Intracellular Elements Research Articles

Enhanced resistance of yeast mutants deficient in low-affinity iron and zinc transporters to stannous-induced toxicity

Tin or stannous (Sn 2+) compounds are used as catalysts, stabilizers in plastic industries, wood preservatives, agricultural biocides and nuclear medicine. In order to verify the Sn 2+ up-take and toxicity in yeast cells we utilized a multi-elemental analysis known as particle-induced X-ray emission (PIXE) along with cell survival assays and quantitative real-time PCR. The detection of Sn 2+ by PIXE was possible only in yeast cells in stationary phase of growth (STAT cells) that survive at 25 mM Sn 2+ concentration. Yeast cells in exponential phase of growth (LOG cells) tolerate only micro-molar Sn 2+ concentrations that result in intracellular concentration below of the method detection limit. Our PIXE analysis showed that STAT XV185-14c yeast cells demonstrate a significant loss of intracellular elements such as Mg, Zn, S, Fe and an increase in P levels after 1 h exposure to SnCl 2. The survival assay showed enhanced tolerance of LOG yeast cells lacking the low-affinity iron and zinc transporters to stannous treatment, suggesting the possible involvement in Sn 2+ uptake. Moreover, our qRT-PCR data showed that Sn 2+ treatment could generate reactive oxygen species as it induces activation of many stress-response genes, including SOD1, YAP1, and APN1.

The Dystrophin-associated Protein Complex Maintains Muscle Excitability by Regulating Ca2+-dependent K+ (BK) Channel Localization

The dystrophin-associated protein complex (DAPC) consists of several transmembrane and intracellular scaffolding elements that have been implicated in maintaining the structure and morphology of the vertebrate neuromuscular junction (NMJ). Genetic linkage analysis has identified loss-of-function mutations in DAPC genes that give rise to degenerative muscular dystrophies. Although much is known about the involvement of the DAPC in maintaining muscle integrity, less is known about the precise contribution of the DAPC in cell signaling events. To better characterize the functional role of the DAPC at the NMJ, we used electrophysiology, immunohistochemistry, and fluorescent labeling to directly assess cholinergic synaptic transmission, ion channel localization, and muscle excitability in loss-of-function (lf) mutants of Caenorhabditis elegans DAPC homologues. We found that all DAPC mutants consistently display mislocalization of the Ca(2+)-gated K(+) channel, SLO-1, in muscle cells, while ionotropic acetylcholine receptor (AChR) expression and localization at the NMJ remained unaltered. Synaptic cholinergic signaling was also not significantly impacted across DAPC(lf) mutants. Consistent with these findings and the postsynaptic mislocalization of SLO-1, we observed an increase in muscle excitability downstream of cholinergic signaling. Based on our results, we conclude that the DAPC is not involved in regulating AChR architecture at the NMJ, but rather functions to control muscle excitability, in an activity-dependent manner, through the proper localization of SLO-1 channels.

“Biofilmology”: a multidisciplinary review of the study of microbial biofilms

The observation of biofilm formation is not a new phenomenon. The prevalence and significance of biofilm and aggregate formation in various processes have encouraged extensive research in this field for more than 40years. In this review, we highlight techniques from different disciplines that have been used to successfully describe the extracellular, surface and intracellular elements that are predominant in understanding biofilm formation. To reduce the complexities involved in studying biofilms, researchers in the past have generally taken a parts-based, disciplinary specific approach to understand the different components of biofilms in isolation from one another. Recently, a few studies have looked into combining the different techniques to achieve a more holistic understanding of biofilms, yet this approach is still in its infancy. In order to attain a global understanding of the processes involved in the formation of biofilms and to formulate effective biofilm control strategies, researchers in the next decade should recognise that the study of biofilms, i.e. biofilmology, has evolved into a discipline in its own right and that mutual cooperation between the various disciplines towards a multidisciplinary research vision is vital in this field.

Electron probe X-ray microanalysis of cisplatin-induced cell death in rat pheochromocytoma PC12 cells.

Several lines of evidence suggest that cisplatin-induced cell death is not always the result of apoptosis. A distinctive feature between apoptosis and necrosis is the alteration in cell volume regulation and ion homeostasis. Here we analyzed the changes in intracellular element content during cell death induced by exposure to therapeutic concentrations of cisplatin in the PC12 cell line. To quantitate Na, Cl and K content, electron probe X-ray microanalysis (EPXMA) was performed in whole freeze-dried cells. We also traced the alterations in morphological features with fluorescence and transmission electron microscopy. EPXMA demonstrated progressive derangement of the absolute intracellular Na, Cl and K contents. Cisplatin-treated cells showed two microanalytical patterns: 1) cells with alterations in elemental content typical of apoptosis, i.e., an increase in intracellular Na and a decrease in intracellular Cl and K, and 2) cells characterized by an increase in Na content and a decrease in K content, with no changes in Cl content. This intracellular profile for Na, Cl, and K was not typical of necrosis or apoptosis. Morphological analysis revealed two cellular phenotypes: 1) cells characterized by a phenotype typical of apoptosis, and 2) cells characterized by a hybrid phenotype combining variable features of apoptosis and necrosis. Taken together, our findings suggest that therapeutic concentrations of cisplatin may cause a hybrid type of cell death characterized by concurrent apoptosis and necrosis in the same individual PC12 cell.

Estado actual en el tratamiento de la enfermedad de Chagas

Efficient drugs against Chagas' disease must have an effect on the amastigote forms or intracellular reproduction elements of Trypanosoma cruzi (T. cruzi). Trypomastigote and epimastigote forms derive from the former and their response to medications is less marked. The only drugs used in humans are nifurtimox (NF) and benznidazole (BNZ). Other useful medications are allopurinol and itraconazole. NF acts producing free radicals and BNZ inhibits the synthesis of macromolecules. There is consensus that Chagas' disease must be treated in all its periods, since T.cruzi DNA is detected by polymerase chain reaction in chronic cases, even when microscopy is negative. The pharmacological treatment modifies the natural evolution of the disease. It also helps to solve a public health problem, considering that there is a high number of subjects with Chagas' disease. Subjects with chronic chagasic cardiomyopathy with terminal heart failure are the only cases without indication for treatment. Due to the digestive and skin secondary effects of the drugs, treated patients must be controlled clinically and with complete blood counts and hepatic proiles before, during and after the therapy. Approximately 30% of patients will experience secondary effects. Children have a better tolerance to the drugs. Congenital or acquired acute, intermediate and chronic cases should be treated.

Update on the treatment of Chagas' disease

Efficient drugs against Chagas' disease must have an effect on the amastigote forms or intracellular reproduction elements of Trypanosoma cruzi (T. cruzi). Trypomastigote and epimastigote forms derive from the former and their response to medications is less marked. The only drugs used in humans are nifurtimox (NF) and benznidazole (BNZ). Other useful medications are allopurinol and itraconazole. NF acts producing free radicals and BNZ inhibits the synthesis of macromolecules. There is consensus that Chagas' disease must be treated in all its periods, since T.cruzi DNA is detected by polymerase chain reaction in chronic cases, even when microscopy is negative. The pharmacological treatment modifies the natural evolution of the disease. It also helps to solve a public health problem, considering that there is a high number of subjects with Chagas' disease. Subjects with chronic chagasic cardiomyopathy with terminal heart failure are the only cases without indication for treatment. Due to the digestive and skin secondary effects of the drugs, treated patients must be controlled clinically and with complete blood counts and hepatic proiles before, during and after the therapy. Approximately 30% of patients will experience secondary effects. Children have a better tolerance to the drugs. Congenital or acquired acute, intermediate and chronic cases should be treated.

Spirostomum ambiguum: a protozoan model for primordial musculoskeletal exchange?

Event Abstract Back to Event Spirostomum ambiguum: a protozoan model for primordial musculoskeletal exchange? PE Garner1*, PE Garner2, V Fallon1 and JE Aaron1 1 University of Leeds, IMSB, FBS, United Kingdom 2 University of Leeds, IMBE, United Kingdom The skeleton is responsive to mechanical usage, yet the basis for its remarkable sensitivity remains uncertain. Abundant osteocytes seem central. Their cytoplasmic syncitium is pervasive and well placed to bridge the gap between mechanical signal transduction and cellular. However, its calcified entombment limits accessibility, while isolation or manipulation may alter its specific threshold characteristics. Insight into the rudiments of musculoskeletal exchange may be found in certain protozoa from which the metazoan pathway apparently evolved (Pautard 1960, 1970; Ruffalo 1978). In particular is the organism Spirostomum ambiguum (a cigar-shaped creature visible to the naked eye) which fabricates and accumulates calcium phosphate particles resembling those found in bone. Moreover, their intracellular, golgi-directed synthesis (Fallon, Garner and Aaron, in press) is determined by their active life-cycle. This modulates between a free-swimming state when calcified particles are few and a burrowing stage when calcified particles are many. Thus when the mineral of cultured S. ambiguum was labelled with the fluorochrome tetracycline the green fluorescence intensity (AU), mapped using laser confocal microscopy, recorded a high mineral level in the burrowing animals (138.0 + SD4.0) compared with the free-swimmers (89.7 SD 3.3). Similarly when the live organisms were transfected with a GFP construct (Fallon, 2006) the resulting mannosidase II enzyme, as an expression of Golgi activity, differed significantly (p<0.0001) between tunnelling (104.6 SD 2.7) and free-swimming (74.5 6.7) by the two-sample t-test. Also it was observed that the distribution of the calcified particles was not random. A proportion related in disposition to a regular and well-defined pattern of contractile muscle myonemes, the fibres of which were arranged longitudinally within the high stress burrowers in contrast to their transverse alignment in the low stress swimmers. The capacity exhibited by this animal model not only to package bone-like mineral in response to changing environmental pressures, but also to relate them to their intracellular contractile elements may suggest an early integrated musculoskeletal system that substantially predated the vertebrates which eventually exploited this major advantage. The protozoan model described may therefore serve as a valuable tool for future fundamental investigation of osteocyte ancestry, mechanotransduction, perception and response. Keywords: Bones, Bone Research Conference: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society, Cambridge, United Kingdom, 27 Jun - 29 Jun, 2011. Presentation Type: Oral Topic: Abstracts Citation: Garner P, Garner P, Fallon V and Aaron J (2011). Spirostomum ambiguum: a protozoan model for primordial musculoskeletal exchange?. Front. Endocrinol. Conference Abstract: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society. doi: 10.3389/conf.fendo.2011.02.00021 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2011; Published Online: 30 Sep 2011. * Correspondence: Prof. PE Garner, University of Leeds, IMSB, FBS, United Kingdom, bms2peg@leeds.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers PE Garner PE Garner V Fallon JE Aaron Google PE Garner PE Garner V Fallon JE Aaron Google Scholar PE Garner PE Garner V Fallon JE Aaron PubMed PE Garner PE Garner V Fallon JE Aaron Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

1SI-04 Visualization of intracellular elements by scanning X-ray fluorescence microscopy(1SI Biophysics to be explored using the fourth-generation light sources, x-ray free electron lasers,The 49th Annual Meeting of the Biophysical Society of Japan)

1SI-04 Visualization of intracellular elements by scanning X-ray fluorescence microscopy(1SI Biophysics to be explored using the fourth-generation light sources, x-ray free electron lasers,The 49th Annual Meeting of the Biophysical Society of Japan)

Cadmium chloride-induced oxidative stress and DNA damage in the human Jurkat T cell line is not linked to intracellular trace elements depletion

Cadmium (Cd) is a widespread environmental contaminant. Cd affects the cellular homeostasis and generates damage via complex mechanisms involving interactions with other metals, induction of oxidative stress and apoptotic or necrotic cell death, depending on the cell type and the concentration. The goal of the present study was to investigate the effect of exposure to CdCl 2 on the intracellular trace elements levels, the antioxidant enzyme activities and on DNA damage in the Jurkat T cell line. Cells were exposed to 5, 25 and 50 μM of CdCl 2 for 24 h. Cd significantly reduced the viability of Jurkat T cells and induced a dose-dependent increase in DNA damage with statistically significant differences relative to controls ( p < 0.001); the superoxide dismutase and glutathione peroxidase activities were significantly decreased. Lipid peroxidation and protein carbonyl levels were significantly increased while glutathione and the total intracellular sulfhydryl groups were decreased showing clearly that an oxidative stress was generated by Cd. Surprisingly the treatment with Cd induced a significant increase in the intracellular levels of all the trace elements measured. The results indicate that cellular pro-oxidative stress induced by Cd is most likely mediated by disruption of redox homeostasis associated to a mishandling of redox-active transition metals and causes lipid and protein oxidation and oxidative DNA damage in Jurkat T cells.

Extra-cellular release and blood diffusion of BART viral micro-RNAs produced by EBV-infected nasopharyngeal carcinoma cells

BackgroundNasopharyngeal carcinoma (NPC) is a human epithelial malignancy consistently associated with the Epstein-Barr virus. The viral genome is contained in the nuclei of all malignant cells with abundant transcription of a family of viral microRNAs called BART miRNAs. MicroRNAs are well known intra-cellular regulatory elements of gene expression. In addition, they are often exported in the extra-cellular space and sometimes transferred in recipient cells distinct from the producer cells. Extra-cellular transport of the microRNAs is facilitated by various processes including association with protective proteins and packaging in secreted nanovesicles called exosomes. Presence of microRNAS produced by malignant cells has been reported in the blood and saliva of tumor-bearing patients, especially patients diagnosed with glioblastoma or ovarian carcinoma. In this context, it was decided to investigate extra-cellular release of BART miRNAs by NPC cells and their possible detection in the blood of NPC patients. To address this question, we investigated by quantitative RT-PCR the status of 5 microRNAs from the BART family in exosomes released by NPC cells in vitro as well as in plasma samples from NPC xenografted nude mice and NPC patients.ResultsWe report that the BART miRNAs are released in the extra-cellular space by NPC cells being associated, at least to a large extent, with secreted exosomes. They are detected with a good selectivity in plasma samples from NPC xenografted nude mice as well as NPC patients.ConclusionsViral BART miRNAs are secreted by NPC cells in vitro and in vivo. They have enough stability to diffuse from the tumor site to the peripheral blood. This study provides a basis to explore their potential as a source of novel tumor biomarkers and their possible role in communications between malignant and non-malignant cells.

Modulation of Agrin-induced Acetylcholine Receptor Clustering by Extracellular Signal-regulated Kinases 1 and 2 in Cultured Myotubes

Agrin released by motoneurons induces and/or maintains acetylcholine receptor (AChR) clustering and other aspects of postsynaptic differentiation at the vertebrate neuromuscular junction. Agrin acts by binding and activating a receptor complex containing LDL receptor protein 4 (Lrp4) and muscle-specific kinase (MuSK). Two critical downstream components of this signaling cascade, Dox-7 and rapsyn, have been identified. However, additional intracellular essential elements remain unknown. Prior observations by others and us suggested antagonistic interactions between agrin and neuregulin-1 (Nrg-1) signaling in cultured myotubes and developing muscle fibers in vivo. A hallmark of Nrg-1 signaling in skeletal muscle cells is the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2 are also activated in most cells by phorbol 12-myristate 13-acetate, a classical inhibitor of agrin-induced AChR clustering in myotubes. Here, it was investigated whether agrin activates ERK1/2 directly and whether such activation modulates agrin-induced AChR clustering. Agrin induced a rapid but transient activation of ERK1/2 in myotubes that was Lrp4/MuSK-dependent. However, blocking this ERK1/2 activation did not prevent but potentiated AChR clustering induced by agrin. ERK1/2 activation was dispensable for Nrg-1-mediated inhibition of the AChR clustering activity of agrin, but was indispensable for such activity by phorbol 12-myristate 13-acetate. Together, these results suggest agrin-induced activation of ERK1/2 is a negative modulator of agrin signaling in skeletal muscle cells.

The Sodium Channel β3-Subunit Induces Multiphasic Gating in NaV1.3 and Affects Fast Inactivation via Distinct Intracellular Regions

Electrical excitability in neurons depends on the activity of membrane-bound voltage gated sodium channels (Na(v)) that are assembled from an ion conducting α-subunit and often auxiliary β-subunits. The α-subunit isoform Na(v)1.3 occurs in peripheral neurons together with the Na(v) β3-subunit, both of which are coordinately up-regulated in rat dorsal root ganglion neurons after nerve injury. Here we examine the effect of the β3-subunit on the gating behavior of Na(v)1.3 using whole cell patch clamp electrophysiology in HEK-293 cells. We show that β3 depolarizes the voltage sensitivity of Na(v)1.3 activation and inactivation and induces biphasic components of the inactivation curve. We detect both a fast and a novel slower component of inactivation, and we show that the β3-subunit increases the fraction of channels inactivating by the slower component. Using CD and NMR spectroscopy, we report the first structural analysis of the intracellular domain of any Na(v) β-subunit. We infer the presence of a region within the β3-subunit intracellular domain that has a propensity to form a short amphipathic α-helix followed by a structurally disordered sequence, and we demonstrate a role for both of these regions in the selective stabilization of fast inactivation. The complex gating behavior induced by β3 may contribute to the known hyperexcitability of peripheral neurons under those physiological conditions where expression of β3 and Na(v)1.3 are both enhanced.

ZFP423 Coordinates Notch and Bone Morphogenetic Protein Signaling, Selectively Up-regulating Hes5 Gene Expression

Zinc finger protein 423 encodes a 30 Zn-finger transcription factor involved in cerebellar and olfactory development. ZFP423 is a known interactor of SMAD1-SMAD4 and of Collier/Olf-1/EBF proteins, and acts as a modifier of retinoic acid-induced differentiation. In the present article, we show that ZFP423 interacts with the Notch1 intracellular domain in mammalian cell lines and in Xenopus neurula embryos, to activate the expression of the Notch1 target Hes5/ESR1. This effect is antagonized by EBF transcription factors, both in cultured cells and in Xenopus embryos, and amplified in vitro by BMP4, suggesting that ZFP423 acts to integrate BMP and Notch signaling, selectively promoting their convergence onto the Hes5 gene promoter.

Notch and Transforming Growth Factor-β (TGFβ) Signaling Pathways Cooperatively Regulate Vascular Smooth Muscle Cell Differentiation

Notch and transforming growth factor-beta (TGFbeta) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFbeta1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle alpha-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFbeta1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-alpha (SM22alpha) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFbeta1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFbeta1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFbeta1 treatment led to synergistic activation of a TGFbeta-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22alpha promoters. Thus, Notch and TGFbeta coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.

Repression of Cellular Retinoic Acid-binding Protein II during Adipocyte Differentiation

In preadipocytes, retinoic acid (RA) regulates gene expression by activating the nuclear RA receptor (RAR) and its cognate intracellular lipid-binding protein CRABP-II. It was previously reported that RA inhibits adipocyte differentiation but only when administered early during the differentiation program. The data presented here indicate that the diminished ability of RA to activate RAR following induction of differentiation stems from down-regulation of CRABP-II. The observations show that expression of CRABP-II in preadipocytes is repressed by all three components of the classical hormonal mixture that induces adipocyte differentiation, i.e. isobutylmethylxanthine, insulin, and dexamethasone. Isobutylmethylxanthine-dependent activation of protein kinase A triggered the phosphorylation of the transcription factor cAMP-response element-binding protein, which induced the expression of the cAMP-response element-binding protein family repressor cAMP-response element modulator. In turn, cAMP-response element modulator was found to associate with a cognate response element in the CRABP-II promoter and to repress CRABP-II expression. The data further show that CRABP-II is a direct target gene for the glucocorticoid receptor and that it is subjected to dexamethasone-induced glucocorticoid receptor-mediated repression during adipogenesis. Finally, the observations demonstrate that permanent repression of CRABP-II in mature adipocytes is exerted by the master regulator of adipocyte differentiation CCAAT/enhancer-binding protein alpha and is directly mediated through CCAAT/enhancer-binding protein alpha-response elements in the CRABP-II promoter. Taken together, the observations emphasize the important role of CRABP-II in regulating the transcriptional activity of RA through RAR, and they demonstrate that repression of this gene is critical for allowing adipogenesis to proceed.

Evidence that gC1qR and DC-SIGN associate on the surface of immature dendritic cells (136.24)

Abstract Complement protein C1q is involved in many immunoregulatory functions, such as modulating T-cell proliferation and regulating dendritic cell (DC) differentiation and maturation. C1q has at least two multi-functional cell surface receptors, cC1qR and gC1qR. While these receptors ostensibly lack a direct conduit into intracellular elements, they are known to form docking/signaling complexes with transmembrane partners. DC-SIGN (dendritic cell-specific-ICAM-grabbing-non integrin) is a C-type lectin expressed on the surface of DCs and involved in T-cell activation and endocytosis. Because SIGN-R1, a C-type lectin related to DC-SIGN has been shown to bind directly to C1q, we investigated whether DC-SIGN also binds to C1q and if DC-SIGN may act as a transmembrane partner for gC1qR and/or cC1qR. Using flow cytometry, we found that DC-SIGN, gC1qR and cC1qR are all present on the surface of human immature DCs (iDC) grown in culture. While antigen-capture ELISA experiments using iDC lysates showed that C1q, gC1qR and cC1qR directly or indirectly associate with DC-SIGN, immunofluorescence microscopy revealed that C1q and gC1qR, but not cC1qR co-localize with DC-SIGN on the surface of iDCs. The data suggest that DC-SIGN and gC1qR may directly associate on the surface of iDCs and regulate DC differentiation in a stage specific manner. Since both gC1qR and DC-SIGN have been shown to bind HIV, it is possible that the two receptors may play a role in the pathogenesis of HIV-AIDS.

Can ionic imbalance in HIV disease be attributed to certain underlying opportunistic infections?

,Department of Infectious Diseases,YRG Centre for AIDS Research and Education (YRG CARE),VHS Hospital Campus, IT Corridor, Taramani,Chennai 600 113, India.Tel.: +91 (44) 22542929E-mail: sundaram@yrgcare.orgDear Editor,Electrolyte disturbance can be associated with numerousmorbid conditions especially among HIV-infected individualsreceiving highly active antiretroviral therapy (HAART) (1- 4).Electrolyte imbalance is described to be the prime cause ofdeath due to myriad infectious ailments involving thegastrointestinal tract, exemplified by Asiatic cholera, rota virusinfection, shigellosis, salmonellosis, leptospirosis, malaria andparasitic diarrhoea. Unless restored, electrolyte imbalance canbe serious and might interfere with a patient’s immuneresponse. T erminal HIV disease is characterized by the onsetof numerous opportunistic infections (OI) and neoplasms andindividuals in terminal AIDS are vulnerable to numerousintracellular infections, where the corresponding aetiologicalagents reportedly use the intracellular elements (e.g. iron) andmetabolites to survive and replicate, which likely alters theevents related to cellular metabolism (5). This couldsubsequently lead to a plethora of functional sabotage eventsin cellular and immune functions (5). Maintenance of anoptimum amount of electrolyte (ionic homeostasis) in theintracellular as well as the extracellular compartment (blood,interstitial fluid etc.) is paramount to healthy living. Electrolyteimbalance could be attributed to numerous factors both fromthe host as well as from the parasite point of view andprincipally reflects a disturbance in the host’s cellular metabolicpathways possibly after establishment of an infection.In the post-HAART era, information of the prevalence ofelectrolyte imbalance is limited both in developed world aswell as in resource-limited settings and has never beenadequately investigated. One study has identified abnormallylow bicarbonate levels (1) and another documented HIV-associated nephropathy (2) among HAART-treated HIVpositive persons. Few others have documented that HIV-infected individual with underlying OIs treated with HAARTregimens developed biological abnormalities includinghyponatraemia, hyperkalaemia or hypokalaemia and acidosis(3, 4). Besides, sodium and potassium imbalances arebelieved to be associated with a plethora of chronic ailments,such as malnutrition, alcoholism, diabetes mellitus and otherinfectious causes especially in AIDS. A detailed literaturesurvey revealed the availability of very limited past data onthe prevalence of electrolyte imbalances in HIV infectedindividuals. Further, the underlying risk factors for biologicalabnormalities are poorly defined. Hence in a cross-sectionalstudy we investigated the proportion of HIV infected personswith abnormal serum electrolytes and identified the possibleco-factors of these biological abnormalities among personsattending YR Gaitonde Centre for AIDS Research andEducation (YRG CARE), a tertiary referral centre in SouthernIndia, between January 2004 and July 2006. Data werecollected with the approval of YRG CARE’s freestandinginstitutional review board.YRG CARE, since 1993, has provided medical andpsychosocial care and support to over 12,000 HIV-infectedpersons in the south of India. The study was approved by theInstitutional Review Board (IRB) of YRG CARE. Writteninformed consent was obtained from all the participants. Weinvestigated 464 HIV-infected persons who were either HAARTnaive or HAART experienced. Demographics, clinicalcharacteristics and co-factors (age, gender, hepatitis B and Cstatus and exposure to HAART) were included in the analysis.Laboratory investigations such as electrolytes (sodium,potassium, chloride and bicarbonate) and absolute CD

Secreted phospholipase A2‐IIA modulates key regulators of proliferation on astrocytoma cells

Human group IIA secreted phospholipase A(2) (sPLA(2)-IIA) has been characterized in numerous inflammatory and neoplastic conditions. sPLA(2)-IIA can either promote or inhibit cell growth depending on the cellular type and the specific injury. We have previously demonstrated that exogenous sPLA(2)-IIA, by engagement to a membrane structure, induces proliferation and activation of mitogen-activated protein kinases cascade in human astrocytoma cells. In this study, we used human astrocytoma 1321N1 cells to investigate the key molecules mediating sPLA(2)-IIA-induced cell proliferation. We found that sPLA(2)-IIA promoted reactive oxygen species (ROS) accumulation, which was abrogated in the presence of allopurinol and DPI, but not by rotenone, discarding mitochondria as a ROS source. In addition, sPLA(2)-IIA triggered Ras and Raf-1 activation, with kinetics that paralleled ERK phosphorylation, and co-immunoprecipitation assays indicated an association between Ras, Raf-1 and ERK. Additionally, Akt, p70 ribosomal protein S6 kinase, and S6 ribosomal protein were also phosphorylated upon sPLA(2)-IIA treatment, effect that was abrogated by N-acetylcysteine or LY294002 treatment indicating that ROS and phosphatidylinositol 3 kinase are upstream signaling regulators. As the inhibitors N-acetylcysteine, PD98059, LY294002 or rapamycin blocked sPLA(2)-IIA-induced proliferation without activation of the apoptotic program, we suggest that inhibition of these intracellular signal transduction elements may represent a mechanism of growth arrest. Our results reveal new potential targets for therapeutic intervention in neuroinflammatory disorders and brain cancer in particular.

Human BAMBI Cooperates with Smad7 to Inhibit Transforming Growth Factor-β Signaling

Transforming growth factor beta (TGF-beta) and related growth factors are essential regulators of embryogenesis and tissue homeostasis. The signaling pathways mediated by their receptors and Smad proteins are precisely modulated by various means. Xenopus BAMBI (bone morphogenic protein (BMP) and activin membrane-bound inhibitor) has been shown to function as a general negative regulator of TGF-beta/BMP/activin signaling. Here, we provide evidence that human BAMBI (hBAMBI), like its Xenopus homolog, inhibits TGF-beta- and BMP-mediated transcriptional responses as well as TGF-beta-induced R-Smad phosphorylation and cell growth arrest, whereas knockdown of endogenous BAMBI enhances the TGF-beta-induced reporter expression. Mechanistically, in addition to interfering with the complex formation between the type I and type II receptors, hBAMBI cooperates with Smad7 to inhibit TGF-beta signaling. hBAMBI forms a ternary complex with Smad7 and the TGF-beta type I receptor ALK5/TbetaRI and inhibits the interaction between ALK5/TbetaRI and Smad3, thus impairing Smad3 activation. These findings provide a novel insight to understand the molecular mechanism underlying the inhibitory effect of BAMBI on TGF-beta signaling.

Repeated antidepressant therapy increases cyclic GMP signaling in rat hippocampus

Cyclic adenosine monophosphpate (cAMP) signaling is thought to be involved in the pathophysiology of major depressive disorder and antidepressant action; however, relatively little is known about the possible role of cyclic guanosine monophosphate (cGMP) signaling. Accumulating evidence suggests that crosstalk occurs between cAMP and cGMP pathways. There is a need to clarify the trajectory of cAMP and cGMP concentrations, their synthesis by cyclases, and degradation by phosphodiesterases (PDEs) to understand the role of cyclic mononucleotide signaling in the effect of chronic antidepressant therapy. We used quantitative real-time PCR and enzyme immunoassay to systematically investigate the expression of intracellular signaling cascade elements in the hippocampus of rats chronically treated with the antidepressants fluoxetine and amitriptyline. We found increased cGMP levels, which were consistent with our findings of decreased PDE gene expression. Immunoassay results showed unchanged cAMP levels. We conclude that increased cGMP signaling might underlie the efficacy of chronic antidepressant treatment.