Protein expression changes induced in murine peritoneal macrophages by Group B Streptococcus

Protein expression changes induced in thioglycolate‐elicited peritoneal murine macrophages (MΦ) by infection with type III Group B Streptococcus (GBS) are described. Proteins from control MΦ and MΦ incubated 2 h with live or heat‐inactivated GBS were separated by 2‐DE. Proteins whose expression was significantly different in infected MΦ, as compared with control cells, were identified by MS/MS analysis. Changes in the expression level of proteins involved in both positive and negative modulation of phagocytic functions, stress response and cell death were induced in MΦ by GBS infection. In particular, expression of enzymes playing a key role in production of reactive oxygen species was lowered in GBS‐infected MΦ. Significant alterations in the expression of some metabolic enzymes were also observed, most of the glycolytic and of the pentose‐cycle enzymes being down‐regulated in MΦ infected with live GBS. Finally, evidence was obtained that GBS infection affects the expression of enzymes or enzyme subunits involved in ATP synthesis and in adenine nucleotides interconversion processes.     

Bacterial and Protozoal Communities and Fatty Acid Profile in the Rumen of Sheep Fed a Diet Containing Added Tannins

This study evaluated the effects of tannins on ruminal biohydrogenation (BH) due to shifts in the ruminal microbial environment in sheep. Thirteen lambs (45 days of age) were assigned to two dietary treatments: seven lambs were fed a barley-based concentrate (control group) while the other six lambs received the same concentrate with supplemental quebracho tannins (9.57% of dry matter). At 122 days of age, the lambs were slaughtered, and the ruminal contents were subjected to fatty acid analysis and sampled to quantify populations of Butyrivibrio fibrisolvens, which converts C_18:2 c9-c12 (linoleic acid [LA]) to C_18:2 c9-t11 (rumenic acid [RA]) and then RA to C_18:1 t11 (vaccenic acid [VA]); we also sampled for Butyrivibrio proteoclasticus, which converts VA to C_18:0 (stearic acid [SA]). Tannins increased (P < 0.005) VA in the rumen compared to the tannin-free diet. The concentration of SA was not affected by tannins. The SA/VA ratio was lower (P < 0.005) for the tannin-fed lambs than for the controls, suggesting that the last step of the BH process was inhibited by tannins. The B. proteoclasticus population was lower (−30.6%; P < 0.1), and B. fibrisolvens and protozoan populations were higher (+107% and +56.1%, respectively; P < 0.05) in the rumen of lambs fed the tannin-supplemented diet than in controls. These results suggest that quebracho tannins altered BH by changing ruminal microbial populations.The fatty acid profile of the meat and milk of ruminants is strongly affected by diet (2, 15). When ingested, the dietary polyunsaturated fatty acids (PUFA) undergo a process known as biohydrogenation (BH) carried out by ruminal microorganisms (20). During the BH of C_18:2(n-6) (linoleic acid [LA]) and C_18:3(n-3) (linolenic acid [LNA]) a number of C_18:1 and C_18:2 isomers are formed (6). The last step in the BH process leads to the formation of C_18:0 (stearic acid [SA]). Among the intermediate products formed during this process, the isomer C_18:2 c9t11 (rumenic acid [RA]) is active in preventing cancer in mammals (17). Only a small amount of the RA found in meat and milk originates during BH. It is produced to a larger extent in muscle and mammary glands from the desaturation of C_18:1 t11 (vaccenic acid [VA], another intermediate of ruminal BH) by the action of Δ⁹-desaturase enzyme (41, 43).Ruminal BH is carried out mostly by bacteria belonging to the Butyrivibrio genus (38). Butyrivibrio fibrisolvens has the capacity to convert LA to RA and RA to VA, while Butyrivibrio proteoclasticus (previously classified as Clostridium proteoclasticum [35]) hydrogenates VA to SA (38, 39). According to Or-Rashid et al. (37), ruminal protozoa also play a role in BH by converting LA to RA. However, this issue is still controversial, as Devillard et al. (11) have reported that protozoa do not have the capability of hydrogenating LA. The proportion of BH intermediates in the rumen can vary depending on changes in ruminal microbial populations (7, 51). Changes in ruminal fatty acid profiles are also reflected in intramuscular fatty acid composition (48, 52).Tannins are phenolic compounds that are widespread in plants. When ingested by ruminants in large amounts, tannins can reduce the activity and the proliferation of ruminal microorganisms (34). Tannins from Lotus corniculatus (33) or from Acacia spp. (12) reduce the proliferation of B. proteoclasticus B316^T and B. proteoclasticus P18, respectively. Durmic et al. (12) reported that VA increased and SA decreased when extracts from Acacia iteaphylla, which contains condensed tannins (1), were incubated in vitro with sheep ruminal fluid inoculated with B. fibrisolvens JW11 and B. proteoclasticus P18 strains. In two recent in vitro studies, the inclusion of tannins in fermentor systems containing bovine ruminal fluid inhibited the conversion of VA to SA, while no effect was detected on RA production (21, 47). These results have been also confirmed in vivo in the rumen of sheep fed a diet with 4.0% dry matter (DM) quebracho tannin (48). However, to date there is no in vivo study focusing on the effects of dietary tannins on the proliferation of the microorganisms involved in ruminal BH.We assessed whether dietary tannins may affect the BH pathway via changes in bacterial and protozoal ruminal populations. We gave particular emphasis to B. fibrisolvens and B. proteoclasticus. We also assayed the production of conjugated linoleic acids (CLAs) by linoleic acid isomerase (LA-I) enzyme.     

Surface adsorption of protein corona controls the cell internalization mechanism of DC-Chol-DOPE/DNA lipoplexes in serum

Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol-DOPE/DNA lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol-DOPE/DNA lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol-DOPE/DNA lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a ‘protein corona’. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications.     

Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1

Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. We have identified a mammalian analog of this caspase-regulatory system in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-X(L) bind and suppress NALP1, reducing caspase-1 activation and interleukin-1beta (IL-1beta) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1beta production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1beta production. The findings reveal an interaction of host defense and apoptosis machinery.     

Helicobacter pylori EstV: identification, cloning, and characterization of the first lipase isolated from an epsilon-proteobacterium

Bacterial lipases are attracting an enormous amount of attention due to their wide biotechnological applications and due to their roles as virulence factors in some bacteria. Helicobacter pylori is a significant and widespread pathogen which produces a lipase(s) and phospholipases that seem to play a role in mucus degradation and the release of proinflammatory and cytotoxic compounds. However, no H. pylori lipase(s) has been isolated and described previously. Therefore, a search for putative lipase-encoding genes was performed by comparing the amino acid sequences of 53 known lipolytic enzymes with the deduced proteome of H. pylori. As a result, we isolated, cloned, purified, and characterized EstV, a novel lipolytic enzyme encoded by open reading frame HP0739 of H. pylori 26695, and classified it in family V of the bacterial lipases. This enzyme has the properties of a small, cell-bound carboxylesterase (EC 3.1.1.1) that is active mostly with short-chain substrates and does not exhibit interfacial activation. EstV is stable and does not require additional cofactors, and the maximum activity occurs at 50 degrees C and pH 10. This unique enzyme is the first lipase isolated from H. pylori that has been described, and it might contribute to ulcer development, as inhibition by two antiulcer substances (beta-aescin and glycyrrhizic acid) suggests. EstV is also the first lipase from an epsilon-proteobacterium to be described. Furthermore, this enzyme is a new member of family V, probably the least-known family of bacterial lipases, and the first lipase of this family for which kinetic behavior, inhibition by natural substances, and other key biochemical features are reported.     

Cougar Predation on Black-and-Gold Howlers on Mutum Island,Southern Brazil<Superscript>1</Superscript>

Researchers consider predation rates by terrestrial animals to be lower in the case of arboreal primates, particularly among large-bodied species. We recorded the consumption of black-and-gold howlers (Alouatta caraya) by cougars (Puma concolor) as evidence of predation on an island of the upper Paraná River. We collected and processed fecal samples of the felid in 2004 and 2005. We identified items in the laboratory by comparison with museum specimens. We considered each species in a fecal sample as a single occurrence. Based on analysis of the cuticle scale pattern, we identified the felid as cougar. Howlers occurred in 4 out of the 8 fecal samples (40% of the occurrences). In addition to howlers, we also recorded 5 occurrences of agouti (Dasyprocta azarae; 50%) and a small unidentified sigmodontine rodent (10%). The abundance of howlers and the low forest canopy in a successional vegetation might have facilitated the predation of the large primates by a primarily terrestrial predator. The versatility of cougars is corroborated by the consumption of prey species that were abundant in the region and that were available in different forest strata, such as howlers and agoutis.     

Baroreflex dysfunction in rats submitted to protein restriction

Earlier studies from the authors' laboratory showed that malnourishment induces alterations in the cardiovascular homeostasis increasing the basal mean arterial pressure and heart rate. In this study, the authors evaluated whether the sympathetic and parasympathetic efferent activities contribute to changes in the cardiovascular homeostasis through altered modulation of the arterial baroreflex of malnourished rats. After weaning, male Fischer rats were given 15% (Normal Protein--NP) or 6% (Low Protein--LP) protein diet for 35 d. The baroreflex gain and latency were evaluated before and after selective autonomic blockades in control and malnourished rats. It was observed that malnourishment affected the baroreflex gain in response to activation and deactivation of the arterial baroreflex. Moreover, malnourished rats showed increased baroreflex latency as compared to that of control rats. Regarding the autonomic efferent activity directed to the heart, the data showed increased sympathetic and decreased parasympathetic efferent activities in malnourished rats, and such alterations could be related to the observed changes in the arterial baroreflex gain as well as in the basal mean arterial pressure and heart rate.     

Glutathione S-transferases in Festuca arundinacea: identification, characterization and inducibility by safener benoxacor

Over recent years it has emerged how certain no crop-species can be employed in phytoremediating contaminated soils or preventing herbicide pollution; in this contest Festuca arundinacea was investigated. Shoots of Festuca were submitted to fast protein liquid chromatography in order to identify their glutathione S-transferases (GST; EC 2.5.1.18), by a combination of anionic, affinity and RP-HPLC chromatography. The chromatographic procedure revealed satisfactory yield and four GSTs were identified: they were named FaGST I, FaGST II, FaGST III and FaGST IV. Among these, significant differences were observed in the chromatographic behaviours, structure, activity toward a "model" substrate, 1-chloro-2,4-dinitrobenzene, and responsiveness to the herbicide safener benoxacor. FaGST I showed the highest activity toward the above substrate, and this activity was up-regulated by the herbicide safener. Therefore, FaGST I was purified till homogeneity and was determined to be an heterodimer consisting of two subunits of 28.0 and 27.2kDa. Each subunit of FaGST I was further characterized by means of LC-ESI-MS/MS and immunoblotting analysis, which revealed that both the subunits belong to the tau subclass.