Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.

Ovarian expression of insulin-like peptide 3 (INSL3) and its receptor (RXFP2) during development of bovine antral follicles and corpora lutea and measurement of circulating INSL3 levels during synchronized estrous cycles

Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna (TIC) and granulosa (GC) compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than GC and increased progressively during follicle maturation with INSL3 peaking in large (11-18mm) estrogen-active follicles and RXFP2 peaking in 9-10mm follicles before declining in larger (11-18mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly auto-/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17β followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant pre-ovulatory follicle, is detectable in peripheral blood of cattle and expression is down-regulated during luteinisation induced by the pre-ovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, whilst raising doubts about its potential contribution to CL function.

Low CD4(+) T-Cell Levels and B-Cell Apoptosis in Vertically HIV-exposed Noninfected Children and Adolescents

Lymphocyte subsets, activation markers and apoptosis were assessed in 20 HIV-exposed noninfected (ENI) children born to HIV-infected women who were or not exposed to antiretroviral (ARV) drugs during pregnancy and early infancy. ENI children and adolescents were aged 6-18 years and they were compared to 25 age-matched healthy non-HIV-exposed children and adolescents (Control). ENI individuals presented lower CD4(+) T cells/mm(3) than Control group (control: 1120.3 vs. ENI: 876.3; t-test, p=0.030). ENI individuals had higher B-cell apoptosis than Control group (Control: 36.6%, ARV exposed: 82.3%, ARV nonexposed: 68.5%; Kruskal-Wallis, p < 0.05), but no statistical difference was noticed between those exposed and not exposed to ARV. Immune activation in CD4(+) T, CD8(+) T and in B cells was comparable in ENI and in Control children and adolescents. Subtle long-term immune alterations might persist among ENI individuals, but the clinical consequences if any are unknown, and these children require continued monitoring.

A Metal-Repressed Promoter From Gram-Positive Bacillus subtilis Is Highly Active and Metal-Induced in Gram-Negative Cupriavidus metallidurans

A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation. Biotechnol. Bioeng. 2010; 107: 469-477. (C) 2010 Wiley Periodicals, Inc.

Leptin and the Immune Response An Active Player or an Innocent Bystander?

Leptin is involved in the control of energy storage by the body. Low serum leptin levels, as seen in starvation, are associated with impaired inflammatory T cell responses that can be reversed by exogenous leptin. Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia and recurrent infections. Several defects in T cell function have also been described, and allergy, autoimmune disease, and lymphomas or other malignancies can be present. Previous studies in Brazilian CVID patients have shown that, in contrast with mononuclear cells from healthy controls, CVID cells cultured with phytohemagglutinin and added leptin increased the proliferative response and decreased activation-induced apoptosis. Interleukin (IL)-2 and especially IL-4 production also increased significantly, although the effects of exposure to leptin were not observed uniformly in CVID patients. The majority, however, responded in some degree, and some exhibited completely restored values of the four parameters. These remarkable results indicate leptin could be used to improve immune function in these patients. On the other hand, we found no specific correlation between serum leptin levels and the number of infectious events over a 24-month period, presence of autoimmunity, allergies, or cancer in these patients. The results suggest that the absolute value of serum leptin does not determine the clinical behavior of patients or responses to leptin in vitro. Of note is the divergence between serum leptin, response to leptin in vitro, and the presence of autoimmunity, indicating the need to identify the cellular and molecular players involved in the regulation of the immune response by leptin in CVID.

Two- and three-dimensional shape fabric analysis by the intercept method in grey levels

The count intercept is a robust method for the numerical analysis of fabrics Launeau and Robin (1996). It counts the number of intersections between a set of parallel scan lines and a mineral phase, which must be identified on a digital image. However, the method is only sensitive to boundaries and therefore supposes the user has some knowledge about their significance. The aim of this paper is to show that a proper grey level detection of boundaries along scan lines is sufficient to calculate the two-dimensional anisotropy of grain or crystal distributions without any particular image processing. Populations of grains and crystals usually display elliptical anisotropies in rocks. When confirmed by the intercept analysis, a combination of a minimum of 3 mean length intercept roses, taken on 3 more or less perpendicular sections, allows the calculation of 3-dimensional ellipsoids and the determination of their standard deviation with direction and intensity in 3 dimensions as well. The feasibility of this quick method is attested by numerous examples on theoretical objects deformed by active and passive deformation, on BSE images of synthetic magma flow, on drawing or direct analysis of thin section pictures of sandstones and on digital images of granites directly taken and measured in the field. (C) 2010 Elsevier B.V. All rights reserved.

Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of similar to 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K(M) was 42 mM, and V(max) was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence. (C) 2008 Elsevier Inc. All rights reserved.

Lithium reduces Gsk3b mRNA levels: implications for Alzheimer Disease

There is evidence of increased systemic expression of active GSK3B in Alzheimer`s disease patients, which apparently is associated with the formation of senile plaques and neurofibrillary tangles. Due to its central role in the pathogenesis of AD, GSK3B is currently a promising target of the pharmaceutical industry. Whilst trials with specific GSK inhibitors in AD are under way, major attention has been focused on the neuroprotective effects of lithium. Whereas the direct and indirect inhibitory effects of lithium over GSK3 activity have been documented by several groups, its effects over Gsk3 transcription have not yet been addressed. We used quantitative PCR to evaluate the transcriptional regulation of Gsk3a and Gsk3b in lithium-treated primary cultures of rat cortical and hippocampal neurons. We found a significant and dose-dependent reduction in the expression of Gsk3b, which was specific to hippocampal cells. This same effect was further confirmed in vivo by measuring Gsk3 expression in different brain regions and in peripheral leukocytes of adult rats treated with lithium. Our studies show that LiCl can modulate Gsk3b transcription in vitro and in vivo. This observation suggest new regulatory effects of lithium over Gsk3b, contributing to the better understanding of its mechanisms of action, offering a new and complementary explanation for Gsk3b modulation and reinforcing its potential for the inhibition of key pathological pathways in Alzheimer`s disease.

Lipodystrophy in HIV/AIDS patients with different levels of physical activity while on antiretroviral therapy

INTRODUÇÃO: A lipodistrofia relacionada ao uso de terapia antirretroviral (TARV) pode causar estigma estético e elevar o risco de doenças cardiovasculares. A atividade física pode ser uma alternativa válida para o tratamento e prevenção da lipodistrofia. Entretanto, poucos estudos tratam dessa temática. O objetivo deste estudo foi verificar a ocorrência de lipodistrofia relacionada ao uso de TARV em portadores de HIV/AIDS, com diferentes hábitos de atividades físicas. MÉTODOS: A casuística foi formada por 42 portadores de HIV em uso de TARV, do Centro de Testagem e Aconselhamento de Presidente Prudente. Para obtenção do nível de atividade física aplicou-se o Questionário Internacional de Atividade Física (IPAQ); a lipodistrofia foi diagnosticada pelo autorrelato do paciente e a confirmação médica. O percentual de gordura de tronco foi estimado pela absortometria por raio-X de dupla energia (DEXA). Foram coletados também dados referentes a sexo, idade, tempo de uso de TARV, valores de CD4 e carga viral. RESULTADOS: Verificou-se maior ocorrência de lipodistrofia no grupo sedentário quando comparado ao ativo, além de fator protetor da prática da atividade física em relação à ocorrência da lipodistrofia. O grupo com valores mais elevados de CD4 também apresentou maior proporção de sujeitos com lipodistrofia, além de maior proporção de ativos e de indivíduos com menor faixa etária. Os acometidos pela lipodistrofia apresentaram maiores valores de percentual de gordura de tronco, bem como, os sedentários em relação aos ativos. CONCLUSÕES: O estilo de vida fisicamente ativa resultou em efeito protetor para ocorrência da lipodistrofia relacionada ao uso da TARV.

Evidence for conformational changes in the yeast deoxyhypusine hydroxylase Lia1 upon iron displacement from its active site

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Abnormalities in apolipoprotein and lipid levels in an HIV-infected Brazilian population under different treatment profiles: the relevance of apolipoprotein E genotypes and immunological status

HIV infection is associated with disturbances in lipid metabolism due to a host's response mechanism and the current antiretroviral therapy. The pathological appearance and progression of atherosclerosis is dependent on the presence of injurious agents in the vascular endothelium and variations in different subsets of candidate genes. Therefore, the Hha I polymorphism in the apolipoprotein E gene was evaluated in addition to triglycerides, total cholesterol, very low-density lipoprotein (VLDL), LDL, high-density lipoprotein (HDL), and apolipoprotein (apo) Al, B and E levels in 86 Brazilian HIV-infected patients and 29 healthy controls. The allele frequency for apoE in the HIV-infected group and controls was in agreement with data on the Brazilian population. Dyslipidemia was observed in the HIV group and verified by increased levels of triglycerides, VLDL and apoE, and decreased levels of HDL and apoAl. The greatest abnormalities in these biochemical variables were shown in the HIV-infected individuals whose immune function was more compromised. The effect of the genetic variation at the APOE gene on biochemical variables was more pronounced in the HIV-infected individuals who carried the apoE2/3 genotype. The highly active antiretroviral therapy (HAART)-receiving group presented increased levels of total cholesterol and apoE. Dyslipidemia was a predictable consequence of HIV infection and the protease inhibitors intensified the increase in apoE values.

Hydroxymethylnitrofurazone Is Active in a Murine Model of Chagas' Disease

The addition of a hydroxymethyl group to the antimicrobial drug nitrofurazone generated hydroxymethylnitrofurazone (NFOH), which had reduced toxicity when its activity against Trypanosoma cruzi was tested in a murine model of Chagas' disease. Four groups of 12 Swiss female mice each received 150 mg of body weight/kg/day of NFOH, 150 mg/kg/day of nitrofurazone (parental compound), 60 mg/kg/day of benznidazole (BZL), or the solvent as a placebo. Treatments were administered orally once a day 6 days a week until the completion of 60 doses. NFOH was as effective as BZL in keeping direct parasitemia at undetectable levels, and PCR results were negative. No histopathological lesions were seen 180 days after completion of the treatments, a time when the levels of anti-T. cruzi antibodies were very low in mice treated with either NFOH or BZL. Nitrofurazone was highly toxic, which led to an overall rate of mortality of 75% and necessitated interruption of the treatment. In contrast, the group treated with its hydroxymethyl derivative, NFOH, displayed the lowest mortality (16%), followed by the BZL (33%) and placebo (66%) groups. The findings of histopathological studies were consistent with these results, with the placebo group showing the most severe parasite infiltrates in skeletal muscle and heart tissue and the NFOH group showing the lowest. The present evidence suggests that NFOH is a promising anti-T. cruzi agent.

Novel active and passive anti-vibration mountings

This paper investigates a novel design approach for a vibration isolator for use in space structures. The approach used can particularly be applicable for aerospace structures that support high precision instrumentation such as satellite payloads. The isolator is a space-frame structure that is folded in on itself to act as a mechanical filter over a defined frequency range. The absence of viscoelastic elements in such a mounting makes the design suitable for use in a vacuum and in high temperature or harsh environments with no risk of drift in alignment of the structure. The design uses a genetic algorithm based geometric optimisation routine to maximise passive vibration isolation, and this is hybridised with a geometric feasibility search. To complement the passive isolation system, an active system is incorporated in the design to add damping. Experimental work to validate the feasibility of the approach is also presented, with the active/passive structure achieving transmissibility of about 19 dB over a range of 1-250 Hz. It is shown here that the use of these novel anti-vibration mountings has no or little consequent weight and cost penalties whilst maintaining their effectiveness with the vibration levels. The approach should pave the way for the design of anti-vibration mountings that can be used between most pieces of equipment and their supporting structure. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.

Assessment of cytokine values in serum by RT-PCR in HIV-1 infected individuals with and without highly active anti-retroviral therapy (HAART)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of soluble fiber on hypertriglyceridemia and immune profile in HIV-positive individuals undergoing highly active antiretroviral therapy

The advent of highly active antiretroviral therapy (HAART), since 1996, represented a profound impact on the natural history of HIV-infection by promoting important and sustainable viral replication suppression and increasing survival and quality of life among seropositive patients. Nonetheless, antiretroviral therapy has been observed to be accompanied by metabolic alterations such as dyslipidemia, especially hypertriglyceridemia, insulin resistance, hyperglycemia and lipodystrophy (body fat redistribution). Epidemiological studies have demonstrated a correlation between high triglyceride (TG) levels and higher incidence of coronary artery disease (CAD). Some investigators suggest dietary intervention as part of hyperlipidemia treatment, including an increase in soluble fiber intake (10-25g/day). Whereas some studies have demonstrated that both cholesterol and serum triglyceride levels decrease with the use of food fiber, others have shown just a serum triglyceride decrease, and others failed to observe any alteration in lipid metabolism. The purpose of this study was to assess the effect of soluble fiber (R) (partially hydrolyzed guar gum) supplementation on hypertriglyceridemia and immune profile in HIVpositive individuals on HAART. Nineteen HIV-positive individuals with hypertriglyceridemia (serum levels >= 150 to < 500mg/dl) were studied. of these individuals, 63.16% were males and 36.84% females, with mean age of 43.52 +/- 9.22 years. These individuals had been on the same HAART regimen for at least six months, had no change in therapy during the study and received 20g/day of soluble fiber for four months at pre-established times. Clinical-nutritional, biochemical (total proteins, albumin, globulin, total cholesterol, LDL-c, HDL-c, TG, TG/HDL-c and LDLc/HDL-c), hematimetric (hemoglobin, hematocrit and total lymphocytes), and immunologic (lymphocytes T CD4(+), T CD8(+); T CD4(+)/CD8(+) ratio, viral load, TNF-alpha and IL-6) parameters were assessed in all patients at three time points (M0: pretreatment, M1: 30 days, and M2: four months after intervention). Significance level was set at 5% for all data statistically analyzed. Serum TG and TG/HDL-c ratio reduction was observed at all time points, but statistical significance was found just at M0 and M2. The remaining biochemical, hematimetric and immunologic parameters (lymphocytes T CD4(+), T CD8(+); T CD4(+)/ CD8(+) ratio, and viral load) showed no significant difference at all times. Regarding serum cytokines, TNF-alpha and IL-6 significantly decreased between M0 and M2, and only IL-6 reduced between M1 and M2. The data collected show that dietary and anthropometric parameters remained unchanged excluding potential confounding factors related with the effect of fiber supplementation on serum TG, TNF-alpha and IL-6. Thus, soluble fiber (R) contributed to an important reduction in hypertriglyceridemia and in the serum levels of the proinflammatory cytokines TNF-alpha and IL-6 in HIV-seropositive individuals on HAART. In addition, soluble fiber (R) might have minimized the process of atherosclerosis in these individuals, given that elevated serum levels of TG, TNF-alpha and IL-6 have been associated with the development of these lesions.