Gamma-tubulin can both nucleate microtubule assembly and self-assemble into novel tubular structures in mammalian cells

alpha-, beta-, and gamma-tubulins are evolutionarily highly conserved members of the tubulin gene superfamily. While the abundant members, alpha- and beta-tubulins, constitute the building blocks of cellular microtubule polymers, gamma-tubulin is a low abundance protein which localized to the pericentriolar material and may play a role in microtubule assembly. To test whether gamma-tubulin mediates the nucleation of microtubule assembly in vivo, and co-assembles with alpha- and beta-tubulins into microtubules or self-assembles into macro- molecular structures, we experimentally elevated the expression of gamma-tubulin in the cell cytoplasm. In most cells, overexpression of gamma-tubulin causes a dramatic reorganization of the cellular microtubule network. Furthermore, we show that when overexpressed, gamma-tubulin causes ectopic nucleation of microtubules which are not associated with the centrosome. In a fraction of cells, gamma-tubulin self-assembles into novel tubular structures with a diameter of approximately 50 nm (named gamma-tubules). Furthermore, unlike microtubules, gamma-tubules are resistant to cold or drug induced depolymerization. These data provide evidence that gamma-tubulin can cause nucleation of microtubule assembly and can self-assemble into novel tubular structures.     

Naturally occurring plasmodium-specific IgA antibody in humans from a malaria endemic area

Blood samples collected from individuals belonging to an endemic area in Uttar Pradesh, were tested for plasmodial antigen specific immunoglobulin A (IgA) by enzyme immuno assay using soluble extract ofPlasmodium falciparum from culture. Among 773 (20.18%,P < 0.0001) samples 156 sera demonstrated a detectable seropositivity for antigen specific IgA. IgA levels were higher among individuals who experienced repeated attacks of malaria compared to acute infected patients. Among seropositive individuals the IgA titers were found increased with the age. Immunoglobulin isolated from sera having high level of IgA showed growth inhibitory effect inPlasmodium falciparum in vitro. A group of sera with high IgA antibody againstPlasmodium falciparum crude antigen showed seronegativity with specific peptides. Statistically, no positive or negative correlations were observed between antigen specific IgG and IgA. However, there was a tendency towards negative correlation between IgA and IgM. Mechanisms for the parasite specific IgA production remain to be established.     

GW bodies and stress granules, two cytoplasmic structures for mRNA degradation and storage in mammalian cells

What does mRNA become at the issue of translation in eukaryotic cells? It can be directly degraded or stored for further use. In some cases, the underlying molecular mechanisms have been studied in detail by biochemical approaches, as examplified by the most recently discovered regulation pathway, RNA interference. However, the cellular context of these regulations has often been ignored, as if these reactions took place diffusely throughout the cytoplasm. Two new structures involved therein have now been described: GW bodies (or P-bodies) and stress granules. The first studies suggested that they were specifically devoted to mRNA degradation and mRNA storage, respectively. This framework is changing rapidly with obvious functional overlapping between both structures.     

Naturally occurring and experimentally transmitted Hepatozoon americanum in coyotes from Oklahoma

Twenty free-ranging coyotes (Canis latrans) in Oklahoma (USA) were examined for the presence of naturally occurring infections with Hepatozoon americanum and to determine if bone lesions attributable to H. americanum were present. Although eight of the 20 free-ranging coyotes were found to be naturally infected with H. americanum, no bone lesions were detected. In addition, two coyote pups were exposed to H. americanum oocysts collected from experimentally infected ticks and the course of the resulting infection was followed. Both experimentally infected coyotes developed hepatozoonosis detectable by specific muscle lesions beginning 4 wk after exposure. Bone lesions were detected grossly and histologically at necropsy. Histologic evidence of periosteal bone proliferation ranged from segmental areas of plump hypercellularity and thickening of the periosteum, with minor degrees of osteogenesis, to extensive proliferation of woven bone and periosteal hypercellularity and thickening. Nymphal Amblyomma maculatum that fed on one of the experimentally infected coyote pups became infected and mature H. americanum oocysts were recovered when the ticks molted to adults. These results demonstrate that coyotes in some parts of Oklahoma are naturally infected with H. americanum, that experimentally infected coyotes can develop clinical disease, including characteristic bone lesions, and that A. maculatum nymphs can acquire infections by feeding on them.     

Chemically induced resistance to heat treatment and stress protein synthesis in cultured mammalian cells

Short exposure (1-2 h) of cultured cells, derived from a transplantable murine mammary carcinoma, to sodium arsenite, 2,4-dinitrophenol (DNP), carbonylcyanide-3-chlorophenylhydrazone (CCP) or disulfiram, induced resistance to a subsequent heat treatment, similar to heat-induced thermotolerance. Optimum resistance to a test heat treatment of 45 min at 45 degrees C after sodium arsenite exposure was obtained at a concentration of 300 microM, after DNP exposure at 3mM, after CCP at 300 microM and after disulfiram exposure in the range 1-30 microM. Exposure of cells to CCP, sodium arsenite or disulfiram led to enhanced synthesis of some proteins with the same molecular weight as 'heat shock' proteins. The pattern of enhanced synthesis of these proteins was agent specific. We could not detect significantly enhanced synthesis of the proteins after DNP using one-dimensional gel electrophoresis. These results suggest that enhanced stress protein synthesis is not a prerequisite for the development of thermal resistance.     

Endoplasmic reticulum stress induced by oxidative stress in decidual cells: a possible mechanism of early pregnancy loss

Early pregnancy loss (EPL) is one of the most common complications of human reproduction. Combined with our previous proteomic studies on villous and decidual tissues of EPL, we found that alterations of the proteins involved in oxidative stress (OS), unfolded protein response (UPR) and proteolysis presented a complex and dynamic interaction at the maternal-fetal interface. In the present study, we developed a cell model of OS using normal decidual cells to examine cell viability and expression levels of proteins related to endoplasmic reticulum stress (ER stress) and UPR. We found that glucose regulated protein 78 (GRP 78) and ubiquitinated proteins were significantly up-regulated in hydrogen peroxide (H(2)O(2)) treated decidual cells in a dose-dependent manner. Excessive OS could influence proper function of UPR by decreasing VCP in decidual cells, thereby leading to cell damage as well as inhibition of cell growth and activation of apoptosis. Furthermore, when pretreated with MG 132, a pharmacological inhibition of the proteasome, the H(2)O(2) treated decidual cells became less viable and could not up-regulate the expression level of GRP 78 to resolve the protein-folding defects, which indicating that malfunction of UPR in decidual cells might aggravate the inhibitory effect of OS in decidual cells. The present results reveal that abnormal protein profiles associated with OS induced ER stress and malfunction of UPR might be involved in the development of EPL, and OS and ER stress are potential targets for pregnant care and prognosis in normal pregnancy and its disorders.     

Proton pump inhibition induces autophagy as a survival mechanism following oxidative stress in human melanoma cells

Proton pump inhibitors (PPI) target tumour acidic pH and have an antineoplastic effect in melanoma. The PPI esomeprazole (ESOM) kills melanoma cells through a caspase-dependent pathway involving cytosolic acidification and alkalinization of tumour pH. In this paper, we further investigated the mechanisms of ESOM-induced cell death in melanoma. ESOM rapidly induced accumulation of reactive oxygen species (ROS) through mitochondrial dysfunctions and involvement of NADPH oxidase. The ROS scavenger N-acetyl--cysteine (NAC) and inhibition of NADPH oxidase significantly reduced ESOM-induced cell death, consistent with inhibition of cytosolic acidification. Autophagy, a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, represents a defence mechanism in cancer cells under metabolic stress. ESOM induced the early accumulation of autophagosomes, at the same time reducing the autophagic flux, as observed by WB analysis of LC3-II accumulation and by fluorescence microscopy. Moreover, ESOM treatment decreased mammalian target of rapamycin signalling, as reduced phosphorylation of p70-S6K and 4-EBP1 was observed. Inhibition of autophagy by knockdown of Atg5 and Beclin-1 expression significantly increased ESOM cytotoxicity, suggesting a protective role for autophagy in ESOM-treated cells. The data presented suggest that autophagy represents an adaptive survival mechanism to overcome drug-induced cellular stress and cytotoxicity, including alteration of pH homeostasis mediated by proton pump inhibition.     

Naturally occurring V1-env region variants mediate simian immunodeficiency virus SIVmac escape from high-titer neutralizing antibodies induced by a protective subunit vaccine

Macaques which developed high-titer neutralizing antibodies (htNAb) after immunization with a virion-derived oligomeric envelope glycoprotein subunit vaccine were protected against a homologous simian immunodeficiency virus SIVmac challenge. Here we demonstrate that the htNAb could be overcome by V1-env region variants isolated ex vivo from an SIVmac-infected macaque. The results further suggest that the development of V1-env region neutralization escape mutants is also necessary for survival of the virus in infected macaques. The immunological capacity of a single variable region to induce neutralizing antibodies in vaccinated and infected macaques initiate new ideas for a successful vaccine strategy.     

Naturally occurring thiophenes: isolation,purification, structural elucidation,and evaluation of bioactivities

Phytochemistry Reviews - Thiophenes are a class of heterocyclic aromatic compounds based on a five-membered ring made up of one sulfur and four carbon atoms. The thiophene nucleus is well...     

Zinc induced damage to kidney proximal tubular cells: Studies on chemical speciation leading to a mechanism of damage

This study was carried out to investigate whether zinc can potentiate renal toxicity using monolayer cultures of kidney proximal tubular cells and if so to establish the chemical species and the mechanism involved.MethodsZinc was prepared as the citrate complex at pH 7.4 in phosphate buffered saline. Monolayers of kidney proximal tubular cells under standard cell culture conditions were exposed to zinc concentrations of 0, 5 10, 20, 50 and 100 μmol/L. To assess cellular damage, thiazol blue (MTT) uptake, NAG and LDH release, DAPI staining and Tunel assay were used. Cytoprotective agents: trolox, cysteine, glutathione, ascorbic acid and sodium selenite were used to investigate if the damage was reversible.ResultsIncubation of kidney cells with zinc citrate showed a dose related reduction in cell viability (p < 0.005) associated with cellular uptake of zinc ions. After 24 h incubation with 100 μmol/L Zn citrate, NAG release was not significantly different compared to the control whereas LDH increased 3 fold. DAPI staining showed apoptotic bodies within the cells confirmed by Tunel assay using flow cytometry. Electron microscopy showed significant morphological changes including loss of brush border, vacuolated cytoplasm and condensed nuclei. Trolox almost completely (>85 ± 5%) and sodium selenite partially recovered (40 ± 4%) the viability of cells exposed to Zn but no protection was observed with other cytoprotectants, e.g. glutathione, cysteine or ascorbic acid.In conclusion zinc can induce damage to kidney cells by a mechanism dependent on zinc ions entering the cell, binding to the cell organelles and disrupting cellular processes rather than damage initiated by free radical and ROS production.     

Naturally secreted amyloid-beta increases mammalian target of rapamycin (mTOR) activity via a PRAS40-mediated mechanism

Reducing the mammalian target of rapamycin (mTOR) activity increases lifespan and health span in a variety of organisms. Alterations in protein homeostasis and mTOR activity and signaling have been reported in several neurodegenerative disorders, including Alzheimer disease (AD); however, the causes of such deregulations remain elusive. Here, we show that mTOR activity and signaling are increased in cell lines stably transfected with mutant amyloid precursor protein (APP) and in brains of 3xTg-AD mice, an animal model of AD. In addition, we show that in the 3xTg-AD mice, mTOR activity can be reduced to wild type levels by genetically preventing Aβ accumulation. Similarly, intrahippocampal injections of an anti-Aβ antibody reduced Aβ levels and normalized mTOR activity, indicating that high Aβ levels are necessary for mTOR hyperactivity in 3xTg-AD mice. We also show that the intrahippocampal injection of naturally secreted Aβ is sufficient to increase mTOR signaling in the brains of wild type mice. The mechanism behind the Aβ-induced mTOR hyperactivity is mediated by the proline-rich Akt substrate 40 (PRAS40) as we show that the activation of PRAS40 plays a key role in the Aβ-induced mTOR hyperactivity. Taken together, our data show that Aβ accumulation, which has been suggested to be the culprit of AD pathogenesis, causes mTOR hyperactivity by regulating PRAS40 phosphorylation. These data further indicate that the mTOR pathway is one of the pathways by which Aβ exerts its toxicity and further support the idea that reducing mTOR signaling in AD may be a valid therapeutic approach.     

Naturally occurring CAR bacillus infection in a laboratory rat colony and epizootiological observations

An epizootic of chronic respiratory disease was found in a rat colony. Lungs of the symptomatic rats showed histopathologically severe peribronchial lymphoid cuffing. Filamentous bacteria were detected on the border of the tracheal and bronchial epithelium by light and electron microscopy. These bacteria did not grown on artificial media but propagated in embryonated chicken eggs. The disease was thus diagnosed as cilia-associated respiratory (CAR) bacillus infection. Epizootiological observations of the natural and experimentally induced cases revealed that the disease was highly contagious, slowly progressive and intractable. Contact infection may play a major role in the transmission of this disease.     

Single-stranded oligonucleotide-mediated gene repair in mammalian cells has a mechanism distinct from homologous recombination repair

Single-stranded DNA oligonucleotide (SSO)-mediated gene repair has great potentials for gene therapy and functional genomic studies. However, its underlying mechanism remains unclear. Previous studies from other groups have suggested that DNA damage response via the ATM/ATR pathway may be involved in this process. In this study, we measured the effect of two ATM/ATR inhibitors caffeine and pentoxifylline on the correction efficiency in SSO-mediated gene repair. We also checked their effect on double-stranded break (DSB)-induced homologous recombination repair (HRR) as a control, which is well known to be dependent on the ATM/ATR pathway. We found these inhibitors could completely inhibit DSB-induced HRR, but could only partially inhibit SSO-mediated process, indicating SSO-mediated gene repair is not dependent on the ATM/ATR pathway. Furthermore, we found that thymidine treatment promotes SSO-mediated gene repair, but inhibits DSB-induced HRR. Collectively, our results demonstrate that SSO-mediated and DSB-induced gene repairs have distinct mechanisms.