Blood pressure variability increases connexin expression in the vascular smooth muscle of rats

Aims Following sinoaortic denervation (SAD), isolated rat aortas present oscillatory contractions and demonstrate a heightened contraction for alpha-adrenergic agonists. Our aim was to verify the effects of SAD on connexin43 (Cx43) expression and phenylephrine-induced contraction in isolated aortas. Methods and results Three days after surgery (SAD or sham operation), isolated aortic rings were exposed to phenylephrine and acetylcholine (0.1-10 mu M) in the presence or absence of the gap junction blocker 18 beta-glycyrrhetinic acid (18 beta-GA, 100 mu M). Vascular reactivity to potassium chloride (KCl, 4.7-120 mM) was also examined. The incidence of rats presenting oscillatory contractions was measured. Effects of SAD on the vascular smooth muscle expression of the Cx43 mRNA by RT-PCR and western blotting for Cx43 protein were examined. Phenylephrine-induced contraction was higher in SAD rat aortas compared with the control. In the presence of 18 beta-GA, the response to phenylephrine was similar in both groups. Oscillatory contractions were observed in 10/10 SAD rat aortas vs. 2/10 controls. Relaxing response to acetylcholine was similar in both groups, but in the presence of 18 beta-GA, the response to acetylcholine decreased significantly in the sham-operated group (82.7 +/- 7.6% reduction of relaxation), whereas a half-maximal relaxation (reduction of 46.2 +/- 5.3%) took place in SAD rat aortas. KCl-induced contraction was similar in both groups. Following SAD, RT-PCR revealed significantly increased levels of Cx43 mRNA (9.85 fold, P < 0.01). Western blot analysis revealed greater levels of Cx43 protein (P < 0.05). Conclusion Blood pressure variability evoked by SAD leads to increased expression of Cx43, which could contribute to enhanced phenylephrine-induced contraction and oscillatory activity in isolated aortas.

Gender-specific vascular effects elicited by chronic ethanol consumption in rats: a role for inducible nitric oxide synthase

Background and purpose: Epidemiological data suggest that the risk of ethanol-associated cardiovascular disease is greater in men than in women. This study investigates the mechanisms underlying gender-specific vascular effects elicited by chronic ethanol consumption in rats. Experimental approach: Vascular reactivity experiments using standard muscle bath procedures were performed on isolated thoracic aortae from rats. mRNA and protein for inducible NO synthase (iNOS) and for endothelial NOS (eNOS) was assessed by RT-PCR or western blotting, respectively. Key results: In male rats, chronic ethanol consumption enhanced phenylephrine-induced contraction in both endothelium-intact and denuded aortic rings. However, in female rats, chronic ethanol consumption enhanced phenylephrine-induced contraction only in endothelium denuded aortic rings. After pre-incubation of endothelium-intact rings with L-NAME, both male and female ethanol-treated rats showed larger phenylephrine-induced contractions in aortic rings, compared to the control group. Acetylcholine-induced relaxation was not affected by ethanol consumption. The effects of ethanol on responses to phenylephrine were similar in ovariectomized (OVX) and intact (non-OVX) female rats. In the presence of aminoguanidine, but not 7-nitroindazole, the contractions to phenylephrine in rings from ethanol-treated female rats were greater than that found in control tissues in the presence of the inhibitors. mRNA levels for eNOS and iNOS were not altered by ethanol consumption. Ethanol intake reduced eNOS protein levels and increased iNOS protein levels in aorta from female rats. Conclusions and implications: Gender differences in the vascular effects elicited by chronic ethanol consumption were not related to ovarian hormones but seemed to involve the upregulation of iNOS.

Immunosuppressive effects of melanoma-derived heavy-chain ferritin are dependent on stimulation of IL-10 production

Cultured melanoma cells release soluble factors that influence immune responses. Screening of a cDNA library with anti-sera from a melanoma patient identified an immunoreactive plaque, which encoded heavy-chain ferritin (H-ferritin), Previous studies have drawn attention to the immunosuppressive effects of this molecule and prompted further studies on its biochemical and functional properties in human melanoma, These studies demonstrated, firstly, that H-ferritin appeared to be secreted by melanoma cells, as shown by immunoprecipitation of a 21.5 kDa band from supernatants. It was also detected in extracts of melanoma cells by Western blotting as 43 and 64 kDa dimers and trimers of the 21.5 kDa fraction. Secondly, flow-cytometric analysis of H- and light-chain ferritin (L-ferritin) expression on melanoma showed a wide variation in L-ferritin expression and consequently of the ratio of H- to L-ferritin expression. Suppression of mitogenic responses of lymphocytes to anti-CD3 showed a correlation with the ratio of H- to L-ferritin in the supernatants and was specific for H-ferritin, as shown by inhibition studies with a monoclonal antibody (MAb) against H-ferritin, Similar results were obtained with H- and L-ferritin from other sources. Suppression of mitogenic responses of lymphocytes to anti-CD3 by H-ferritin was inhibited using a MAb against IL-IO, which suggested that the immunosuppressive effect of H-ferritin was mediated by IL-IO, Assays of cytokine production from anti-CD3-stimulated lymphocytes showed that H-ferritin markedly increased production of IL-10 and IFN-gamma and had only slight effects on IL-2 and IL-4 production, Our results suggest that melanoma cells may be a major source of H-ferritin and that production of the latter may account for some of the immunosuppressive effects of melanoma, (C) 2001 Wiley-Liss. Inc.

Proteomic analysis of normal and malignant prostate tissue to identify novel proteins lost in cancer

BACKGROUND. Alterations of important protein pathways, including loss of prostate secretory granules, and disruption of the prostatic secretory pathway have been identified as early events in malignancy. In this study, proteomics was used to map the differences in protein expression between normal and malignant prostate tissues and to identify and analyze differentially expressed proteins in human prostate tissue with particular regard to the proteins lost in malignancy. METHODS. Small quantities of normal and malignant prostate tissue were taken fresh from 34 radical prostatectomy cases. After histological examination, proteins were solubilized from selected tissues and separated using two-dimensional electrophoresis. Using image analysis, the proteome of normal and malignant tissues were mapped and differentially expressed proteins (present in normal and absent in malignant tissue) were identified and subsequently analyzed using peptide mass finger printing and N-terminal sequencing. Western blotting and immunohistochemistry were performed to examine expression profiles and tissue localization of candidate proteins. RESULTS. Comparison of protein maps of normal and malignant prostate were used to identify 20 proteins which were lost in malignant transformation, including prostate specific antigen (PSA), alpha-l antichymotrypsin (ACT), haptoglobin, and lactoylglutathione lyase. Three of the 20 had not previously been reported in human prostate tissue (Ubiquitin-like NEDD8, calponin, and a follistatin-related protein). Western blotting confirmed differences in the expression profiles of NEDD8 and calponin, and immunohistochemistry demonstrated differences in the cellular localization of these two proteins in normal and malignant prostate glands. CONCLUSIONS. The expression of NEDD8, calponin, and the follistatin-related protein in normal prostate tissues is a novel finding and the role of these important functional proteins in normal prostate and their loss or reduced expression in prostate malignancy warrants further investigations. (C) 2002 Wiley-Liss, Inc.

Development and subunit composition of synaptic NMDA receptors in the amygdala: NR2B Synapses in the adult central amygdala

NMDA receptors are well known to play an important role in synaptic development and plasticity. Functional NMDA receptors are heteromultimers thought to contain two NR1 subunits and two or three NR2 subunits. In central neurons, NMDA receptors at immature glutamatergic synapses contain NR2B subunits and are largely replaced by NR2A subunits with development. At mature synapses, NMDA receptors are thought to be multimers that contain either NR1/NR2A or NR1/NR2A/NR2B subunits, whereas receptors that contain only NR1/NR2B subunits are extrasynaptic. Here, we have studied the properties of NMDA receptors at glutamatergic synapses in the lateral and central amygdala. We find that NMDA receptor-mediated synaptic currents in the central amygdala in both immature and mature synapses have slow kinetics and are substantially blocked by the NR2B-selective antagonists (1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propano and ifenprodil, indicating that there is no developmental change in subunit composition. In contrast, at synapses on pyramidal neurons in the lateral amygdala, whereas NMDA EPSCs at immature synapses are slow and blocked by NR2B-selective antagonists, at mature synapses their kinetics are faster and markedly less sensitive to NR2B-selective antagonists, consistent with a change from NR2B to NR2A subunits. Using real-time PCR and Western blotting, we show that in adults the ratio of levels of NR2B to NR2A subunits is greater in the central amygdala than in the lateral amygdala. These results show that the subunit composition synaptic NMDA receptors in the lateral and central amygdala undergo distinct developmental changes.

Purification of the keratan sulfate proteoglycan expressed in prostatic secretory cells and its identification as lumican

BACKGROUND. Secretory epithelial cells of human prostate contain a keratan sulfate proteoglycan (KSPG) associated with the prostatic secretory granules (PSGs). The proteoglycan has not been identified, but like the PSGs, it is lost in the early stages of malignant transformation. METHODS. Anion exchange and affinity chromatography were used to purify KSPG from human prostate tissue. Enzymatic deglycosylation was used to remove keratan sulfate (KS). The core protein was isolated using 2D gel electrophoresis, digested in-gel with trypsin, and identified by peptide mass fingerprinting (PMF). RESULTS. The purified proteoglycan was detected as a broad smear on Western blots with an apparent molecular weight of 65-95 kDa. The KS moiety was susceptible to digestion with keratanase 11 and peptide N-glycosidase F defining it as highly sulfated and N-linked to the core protein. The core protein was identified, following deglycosylation and PMF, as lumican and subsequently confirmed by Western blotting using an anti-lumican antibody. CONCLUSIONS. The KSPG associated with PSGs in normal prostate epithelium is lumican. While the role of lumican in extracellular matrix is well established, its function in the prostate secretory process is not known. It's potential to facilitate packaging of polyamines in PSGs, to act as a tumor suppressor and to mark the early stages of malignant transformation warrant further investigation. (C) 2003 Wiley-Liss, Inc.

Use of proteoliposome as a vaccine against Trypanosoma cruzi in mice

We have generated proteoliposomes carrying proteins of Topanosoma cruzi for use as immunogens in BALB/c mice. T cruzi trypomastigote and amastigote forms were sonicated and mixed with SDS, with 94% recovery of soluble proteins. To prepare proteoliposomes, we have used a protocol in which dipalmitoylphosphatidylcholine, dipalmitoyl-phosphatidylserine and cholesterol were incubated with the parasite proteins. BALB/c mice immunized with 20 mu g were able to generate antibodies which, in Western blotting, reacted with the proteins of T cruzi. We further investigated the ability of peritoneal cells from immunized mice to arrest the intracellular replication of trypomastigotes, in vitro. After 72h of culture, the number of intracellular parasites in immunized macrophages decreased significantly, as compared to controls. Despite the fact that exposure of mice to T cruzi proteins incorporated into proteoliposomes generate antibodies and activate macrophages, the immunized mice were not protected against T cruzi intraperitoneal challenge. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Structural and Biochemical Correlates of Na(+), K(+)-ATPase Driven Ion Uptake Across the Posterior Gill Epithelium of the True Freshwater Crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+), K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (approximate to 14 mu m(2) membrane per mu m(3) cytoplasm), deep invaginations that house the Na(+), K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 mu m(2) mu m(-2)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 mu m(2) mu m(-2)), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+), K(+)-ATPase specific activity resembles marine crabs but is approximate to 5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two alpha-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4)(+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water. J. Exp. Zool. 313A:508-523, 2010. (C) 2010 Wiley-Liss, Inc.

Inhibition of NF kappa B leads to an intracellular reducing environment in human monocyte-derived immature dendritic cells

Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.

Effect of paricalcitol and calcitriol on aortic wall remodeling in uninephrectomized ApoE knockout mice

Becker LE, Koleganova N, Piecha G, Noronha IL, Zeier M, Geldyyev A, Kokeny G, Ritz E, Gross ML. Effect of paricalcitol and calcitriol on aortic wall remodeling in uninephrectomized ApoE knockout mice. Am J Physiol Renal Physiol 300: F772-F782, 2011. First published December 15, 2010; doi:10.1152/ajprenal.00042.2010.-Despitean only minor reduction in the glomerular filtration rate, uninephrectomy (UNX) markedly accelerates the rate of growth of atherosclerotic plaques in ApoE-/- mice. It has been suggested that vitamin D receptor (VDR) activation exerts an antiproliferative effect on vascular smooth muscle cells, but the side effects may limit its use. To assess a potentially different spectrum of actions, we compared the effects of paricalcitol and calcitriol on remodeling and calcification of the aortic wall in sham-operated and UNX ApoE-/- mice on a diet with normal cholesterol content. Sham-operated and UNX mice were randomly allotted to treatment with solvent, calcitriol (0.03 mu g/kg) or paricalcitol (0.1 mu g/kg) 5 times/wk intraperitoneally for 10 wk. Semithin (0.6 mu m) sections of the aorta were analyzed by 1) morphometry, 2) immunohistochemistry, and 3) Western blotting of key proteins involved in vascular calcification and growth. Compared with sham-operated animals (5.6 +/- 0.24), the wall-to-lumen ratio (x100) of the aorta was significantly higher in solvent-and calcitriol-treated UNX animals (6.64 +/- 0.27 and 7.17 +/- 0.81, respectively, P < 0.05), but not in paricalcitol-treated UNX (6.1 5 +/- 0.32). Similar differences were seen with respect to maximal plaque height. Expression of transforming growth factor (TGF)-beta 1 in aortic intima/plaque was also significantly higher in UNX solvent and UNX calcitriol compared with sham-operated and UNX paricalcitol animals. Treatment with both paricalcitol and calcitriol caused significant elevation of VDR expression in the aorta. While at the dose employed paricalcitol significantly reduced TGF-beta expression in plaques, calcitriol in contrast caused significant vascular calcification and elevated expression of related proteins (BMP2, RANKL, and Runx2).

Molecular evidence of human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) infections in HTLV seroindeterminate individuals from Sao Paulo, Brazil

Background: Using enzyme immunoassays and Western blot (Wb) tests, HTLV serodiagnosis yields indeterminate results in a significant number of cases. Objective: To determine the prevalence of HTLV infection among HTLV-seroindeterminate individuals. Study design: We studied peripheral blood mononuclear cells from 65 anti-HTLV Wb-seroindeterminate individuals by attempting to amplify proviral DNA sequences (tax and pot) to identify HTLV-I and HTLV-2 infections. Results: These 65 specimens exhibited predominantly (43%) anti-HTLV antibodies to gag-coded antigens in the absence of anti-p24 on Wb analysis. Tax proviral sequences were detected in 6 (9.2%) samples. According to restricted fragment polymorphism analysis (RFLP), we identified HTLV-1 proviral DNA in 4 samples. HTLV-2 in one and sequences from both in another. Nested PCR for the pot region was positive in 3 (4.6%) specimens, which were also positive for tax sequences. After hybridization HTLV-1 infection was confirmed in 2 samples (3.1%) and HTLV-2 in another (1.5%). Detection of a single HTLV DNA sequence may be due to infection by defective provirus, but its significance remains undefined. In this cohort, no Wb reactivity pattern was predictive of proviral detection. HTLV-I infection was demonstrated in an individual who had Wb reactivity to gag-coded antigens only. Conclusions: This emphasizes the importance of clinical and laboratory follow-up of HTLV-seroindeterminate individuals from endemic areas. (c) 2009 Elsevier B.V. All rights reserved.

Inhibition of phospholipase A(2) increases Tau phosphorylation at Ser214 in embryonic rat hippocampal neurons

Background: Arachidonic acid is released from cellular membranes by the action of phospholipase A(2) (PLA(2)) and is implicated in microtubule-associated protein Tau phosphorylation. Tau hyperphosphorylation affects its ability to stabilize microtubules. Objective: To determine the effect of PLA(2) inhibition on the phosphorylation state of Tau phosphoepitopes in primary cultures of hippocampal neurons. Methods: 4 DIC neurons were incubated at different concentrations of methyl-arachidonylfluorophosphonate (MAFP), an irreversible inhibitor of cPLA(2) and iPLA(2). Changes on Tau phosphorylation were determined by Western blotting with a panel of anti-Tau antibodies (C-terminal, Ser199/202, Ser202/205, Ser396 and Ser214). Results: The Ser214 site was hyperphosphorylated upon MAFP treatment. Significant differences were observed with 10 mu M (p = 0.01), 50 mu M (p = 0.01) and 100 mu M (p = 0.05) of MAFP. Less-intense changes were found in other phosphoepitopes. Conclusion: The present findings indicate that the phosphorylation of Ser214 is regulated by c- and/or iPLA(2), whereas other phosphoepitopes primarily regulated by GKS3b were not affected. (C) 2009 Elsevier Ltd. All rights reserved.

Human T-cell lymphotropic virus type 1 infective dermatitis emerging in adulthood

Background Infective dermatitis (ID) is a rare dermatologic condition of childhood that has been linked to human T-cell lymphotropic virus type 1 (HTLV-1). Objective To analyze the clinical and laboratory features associated with adult-onset ID linked to HTLV-1. Methods From December 1995 to December 2007, four patients with ID were followed in the dermatology outpatient clinic of the ""Hospital das Clinicas"" of the University of Sao Paulo Medical School, Sao Paulo, Brazil. Epidemiologic data were collected and dermatologic examination was performed. Patients were submitted to histopathologic, hematologic, virologic, and immunologic investigations. Results All patients had a diagnosis of ID according to previously established criteria, despite being adults. HTLV-1 infection was demonstrated by enzyme-linked immunosorbent assay, Western blotting assays, and polymerase chain reaction. The male to female ratio was 1 : 3 and the median age at diagnosis was 42 years. The cutaneous manifestations were erythematous, scaly, and crusted lesions in all patients, and ichthyosis in three of the four cases. Histopathologic study showed lymphocytic epidermotropism in two cases. The median proviral load was 281 copies/10,000 peripheral blood mononuclear cells. Immunodeficiency was not observed in any case. The therapies used were antimicrobials, corticosteroids, and phototherapy. Conclusions Although many authors have considered ID to be a form of childhood dermatitis, we have described four cases that fulfilled the major criteria for ID, except for onset in adulthood.

Inhibition of phospholipase A(2) in rat brain decreases the levels of total Tau protein

The microtubule-associated protein Tau promotes the assembly and stability of microtubules in neuronal cells. Six Tau isoforms are expressed in adult human brain. All six isoforms become abnormally hyperphosphorylated and form neurofibrillary tangles in Alzheimer disease (AD) brains. In AD, reduced activity of phospholipase A(2) (PLA(2)), specifically of calcium-dependent cytosolic PLA(2) (cPLA(2)) and calcium-independent intracellular PLA(2) (iPLA(2)), was reported in the cerebral cortex and hippocampus, which positively correlated with the density of neurofibrillary tangles. We previously demonstrated that treatment of cultured neurons with a dual cPLA(2) and iPLA(2) inhibitor, methyl arachidonyl fluorophosphonate (MAFP), decreased total Tau levels and increased Tau phosphorylation at Ser(214) site. The aim of this study was to conduct a preliminary investigation into the effects of in vivo infusion of MAFP into rat brain on PLA(2) activity and total Tau levels in the postmortem frontal cortex and dorsal hippocampus. PLA(2) activity was measured by radioenzymatic assay and Tau levels were determined by Western blotting using the anti-Tau 6 isoforms antibody. MAFP significantly inhibited PLA(2) activity in the frontal cortex and hippocampus. The reactivity to the antibody revealed three Tau protein bands with apparent molecular weight of close to 40, 43 and 46 kDa in both brain areas. MAFP decreased the 46 kDa band intensity in the frontal cortex, and the 43 and 46 kDa band intensities in the hippocampus. The results indicate that in vivo PLA(2) inhibition in rat brain decreases the levels of total (nonphosphorylated plus phosphorylated) Tau protein and corroborate our previous in vitro findings.

Cellular prion protein (PrP(C)) and superoxide dismutase (SOD) in vascular cells under oxidative stress

The PrP(C) is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP(C) with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP(C) expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrP(C) expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9 +/- 1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP(C) expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP(C) only in cells stimulated for 8 hours with the different stressors. The PrP(C) mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP(C) protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP(C). (C) 2009 Elsevier GmbH. All rights reserved.