Transcription of the Neurospora crassa 70-kDa class heat shock protein genes is modulated in response to extracellular pH changes

Heat shock proteins belong to a conserved superfamily of molecular chaperones found in prokaryotes and eukaryotes. These proteins are linked to a myriad of physiological functions. In this study, we show that the N. crassa hsp70-1 (NCU09602.3) and hsp70-2 (NCU08693.3) genes are preferentially expressed in an acidic milieu after 15 h of cell growth in sufficient phosphate at 30A degrees C. No significant accumulation of these transcripts was detected at alkaline pH values. Both genes accumulated to a high level in mycelia that were incubated for 1 h at 45A degrees C, regardless of the phosphate concentration and extracellular pH changes. Transcription of the hsp70-1 and hsp70-2 genes was dependent on the pacC (+) background in mycelia cultured under optimal growth conditions or at 45A degrees C. The pacC gene encodes a Zn-finger transcription factor that is involved in the regulation of gene expression by pH. Heat shock induction of these two hsp genes in mycelia incubated in low-phosphate medium was almost not altered in the nuc-1 (-) background under both acidic and alkaline pH conditions. The NUC-1 transcriptional regulator is involved in the derepression of nucleases, phosphatases, and transporters that are necessary for fulfilling the cell`s phosphate requirements. Transcription of the hsp70-3 (NCU01499.3) gene followed a different pattern of induction-the gene was depressed under insufficient phosphate conditions but was apparently unaffected by alkalinization of the culture medium. Moreover, this gene was not induced by heat shock. These results reveal novel aspects of the heat-sensing network of N. crassa.

Genomic deletions at 1p and 14q are associated with an abnormal cDNA microarray gene expression pattern in meningiomas but not in schwannomas

The molecular pathology of meningiomas and shwannomas involve the inactivation of the NF2 gene to generate grade I tumors. Genomic losses at 1p and 14q are observed in both neoplasms, although more frequently in meningiomas. The inactivation of unidentified genes located in these regions appears associated with tumor progression in meningiomas, but no clues to its molecular/clinical meaning are available in schwannomas. Recent microarray gene expression studies have demonstrated the existence of molecular subgroups in both entities. In the present study, we correlated the presence of genomic deletions at 1p, 14q, and 22q with the expression patterns of 96 tumor-related genes obtained by cDNA low-density microarrays in a series of 65 tumors including 42 meningiomas and 23 schwannomas. Two expression pattern groups were identified by cDNA mycroarray analysis when compared to the expression pattern in normal control RNA in both meningiomas and schwannomas, each one with patterns similar and different from the normal control. Meningioma and schwannoma subgroups differed in the expression of 38 and 16 genes, respectively. Using MLPA and microsatellites, we identified genomic losses at 1p, 14q, and 22q at nonrandom frequencies (12.5-69%) in meningiomas and schwannomas. Losses at 22q were almost equally frequent in both molecular expression subgroups in both neoplasms. However, deletions at 1p and 14q accumulated in meningiomas with a gene expression pattern different from the normal pattern, whereas the inverse situation occurred in schwannomas. Those anomalies characterized the schwannomas with expression pattern similar to the normal control. These findings suggest that deletions at 1p and 14q enhance the development of an abnormal tumor-related gene expression pattern in meningiomas, but this fact is not corroborated in schwannomas. (C) 2010 Elsevier Inc. All rights reserved.

Allelic status of 1p and 19q in oligodendrogliomas and glioblastomas: multiplex ligation-dependent probe amplification versus loss of heterozygosity

Identification of the 1p/19q allelic status in gliomas, primarily those with a major oligodendroglial component, has become an excellent molecular complement to tumor histology in order to identify those cases sensitive to chemotherapy. In addition to loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), or comparative genomic hybridization (CGH), multiplex ligation-dependent probe amplification (MLPA) has been shown to be an alternative methodology to identify deletions of those chromosome arms. We used MLPA to explore the 1p and 19q glioblastomas, and a series of 76 gliomas: 41 tumors with a major oligodendroglial component, 34 glioblastomas, and one low-grade astrocytoma. We compared the MLPA findings of the oligodendroglial cases with those previously obtained using LOH in the same samples. Thirty-eight of 41 oligodendrogliomas displayed identical findings by both LOH and MLPA, and losses at either 1p and/or 19q were identified in 12 of 35 (34%) astrocytic tumors. These findings agree with data previously reported comparing MLPA versus FISH or CGH in gliomas and suggest that MLPA can be used in the identification 1p/19q allelic deletions on these brain neoplams. (c) 2009 Elsevier Inc. All rights reserved. reserved.

A splice variant of the Neurospora crassa hex-1 transcript, which encodes the major protein of the Woronin body, is modulated by extracellular phosphate and pH changes

The Woronin body, a septal pore-associated organelle specific to filamentous ascomycetes, is crucial for preventing cytoplasmic bleeding after hyphal injury. In this study, we show that T1hex-1 transcript and a variant splicing T2hex-1 transcript are up-regulated at alkaline pH. We also show that both hex-1 transcripts are overexpressed in the preg(c), nuc-1(RIP), and pacC(ko) mutant strains of Neurospora crassa grown under conditions of phosphate shortage at alkaline pH, suggesting that hex-1 transcription may be coregulated by these genes. In addition, we present evidence that N. crassa PacC also has metabolic functions at acidic pH. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Transcriptional changes in the nuc-2A mutant strain of Neurospora crassa cultivated under conditions of phosphate shortage

The molecular mechanism that controls the response to phosphate shortage in Neurospora crassa involves four regulatory genes - nuc-2, preg, pgov, and nuc-1. Phosphate shortage is sensed by the nuc-2 gene, the product of which inhibits the functioning of the PREG-PGOV complex. This allows the translocation of the transcriptional factor NUC-1 into the nucleus, which activates the transcription of phosphate-repressible phosphatases. The nuc-2A mutant strain of N. crassa carries a loss-of-function mutation in the nuc-2 gene, which encodes an ankyrin-like repeat protein. In this study, we identified transcripts that are downregutated in the nuc-2A mutant strain. Functional grouping of these expressed sequence tags allowed the identification of genes that play essential roles in different cellular processes such as transport, transcriptional regulation, signal transduction, metabolism, protein synthesis, protein fate, and development. These results reveal novel aspects of the phosphorus-sensing network in N. crassa. (C) 2009 Elsevier GmbH. All rights reserved.

PULSATILITY INDEX CHANGE OF BRACHIAL ARTERY SHOWS BETTER REPRODUCIBILITY THAN FLOW-MEDIATED VASODILATION

We aim to evaluate intra- and interobserver reproducibility of brachial artery flow-mediated dilation (FMD) and of pulsatility index change (PI-C) 1 min after a 5-min forearm compression in pregnant women. Flow-mediated dilation and PI-C were assessed in 31 healthy pregnant women in 3 consecutive days, by two observers. A broader range of response to the stimulus was observed for PI-C (-58.99%-86.71%) than FMD (2.38%-27.01%). The intraclass correlation coefficients were higher for PI-C than for FMD (intra = 0.90 vs. 0.69, p = 0.047; inter = 0.94 vs. 0.61, p = 0.007). Therefore, PI-C measurements were more reliable than FMD. However, studies comprising of subjects with cardiovascular risk factors relating FMD and PI-C become necessary to validate this method for the study of endothelial dysfunction. (E-mail: wpmartins@gmail.com) (C) 2010 World Federation for Ultrasound in Medicine & Biology.

UTERINE ULTRASONOGRAPHIC CHANGES DURING ENDOMETRIOSIS TREATMENT: A COMPARISON BETWEEN LEVONORGESTREL-RELEASING INTRAUTERINE DEVICES AND A GONADOTROPIN-RELEASING HORMONE AGONIST

We compared the effects of levonorgestrel-releasing intrauterine devices (LNG-IUD) and a gonadotropin-releasing hormone agonist (GnRHa) on uterine volume, uterine arteries pulsatility index (PI) and endometrial thickness before and after six months of endometriosis treatment. Sixty women aged 18-40 y were allocated randomly to one of two groups: LNG-IUDs were inserted in 30 women, and GnRHa monthly injections were performed on the other 30. All 60 women were submitted to transvaginal 2-D ultrasound scans on the day that the treatment started and then six months later. Measurements of uterine arteries PI, uterine volume and endometrial thickness were performed at both evaluations. The use of LNG-IUDs significantly decreased endometrial thickness (pre = 6.08 +/- 3.00 mm, post = 2.7 +/- 0.98 mm; mean +/- SD), as did the use of GnRHa (pre = 6.96 +/- 3.82 mm, post = 3.23 +/- 2.32 mm). The uterine volume decreased in the GnRHa group (pre = 86.67 +/- 28.38 cm(3), Post = 55.27 +/- 25.52 cm(3)), but not in the LNG-IUD group (pre = 75.77 +/- 20.88 cm(3), post = 75.97 +/- 26.62 cm(3)). Uterine arteries PI increased for both groups; however, the increase was higher in the GnRHa group (0.99 +/- 0.84 vs. 0.38 +/- 0.84, p = 0.007; PI increase in GnRHa and in LNG-IUD groups, respectively). In conclusion, levonorgestrel released directly onto the endometrium by the LNG-IUD induced smaller uterine changes than did the hypoestrogenism induced by GnRHa. Nevertheless, both promoted similar effects on endometrial thickness. (E-mail: wpmartins@gmail.com) (C) 2008 World Federation for Ultrasound in Medicine & Biology.

Brachial artery pulsatility index change 1 minute after 5-minute forearm compression - Comparison with flow-mediated dilatation

Objective. Endothelial impairment evaluation by sonographic measurement of flow-mediated dilatation (FMD) has become broadly used. However, this method has 2 main caveats: the dilatation depends on the baseline arterial diameter, and a high precision level is required. Vasodilatation leads to an amplified fall in impedance. We hypothesized that assessment of the pulsatility index change (PI-C) 1 minute after 5-minute forearm compression might evaluate that fall in impedance. The aim of this study was to compare the PI-C with FMD. Methods. Flow-mediated dilatation and the PI-C were assessed in 51 healthy women aged between 35.1 and 67.1 years. We correlated both FMD and the PI-C with age, body mass index, waist circumference, cholesterol level, high-density lipoprotein level, glucose level, systolic and diastolic blood pressure, pulse pressure, brachial artery diameter, simplified Framingham score, intima-media thickness, and carotid stiffness index. Intraclass correlation coefficients between 2 FMD and PI-C measurements were also examined. Results. Only FMD correlated with baseline brachial diameter (r=-0.53). The PI-C had a high correlation with age, body mass index, waist circumference, cholesterol level, systolic blood pressure, pulse pressure, simplified Framingham score, and intima-media thickness. The correlation between FMD and the PI-C was high (r=-0.66). The PI-C had a higher intraclass correlation coefficient (0.991) than FMD (0.836) but not brachial artery diameter (0.989). Conclusions. The PI-C had a large correlation with various markers of cardiovascular risk. Additionally, PI-C measurement does not require offline analysis, extra software, or electrocardiography We think that the PI-C could be considered a marker of endothelial function. However, more studies are required before further conclusions.

Cigarette Smoking Exacerbates Nonalcoholic Fatty Liver Disease in Obese Rats

The prevalence of cigarette smoking (CS) is increased among obese subjects, who are susceptible to develop nonalcoholic fatty liver disease (NAFLD). We investigated the hepatic effects of CS in control and obese rats. Control and obese Zucker rats were divided into smokers and nonsmokers (n = 12 per group). Smoker rats were exposed to 2 cigarettes/day, 5 days/week for 4 weeks. The effects of CS were assessed by biochemical analysis, hepatic histological examination, immunohistochemistry, and gene expression analysis. Phosphorylation of AKT and extracellular signal-regulated kinase (ERK) and quantification of carbonylated proteins were assessed by western blotting. As expected, obese rats showed hypercholesterolemia, insulin resistance, and histological features of NAFLD. Smoking did not modify the lipidic or glucidic serum profiles. Smoking increased alanine aminotransferase serum levels and the degree of liver injury in obese rats, whereas it only induced minor changes in control rats. Importantly, CS increased the histological severity of NAFLD in obese rats. We also explored the potential mechanisms involved in the deleterious effects of CS. Smoking increased the degree of oxidative stress and hepatocellular apoptosis in obese rats, but not in controls. Similarly, smoking increased the hepatic expression of tissue inhibitor of metalloproteinase-1 and procollagen-alpha2(I) in obese rats, but not in controls. Finally, smoking regulated ERK and AKT phosphorylation. The deleterious effects of CS were not observed after a short exposure (5 days). Conclusion: CS causes oxidative stress and worsens the severity of NAFLD in obese rats. Further studies should assess whether this finding also occurs in patients with obesity and NAFLD. (HEPATOLOGY 2010;51:1567-1576.)

An Experimental Model of Meningoencephalomyelitis by Rocio Flavivirus in Balb/C Mice: Inflammatory Response, Cytokine Production, and Histopathology

Rocio virus (ROCV) is a flavivirus, probably transmitted by Culex mosquitoes and maintained in nature as a zoonosis of wild birds. Rocio virus caused a human epidemic of severe encephalitis that lasted from 1973 to 1980 in the Ribeira valley, in the southeastern coast of Brazil. After this outbreak, serologic evidence of ROCV circulation has been reported and public health authorities are concerned about a return of ROCV outbreaks in Brazil. We show here a study on the pathogenesis and the physiopathology of ROCV disease in the central nervous system of a Balb/C young adult mice experimental model. The animals were intraperitoneally infected by ROCV and followed from 0 to 9 days after infection, when all of them died. Nervous tissue samples were collected from infected animals for immunohistochemistry and molecular biology analysis. We observed the virus in the central nervous system, the inflammatory changes induced by Th1 and Th2 cytokines, and the final irreversible damage of nervous tissues by neuronal degeneration and apoptosis. These findings can help to better understand the pathogenesis and physiopathology of the human meningoencephalomyelitis by ROCV and other flaviviruses.

Effects of Caffeoylquinic Acid Derivatives and C-Flavonoid from Lychnophora ericoides on in vitro Inflammatory Mediator Production

In this study we aimed at evaluating the effect of the major polar constituents of the medicinal plant Lychnophora ericoides on the production of inflammatory mediators produced by LPS-stimulated U-937 cells. The 6,8-di-C-beta-glucosylapigenin (vicenin-2) presented no effect on tumor necrosis factor (TNF)-alpha production, but inhibited, in a dose-dependent manner, the production of prostaglandin (PG) E(2) without altering the expression of cyclooxygenase (COX) -2 protein. 3,5-Dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, at lower concentrations, had small but significant effects on reducing PG E, levels; at higher doses these compounds stimulated PGE(2) and also TNF-alpha production by the cells. All the caffeoylquinic acid derivatives, in a dose-dependent fashion, were able to inhibit monocyte chemoattractant protein-3 synthesis/release, with 4,5-DCQ being the most potent at the highest tested concentration. These results add important information on the effects of plant natural polyphenols, namely vicenin-2 and caffeoylquinic acid derivatives, on the production of inflammatory mediators by cultured cells.

C-Peptide Levels and Insulin Independence Following Autologous Nonmyeloablative Hematopoietic Stem Cell Transplantation in Newly Diagnosed Type 1 Diabetes Mellitus

Context In 2007, the effects of the autologous nonmyeloablative hematopoietic stem cell transplantation (HSCT) in 15 patients with type 1 diabetes mellitus (DM) were reported. Most patients became insulin free with normal levels of glycated hemoglobin A(1c) (HbA(1c)) during a mean 18.8-month follow-up. To investigate if this effect was due to preservation of beta-cell mass, continued monitoring was performed of C-peptide levels after stem cell transplantation in the 15 original and 8 additional patients. Objective To determine C-peptide levels after autologous nonmyeloablative HSCT in patients with newly diagnosed type 1 DM during a longer follow-up. Design, Setting, and Participants A prospective phase 1/2 study of 23 patients with type 1 DM(aged 13-31 years) diagnosed in the previous 6 weeks by clinical findings with hyperglycemia and confirmed by measurement of serum levels of anti glutamic acid decarboxylase antibodies. Enrollment was November 2003-April 2008, with follow-up until December 2008 at the Bone Marrow Transplantation Unit of the School of Medicine of Ribeirao Preto, Ribeirao Preto, Brazil. Hematopoietic stem cells were mobilized via the 2007 protocol. Main Outcome Measures C-peptide levels measured during the mixed-meal tolerance test, before, and at different times following HSCT. Secondary end points included morbidity and mortality from transplantation, temporal changes in exogenous insulin requirements, and serum levels of HbA1c. Results During a 7- to 58-month follow-up (mean, 29.8 months; median, 30 months), 20 patients without previous ketoacidosis and not receiving corticosteroids during the preparative regimen became insulin free. Twelve patients maintained this status for a mean 31 months (range, 14-52 months) and 8 patients relapsed and resumed insulin use at low dose (0.1-0.3 IU/kg). In the continuous insulin-independent group, HbA(1c) levels were less than 7.0% and mean (SE) area under the curve (AUC) of C-peptide levels increased significantly from 225.0 (75.2) ng/mL per 2 hours pretransplantation to 785.4 (90.3) ng/mL per 2 hours at 24 months posttransplantation (P<.001) and to 728.1 (144.4) ng/mL per 2 hours at 36 months (P=.001). In the transient insulin-independent group, mean (SE) AUC of C-peptide levels also increased from 148.9 (75.2) ng/mL per 2 hours pretransplantation to 546.8 (96.9) ng/mL per 2 hours at 36 months (P=.001), which was sustained at 48 months. In this group, 2 patients regained insulin independence after treatment with sitagliptin, which was associated with increase in C-peptide levels. Two patients developed bilateral nosocomial pneumonia, 3 patients developed late endocrine dysfunction, and 9 patients developed oligospermia. There was no mortality. Conclusion After a mean follow-up of 29.8 months following autologous nonmyeloablative HSCT in patients with newly diagnosed type 1 DM, C-peptide levels increased significantly and the majority of patients achieved insulin independence with good glycemic control. Trial Registration clinicaltrials.gov Identifier: NCT00315133

Chemokines expression during Leptospira interrogans serovar Copenhageni infection in resistant BALB/c and susceptible C3H/HeJ mice

The role of innate immune responses in protection against leptospirosis remains unclear. We examined the expression of the chemokines CCL2/JE (MCP-1), CCL3/MIP-1 alpha (MIP-1 alpha) and CXCL1/KC (IL-8) regarding resistance and susceptibility to leptospirosis in experimental mice models BALB/c and C3H/HeJ, respectively. A virulent strain of Leptospira interrogans serovar Copenhageni was used in this study. Twenty-five animals of each mouse strain of C3H/HeJ and BALB/c, were infected intraperitoneally with 106 cells. Five un-infected animals of each strain were kept as control. Mortality of C3H/HeJ mouse was observed while BALB/c mice were asymptomatic. The presence of leptospire DNA in tissues of infected animals was demonstrated by PCR. Chemokines were measured in serum, spleen, liver, kidney and lung of both strains of animals using immunoenzymatic assay (ELISA). Elevations in the levels of chemokines MCP-1 and IL-8 occurred in all organs and sera of C3H/HeJ and BALB/c infected mice. The levels of MIP-1 alpha were lower when compared to MCP-1 and IL-8 in all analyzed organs, with a slight increase in liver and kidney. Our results indicate that the expression of inflammatory mediators can vary greatly, depending on the tissue and mouse strains. It is possible that the resistance to Leptospira can be partially correlated to the increase of MIP-1 alpha observed in BALB/c mice, while an increasing and a sustained expression of MCP-1 and IL-8 in the lungs of C3H/HeJ mice can be correlated to the severity and progression of leptospirosis. (C) 2009 Elsevier Ltd. All rights reserved.

FURTHER INSIGHTS TOWARD VITAMIN C DETERMINATION AND STABILITY. PROPOSAL OF A NEW QUANTIFICATION METHOD

This work aimed at an evaluation of the classical iodine method for quantification of vitamin C (L-ascorbic acid) in fruit juices, as well as at a search into the stability of this so popular vitamin under different conditions of pH, temperature and light exposition, in addition to a proposal of a new quantification method. Our results point to the persistent reversibility of the blue color of the starch-triiodide complex at the end point when using the classical iodine titration, and the overestimation of the true vitamin concentration in fruit juices. A new quantification method is proposed in order to overcome this problem. Surprising conclusions were obtained regarding the controversial stability of L-ascorbic acid toward atmospheric oxygen, at low pH, even in fruit juice and at room temperature, showing that the major problem concerned with aging of fruit juices is proliferation of microorganisms rather than expontaneous oxidation of L-ascorbic acid.

Vaccinating the vampire bat Desmodus rotundus against rabies

The objective of this study was to extend the previous work of indirect oral rabies immunization of vampire bats (Desmodus rotundus) maintained in captivity, which demonstrated the immunogenicity of the V-RG vaccine (Vaccinia-Rabies Glycoprotein) and indicated that although the results had been encouraging, a new method for concentrating the vaccine should be tested in order to avoid vaccine loss and increase the survival proportion of bats after rabies challenge. In this study, three groups of seven bats each were tested with vaccine concentrated by ultrafiltration through a cellulose membrane. The vaccine was homogenized in Vaseline paste and applied to the back of one vector bat, which was then reintroduced into its group. A dose of 10(5.0) MICLD50 rabies virus was used by intramuscular route to challenge the bats postvaccination. The survival proportion in the three groups after the challenge was 71.4%, 71.4% and 100%. (c) 2008 Elsevier B.V. All rights reserved.