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kDa Isoform (kda + isoform)
Selected AbstractsEffects of early weaning on anxiety and prefrontal cortical and hippocampal myelination in male and female wistar ratsDEVELOPMENTAL PSYCHOBIOLOGY, Issue 4 2008Yuka Kodama Abstract We investigated developmental changes in myelin formation in the prefrontal cortex and the hippocampus, and behavioral effects of early weaning in Wistar rats. Early-weaned rats showed decreased numbers of open-arm entries in an elevated plus-maze in both sexes at 4 weeks old; this effect persisted in males, but ceased in females after this age. Expression of myelin basic protein (MBP) showed both age-dependent increases and sex differences; 4-week-old males exhibited higher MBP levels in the hippocampus, whereas 7-week-old males showed lower MBP levels in the prefrontal cortex compared to females of the same age. There was a tendency for group differences from weaning for the 21.5-kDa isoform in the prefrontal cortex. Although these results suggest that male rats are more vulnerable than females to early-weaning effects on anxiety-related behaviors, further detailed analysis is needed to clarify the functional relationship between myelination and anxiety-related behaviors. © 2008 Wiley Periodicals, Inc. Dev Psychobiol 50: 332,342, 2008. [source] Malonyl-CoA decarboxylase (MCD) is differentially regulated in subcellular compartments by 5,AMP-activated protein kinase (AMPK)FEBS JOURNAL, Issue 13 2004AMPK by adenoviral gene transfer technique, Studies using H9c2 cells overexpressing MCD Malonyl-CoA, a potent inhibitor of carnitine pamitoyl transferase-I (CPT-I), plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCD has several Ser/Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial. We therefore overexpressed MCD (Ad.MCD) and constitutively active AMPK (Ad.CA-AMPK) in H9c2 cells, using an adenoviral gene delivery approach in order to examine if MCD is regulated by AMPK. Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in AMPK activity as compared with control cells expressing green fluorescent protein (Ad.GFP). MCD activity increased 40- to 50-fold in Ad.MCD + Ad.GFP cells when compared with Ad.GFP control. Co-expressing AMPK with MCD further augmented MCD expression and activity in Ad.MCD + Ad.CA-AMPK cells compared with the Ad.MCD + Ad.GFP control. Subcellular fractionation further revealed that 54.7 kDa isoform of MCD expression was significantly higher in cytosolic fractions of Ad.MCD + Ad.CA-AMPK cells than of the Ad.MCD +Ad.GFP control. However, the MCD activities in cytosolic fractions were not different between the two groups. Interestingly, in the mitochondrial fractions, MCD activity significantly increased in Ad.MCD + Ad.CA-AMPK cells when compared with Ad.MCD + Ad.GFP cells. Using phosphoserine and phosphothreonine antibodies, no phosphorylation of MCD by AMPK was observed. The increase in MCD activity in mitochondria-rich fractions of Ad.MCD + Ad.CA-AMPK cells was accompanied by an increase in the level of the 50.7 kDa isoform of MCD protein in the mitochondria. This differential regulation of MCD expression and activity in the mitochondria by AMPK may potentially regulate malonyl-CoA levels at sites nearby CPT-I on the mitochondria. [source] Altered conditioned fear behavior in glutamate decarboxylase 65 null mutant miceGENES, BRAIN AND BEHAVIOR, Issue 2 2003O. Stork We investigated the involvement of the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and GAD65-mediated ,-aminobutyric acid (GABA) synthesis in the formation and expression of Pavlovian fear memory. To this end, behavioral, endocrine and autonomic parameters were examined during conditioned fear retrieval of mice with targeted ablation of the GAD65 gene (GAD65,/, mice). These mutant mice were found to display specific fear behavior (freezing, escape), as well as autonomic (increased defecation) and endocrine activation (increased plasma corticosterone) during fear memory retrieval. However, freezing was reduced and flight and escape behavior were increased in GAD65,/, mice compared to their wild type and heterozygous littermates, while corticosterone levels and defecation rates did not differ between genotypes. Active defensive behavior of GAD65,/, mice was observed during both auditory cued and contextual retrieval of fear memory, as well as immediately after conditioning. These data indicate a selectively altered behavioral fear response in GAD65,/, mice, most likely due to deficits in threat estimation or the elicitation of appropriate conditioned fear behavior, and suggest that GAD65 is a genetic determinant of conditioned fear behavior. GAD65,/, mice provide a valuable tool to further dissect the GABAergic mechanisms involved in fear and anxiety and to model GABA-related neurological and psychiatric disorders. [source] Modulation of diabetes in NOD mice by GAD65-specific monoclonal antibodies is epitope specific and accompanied by anti-idiotypic antibodiesIMMUNOLOGY, Issue 4 2008Tyler R. Hall Summary Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells. Here we show that administration of a human monoclonal antibody (b96·11) specific to the 65-kDa isoform of glutamate decarboxylase (GAD65) to prediabetic non-obese diabetic (NOD) mice significantly delays the onset of autoimmune diabetes. We found this effect to be epitope-specific, as only b96·11 showed this therapeutic property, while a GAD65-specific human monoclonal control antibody (b78) derived from the same patient, but specific to a different determinant of GAD65, had no significant effect on the progression of disease. Administration of b96·11 or b78 to NOD mice was accompanied by the generation of anti-idiotypic antibodies. Importantly, the induced anti-idiotypic antibodies were specific for the immunizing antibody and blocked the binding of GAD65 by the respective antibody. These findings suggest a potential role for the internal image of the GAD65 determinant recognized by b96·11 in the anti-idiotypic antibody, supporting an immunomodulatory role for GAD65-specific autoantibodies, as originally postulated by Jerne. [source] Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutantPLANT BIOTECHNOLOGY JOURNAL, Issue 8 2010Linda Avesani Summary The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM. [source] Quantitative analysis of neuronal diversity in the mouse olfactory bulbTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 6 2007S. Parrish-Aungst Abstract Olfactory sensory information is processed and integrated by circuits within the olfactory bulb. Golgi morphology suggests the olfactory bulb contains several major neuronal classes. However, an increasingly diverse collection of neurochemical markers have been localized in subpopulations of olfactory bulb neurons. While the mouse is becoming the animal model of choice for olfactory research, little is known about the proportions of neurons expressing and coexpressing different neurochemical markers in this species. Here we characterize neuronal populations in the mouse main olfactory bulb, focusing on glomerular populations. Immunofluorescent labeling for: 1) calretinin, 2) calbindin D-28K (CB), 3) parvalbumin, 4) neurocalcin, 5) tyrosine hydroxylase (TH), 6) the 67-kDa isoform of GAD (GAD67), and 7) the neuronal marker NeuN was performed in mice expressing green fluorescent protein under the control of the glutamic acid decarboxylase 65kDa (GAD65) promoter. Using unbiased stereological cell counts we estimated the total numbers of cells and neurons in the bulb and the number and percentage of neurons expressing and coexpressing different neurochemical populations in each layer of the olfactory bulb. Use of a genetic label for GAD65 and immunohistochemistry for GAD67 identified a much larger percentage of GABAergic neurons in the glomerular layer (55% of all neurons) than previously recognized. Additionally, while many glomerular neurons expressing TH or CB coexpress GAD, the majority of these neurons preferentially express the GAD67 isoform. These data suggest that the chemospecific populations of neurons in glomeruli form distinct subpopulations and that GAD isoforms are preferentially regulated in different neurochemical cell types. J. Comp. Neurol. 501:825,836, 2007. © 2007 Wiley-Liss, Inc. [source] Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiationTHE JOURNAL OF PHYSIOLOGY, Issue 23 2008Dina Simkin TREK-2 expressed in mammalian cells exhibits small (,52 pS) and large (,220 pS) unitary conductance levels. Here we tested the role of the N-terminus (69 amino acids long) in the control of the unitary conductance, and role of the alternative translation initiation as a mechanism that produces isoforms of TREK-2 that show different conductance levels. Deletion of the first half (,1,36) of the N-terminus had no effect. However, deletion of most of the N-terminus (,1,66) resulted in the appearance of only the large-conductance channel (,220 pS). In support of the critical function of the distal half of the N-terminus, the deletion mutants ,1,44 and ,1,54 produced ,90 pS and 188 pS channels, respectively. In Western blot analysis, TREK-2 antibody detected two immunoreactive bands at ,54 kDa and ,60 kDa from cells expressing wild-type TREK-2 that has three potential translation initiation sites (designated M1M2M3) within the N-terminus. Mutation of the second and third initiation sites from Met to Leu (M1L2L3) produced only the ,60 kDa isoform and the small-conductance channel (,52 pS). Mutants designed to produce translation from the second (M2L3) or third (M3) initiation site produced the ,54 kDa isoform, and the large conductance channel (,185,224 pS). M1L2L3, M2L3 and M3 were relatively selectively permeable to K+, as judged by the 51,55 mV shifts in reversal potential following a 10-fold change in [K+]o. PNa/PK values were also similar for M1L2L3 (,0.02), M2L3 (,0.02) and M3 (,0.03). Arachidonic acid, proton and membrane stretch activated, whereas dibutyryl-cAMP inhibited all three isoforms of TREK-2, indicating that deletion of the N-terminus does not abolish modulation. These results show that the small and large conductance TREK-2 channels are produced as a result of alternative translation initiation, producing isoforms with long and short N-termini, and that the distal half of the N-terminus controls the unitary conductance. [source] Expression and distribution of distinct variants of E-MAP-115 during proliferation and differentiation of human intestinal epithelial cellsCYTOSKELETON, Issue 4 2003Marie-Thérèse Vanier Abstract Epithelial cell proliferation and differentiation occur concomitant with striking remodeling of the cytoskeleton. Microtubules (MTs) play important roles in these processes, during which the MTs themselves are reorganized and stabilized by microtubule-associated proteins (MAPs). Among the proteins classified as structural MAPs, E-MAP-115 (also named ensconsin) is preferentially expressed in cells of epithelial origin. The aims of this study were, first, to determine if E-MAP-115, like other MAPs, is expressed as different isoforms during differentiation and, second, to perform a detailed analysis of the expression and distribution of any E-MAP-115 variants detected in intestinal epithelial cells during their polarization/differentiation. It was our expectation that these data would help us to develop hypotheses concerning the role of this MAP in epithelial development. We report the expression of three E-MAP-115 transcripts encoding isoforms of 115, 105, and 95 kDa; two display an expression gradient inverse to the third one as Caco-2 cells progress from proliferation through the stages of differentiation. To monitor the proteins produced from each transcript, we used purified polyclonal antibodies against synthetic peptides contained within the 115, 105, and 95 kDa isoforms to assay proliferating and differentiating CaCo-2 cells. Our results indicate that the expression and MT-binding capacity of the 115, 105, and 95 kDa isoforms vary upon proliferation/differentiation of the cells. E-MAP-115 proteins colocalize with MTs in proliferative and differentiated Caco-2 cells; in vivo, they are expressed in both crypt and villus epithelial cells where they are mainly concentrated at the apical pole of the cells. Cell Motil. Cytoskeleton 55:221,231, 2003. © 2003 Wiley-Liss, Inc. [source] Effects of Ethanol and Transforming Growth Factor , (TGF,) on Neuronal Proliferation and nCAM ExpressionALCOHOLISM, Issue 8 2002Michael W. Miller Background Developmental events targeted by ethanol are cell proliferation, neuronal migration, and neurite outgrowth; the latter processes being mediated by neural cell adhesion molecule (nCAM). TGF,1 affects all three of these events. Therefore, the effects of ethanol on transforming growth factor (TGF) ,1 mediated activities in neocortical neurons in vitro were examined. Methods Primary cultures of cortical neurons were obtained from 16-day-old fetuses and were treated with TGF,1 (0 or 10 ng/ml) and ethanol (0 or 400 mg/dl) for 48 hr. The effects of these substances on cell numbers, [3H]thymidine incorporation, and the expression of nCAM were determined. Results Both cell growth (the change in cell numbers over time) and cell proliferation were inhibited by TGF,1 and ethanol. The action of these two anti-mitogenic factors was additive. In contrast, TGF,1 also promoted the expression of three isoforms of nCAM. Likewise, ethanol also up-regulated nCAM expression. On the other hand, ethanol blocked TGF,1-mediated nCAM expression, particularly of the 120 and 180 kDa isoforms. Conclusions TGF, ligands inhibit neuronal proliferation and stimulate the expression of cell adhesion proteins that promote the movement of postmitotic neurons and process outgrowth. Ethanol alters these phenomena as well. Thus, in neurons, as in astrocytes, TGF,1 and ethanol may interact. [source] Susceptibility to oxidative stress: proteomic analysis of bronchoalveolar lavage from ozone-sensitive and ozone-resistant strains of micePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2003Ruddy Wattiez Abstract Previous studies have shown that the pulmonary response to ozone (O3) varies greatly among strains of mice, but the factor(s) and the mechanism(s) that are responsible for this differential susceptibility have not yet been clearly identified. The present study explores the molecular bases for this differential O3 susceptibility by studying the expression of proteins associated to the epithelial lining fluid (ELF) from two strains of mice, C57BL/6J and the C3H/HeJ, respectively described as O3 -sensitive and O3 -resistant. The ELF proteins of these two strains were displayed by two-dimensional gel electrophoresis (2-DE) of bronchoalveolar lavage fluids (BALFs) and the protein patterns obtained with BALF samples of both strains were compared. Two major differences were observed between the BALF 2-DE protein maps obtained from C57BL/6J and C3H/HeJ strains. First, two isoforms of the antioxidant protein 2 (AOP2) were detected in a strain-dependent manner: C3J/HeJ possesses only AOP2a (isoelectric point 5.7) and C57BL/6J exhibits only AOP2b (isoelectric point 6.0). Second, the levels of anti-inflammatory and immunosuppressive Clara cell protein-16 (CC16) were 1.3 times higher in the BALF from resistant C3H/HeJ than from sensitive C57BL/6 mice. Moreover, two 6 kDa isoforms of CC16 with isoelectric points of 4.9 (CC16a) and 5.2 (CC16b) are detected in both strains. Interestingly, the C57BL/6J strain had a twice decreased level of the acidic isoform of CC16 compared to C3H/HeJ. Our results suggest that AOP2 and CC16 might participate in the protection of the pulmonary tract to O3 -induced lung injury. The possible differential contribution of specific protein isoforms in the differential susceptibility to oxidative stress is discussed. [source] | ||||||||||||||||