Vaginal bacterial microflora modifications during the growth of healthy cows

The aim of this work was first, to determine the predominant groups capable of colonizing the vagina and maintaining high numbers with time. The normal microbial flora of the cow's vagina and its evolution from weaning to service was then studied using standard microbiological methods. The results show that the most dominant bacteria belong to the streptococci, followed by the staphylococci, with similar levels during the whole study period. Enterobacteriaceae and lactobacilli were present at very low levels, the latter increasing during the cow's growth, suggesting some kind of hormonal influence. The results will allow the selection of micro-organisms with probiotic characteristics, classified as GRAS (Generally Regarded as Safe), to be used in the prevention of infections in the vaginal tract of cows, such as metritis, which produces delayed periods between partum and conception, and consequent economic losses.     

Chaperone mediated autophagy contributes to the newly synthesized histones H3 and H4 quality control

Although there are several pathways to ensure that proteins are folded properly in the cell, little is known about the molecular mechanisms regulating histone folding and proteostasis. In this work, we identified that chaperone-mediated autophagy (CMA) is the main pathway involved in the degradation of newly synthesized histones H3 and H4. This degradation is finely regulated by the interplay between HSC70 and tNASP, two histone interacting proteins. tNASP stabilizes histone H3 levels by blocking the direct transport of histone H3 into lysosomes. We further demonstrate that CMA degrades unfolded histone H3. Thus, we reveal that CMA is the main degradation pathway involved in the quality control of histone biogenesis, evidencing an additional mechanism in the intricate network of histone cellular proteostasis.     

Fatty acid-binding protein 4 impairs the insulin-dependent nitric oxide pathway in vascular endothelial cells

ABSTRACT: BACKGROUND: Recent studies have shown that fatty acid-binding protein 4 (FABP4) plasma levels are associated with impaired endothelial function in type 2 diabetes (T2D). In this work, we analysed the effect of FABP4 on the insulin-mediated nitric oxide (NO) production by endothelial cells in vitro. METHODS: In human umbilical vascular endothelial cells (HUVECs), we measured the effects of FABP4 on the insulin-mediated endothelial nitric oxide synthase (eNOS) expression and activation and on NO production. We also explored the impact of exogenous FABP4 on the insulin-signalling pathway (insulin receptor substrate 1 (IRS1) and Akt). RESULTS: We found that eNOS expression and activation and NO production are significantly inhibited by exogenous FABP4 in HUVECs. FABP4 induced an alteration of the insulin-mediated eNOS pathway by inhibiting IRS1 and Akt activation. These results suggest that FABP4 induces endothelial dysfunction by inhibiting the activation of the insulin-signalling pathway resulting in decreased eNOS activation and NO production. CONCLUSION: These findings provide a mechanistic linkage between FABP4 and impaired endothelial function in diabetes, which leads to an increased cardiovascular risk.     

Annexin A2 Reduces PCSK9 Protein Levels via a Translational Mechanism and Interacts with the M1 and M2 Domains of PCSK9

Annexin A2 (AnxA2) was reported to be an extracellular endogenous inhibitor of proprotein convertase subtilisin kexin type 9 (PCSK9) activity on cell-surface LDL receptor degradation. In this study, we investigated the effect of silencing the expression of AnxA2 and PCSK9 in HepG2 and Huh7 cells to better define the role of AnxA2 in PCSK9 regulation. AnxA2 knockdown in Huh7 cells significantly increased PCSK9 protein levels as opposed to AnxA2 knockdown in HepG2 cells. However, HepG2 cells overexpressing AnxA2 had lower levels of PCSK9 protein. Overall, our data revealed a plausible new role of AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Moreover, the C-terminal Cys/His-rich domain of PCSK9 is crucial in the regulation of PCSK9 activity, and we demonstrated by far-Western blot assay that the M1 and M2 domains are necessary for the specific interaction of PCSK9''s C-terminal Cys/His-rich domain and AnxA2. Finally, we produced and purified recombinant PCSK9 from humans and mice, which was characterized and used to perform 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate LDL cell-based assays on the stable knockdown HepG2 and Huh7 cells. We also demonstrated for the first time the equipotency of human and mouse PCSK9 R218S on human cells.     

Fermentation of cashew apple juice to produce high added value products

The use of agriculture substrates in industrial biotechnological processes has been increasing because of its low cost. Cashew apples are considered an agriculture low cost product in the Brazilian Northeast because the cashew cultivation is done mainly to produce cashew nuts. About 90% of the cashew apples production is lost in the field after removing the nut. In this work, the use of clarified cashew apple juice as substrate for microbial cultivation was investigated. The results showed that cashew apple juice is a good source of reducing sugars and can be used to grow Leuconostoc mesenteroides to produce high added value products such as dextran, lactic acid, mannitol and oligosaccharides.     

Depletion with PC61 mAb before Toxoplasma gondii infection eliminates mainly Tregs in BALB/c mice, but activated cells in C57BL/6J mice

Analysis of regulatory T cells (Tregs) in vivo during infection is crucial for the understanding of immune response modulation. Depletion experiments using anti-CD25 monoclonal antibody (mAb) in order to eliminate Tregs have been widely used for this purpose despite the fact that this approach may also lead to the elimination of activated T cells. We show in this paper that treatment with anti-CD25 mAb before Toxoplasma gondii infection eliminates a different pattern of cell subsets in the resistant BALB/c and the susceptible C57BL/6J mouse strain. Injection with PC61 mAb leads to the elimination of most Tregs in BALB/c mice, while in C57BL/6J animals, treatment depletes other activated subsets [natural killer (NK), B and CD4(+) T cells]. This difference is a consequence of the dramatic cell activation observed in the latter, but not in the former strain. The different effect of the depletion reported here demonstrates that careful analysis in each model is mandatory in order to avoid misleading conclusions.     

Effect of nitric oxide on neointimal hyperplasia based on sex and hormone status

Nitric oxide (NO)-based therapies decrease neointimal hyperplasia; however, studies have been performed only in male animal models. Thus, we sought to evaluate the effect of NO on vascular smooth muscle cells (VSMC) in vitro and neointimal hyperplasia in vivo based on sex and hormone status. In hormone-replete medium, male VSMC proliferated at greater rates than female VSMC. In hormone-depleted medium, female VSMC proliferated at greater rates than male VSMC. However, in both hormone environments, NO inhibited proliferation and migration to a greater extent in male compared to female VSMC. These findings correlated with greater G₀/G₁ cell cycle arrest and changes in cell cycle protein expression in male compared to female VSMC after exposure to NO. Next, the rat carotid artery injury model was used to assess the effect of NO on neointimal hyperplasia in vivo. Consistent with the in vitro data, NO was significantly more effective at inhibiting neointimal hyperplasia in hormonally intact males compared to females using weight-based dosing. An increased weight-based dose of NO in females was able to achieve efficacy equal to that in males. Surprisingly, NO was less effective at inhibiting neointimal hyperplasia in castrated animals of both sexes. In conclusion, these data suggest that NO inhibits neointimal hyperplasia more effectively in males compared to females and in hormonally intact compared to castrated rats, indicating that the effects of NO in the vasculature may be sex- and hormone-dependent.     

Pyrrolidon carboxypeptidase activities in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-ovary axes of rats with mammary gland cancer induced by N-methyl nitrosourea.

Pyrrolidon carboxypeptidase is an omega-peptidase that hydrolyses N-terminal pyroglutamyl residues from biologically active peptides such as gonadotropin-releasing and thyrotrophin-releasing hormones. We previously described a decrease in both rat and human pyrrolidon carboxypeptidase activity with breast cancer, suggesting that gonadotropin-releasing hormone may be an important local intracrine, autocrine and/or paracrine hormonal factor in the pathogenesis of breast cancer while playing a role in the tumoral process. However, the other susceptible substrate of pyrrolidon carboxypeptidase, thyrotrophin-releasing hormone, may also be modified with breast cancer, supporting an association between breast cancer and thyroid disorders. The present work analyses soluble and membrane-bound pyrrolidon carboxypeptidase activities in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-ovary axes in N-methyl nitrosourea-induced breast cancer in rats. Our aim was to determine the possible relationship between gonadotropin-releasing hormone and thyrotrophin-releasing hormone regulation through pyrrolidon carboxypeptidase activity. We propose that pyrrolidon carboxypeptidase activity dysregulation at various local and systemic levels may participate in the initiation, promotion and progression of breast cancer induced in rat by N-methyl nitrosourea through the increase in gonadotropin-releasing hormone. Since pyrrolidon carboxypeptidase activity also acts on thyrotrophin-releasing hormone, the dysregulation of this enzyme's activity could indirectly affect hypothalamus-pituitary-thyroid axis function, and thus potentially represent a link between the diseases of thyroid and breast cancer.     

Conversion of a polysaccharide to nitric oxide-releasing form. Dual-mechanism anticoagulant activity of diazeniumdiolated heparin

We describe heparin/diazeniumdiolate conjugates that generate nitric oxide (NO) at physiological pH. Like the heparin from which they were prepared, they inhibit thrombin-induced blood coagulation. Unlike heparin, they can also inhibit and reverse ADP-induced platelet aggregation (as expected for an NO-releasing agent), suggesting potential utility as dual-action antithrombotics.