Latrunculin B or ATP depletion induces cofilin-dependent translocation of actin into nuclei of mast cells

Increasing cellular G-actin, using latrunculin B, in either intact or permeabilized rat peritoneal mast cells, caused translocation of both actin and an actin regulatory protein, cofilin, into the nuclei. The effect was not associated with an increase in the proportion of apoptotic cells. The major part of the nuclear actin was not stained by rhodamine-phalloidin but could be visualized with an actin antibody, indicating its monomeric or a conformationally distinct state, e.g. cofilin-decorated filaments. Introduction of anti-cofilin into permeabilized cells inhibited nuclear actin accumulation, implying that an active, cofilin-dependent, import exists in this system. Nuclear actin was localized outside the ethidium bromide-stained region, in the extrachromosomal nuclear domain. In permeabilized cells, the appearance of nuclear actin and cofilin was not significantly affected by increasing [Ca(2+)] and/or adding guanosine 5'-O-(3-thiotriphosphate), but was greatly promoted when ATP was withdrawn. Similarly, ATP depletion in intact cells also induced nuclear actin accumulation. In contrast to the effects of latrunculin B, ATP depletion was associated with an increase in cortical F-actin. Our results suggest that the presence of actin in the nucleus may be required for certain stress-induced responses and that cofilin is essential for the nuclear import of actin.     

Effects of latrunculin reveal requirements for the actin cytoskeleton during secretion from mast cells

To investigate the role of the actin cytoskeleton in exocytosis, we have tested the effects of latrunculin B, a microfilament-disrupting drug, on secretion from intact and permeabilised rat peritoneal mast cells. The toxin strongly inhibited secretion from intact cells (attached or in suspension) responding to a polybasic agonist, compound 48/80. However, this effect was revealed only after a profound depletion of actin filaments. This was achieved by a long (1 h) exposure of cells to the drug before activation, together with its presence during activation. Maximal inhibition of secretion by such treatment was 85% at 40 microgram/ml latrunculin B. These results indicate that minimal actin structures are essential for the exocytotic response. In contrast, stimulus-induced cell spreading was prevented by latrunculin (5 microgram/ml) applied either before or after activation. The effects of the toxin on intact cells were fully reversible. The responses of permeabilised cells were affected differentially: secretion induced by calcium was more sensitive to latrunculin than that induced by GTP-gamma-S. The calcium response, therefore, is more dependent upon the integrity of the actin cytoskeleton than the response induced by GTP-gamma-S. Again, maximal inhibitory effects (approximately 65 and 25% at 40 microgram/ml) were observed only when cells were exposed to the toxin both before and after permeabilisation. Since the permeabilised cells system focuses on the final steps of exocytosis, the incomplete inhibition suggests that actin plays a modulatory rather than a central role at this stage.     

DEVELOPMENT OF DISPERSAL, MOVEMENT PATTERNS, AND HAUL-OUT USE BY PUP AND JUVENILE STELLER SEA LIONS (EUMETOPIAS JUBATUS) IN ALASKA

Population declines of Steller sea lions ( Eumetopias juhatus ) in western Alaska (west of 144°W) may be a result of reduced juvenile survival. We used satellite telemetry to study the at-sea distribution and movement patterns of pup (1.6–11.9 mo) and juvenile (12.0–35.1 mo) Steller sea lions. We studied trip distance, duration, and interhaul-out movements of sea lions in relation to age, sex, and month of year in the decreasing western population (WP; Prince William Sound, Kodiak, Aleutian Islands, Alaska) and the increasing eastern population (EP; Southeast Alaska). We deployed 103 satellite transmitters (29 WP; 74 EP) on sea lions between 1998 and 2001. Round trip distance and duration increased with age, trip distance was greater in the WP than the EP, trip duration was greater for females than males, and haul-out use was clustered. Changes in round trip distance and duration occurred from April to June for all age classes studied indicating that the annual timing of weaning may be less variable than the age of weaning. Overall, 90% of round trips were ≤ 15 km from haul-outs and 84% were <20 h, indicating nearshore areas adjacent to haulouts are critical to the developing juvenile.     

Tumor necrosis factor-alpha reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells

Endothelial dysfunction associated with elevated serum levels of TNF-alpha observed in diabetes, obesity, and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, substrate of endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment with TNF-alpha (10 ng/ml) suppresses not only eNOS expression but also the availability of arginine via the coordinate suppression of argininosuccinate synthase (AS) expression in aortic endothelial cells. Western blot and real-time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF-alpha with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF-alpha suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies suggested that the repression of AS expression by TNF-alpha may be mediated, in part, via the NF-kappaB signaling pathway. These findings demonstrate that TNF-alpha coordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNF-alpha is an added insult to endothelial function because of its important role in NO production and in endothelial viability.     

Characterisation and carriage ratio of Clostridium difficile strains isolated from a community-dwelling elderly population in the United Kingdom

Background

Community-associated Clostridium difficile infection (CDI) appears to be an increasing problem. Reported carriage rates by C.difficile are debatable with suggestions that primary asymptomatic carriage is associated with decreased risk of subsequent diarrhoea. However, knowledge of potential reservoirs and intestinal carriage rates in the community, particularly in the elderly, the most susceptible group, is limited. We have determined the presence of C.difficile in the faeces of a healthy elderly cohort living outside of long-term care facilities (LCFs) in the United Kingdom.

Methods

Faecal samples from 149 community-based healthy elderly volunteers (median age 81 years) were screened for C.difficile using direct (Brazier''s CCEY) and enrichment (Cooked Meat broth) culture methods and a glutamate dehydrogenase (GDH) immunoassay. Isolates were PCR-ribotyped and analysed for toxin production and the presence of toxin genes.

Results

Of 149 faecal samples submitted, six (4%) were found to contain C.difficile. One particular sample was positive by both the GDH immunoassay and direct culture, and concurrently produced two distinct strain types: one toxigenic and the other non-toxigenic. The other five samples were only positive by enrichment culture method. Overall, four C.difficile isolates were non-toxigenic (PCR-ribotypes 009, 026 (n = 2) and 039), while three were toxigenic (PCR-ribotypes 003, 005 and 106). All individuals who had a positive culture were symptom-free and none of them had a history of CDI and/or antibiotics use in the 3 month period preceding recruitment.

Conclusions

To our knowledge, this is the first study of the presence of C.difficile in healthy elderly community-dwelling individuals residing outside of LCFs. The observed carriage rate is lower than that reported for individuals in LCFs and interestingly no individual carried the common epidemic strain PCR-ribotype 027 (NAP1/BI). Further follow-up of asymptomatic carriers in the community, is required to evaluate host susceptibility to CDI and identify dynamic changes in the host and microbial environment that are associated with pathogenicity.     

Anti-neoplastic activity of two flavone isomers derived from Gnaphalium elegans and Achyrocline bogotensis

Over 4000 flavonoids have been identified so far and among these, many are known to have antitumor activities. The basis of the relationships between chemical structures, type and position of substituent groups and the effects these compounds exert specifically on cancer cells are not completely elucidated. Here we report the differential cytotoxic effects of two flavone isomers on human cancer cells from breast (MCF7, SK-BR-3), colon (Caco-2, HCT116), pancreas (MIA PaCa, Panc 28), and prostate (PC3, LNCaP) that vary in differentiation status and tumorigenic potential. These flavones are derived from plants of the family Asteraceae, genera Gnaphalium and Achyrocline reputed to have anti-cancer properties. Our studies indicate that 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays potent activity against more differentiated carcinomas of the colon (Caco-2), and pancreas (Panc28), whereas 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) cytototoxic action is observed on poorly differentiated carcinomas of the colon (HCT116), pancreas (Mia PaCa), and breast (SK-BR3). Both flavones induced cell death (>50%) as proven by MTT cell viability assay in these cancer cell lines, all of which are regarded as highly tumorigenic. At the concentrations studied (5-80 μM), neither flavone demonstrated activity against the less tumorigenic cell lines, breast cancer MCF-7 cells, androgen-responsive LNCaP human prostate cancer line, and androgen-unresponsive PC3 prostate cancer cells. 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays activity against more differentiated carcinomas of the colon and pancreas, but minimal cytotoxicity on poorly differentiated carcinomas of these organs. On the contrary, 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) is highly cytotoxic to poorly differentiated carcinomas of the colon, pancreas, and breast with minimal activity against more differentiated carcinomas of the same organs. These differential effects suggest activation of distinct apoptotic pathways. In conclusion, the specific chemical properties of these two flavone isomers dictate mechanistic properties which may be relevant when evaluating biological responses to flavones.     

The non-synonymous SNP, R1150W, in SCN9A is not associated with chronic widespread pain susceptibility

Background

Chronic pain conditions are characterized by significant individual variability complicating the identification of pathophysiological markers. Leukocyte telomere length (TL), a measure of cellular aging, is associated with age-related disease onset, psychosocial stress, and health-related functional decline. Psychosocial stress has been associated with the onset of chronic pain and chronic pain is experienced as a physical and psychosocial stressor. However, the utility of TL as a biological marker reflecting the burden of chronic pain and psychosocial stress has not yet been explored.

Findings

The relationship between chronic pain, stress, and TL was analyzed in 36 ethnically diverse, older adults, half of whom reported no chronic pain and the other half had chronic knee osteoarthritis (OA) pain. Subjects completed a physical exam, radiographs, health history, and psychosocial questionnaires. Blood samples were collected and TL was measured by quantitative polymerase chain reaction (qPCR). Four groups were identified characterized by pain status and the Perceived Stress Scale scores: 1) no pain/low stress, 2) no pain/high stress, chronic pain/low stress, and 4) chronic pain/high stress. TL differed between the pain/stress groups (p = 0.01), controlling for relevant covariates. Specifically, the chronic pain/high stress group had significantly shorter TL compared to the no pain/low stress group. Age was negatively correlated with TL, particularly in the chronic pain/high stress group (p = 0.03).

Conclusions

Although preliminary in nature and based on a modest sample size, these findings indicate that cellular aging may be more pronounced in older adults experiencing high levels of perceived stress and chronic pain.     

Characterization of osteoblastic properties of 7F2 and UMR-106 cultures after acclimation to reduced levels of fetal bovine serum

Estrogen plays an important role in skeletal physiology by maintaining a remodeling balance between the activity of osteoblasts and osteoclasts. In an attempt to decipher the mechanism through which estrogen elicits its action on osteoblasts, experimentation necessitated the development of a culturing environment reduced in estrogenic compounds. The selected medium (OPTI-MEM) is enriched to sustain cultures under reduced fetal bovine serum (FBS) conditions and is devoid of the pH indicator phenol red, a suspected estrogenic agent. This protocol reduced the concentration of FBS supplementation to 0% through successive 24 h incubations with diminishing amounts of total FBS (1%, 0.1%, and 0%). The protocol does not appear to alter the viability, cell morphology, or osteoblast-like phenotype of 7F2 and UMR-106 cell lines when compared with control cells grown in various concentrations of FBS. Although the rate of mitotic divisions declined, the 7F2 and UMR-106 cultures continued to express osteoblast-specific markers and exhibited estrogen responsiveness. These experimental findings demonstrate that the culture protocol developed did not alter the osteoblast nature of the cell lines and provides a model system to study estrogen's antiresorptive role on skeletal turnover.     

MOLTING PHENOLOGY OF HARBOR SEALS ON TUGIDAK ISLAND, ALASKA

We documented the progression and timing of the annual molt of harbor seals on Tugidak Island, Alaska, from 1997 to 1999. In all years the timing of molting differed among age-sex classes. Yearlings molted first, subadults second, adult females third, and lastly adult males. Timing of molting was nearly identical in 1997–1998, whereas in 1999 molting occurred three to six days later for all age-sex classes except yearlings. Estimated dates when peak proportions of each age-sex class were molting ranged from 2 August (yearlings) to 2 September (adult males). The number of seals hauled out was positively related to the proportion of seals in the molt and negatively related to the proportion of seals in the postmolt. Population trend estimates, based on aerial counts conducted during a narrow window within the molting period, are likely biased toward certain age-sex classes. Statistical models used to estimate trend include covariates to help account for within-year variation in seal numbers, but do not account for compositional changes that occur during molting. Population modeling may elucidate the effects of within-year population structure on trend estimates. Monitoring molting phenology at additional sites is necessary to determine the extent of geographic variation in molting.