Time-Dependent Effects of Progressive Gamma -Linolenate Feeding on Hyperphagia,Weight Gain,and Erythrocyte Fatty Acid Composition During Growth of Zucker Obese Rats

Obese Zucker rats (fa/fa) have low levels of arachidonic acid (AA) in liver phospholipids (PL). We have previously shown that a 70% gamma-linolenate concentrate (GLA; an AA intermediate) fed at a fixed dose (0.07 g/day) normalized hepatic PL AA and reduced weight gain selectively in the obese animals. In a follow-up study, 16 obese (fa/fa) and 16 lean (Fa/Fa) 4-week-old male rats were randomized into 4 groups of 8 each and gavaged daily with soybean oil (SOY) containing 55% 18:2ω6 (an AA precursor) or GLA, using a progressive dose (≤ 5% of total calories) based on body weight. A defined diet with 11% of energy as SOY was fed ad libitum for 60 days. GLA obese had lower body weight (p<0.0001) and 60-day cumulative food intake (p<0.05) compared to SOY obese, but neither parameter differed between the lean groups. For the last twenty days cumulative food intake was identical for GLA obese and SOY lean, whereas SOY obese consumed 18% more (p<0.05). Thus the progressive dose of GLA selectively suppressed hyperphagia in obese Zucker rats. Erythrocytes collected at 15-day intervals showed parallel increases in AA in both genotypes over time, suggesting normal AA availability during rapid growth. Thus, the reduced PL AA in the livers from the obese rats probably reflects impaired distribution in selected tissues rather than reduced hepatic production. Due to the potential health risks of enriching tissue lipids with AA, great caution is advised in considering GLA as therapy for human obesity.     

Functional and biochemical disposition of 7,12-dimethylbenz[a]anthracene in murine lymphoid cells

The metabolism of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) was studied in murine lymphocytes. This carcinogen has previously been shown to be immunosuppressive to lymphocytes regardless of their ability to be induced via the Ah locus and receptor. Experiments were designed to quantify the generation of metabolites of DMBA by lymphocytes incubated with [14C]DMBA and to ascertain whether radioactivity was covalently bound to cellular macromolecules in DMBA-exposed lymphocytes. No significant metabolism of DMBA was detected in culture supernatants, except when cultures were incubated in the presence of Arochlor-induced rat liver 9000 x g supernatants (S9). Covalent binding of 14C to cellular macromolecules was enhanced approximately eightfold in the presence of S9. Inhibition of monooxygenase activity by alpha-naphthoflavone did not modulate the immunosuppressive character of DMBA. Furthermore, addition of S9 did not amplify or ablate DMBA-mediated suppression of lymphocyte proliferation to the mitogen concanavalin A (Con A). Selected metabolites of DMBA were evaluated for immunosuppressive effects in cultures stimulated with mitogens and cellular alloantigens. 7-Hydroxymethyl-12-methylbenz[a]anthracene (OHMe) and 5,6-dihydro-5,6-dihydroxybenz[a]anthracene (Diol) were found to cause only slightly greater suppression of lymphocyte responses than DMBA. Thus, it appears that metabolites of DMBA were not responsible for the immunosuppression observed in lymphocyte cultures and that lymphocytes were not equipped to metabolize any significant amount of DMBA. These data lend support to the hypothesis that parent compound alone is responsible for the immunosuppressive effects observed in murine lymphocyte culture.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Effect of weaning time and associated management practices on postweaning chronic diarrhea in captive rhesus monkeys (Macaca mulatta)

OBJECTIVE: Our purpose was to assess the extent to which early weaning and other weaning-management factors affect development of postweaning chronic diarrhea in captive rhesus monkeys at the California Regional Primate Research Center between 1992 and 1995. METHODS: Data for weaning, management, and onset of diarrhea were obtained from daily records. The Cox proportional hazard model was used to assess whether the risk of chronic diarrhea was related to early weaning. RESULTS: Monkeys that were lighter at weaning had a threefold increase in risk of postweaning chronic diarrhea (P = 0.07), compared with that in heavier monkeys. An episode of preweaning diarrhea increased the risk of postweaning chronic diarrhea twofold (P = 0.08). Relocation of monkeys to outdoor facilities in the fall was associated with a fivefold decrease in risk (P < 0.001), compared with that of other seasons, and weaning in 1993 was associated with a twofold decrease in risk, compared with that of other years (P = 0.04). CONCLUSIONS: Multiple factors need to be considered for prevention of postweaning chronic diarrhea, including weaning weight, preweaning diarrhea, season weaned, and weaning conditions that change from year to year.     

Subdomains in the F and G helices of bacteriorhodopsin regulate the conformational transitions of the reprotonation mechanism

We have performed cysteine scanning mutagenesis of the bacteriorhodopsin mutant D85N to explore the role of individual amino acids in the conformational transitions of the reprotonation mechanism. We have used whole-cell reflectance spectroscopy to evaluate the spectral properties of the 59 mutants generated during a scan of the entire F and G helices and the intervening loop region. Cys mutants were grouped into one of six phenotypes based on the spectral changes associated with their M <--> N <--> O intermediate-state transitions. Mutations that produced similar phenotypes were found to cluster in discrete molecular domains and indicate that M, N, and O possess distinct structures and that unique molecular interactions regulate the transitions between them. The distribution of these domains suggests that 1) the extramembranous loop region is involved in the stabilization of the N and M intermediates, 2) lipid-protein interactions play a key role in the accumulation of N, and 3) the amino acid side-chain interactions in the extracellular portion of the interface between helices G and A participate in the accumulation of M.     

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction  

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.