The role of matrix metalloproteinases 2 and 9 during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Matrix metalloproteinases (MMPs) are implicated in a wide range of physiological and pathological processes, including morphogenesis, wound healing, angiogenesis, inflammation, and cancer. The purpose of this study was to characterize the role of MMPs as depicted by the expression of MMP-2 and MMP-9 during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis. Male Wistar rats were distributed into three groups of 10 animals each and treated with 4-nitroquinoline 1-oxide solution at 50 ppm through their drinking water for 4, 12, and 20 weeks. Ten animals were used as control group. No histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure; however, immunoexpression of MMP-2 was noticed. The same picture occurred to MMP-9, in which positive expression was detected for this immunomarker. MMP-2 and MMP-9 showed positive expression either in pre-neoplastic lesions at 12 weeks following carcinogen exposure or in well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO. Taken together, our results support the belief that MMP-2 and MMP-9 play important role during malignant transformation and conversion of oral mucosa as assessed by immunohistochemistry.     

Madin–Darby canine kidney cells are increased in aerobic glycolysis when cultured on flat and stiff collagen‐coated surfaces rather than in physiological 3‐D cultures

We investigate the influence of the dimensionality and the biochemistry of the culture system on the cellular functionality by analyzing the protein expression levels in Madin–Darby canine kidney (MDCK) cells grown in 3‐D and 2‐D substrates. We cultured MDCK cells on a hard and flat 2‐D uncoated plastic surface, on a 2‐D collagen‐coated plastic surface and in 3‐D collagen gel and employed 2‐D gel electrophoresis, MALDI‐TOF‐MS, and LC‐MS/MS analysis to identify the differentially regulated proteins. We found significant differences in the expression of antioxidant proteins, actin‐binding proteins, glycolytic enzymes, and heat‐shock proteins/chaperons among the three types of cultures. While MDCK cells cultured in 3‐D collagen up‐regulate antioxidant proteins and proteins involved in the dynamic remodeling of the actin cytoskeleton, 2‐D collagen‐coated plastic surfaces induce the up‐regulation of glycolytic enzymes. Our data shows that the culture conditions have profound effects on the physiology of the cell. Culture in 3‐D collagen induces a differentiated polarized phenotype. In contrast, collagen‐coated 2‐D substrates favor a tumor‐like phenotype with increased glycolysis. Thus, the suitability of 2‐D cultures to study the physiological behavior of cells, especially in drug discovery, bioprocessing, and toxicology, should be carefully reconsidered.     

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.     

The <Emphasis Type="Italic">lethal giant larvae</Emphasis>tumour suppressor mutation requires dMyc oncoprotein to promote clonal malignancy

Background  

Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. Precancerous cells are often removed by cell death from normal tissues in the early steps of the tumourigenic process, but the molecules responsible for such a fundamental safeguard process remain in part elusive. With the aim to investigate the molecular crosstalk occurring between precancerous and normal cells in vivo, we took advantage of the clonal analysis methods that are available in Drosophila for studying the phenotypes due to lethal giant larvae (lgl) neoplastic mutation induced in different backgrounds and tissues.     

Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization

Background

Porcine reproductive and respiratory syndrome (PRRS) has now been widely recognized as an economically important disease. The objective of this study was to compare the molecular and biological characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) field isolates in China to those of the modified live virus (MLV) PRRS vaccine and its parent strain (ATCC VR2332).

Results

Five genes (GP2, GP3, GP4, GP5 and NSP2) of seven isolates of PRRSV from China, designated LS-4, HM-1, HQ-5, HQ-6, GC-2, GCH-3 and ST-7/2008, were sequenced and analyzed. Phylogenetic analyses based on the nucleotide sequence of the ORF2-5 and NSP2 showed that the seven Chinese isolates belonged to the same genetic subgroup and were related to the North American PRRSV genotype. Comparative analysis with the relevant sequences of another Chinese isolate (BJ-4) and North American (VR2332 and MLV) viruses revealed that these isolates have 80.8-92.9% homology with VR-2332, and 81.3-98.8% identity with MLV and 80.7-92.9% with BJ-4. All Nsp2 nonstructural protein of these seven isolates exhibited variations (a 29 amino acids deletion) in comparison with other North American PRRSV isolates. Therefore, these isolates were novel strain with unique amino acid composition. However, they all share more than 97% identity with other highly pathogenic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and the Nsp2 protein when compared with the previous isolates.

Conclusions

These results might be useful to study the genetic diversity of PRRSV in China and to track the infection sources as well as for vaccines development.     

First report on polysaccharides of Asterochloris and their potential role in the lichen symbiosis

A structural study of the carbohydrates from the aposymbiotically cultured Asterochloris sp., the algal symbiont of the lichen Cladina confusa was carried out for the first time. A xylorhamnogalactofuranan was purified and was predominated by (1-->3)-linked galactofuranosyl units with sidechains in position 6 on approximately 6.4% of the units. The sidechains have galactofuranosyl units 5-O and 6-O-substituted, as well rhamnopyranosyl units 2-O, 3-O and 2,3-di-O-substituted. Xylose was detected only as nonreducing end units, together with galactofuranosyl units. Amylose and a beta-(1-->4)-xylan were also present. These polysaccharides have not been found in the symbiotic thallus of C. confusa, which contained only glucans, galactomannoglucan and galactoglucomannan. A potential role of these carbohydrates in lichen recognition proccess is also discussed.     

The GATC-binding protein SeqA is required for bile resistance and virulence in Salmonella enterica serovar typhimurium

Disruption of the seqA gene of Salmonella enterica serovar Typhimurium causes defects similar to those described in E. coli: filament formation, aberrant nucleoid segregation, induction of the SOS response, envelope instability, and increased sensitivity to membrane-damaging agents. Differences between SeqA⁻ mutants of E. coli and S. enterica, however, are found. SeqA⁻ mutants of S. enterica form normal colonies and do not exhibit alterations in phage plaquing morphology. Lack of SeqA causes attenuation of S. enterica virulence by the oral route but not by the intraperitoneal route, suggesting a virulence defect in the intestinal stage of infection. However, SeqA⁻ mutants are fully proficient in the invasion of epithelial cells. We hypothesize that attenuation of SeqA⁻ mutants by the oral route may be caused by bile sensitivity, which in turn may be a consequence of envelope instability.     

Pim-1 regulates cardiomyocyte survival downstream of Akt

The serine-threonine kinases Pim-1 and Akt regulate cellular proliferation and survival. Although Akt is known to be a crucial signaling protein in the myocardium, the role of Pim-1 has been overlooked. Pim-1 expression in the myocardium of mice decreased during postnatal development, re-emerged after acute pathological injury in mice and was increased in failing hearts of both mice and humans. Cardioprotective stimuli associated with Akt activation induced Pim-1 expression, but compensatory increases in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload did not protect the myocardium in Pim-1-deficient mice. Transgenic expression of Pim-1 in the myocardium protected mice from infarction injury, and Pim-1 expression inhibited cardiomyocyte apoptosis with concomitant increases in Bcl-2 and Bcl-X(L) protein levels, as well as in Bad phosphorylation levels. Relative to nontransgenic controls, calcium dynamics were significantly enhanced in Pim-1-overexpressing transgenic hearts, associated with increased expression of SERCA2a, and were depressed in Pim-1-deficient hearts. Collectively, these data suggest that Pim-1 is a crucial facet of cardioprotection downstream of Akt.