Clearing factor pancreático

Dosou-se o "clearing factor" (lipase lipoproteica), em pâncreas de ratos normais após serem submetidos a algumas condições experimentais. As seguintes conclusões podem ser extraídas: a) O pãncreas é o órgão mais rico nesta enzima (8 vezes mais que o tecido adiposo, até então considerado o mais rico em CF); b) O jejum total de 48 horas diminui significamente a atividade de CF; c) O complamin, o derivado do ácido nicotínico, agindo sobre a arteriosclerose experimental, não afeta a atividade da enzima; d) O ácido épsilon-aminocapróico (AEAC) potente antagonista da heparina, inibe a ativdade do Cf pancreático; e) Ratos com arteriosclerose experimental apresentam níveis diminuídos desta enzima no pâncreas.

The retained capacity of Lutzomya longipalpis (Lutz & Neiva) to transmit Leishmania chagasi (Cunha & Chagas) after eight years (64 generations in a closed laboratory colony

A closed Lutzomyia longipalpis colony, from Ceará has been used to transmit Leishmania chagasi isolated from a fox in Pará state. The last time this colony was successfully used in similar transmission experiments was eight years (64 generations) ago indicating that this colony of Lu. longipalpis has fully maintained its vectorial capacity in spite of such a long period of maintainance in the laboratory.

Generalized factor models: a bayesian approach

There is recent interest in the generalization of classical factor models in which the idiosyncratic factors are assumed to be orthogonal and there are identification restrictions on cross-sectional and time dimensions. In this study, we describe and implement a Bayesian approach to generalized factor models. A flexible framework is developed to determine the variations attributed to common and idiosyncratic factors. We also propose a unique methodology to select the (generalized) factor model that best fits a given set of data. Applying the proposed methodology to the simulated data and the foreign exchange rate data, we provide a comparative analysis between the classical and generalized factor models. We find that when there is a shift from classical to generalized, there are significant changes in the estimates of the structures of the covariance and correlation matrices while there are less dramatic changes in the estimates of the factor loadings and the variation attributed to common factors.

Nuclear factor I-C links platelet-derived growth factor and transforming growth factor beta1 signaling to skin wound healing progression.

Transforming growth factor beta (TGF-beta) and platelet-derived growth factor A (PDGFAlpha) play a central role in tissue morphogenesis and repair, but their interplay remain poorly understood. The nuclear factor I C (NFI-C) transcription factor has been implicated in TGF-beta signaling, extracellular matrix deposition, and skin appendage pathologies, but a potential role in skin morphogenesis or healing had not been assessed. To evaluate this possibility, we performed a global gene expression analysis in NFI-C(-/-) and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-beta1. Misregulated genes were prominently overrepresented by regulators of connective tissue inflammation and repair. In vivo skin healing revealed a faster inflammatory stage and wound closure in NFI-C(-/-) mice. Expression of PDGFA and PDGF-receptor alpha were increased in wounds of NFI-C(-/-) mice, explaining the early recruitment of macrophages and fibroblasts. Differentiation of fibroblasts to contractile myofibroblasts was also elevated, providing a rationale for faster wound closure. Taken together with the role of TGF-beta in myofibroblast differentiation, our results imply a central role of NFI-C in the interplay of the two signaling pathways and in regulation of the progression of tissue regeneration.

Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting beta cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet beta cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.

Dilated cardiomyopathy and impaired cardiac hypertrophic response to angiotensin II in mice lacking FGF-2.

FGF-2 has been implicated in the cardiac response to hypertrophic stimuli. Angiotensin II (Ang II) contributes to maintain elevated blood pressure in hypertensive individuals and exerts direct trophic effects on cardiac cells. However, the role of FGF-2 in Ang II-induced cardiac hypertrophy has not been established. Therefore, mice deficient in FGF-2 expression were studied using a model of Ang II-dependent hypertension and cardiac hypertrophy. Echocardiographic measurements show the presence of dilated cardiomyopathy in normotensive mice lacking FGF-2. Moreover, hypertensive mice without FGF-2 developed no compensatory cardiac hypertrophy. In wild-type mice, hypertrophy was associated with a stimulation of the c-Jun N-terminal kinase, the extracellular signal regulated kinase, and the p38 kinase pathways. In contrast, mitogen-activated protein kinase (MAPK) activation was markedly attenuated in FGF-2-deficient mice. In vitro, FGF-2 of fibroblast origin was demonstrated to be essential in the paracrine stimulation of MAPK activation in cardiomyocytes. Indeed, fibroblasts lacking FGF-2 expression have a defective capacity for releasing growth factors to induce hypertrophic responses in cardiomyocytes. Therefore, these results identify the cardiac fibroblast population as a primary integrator of hypertrophic stimuli in the heart, and suggest that FGF-2 is a crucial mediator of cardiac hypertrophy via autocrine/paracrine actions on cardiac cells.

Regulació del factor de transcripció NFAT5 per les quinases WNK1, SPAK i OSR1 i cerca de quinases activadores de la pròpia WNK1 en resposta a estrés osmòtic

Estudi elaborat a partir d’una estada a la School of Life Sciences de la University of Dundee, Gran Bretanya, entre gener i març del 2007.L'estrès osmòtic causa rà pidament l'activació de la quinasa WNK1, que fosforila i activa a continuació les quinases SPAK i OSR1, que alhora regulen canals i transportadors d’ions preexistents a la membrana cel•lular. El factor de transcripció NFAT5 és el principal regulador de la resposta cel•lular transcripcional secundà ria a hipertonicitat i s’ha descrit que les quinases p38, Fyn, PKA, ERK/MEK i ATM estan involucrades en la seva regulació post-traduccional. No obstant, com que la funció d’aquestes quinases no explica totalment els mecanismes d'activació de NFAT5, s’ha estudiat si l’activitat transcripcional de NFAT5 pot estar regulada per WNK1, SPAK o OSR1. Així doncs, es va observar que l’activitat d’un reporter dependent de NFAT5 no es veu afectada per la presència de cap de les quinases anteriors, en la seva forma wild-type o dominant negatiu. D’altra banda, es va estudiar quin domini de WNK1 és necessari per a que pugui respondre a hipertonicitat i quines quinases poden estar involucrades en la fosforilació de la serina 382 de WNK1. En conclusió, les dades obtingudes apunten que l’activació de WNK1 en resposta a estrès osmòtic requereix la seva fosforilació en la serina 382 per quinases upstream com PAK2 o RSK i que també és necessari un dels seus dominis coiled-coil, almenys els aminoà cids 558 i 561. Aquests processos, però, semblen ser independents de l’activació de NFAT5 en resposta a hipertonicitat.   

Epidermal growth factor receptor: a re-emerging target in glioblastoma.

PURPOSE OF REVIEW: Amplification and overexpression of the epidermal growth factor receptor (EGFR) gene are a hallmark of primary glioblastoma (45%), making it a prime target for therapy. In addition, these amplifications are frequently associated with oncogenic mutations in the extracellular domain. However, efforts at targeting the EGFR tyrosine kinase using small molecule inhibitors or antibodies have shown disappointing efficacy in clinical trials for newly diagnosed or recurrent glioblastoma. Here, we review recent insights into molecular mechanisms relevant for effective targeting of the EGFR pathway. RECENT FINDINGS: Molecular workup of glioblastoma tissue of patients under treatment with small molecule inhibitors has established drug concentrations in the tumor tissue, and has shed light on the effectiveness of target inhibition and respective effects on pathway signaling. Further, functional analyses of interaction of small molecule inhibitors with distinct properties to bind to the active or inactive form of EGFR have provided new insights that will impact the choice of drugs. Finally, vaccination approaches targeting the EGFRvIII mutant featuring a tumor-specific antigen have shown promising results that warrant larger controlled clinical trials. SUMMARY: A combination of preclinical and clinical studies at the molecular level has provided new insights that will allow refining strategies for targeting the EGFR pathway in glioblastoma.

Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.

Decay accelerating factor (DAF) as the host antigen with protective activity to complement killing of schistosomula

The acquisition of host antigens by Schistosoma mansoni was studied by evaluating the resistance of schistosomula to the complement attack mediated by lethal antibody. Schistosomula cultured for 24 hours with intact human erythrocytes (N-HuE) or ghosts of any type of ABO or Rh blood group, showed a marked resistance to complement damage. Sheep red blood cells, pronase-treated N-HuE or erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, which are complement-sensitive cells, were unable to protect schistosomula. Schistosomula protected by N-HuE became again susceptible to complement killing after incubation with a monoclonal antibody anti-DAF. These results indicate that, in vitro, host DAF from N-HuE can be acquired by schistosomula surface in a biological active form that protects the parasite from the complement lesion.

Reduced capacity of peritoneal exudate cells for oxidative killing of Schistosoma mansoni schistosomula

Peritoneal exudate cells from mice infected with Schistosoma mansoni (S-PEC) can kill schistosomula in vitro in the presence of immune serum. S-PEC produce a low level of respiratory burst, and schistosomula mortality in their presence is not reduced when exogenous antioxidants are added, suggesting that with S-PEC, oxidative killing is not important. Hydrogen peroxide (H2O2) and superoxide production by S-PEC, and cells from BCG and thioglycollate (THGL) injected non-infected mice, non-specifically stimulated with opsonized zymosan, were measured. Levels of H2O2 produced by S-PEC were significantly lower than BCG or THGL PEC, and were below the H2O2 threshold for schistosomula killing. This resulted in lower levels of cell-mediated killing of schistosomula in vitro by S-PEC than by BCG or THGL PEC. Superoxide levels, however, were similar between the three cell populations. The efficiency of PEC to kill schistosomules in vitro correlated with H2O2 rather than superoxide levels. The lower tolerance of schistosomula, compared to adult S. mansoni to GSH depleting agents increases their sensitivity to oxidative attack and resulted in higher levels of cell-mediated killing in vitro.