activity in locomotive cells. srGAP1 possesses a GAP activity specific to Rac1 and is recruited to lamellipodia in a Rac1-dependent manner. srGAP1 limits Rac1 activity and allows concomitant activation of Rac1 and RhoA, which are mutually inhibitory. When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility. Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent. Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.     

Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1^K29R-K31R-GFP remained. The HPG1-1 (Rsp5^P514T) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.     

Effect of Caffeine-Containing Beverage Consumption on Serum Alanine Aminotransferase Levels in Patients with Chronic Hepatitis C Virus Infection: A Hospital-Based Cohort Study

Introduction

To date, there have been no prospective studies examining the effect of coffee consumption on serum alanine aminotransferase (ALT) level among individuals infected with the hepatitis C virus (HCV). We conducted a hospital-based cohort study among patients with chronic HCV infection to assess an association between baseline coffee consumption and subsequent ALT levels for 12 months.

Materials and Methods

From 1 August 2005 to 31 July 2006, total 376 HCV-RNA positive patients were recruited. A baseline questionnaire elicited information on the frequency of coffee consumption and other caffeine-containing beverages. ALT level as a study outcome was followed through the patients’ medical records during 12 months. The association between baseline beverage consumption and subsequent ALT levels was evaluated separately among patients with baseline ALT levels within normal range (≤45 IU/L) and among those with higher ALT levels (>45 IU/L).

Results

Among 229 patients with baseline ALT levels within normal range, 186 (81%) retained normal ALT levels at 12 months after recruitment. Daily drinkers of filtered coffee were three times more likely to preserve a normal ALT level than non-drinkers (OR=2.74; P=0.037). However, decaffeinated coffee drinkers had a somewhat inverse effect for sustained normal ALT levels, with marginal significance (OR=0.26; P=0.076). In addition, among 147 patients with higher baseline ALT levels, 39 patients (27%) had ALT reductions of ≥20 IU/L at 12 months after recruitment. Daily drinkers of filtered coffee had a significantly increased OR for ALT reduction (OR=3.79; P=0.034). However, in decaffeinated coffee drinkers, OR could not be calculated because no patients had ALT reduction.

Conclusion

Among patients with chronic HCV infection, daily consumption of filtered coffee may have a beneficial effect on the stabilization of ALT levels.     

Saffold Virus Type 3 (SAFV-3) Persists in HeLa Cells

Saffold virus (SAFV) was identified as a human cardiovirus in 2007. Although several epidemiological studies have been reported, they have failed to provide a clear picture of the relationship between SAFV and human diseases. SAFV genotype 3 has been isolated from the cerebrospinal fluid specimen of patient with aseptic meningitis. This finding is of interest since Theiler’s murine encephalomyelitis virus (TMEV), which is the closely related virus, is known to cause a multiple sclerosis-like syndrome in mice. TMEV persistently infects in mouse macrophage cells in vivo and in vitro, and the viral persistence is essential in TMEV-induced demyelinating disease. The precise mechanism(s) of SAFV infection still remain unclear. In order to clarify the SAFV pathogenicity, in the present study, we studied the possibilities of the in vitro persistent infection of SAFV. The two distinct phenotypes of HeLa cells, HeLa-N and HeLa-R, were identified. In these cells, the type of SAFV-3 infection was clearly different. HeLa-N cells were lyticly infected with SAFV-3 and the host suitable for the efficient growth. On the other hand, HeLa-R cells were persistently infected with SAFV-3. In addition, the SAFV persistence in HeLa-R cells is independent of type I IFN response of host cells although the TMEV persistence in mouse macrophage cells depends on the response. Furthermore, it was suggested that SAFV persistence may be influenced by the expression of receptor(s) for SAFV infection on the host cells. The present findings on SAFV persistence will provide the important information to encourage the research of SAFV pathogenicity.