Compaction and Destruction Cross-Sections for α-Glycine from Radiolysis Process via 1.0 keV Electron Beam as a Function of Temperature.

Glycine is an amino acid that has already been detected in space. It is relevant to estimate its resistance under cosmic radiation. In this way, a sublimate of glycine in α-form on KBr substrate was exposed in the laboratory to a 1.0 keV electron beam. The radiolysis study was performed at 40 K, 80 K, and 300 K sample temperatures. These temperatures were chosen to cover characteristics of the outer space environment. The evolution of glycine compaction and degradation was monitored in real time by infrared spectroscopy (Fourier-transform infrared) by investigation in the spectral ranges of 3500-2100, 1650-1200, and 950-750 cm-1. The compaction cross-section increases as the glycine temperature decreases. The glycine film thickness layer of ∼160 nm was depleted completely after ∼15 min at 300 K under irradiation with ∼1.4 µA beam current on the target, whereas the glycine depletion at 40 K and 80 K occurred after about 4 h under similar conditions. The destruction cross-section at room temperature is found to be (13.8 ± 0.2) × 10-17 cm2, that is, about 20 times higher than the values for glycine depletion at lower temperatures (<80 K). Emerging and vanishing peak absorbance related to OCN- and CO bands was observed in 2230-2100 cm-1 during the radiolysis at 40 K and 80 K. The same new IR bands appear in the range of 1600-1500, 1480-1370, and 1350-1200 cm-1 after total glycine depletion for all temperature configurations. A strong N-H deformation band growing at 1510 cm-1 was observed only at 300 K. Finally, the destruction cross-section associated to tholin decay at room temperature is estimated to be (1.30 ± 0.05) × 10-17 cm2. In addition, a correlation between the formation cross-sections for daughter and granddaughter molecules at 300 K is also obtained from the experimental data.

Taro flour (Colocasia esculenta) increases testosterone levels and gametogenic epithelium of Wistar rats.

This study evaluated the effects of diet containing taro flour on hormone levels and the seminiferous tubules morphology of rats. After weaning, the male rats were divided into two groups (n=12 each): control group (CG) treated with control diet and taro group (TG), fed with 25% taro flour for 90 days. Food, caloric intake, mass and body length were evaluated at experiment end. Testis followed the standard histological processing. Immunostaining was performed using an anti-vimentin antibody to identify Sertoli cells. In histomorphometry, total diameter, total area, epithelial height, luminal height and luminal area were analyzed. The testosterone levels were performed using the radioimmunoassay method. Group TG presented (P<0.05): increase in mass, body length, testicular weight, histomorphometric parameters and hormonal levels. Food intake, calorie and Sertoli cells not presented statistical differences. The taro promoted increase in the testicles parameters and hormones.

Body adiposity and bone parameters of male rats from mothers fed diet containing flaxseed flour during lactation.

Obesity and osteoporosis may have their origins in early postnatal life. This study was designed to evaluate whether flaxseed flour use during lactation period bears effect on body adiposity and skeletal structure of male rat pups at weaning. At birth, male Wistar rats were randomly assigned to control and experimental (FF) groups, whose dams were treated with control or flaxseed flour diet, respectively, during lactation. At 21 days of age, pups were weaned to assess body mass, length and composition by dual-energy X-ray absorptiometry. The animals were then sacrificed to carry out analysis of serum profile, intra-abdominal adipocyte morphology and femur characteristics. Differences were considered significant when P<0.05. The FF group displayed the following characteristics (P<0.05): higher body mass, length, bone mineral content, bone area and concentrations of osteoprotegerin, osteocalcin and high-density lipoprotein cholesterol; higher levels of stearic, α-linolenic, eicosapentaenoic and docosapentaenoic acids and lower levels of arachidonic acid and cholesterol; smaller adipocyte area; and higher mass, epiphysis distance, diaphysis width, maximal load, break load, resilience and stiffness of femur. Flaxseed flour intake during lactation period promoted adipocyte hypertrophy down-regulation and contributed to pup bone quality at weaning.

Body composition in male rats subjected to early weaning and treated with diet containing flour or flaxseed oil after 21 days until 60 days.

The aim of this study was analyzed if the flour or flaxseed oil treatment contributes to body composition in male rats subjected to early weaning. Pups were weaned for separation from mother at 14 (early weaning, EW) and 21 days (control, C). At 21 days, part of the pups was evaluated (C21 v. EW21). After 21 days, control (C60) was fed with control diet. EW was divided in control (EWC60); flaxseed flour (EWFF60); flaxseed oil (EWFO60) diets until 60 days. Body mass, length and body composition by dual-energy X-ray absorptiometry were determined. EW21 (v. C21) and EWC60 (v. C60 and EWFF60) showed lower (P<0.05) mass, length and body composition. EWFO60 (v. C60 and EWFF60) showed lower (P<0.05) body mass and length, body and trunk lean mass, bone mineral density and content and bone area. Flaxseed flour, in comparison with flaxseed oil, contributes to recovery of body composition after early weaning.

Brain development in male rats subjected to early weaning and treated with diet containing flour or flaxseed oil after 21 days until 60 days.

The precocious interruption of lactation is a prime factor for developmental plasticity. Here we analyzed whether flour or flaxseed oil treatment contributes to body and brain mass in male rats subjected to early weaning. Pups were weaned for separation from their mother at 14 (early weaning, EW) and 21 days (control, C). At 21 days, some of the pups were evaluated (C21 v. EW21). After 21 days, control pups (C60) were fed a control diet. EW pups were divided into those fed a control diet (EWC60), those given flaxseed flour (EWFF60), and those given flaxseed oil (EWFO60) until 60 days. EW21 showed lower body and absolute brain mass and higher relative brain mass. At 60 days, EWC60 and EWFO60 had lower body mass. With regard to relative brain mass, EWC60 was heavier; EWFO60 had lower values compared with EWC60 and higher values compared with C60 and EWFF60. These results indicated that flaxseed flour, in comparison with flaxseed oil, contributes to brain development after EW.

High-fat diets containing soybean or canola oil affect differently pancreas function of young male rats.

The excessive fat intake generally might induce obesity and metabolic disturbances. Thus, the goal of the study was to assess the role of high-fat diets containing soybean or canola oil on intra-abdominal adiposity and pancreatic morphology and function of young rats. After weaning, rats were fed with a control diet (7S) or a high-fat diet containing soybean oil (19S) or canola oil (19C) until they were 60 days old, when they were sacrificed. Food intake (g/day), body mass and length, retroperitoneal and epididymal fat mass, HOMA-IR, HOMA-ß and area of pancreatic islets were assessed. The results were considered different with a significant level of p<0.05. Both 19S and 19C groups showed higher body mass, length, and retroperitoneal fat mass. The 19C group showed higher HOMA-IR (+43% and +78%) and HOMA-ß (+40% and +59%) than 19S and 7S groups, respectively. Both 19S and 19C groups showed lower pancreatic islets area in relation to 7S group. Meantime, 19C presented lower percentage of pancreatic islets area in comparison to 19S (-41%) and 7S group (-70%, p<0.0001). Independent of soybean or canola oil, the high fat diet promoted development of the obesity. Comparing 19C and 19S groups, the higher concentrations of monounsaturated fatty acids, present in the canola oil were worse than higher concentrations of polyunsaturated fatty acids, present in the soybean oil.

Two models of early weaning decreases bone structure by different changes in hormonal regulation of bone metabolism in neonate rat.

During the last decade a great concern has developed for determining what factors influence bone mineral accretion in healthy children. Mother's milk represents the primary source of calcium and other nutrients in the neonate. The development of bone and adipose tissue has common origins. Since early weaning decreases adipogenesis in neonate, our aim was to evaluate bone metabolism in 2 models of early weaning (EW) in neonate rats. Lactating rats were separated into 3 groups: control: pups had free access to milk; MEW: dams were involved with a bandage mechanically (M) interrupting lactation in the last 3 days; and PEW: dams were pharmacologically (P) treated to block prolactin (0.5 mg bromocryptine/twice a day) 3 days before standard weaning. Significant difference had p<0.05. At weaning, MEW and PEW pups presented lower body weight (-18% and -15%), total body fat (-26% and -27%), total bone mineral density (-7% and -6%), total bone mineral content (-30% and -32%), bone area (-28% and -30%), serum osteocalcin (-20% and -55%), and higher C-terminal cross-linked telopeptide of type I collagen (CTX-I) (1.3 and 1.1-fold increase). However, serum ionized calcium was lower only in MEW pups (-34%), 25-hydroxyvitamin D was higher (1.4-fold increase), and PTH was lower (-26%) only in PEW group. The present study shows that both early weaning models leads to an impairment of osteogenesis associated with lower adipogenesis by different mechanisms, involving mainly changes in vitamin D and PTH.

The caspase 6 derived N-terminal fragment of DJ-1 promotes apoptosis via increased ROS production.

In pathological conditions, the amount of DJ-1 determines whether a cell can survive or engage a cell death program. This is exemplified in epithelial cancers, in which DJ-1 expression is increased, while autosomal recessive early onset Parkinson's disease mutations of DJ-1 generally lead to decreased stability and expression of the protein. We have shown previously that DJ-1 is cleaved by caspase-6 during induction of apoptosis. We demonstrate here that the N-terminal cleaved fragment of DJ-1 (DJ-1 Nt) is specifically expressed in the nucleus and promotes apoptosis in SH-SY5Y neuroblastoma cell lines. In addition, overexpression of DJ-1 Nt in different cell lines leads to a loss of clonogenic potential and sensitizes to staurosporin and 1-methyl-4-phenylpyridinium (MPP+)-mediated caspase activation and apoptosis. Importantly, inhibition of endogenous DJ-1 expression with sh-RNA or DJ-1 deficiency mimics the effect of DJ-1 Nt on cell growth and apoptosis. Moreover, overexpression of DJ-1 Nt increases reactive oxygen species (ROS) production, and sensitizes to MPP+-mediated apoptosis and DJ-1 oxidation. Finally, specific exclusion of DJ-1 Nt from the nucleus abrogates its pro-apoptotic effect. Taken together, our findings identify an original pathway by which generation of a nuclear fragment of DJ-1 through caspase 6-mediated cleavage induces ROS-dependent amplification of apoptosis.

Endogenous beta-amyloid production in presenilin-deficient embryonic mouse fibroblasts.

Genetic and biochemical evidence have led to the suggestion that presenilins could be the long-searched-for gamma-secretase, the proteolytic activity that generates the carboxy terminus of amyloid beta-peptides. This activity is also thought to be responsible for the release of the Notch intracellular domain (NICD) from Notch. Here, we report the production of endogenous secreted and intracellular 40- and 42-amino-acid Abeta peptides in mouse fibroblasts deficient in presenilin 1, presenilin 2 or both. We show that the endogenous production of Abeta40 and Abeta42 was not altered by presenilin deficiency. By contrast, inactivating presenilin genes fully abolished NICD production. These data indicate that Abeta and NICD production are distinct catabolic events. Also, even though NICD formation is indeed presenilin dependent, endogenous secreted and intracellular beta-amyloid peptides are still generated in absence of presenilins, indicating that there is a gamma-secretase activity distinct from presenilins, at least in murine fibroblasts.

The caspase-derived C-terminal fragment of betaAPP induces caspase-independent toxicity and triggers selective increase of Abeta42 in mammalian cells.

During its physiopathological maturation, the beta-amyloid precursor protein undergoes several distinct proteolytic events by activities called secretases. In Alzheimer's disease, the main histological hallmark called senile plaque is clearly linked to the overproduction of the amyloid peptides Abeta40 and Abeta42, two highly aggregable betaAPP-derived fragments generated by combined cleavages by beta- and gamma-secretases. Recently, an alternative hydrolytic pathway was described, involving another category of proteolytic activities called caspases, responsible for the production of a 31 amino acids betaAPP C-terminal fragment called C31. C31 was reported to lower the viability of N2a cells but the exact mechanisms mediating C31-toxicity remained to be established. Here we show that the transient transfection of pSV2 vector encoding C31 lowers by about 80% TSM1 neuronal cells viability. Arguing against a C31-stimulated apoptotic response, we demonstrate by combined enzymatic and immunological approaches that C31 expression did not modulate basal or staurosporine-induced caspase 3-like activity and pro-caspase-3 activation. Furthermore, C31 did not modify Bax and p53 expressions, poly-(ADP-ribose)-polymerase cleavage and cytochrome c translocation into the cytosol. However, we established that C31 overexpression triggers selective increase of Abeta42 but not Abeta40 production by HEK293 cells expressing wild-type betaAPP751. Altogether, our data demonstrate that C31 induces a caspase-independent toxicity in TSM1 neurons and potentiates the pathogenic betaAPP maturation pathway by increasing selectively Abeta42 species in wild type-betaAPP-expressing human cells.

Role of the proteasome in Alzheimer's disease.

The proteasome is a multicatalytic complex involved in the degradation of polyubiquitinated proteins. Here we review the clues of a possible involvement of the proteasome in Alzheimer's disease neuropathology. Thus, we discuss the fact that the proteasome modulates the intracellular concentrations of presenilins 1 and 2. These two proteins, when mutated, appear responsible for most of early onset forms of Alzheimer's disease and this is thought to be due to the exacerbation of the pathogenic pathway of the maturation of the beta-amyloid precursor protein. Controlling presenilins concentrations could have drastic repercussions on cell physiology as suggested by the fact that proteasome inhibitors drastically potentiate the 'normal' or 'pathogenic' presenilins phenotype related with betaAPP processing. The possibility of considering the proteasome as a potential target for therapeutic intervention in Alzheimer's disease is discussed.

Wild-type but not Parkinson's disease-related ala-53 --> Thr mutant alpha -synuclein protects neuronal cells from apoptotic stimuli.

Recent works suggest that alpha-synuclein could play a central role in Parkinson's disease (PD). Thus, two mutations were reported to be associated with rare autosomal dominant forms of the disease. We examined whether alpha-synuclein could modulate the caspase-mediated response and vulnerability of murine neurons in response to various apoptotic stimuli. We established TSM1 neuronal cell lines overexpressing wild-type (wt) alpha-synuclein or the PD-related Ala-53 --> Thr mutant alpha-synuclein. Under basal conditions, acetyl-Asp-Glu-Val-Asp-aldehyde-sensitive caspase activity appears significantly lower in wt alpha-synuclein-expressing cells than in neurons expressing the mutant. Interestingly, wt alpha-synuclein drastically reduces the caspase activation of TSM1 neurons upon three distinct apoptotic stimuli including staurosporine, etoposide, and ceramide C(2) when compared with mock-transfected cells. This inhibitory control of the caspase response triggered by apoptotic agents was abolished by the PD-related pathogenic mutation. Comparison of wild-type and mutated alpha-synuclein-expressing cells also indicates that the former exhibits much less vulnerability in response to staurosporine and etoposide as measured by the sodium 3'-[1-(phenylaminocarbonyl)-3, 4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid assay. Altogether, our study indicates that wild-type alpha-synuclein exerts an antiapoptotic effect in neurons that appears to be abolished by the Parkinson's disease-related mutation, thereby suggesting a possible mechanism underlying both sporadic and familial forms of this neurodegenerative disease.

C-terminal maturation fragments of presenilin 1 and 2 control secretion of APP alpha and A beta by human cells and are degraded by proteasome.

BACKGROUND: Most early-onset forms of Alzheimer's disease are due to missense mutations located on two homologous proteins named presenilin 1 and 2 (PS1 and PS2). Several lines of evidence indicate that PS1 and PS2 undergo various post-transcriptional events including endoproteolytic cleavages, giving rise to 28-30 kD N-terminal (NTF) and 18-20 kD C-terminal (CTF) fragments that accumulate in vivo. Whether the biological activity of presenilins is borne by the processed fragments or their holoprotein precursor remains in question. We have examined the putative control of beta APP maturation by CTF-PS1/PS2 and the catabolic process of the latter proteins by the multicatalytic complex, proteasome. MATERIALS AND METHODS: We transiently and stably transfected HEK293 cells with CTF-PS1 or CTF-PS2 cDNA. We examined these transfectants for their production of A beta 40, A beta 42, and APP alpha by immunoprecipitation using specific polyclonals. The effect of a series of proteases inhibitors on the immunoreactivity of CTF-PS1/PS2 was examined by Western blot. Finally, the influence of proteasome inhibitors on the generation of beta APP fragments by CTF-expressing cells was assessed by combined immunoprecipitation and densitometric analyses. RESULTS: We showed that transient and stable transfection of CTF-PS1 and CTF-PS2 cDNAs in human cells leads to increased secretion of APP alpha and A beta, the maturation products of beta APP. Furthermore, we demonstrated that two proteasome inhibitors, lactacystin and Z-IE(Ot-Bu)A-Leucinal, prevent the degradation of both CTFs. Accordingly, we established that proteasome inhibitors drastically potentiate the phenotypic increased production of APP alpha and A beta elicited by CTF-PS1/PS2. CONCLUSION: Our data establish that the C-terminal products of PS1 and PS2 maturation exhibit biological activity and in particular control beta APP maturation upstream to alpha-and beta/gamma-secretase cleavages. This function is directly controlled by the proteasome that modulates the intracellular concentration of CTFs.

Assessment of immunity induced in mice by glycoproteins derived from different strains and species of Leishmania.

A comparative study was undertaken on the immunogenic properties of 63kDa glycoproteins obtained from five different strains/species of Leishmania and assessed in C57BL/10 mice. The humoral immune response was assessed by ELISA against the five different antigens of the immunized animals. The cellular immune response was derived from Leishmania. The response was found to be species-specific in all of determined by means of the cytokine profiles secreted by the spleen cells of immunized animals. The presence of gamma-IFN and IL-2, and the absence of IL-4 in the supernatants of cells stimulated by L. amazonensis antigen established that the cellular response is of Th1 type. The five glycoproteins tested were equally effective in protecting C57BL/10 mice against challenge by L. amazonensis. About 50% of the immunized animals were protected for six months.

Characterization of human T lymphocyte-mediated immune responses induced by a vaccine against American tegumentary leishmaniasis.

Forty-three Brazilians were immunized against American tegumentary leishmaniasis using a vaccine made of whole antigens from killed promastigotes of five American dermotropic Leishmania strains. None of the immunized subjects had a positive reaction in the Montenegro skin test (leishmanin) before vaccination, and 74% developed positive reactions in the skin test after vaccination. The proliferative responses of peripheral blood mononuclear cells (PBMC) induced by antigens from dermotropic Leishmania species were significantly higher after vaccination than before vaccination. However, with antigens from L. chagasi (a causative agent of American visceral leishmaniasis), there was no significant difference between the proliferative responses obtained before and after vaccination. Interferon-gamma was detected in the supernatants of L. braziliensis antigen-stimulated PBMC cultures after vaccination (but not before vaccination). One year after vaccination, PBMC were obtained from eight of the immunized individuals and stimulated with L. braziliensis antigens in proliferative response assays. In all cases, the majority of the responding cells were CD8+ T cells, in contrast to the results of a group of patients with active lesions of tegumentary leishmaniasis, whose L. braziliensis-reactive cells were mainly of the CD4+ T cell phenotype.