Bacillusanthracis Surface-Layer Proteins Assemble by Binding to the Secondary Cell Wall Polysaccharide in a Manner that Requires csaB and tagO

Bacillus anthracis, the causative agent of anthrax, requires surface (S)-layer proteins for the pathogenesis of infection. Previous work characterized S-layer protein binding via the surface layer homology domain to a pyruvylated carbohydrate in the envelope of vegetative forms. The molecular identity of this carbohydrate and the mechanism of its display in the bacterial envelope are still unknown. Analyzing acid-solubilized, purified carbohydrates by mass spectrometry and NMR spectroscopy, we identify secondary cell wall polysaccharide (SCWP) as the ligand of S-layer proteins. In agreement with the model that surface layer homology domains bind to pyruvylated carbohydrate, SCWP was observed to be linked to pyruvate in a manner requiring csaB, the only structural gene known to be required for S-layer assembly. B. anthracis does not elaborate wall teichoic acids; however, its genome harbors tagO and tagA, genes responsible for the synthesis of the linkage unit that tethers teichoic acids to the peptidoglycan layer. The tagO gene appears essential for B. anthracis growth and complements the tagO mutant phenotypes of staphylococci. Tunicamycin-mediated inhibition of TagO resulted in deformed, S-layer-deficient bacilli. Together, these results suggest that tagO-mediated assembly of linkage units tethers pyruvylated SCWP to the B. anthracis envelope, thereby enabling S-layer assembly and providing for the pathogenesis of anthrax infections.     

Modulation of hypothalamic PTP1B in the TNF-α-induced insulin and leptin resistance

We have associated functional and molecular studies of insulin and leptin to investigate the effect of TNF-α on central insulin and leptin signaling in rats pre-treated with PTP1B-ASO. The icv infusion of TNF-α-induced an increase in PTP1B protein expression and activity, and attenuated insulin and leptin sensitivity and signaling in the hypothalamus. However, TNF-α was able to completely blunt the leptin and insulin effect in rats treated with PTP1B-ASO, suggesting that TNF-α does not require PTP1B to fully attenuate the leptin and insulin effects. In addition, our data also show that other mechanisms of insulin and leptin resistance are activated in the hypothalamus by TNF-α.     

Low liver conversion rate of alpha-linolenic to docosahexaenoic acid in awake rats on a high-docosahexaenoate-containing diet

We quantified the rates of incorporation of alpha-linolenic acid (alpha-LNA; 18:3n-3) into "stable" lipids (triacylglycerol, phospholipid, cholesteryl ester) and the rate of conversion of alpha-LNA to docosahexaenoic acid (DHA; 22: 6n-3) in the liver of awake male rats on a high-DHA-containing diet after a 5-min intravenous infusion of [1-(14)C]alpha-LNA. At 5 min, 72.7% of liver radioactivity (excluding unesterified fatty acid radioactivity) was in stable lipids, with the remainder in the aqueous compartment. Using our measured specific activity of liver alpha-LNA-CoA, in the form of the dilution coefficient lambda(alpha-LNA-CoA), we calculated incorporation rates of unesterified alpha-LNA into liver triacylglycerol, phospholipid, and cholesteryl ester as 2,401, 749, and 9.6 nmol/s/g x 10(-4), respectively, corresponding to turnover rates of 3.2, 8.7, and 2.9%/min and half-lives of 8-24 min. A lower limit for the DHA synthesis rate from alpha-LNA equaled 15.8 nmol/s/g x 10(-4) (0.5% of the net in corporation rate). Thus, in rats on a high-DHA-containing diet, rates of beta-oxidation and esterification of alpha-LNA into stable liver lipids are high, whereas its conversion to DHA is comparatively low and insufficient to supply significant DHA to the brain. High incorporation and turnover rates likely reflect a high secretion rate by liver of stable lipids within very low density lipoproteins.     

Trypanosoma cruzi: infectivity modulation of a clone after passages through different hosts

Although Trypanosoma cruzi virulence can be modified through passages in vivo or long-term in vitro culture, the mechanisms involved are poorly understood. Here we report modifications in the infectivity of a T. cruzi clone after passages in different hosts without detectable changes in parasite genetic patterns. A clone was obtained from a T. cruzi IIe isolate and showed to be less virulent than the original isolate (p<0.05). This clone was enzymatically similar to the original isolate as shown by multilocus enzyme electrophoresis. Infection of this clone was compared by successive passages in mice and guinea pigs. The mouse-passaged subline became more virulent for both host species compared to the guinea pig-passaged subline (p<0.05). The clone line displayed similar random amplified polymorphic DNA patterns before and after passages in different hosts suggesting that alterations in virulence could be a result of a differential expression of virulence factors.     

Continuous high expression of XBP1 and GRP78 is important for the survival of bone marrow cells in CCl4-treated cirrhotic liver

We have previously shown that infusion of bone marrow cells (BMC) improves CCl₄-induced cirrhosis. However, it is unclear why the injected BMC are resistant to CCl₄ damage and subsequently improve the local microenvironment in damaged liver. To analyze the cellular phenomena involved in this process, we studied the damaged liver using electron microscopy. We found that CCl₄ caused rough endoplasmic reticula to swell in hepatocytes. To analyze the gene expression patterns associated with this process, we conducted PCR-selected suppressive subtractive hybridization. We found that expression levels of HSP84, HSP40, and XBP1 differed markedly between control liver and liver infused with BMC. Immunohistochemical staining revealed that expression levels of HSP84 and HSP40 were markedly higher in the early phase of differentiation immediately after BMC infusion, but decreased over time. XBP1 expression remained high during the late phase, and GRP78 expression increased with XBP1 activation. We also found that GFP-positive BMC expressed XBP1 and GRP78. XBP1 and GRP78 are associated with ER stress. Thus, continuous high XBP1 and GRP78 expression might be essential for the survival and proliferation of BMC in a CCl₄-induced persistent liver damage environment.     

Leishmania amazonensis: xylitol as inhibitor of macrophage infection and stimulator of macrophage nitric oxide production

Xylitol is a sugar alcohol being explored for clinical uses. The aim was to evaluate the effects of xylitol on Leishmania amazonensis-infected J774A.1 macrophages. Macrophages were infected with L. amazonensis for 3h, washed and incubated with 2.5 or 5.0% xylitol for 24, 48, and 72 h at 37 degrees C. Infection indexes for macrophages incubated only in medium were compared to those treated with xylitol. Cell viability and nitric oxide production were determined each time. Xylitol did not affect L. amazonensis or J774A.1 cell viabilities. Xylitol at 5.0% stimulated nitric oxide production by macrophages at 72 h (p<0.01). At 2.5 and 5.0%, xylitol inhibited nitric oxide production by L. amazonensis at 48 h (p<0.05) when compared to control. Infection indexes were significantly lower at 72 h (p<0.05), (16.9% and 9.6%) in cells cultivated with 2.5 and 5.0% xylitol, respectively, compared to control (38.4%). Results suggest a potential leishmanicidal action of the xylitol on infected macrophages.     

Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics

Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the "gold-standard" Folch or Bligh and Dyer recipes.     

Effects of a Seven‐Day Continuous Infusion of Ropivacaine on Circadian Rhythms in the Rat

The present study was conducted to evaluate the effect of a 7 d continuous infusion of ropivacaine on the 24 h rhythms of body temperature, heart rate, and locomotor activity. After an initial 7 d baseline, rats were randomly divided into two groups of 4 rats each to receive ropivacaine or saline via an osmotic pump for 7 consecutive days. The pumps were removed thereafter and observed during a 7 d recovery span. The studied circadian rhythms were measured by radiotelemetry throughout each of the 7 d periods. An additional group of 4 rats was studied under the same experimental conditions to assess the plasma levels of ropivacaine on days 3 and 8 following pump implantation. Our results indicate that ropivacaine does not induce loss of the circadian rhythms of body temperature, heart rate, or locomotor activity; a prominent period of 24 h was found for all variables in all animals, before, during, and after ropivacaine treatment. However, ropivacaine treatment did modify some characteristics of the rhythms; it increased the MESOR (24 h mean) of the heart rate and locomotor activity rhythms and advanced the acrophase (peak time) of the locomotor activity circadian rhythm. The present study indicates that the circadian rhythms of heart rate and locomotor activity are modified after continuous infusion of ropivacaine, which is of particular interest, given the potential cardiotoxicity of this local anesthetic agent.     

Intraruminal infusion of n-butyric acid induces an increase of ruminal papillae size independent of IGF-1 system in castrated bulls

Abstract

The objective of this study was to explore morphological alterations of rumen papillae induced by n-butyric acid in relation to the insulin-like growth factor (IGF) system in adult castrated bulls. Three animals fitted with rumen cannula were fed twice daily at a low and high nutritional level (LL and HL), i.e., at 1.1 × maintenance (M) and 1.6 × M, respectively. Diets contained artificial dried grass and concentrate (74:26 and 52:48). Bulls received no (B0) or daily intraruminal infusions of 500 g n-butyric acid (B500) over 14 d. The infusion started 1 h after the morning feeding (9:00) and lasted for 3.5 h. Thus, four treatments (B0LL, B500LL, B0HL, and B500HL) were compared. Blood and rumen mucosa samples from the atrium ruminis were taken at the last day of each period. Length, width and surface of rumen papillae were greater (p < 0.001) in B0HL than in B0LL. Treatment with n-butyric acid resulted in an increase of the papillae surface of 20 – 40% (p = 0.047) for both nutritional levels as compared to periods without n-butyric acid treatments. The higher nutritional level and intraruminal n-butyric acid infusion induced epithelial cell death. The percentage of proliferative cells was doubled by n-butyric acid treatment. The mRNA of IGF-1 and IGF type 1 receptor (IGF-1R), as well as IGF-1R binding capacity were unaffected by butyric acid treatments. The abundance of IGF-1 mRNA tended to be lower (p = 0.1) and IGF-1R abundance was lower (p = 0.03) in response to the HL. The plasma IGF-1 concentration was lower with butyric acid treatment (p < 0.01), but was unaffected by the nutritional level. In conclusion, under described experimental preconditions of daily short-time intraruminal n-butyric acid infusion alterations of rumen papillae morphology is not mediated by ruminal IGF type 1 receptor and by local IGF-1 expression in papillae in castrated bulls.