Heart Rate and Systolic Blood Pressure Variability in the Time Domain in Patients with Recent and Long-Standing Diabetes Mellitus

Diabetes Mellitus (DM) affects the cardiovascular response of patients. To study this effect, interbeat intervals (IBI) and beat-to-beat systolic blood pressure (SBP) variability of patients during supine, standing and controlled breathing tests were analyzed in the time domain. Simultaneous noninvasive measurements of IBI and SBP for 30 recently diagnosed and 15 long-standing DM patients were compared with the results for 30 rigorously screened healthy subjects (control). A statistically significant distinction between control and diabetic subjects was provided by the standard deviation and the higher moments of the distributions (skewness, and kurtosis) with respect to the median. To compare IBI and SBP for different populations, we define a parameter, α, that combines the variability of the heart rate and the blood pressure, as the ratio of the radius of the moments for IBI and the same radius for SBP. As diabetes evolves, α decreases, standard deviation of the IBI detrended signal diminishes (heart rate signal becomes more “rigid”), skewness with respect to the median approaches zero (signal fluctuations gain symmetry), and kurtosis increases (fluctuations concentrate around the median). Diabetes produces not only a rigid heart rate, but also increases symmetry and has leptokurtic distributions. SBP time series exhibit the most variable behavior for recently diagnosed DM with platykurtic distributions. Under controlled breathing, SBP has symmetric distributions for DM patients, while control subjects have non-zero skewness. This may be due to a progressive decrease of parasympathetic and sympathetic activity to the heart and blood vessels as diabetes evolves.     

Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv

Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease’s causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli’s nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (E_a, ΔG^#, ΔS^#, ΔH^#) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH.     

Stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1alpha treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1alpha induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important "cross-talk" between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.     

Characterization of thimet- and neurolysin-like activities in Escherichia coli M 3 A peptidases and description of a specific substrate

M 3 A oligopeptidases from Escherichia coli, with hydrolytic properties similar to Zn-dependent mammalian thimet oligopeptidase (EP 24.15) and neurolysin (EP 24.16), were studied aiming at identification of comparative enzyme and substrate specificity, hydrolytic products, and susceptibility to inhibitors. Fluorescent peptides, neurotensin (NT) and bradykinin (BK), were used as substrates for bacterial lysates. Bacterial enzymes were totally inhibited by o-phenanthrolin, JA-2 and partially by Pro-Ile, but not by leupeptin, PMSF, E-64, and Z-Pro-Prolinal, using internally quenched Abz-GFSPFRQ-EDDnp as substrate. The molecular mass of the bacterial oligopeptidase activity (77--78 kDa) was determined by gel filtration, and the effect of inhibitors, including captopril, suggested that it results from a combination of oligopeptidase A (OpdA) and peptidyl dipeptidase Dcp (77.1 and 77.5 kDa, respectively). Recombinant OpdA cloned from the same E. coli strain entirely reproduced the primary cleavage of fluorescent peptides, NT and BK, by the bacterial lysate. Genes encoding these M 3 A enzymes were those recognized in E. coli genome, bearing identity at the amino acid level (25--31%) with mammalian Zn-dependent oligopeptidases. We also describe a substrate, Abz-GFSPFRQ-EDDnp, that differentiates bacterial and mammalian oligopeptidases.     

Constant light during lactation programs circadian and metabolic systems

Exposure to light at night is a disruptive condition for the adult circadian system, leading to arrhythmicity in nocturnal rodents. Circadian disruption is a risk factor for developing physiological and behavioral alterations, including weight gain and metabolic disease. During early stages of development, the circadian system undergoes a critical period of adjustment, and it is especially vulnerable to altered lighting conditions that may program its function, leading to long-term effects. We hypothesized that during lactation a disrupted light-dark cycle due to light at night may disrupt the circadian system and in the long term induce metabolic disorders. Here we explored in pups, short- and long-term effects of constant light (LL) during lactation. In the short term, LL caused a loss of rhythmicity and a reduction in the immunopositive cells of VIP, AVP, and PER1 in the suprachiasmatic nucleus (SCN). In the short term, the affection on the circadian clock in the pups resulted in body weight gain, loss of daily rhythms in general activity, plasma glucose and triglycerides (TG). Importantly, the DD conditions during development also induced altered daily rhythms in general activity and in the SCN. Exposure to LD conditions after lactation did not restore rhythmicity in the SCN, and the number of immunopositve cells to VIP, AVP, and PER1 remained reduced. In the long term, daily rhythmicity in general activity was restored; however, daily rhythms in glucose and TG remained disrupted, and daily mean levels of TG were significantly increased. Present results point out the programming role played by the LD cycle during early development in the function of the circadian system and on metabolism. This study points out the risk represented by exposure to an altered light-dark cycle during early stages of development.

Abbreviations:

AVP: arginine vasopressin peptide; CRY: cryptochrome; DD: constant darkness; DM: dorsomedial; LD: light-dark cycle; LL: constant light; NICUs: neonatal intensive care units; P: postnatal days; PER: period; S.E.M.: standard error of the mean; SCN: suprachiasmatic nucleus; TG: triglycerides; VIP: vasointestinal peptide; VL: ventrolateral; ZT: zeitgeber time.     

The HIV Matrix Protein p17 Promotes the Activation of Human Hepatic Stellate Cells through Interactions with CXCR2 and Syndecan-2

Background

The human immunodeficiency virus type 1 (HIV-1) p17 is a matrix protein involved in virus life''s cycle. CXCR2 and Syndecan-2, the two major coreceptors for the p17 protein, are expressed in hepatic stellate cells (HSCs), a key cell type involved in matrix deposition in liver fibrotic disorders.

Aim

In this report we have investigated the in vitro impact of p17 on HSCs transdifferentiation and function and underlying signaling pathways involved in these processes.

Methods

LX-2 cells, a human HSC line, and primary HSC were challenged with p17 and expressions of fibrogenic markers and of p17 receptors were assessed by qRT-PCR and Western blot. Downstream intracellular signaling pathways were evaluated with qRT-PCR and Western blot as well as after pre-treatment with specific pathway inhibitors.

Results

Exposure of LX2 cells to p17 increases their contractile force, reshapes the cytoskeleton fibers and upregulates the expression of transdifferentiation markers including αSMA, COL1α1 and endothelin-1 through the activation of Jak/STAT and Rho signaling pathways. These effects are lost in HSCs pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide. Confocal laser microscopy studies demonstrates that CXCR2 and syndecan-2 co-associate at the plasma membrane after exposure to p17. Immunostaining of HIV/HCV liver biopsies from co-infected patients reveals that the progression of liver fibrosis correlates with a reduced expression of CXCR2.

Conclusions

The HIV matrix protein p17 is pro-fibrogenic through its interactions both with CXCR2 and syndecan-2 on activated HSCs.     

Changes in T-cell subpopulations during four years of suppression of HIV-1 replication in patients with advanced disease

We compared the number/percentages of naive and memory CD4+ T-cells, CD38+ CD8+ T-cells, and CD28+ CD4+ and CD28+ CD8+ T-cells in patients with advanced HIV disease (baseline CD4+ count < 100) with those with less advanced (baseline CD4+ cell count > 100) HIV disease during 4 years of suppressive highly active antiretroviral therapy. This prospective, longitudinal study included 30 treatment-naive patients and 32 controls. Advanced HIV-infected patients (n = 13) gained more CD4+ T-cells than less advanced patients (n = 11) at 1 month (median: 60 vs. 36 microL(-1)), 3 months (86 vs. 14), 6 months (111 vs. 23), 12 months (174 vs. 47), 24 months (162 vs. 72) and 48 months (257 vs. 123) (P = 0.15, P < 0.001, P = 0.026, P = 0.021, P = 0.1 and P = 0.06, respectively). Advanced patients gained more naive CD4+ T-cells at 48 months compared to less advanced patients (27.3 vs. 11.4%, P = 0.05). The relative gain in memory CD4+ T-cells was greater in advanced vs. less advanced patients at 1 month (median: 6.4 vs. 1.4%), 3 months (4.3 vs. 2.0), 6 months (6.7 vs. 1.6), 12 months (6.9 vs. 2.4), 24 months (7.5 vs. 3.1) and 48 months (11.3 vs. 6.8) (P = 0.002, P = 0.013, P < 0.001, P = 0.004, P = 0.001 and P = 0.015, respectively). At 48 months, CD38+ CD8+ T-cells and naive CD4+ T-cells reached normal values (9.2%, P = 0.869 vs. controls and 47.5%, P = 0.699, respectively) in less advanced patients, as did CD38+ CD8+ T-cells in advanced patients (4.7%, P = 0.309 vs. controls). The kinetics of naive and memory CD4+ T-cell reconstitution is different in less advanced compared to advanced HIV patients.     

Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass

Background

Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world''s second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant.

Results

Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, β-glucosidases, β-xylosidases, endoxylanases, xyloglucanases, and α-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source.

Conclusion

Our work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.     

β-Amyloid1-42, HIV-1Ba-L (Clade B) Infection and Drugs of Abuse Induced Degeneration in Human Neuronal Cells and Protective Effects of Ashwagandha (Withania somnifera) and Its Constituent Withanolide A

Alzheimer''s disease (AD) is characterized by progressive dysfunction of memory and higher cognitive functions with abnormal accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles throughout cortical and limbic brain regions. Withania somnifera (WS) also known as ‘ashwagandha’ (ASH) is used widely in Ayurvedic medicine as a nerve tonic and memory enhancer. However, there is paucity of data on potential neuroprotective effects of ASH against β-Amyloid (1–42) (Aβ) induced neuropathogenesis. In the present study, we have tested the neuroprotective effects of Methanol: Chloroform (3:1) extract of ASH and its constituent Withanolide A (WA) against Aβ induced toxicity, HIV-1_Ba-L (clade B) infection and the effects of drugs of abuse using a human neuronal SK-N-MC cell line. Aβ when tested individually, induced cytotoxic effects in SK-N-MC cells as shown by increased trypan blue stained cells. However, when ASH was added to Aβ treated cells the toxic effects were neutralized. This observation was supported by cellular localization of Aβ, MTT formazan exocytosis, and the levels of acetylcholinesterase activity, confirming the chemopreventive or protective effects of ASH against Aβ induced toxicity. Further, the levels of MAP2 were significantly increased in cells infected with HIV-1_Ba-L (clade B) as well as in cells treated with Cocaine (COC) and Methamphetamine (METH) compared with control cells. In ASH treated cells the MAP2 levels were significantly less compared to controls. Similar results were observed in combination experiments. Also, WA, a purified constituent of ASH, showed same pattern using MTT assay as a parameter. These results suggests that neuroprotective properties of ASH observed in the present study may provide some explanation for the ethnopharmacological uses of ASH in traditional medicine for cognitive and other HIV associated neurodegenerative disorders and further ASH could be a potential novel drug to reduce the brain amyloid burden and/or improve the HIV-1 associated neurocognitive impairments     

澳大利亚轮藻植物LYCHNOTHAMNUS BARBATUS(MEYEN)LEONHARDI藏卵器统计分析及与欧洲同类植物的对比研究

Lychnothamnus(Characeae)为一分布于欧洲和大洋洲的单型属,该类植物日趋衰落。过去十年中,仅在澳大利亚昆士兰州华莱士湾发现数量丰富生长良好的L．barbatus(Meyen)Leonhardi。本文对澳大利亚现生L．barbatus藏卵器进行描述、统计分析和扫描照相,并与其它地区同类现生和化石藏卵器进行对比,结果表明澳大利亚的藏卵器除形状偏长球形、宽度较小、钙化较弱外,其余特征与欧洲植物较为一致。钙化减弱的原因可能与澳大利亚热带地区由季风造成的洪水泛滥降低了水体中离子的浓度有关。