JOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2001
JONI YLOSTALO
ABSTRACT A major tyrosinase isoform was isolated from the cap skins of Portabella mushrooms after chromatography on DEAE cellulose and hydroxylapatite columns. The isolated enzyme had a pI of 4.3 and a subunit molecular weight of 48 kDa while the native size was estimated to be 43 kDa. Western blotting indicated that 48 and 26 kDa cross-reacting proteins were present in the isolated fraction. This tyrosinase isoform had a pH optimum of 7.0 and was most active with catechol, tert-butylcatechol, andpyrogallol as substrates. The enzyme was severely inhibited by erythorbic acid, glutathione, cysteine, tropolone, salicylhydroxamic acid, kcjic acid, and diethyldithiocarbamic acid, but little inhibition was observed using honey extracts, borax, resveratrol, cyclodextrins, or a copper chelating peptide. [source]

Doubly Truncated FosB Isoform (,2,FosB) Induces Osteosclerosis in Transgenic Mice and Modulates Expression and Phosphorylation of Smads in Osteoblasts Independent of Intrinsic AP-1 Activity,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2008
George Sabatakos
Abstract Introduction: Activator protein (AP)-1 family members play important roles in the development and maintenance of the adult skeleton. Transgenic mice that overexpress the naturally occurring ,FosB splice variant of FosB develop severe osteosclerosis. Translation of ,fosb mRNA produces both ,FosB and a further truncated isoform (,2,FosB) that lacks known transactivation domains but, like ,FosB, induces increased expression of osteoblast marker genes. Materials and Methods: To test ,2,FosB's ability to induce bone formation in vivo, we generated transgenic mice that overexpress only ,2,FosB using the enolase 2 (ENO2) promoter-driven bitransgenic Tet-Off system. Results: Despite ,2,FosB's failure to induce transcription of an AP-1 reporter gene, the transgenic mice exhibited both the bone and the fat phenotypes seen in the ENO2-,FosB mice. Both ,FosB and ,2,FosB activated the BMP-responsive Xvent-luc reporter gene and increased Smad1 expression. ,2,FosB enhanced BMP-induced Smad1 phosphorylation and the translocation of phospho-Smad1 (pSmad1) to the nucleus more efficiently than ,FosB and showed a reduced induction of inhibitory Smad6 expression. Conclusions: ,FosB's AP-1 transactivating function is not needed to induce increased bone formation, and ,2,FosB may act, at least in part, by increasing Smad1 expression, phosphorylation, and translocation to the nucleus. [source]

NFAT expression in human osteoclasts

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005
Christopher J. Day
Abstract Nuclear factor of activated T-cells cytoplasmic (NFATc) is a family of transcription factors originally identified in T-cells. The gene family is currently known to have four members (NFATc1 through NFATc4) which have roles both within and outside the immune system. We show that NFATc1 is the major induced NFAT in human osteoclasts, with expression greatly exceeding that of NFATc2 through NFATc4. In macrophage-like cells in culture, NFATc1 through NFATc4 are expressed at similar low levels. NFATc1 is comprised of five mRNA transcript variants known to encode three different protein isoforms. The mRNA encoding isoform C (mRNA variant 3) was the most expressed with 38 copies per nanogram followed by isoform B (mRNA variant 5) with 17 copies per nanogram of total RNA. Isoform A (mRNA variant 1) and mRNA variants 2 and 4 made up less than 1% of the total NFATc1 expressed. NFATc1 is activated by calcineurin after calcium-calmodulin signalling. The induction of NFATc1 in osteoclasts was not altered in the presence of cyclosporin A, an inhibitor of calcineurin, suggesting that NFATc1 does not participate in autoregulatory activation of its own promoter. The NFATc1 variants expressed by human osteoclasts are not those normally expressed by effector T-cells but are similar to those seen in naïve T-cells. © 2005 Wiley-Liss, Inc. [source]

Regulation of Eukaryotic Initiation Factor 4E and Its Isoform: Implications for Antiviral Strategy in Plants

JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 10 2006
Yu-Yang Zhang
Abstract In recent years, biotechnology has permitted regulation of the expression of endogenous plant genes to improve agronomically important traits. Genetic modification of crops has benefited from emerging knowledge of new genes, especially genes that exhibit novel functions, one of which is eukaryotic initiation factor 4E (eIF4E). eIF4E is one of the most important translation initiation factors involved in eukaryotic initiation. Recent research has demonstrated that virus resistance mediated by eIF4E and its isoform eIF (iso)4E occurs in several plant-virus interactions, thus indicating a potential new role for eIF4E/eIF(iso)4E in resistance strategies against plant viruses. In this review, we briefly describe eIF4E activity in plant translation, its potential role, and functions of the eIF4E subfamily in plant-virus interactions. Other initiation factors such as eIF4G could also play a role in plant resistance against viruses. Finally, the potential for developing eIF4E-mediated resistance to plant viruses in the future is discussed. Future research should focus on elucidation of the resistance mechanism and spectrum mediated by eIF4E. Knowledge of a particular plant-virus interaction will help to deepen our understanding of eIF4E and other eukaryotic initiation factors, and their involvement in virus disease control. (Managing editor: Li-Hui Zhao) [source]

Isoform- and subcellular fraction-specific differences in hippocampal 14-3-3 levels following experimentally evoked seizures and in human temporal lobe epilepsy

JOURNAL OF NEUROCHEMISTRY, Issue 2 2006
Clara K. Schindler
Abstract 14-3-3 proteins are a family of signaling molecules involved in diverse cellular functions, which can mediate anti-apoptotic effects. Seizure-induced neuronal death may involve programmed (apoptotic) cell death pathways and is associated with a decline in brain 14-3-3 levels. Presently, we investigated the subcellular localization and effects of seizures on isoforms of 14-3-3 in rat hippocampus, and contrasted these to findings in human temporal lobe epilepsy (TLE). All brain isoforms of 14-3-3 were detected in the cytoplasmic compartment of rat hippocampus, while 14-3-3, and -, were also present in mitochondrial and microsome-enriched fractions. Focally evoked seizures in rats significantly reduced 14-3-3, levels within the microsome-enriched compartment at 4 h, with similar responses for 14-3-3,, while cytoplasm-localized 14-3-3,, -, and -, remained unchanged. Analysis of human autopsy control hippocampus revealed similar 14-3-3 isoform expression profiles. In TLE samples, the microsome-enriched fraction also showed differences, but here 14-3-3, and -, levels were higher than controls. TLE sample 14-3-3 isoform abundance within the cytoplasmic fraction was not different to controls. This study defines the subcellular localization of 14-3-3 isoforms in rat and human hippocampus and identifies the microsome-enriched fraction as the main site of altered 14-3-3 levels in response to acute prolonged and chronic recurrent seizures. [source]

The p110, Isoform of PI3K Differentially Regulates ,1 and ,2 Integrin-Mediated Monocyte Adhesion and Spreading and Modulates Diapedesis

MICROCIRCULATION, Issue 6 2006
ALEXANDER M. FERREIRA
ABSTRACT Objective: Leukocyte diapedesis is misregulated in inflammatory disease and depends on the binding of monocytic LFA-1 and VLA-4 to endothelial ICAM-1 and VCAM-1, respectively. The authors hypothesized that these different molecular interactions elicit specific signaling cascades within monocytes regulating specific steps in adhesion, motility, and diapedesis. Methods: The authors employed the PI3K p110, catalytic subunit specific inhibitor IC87114 (2 , M) and the broad-spectrum PI3K inhibitory agents LY294002 (50 , M) and wortmannin (100 nM), to examine the role of PI3K, in monocyte diapedesis through endothelial monolayers and its role in monocyte adhesion and spreading upon carpets of ICAM-1 or VCAM-1. They further explored the effects of PI3K, inhibition on the activation state of , 1 and , 2 integrins with immunocytochemistry and flow cytometry. Results: In human peripheral blood monocytes IC87114 was as effective as wortmannin and LY294002 at inhibiting diapedesis, however, in THP-1 cells LY294002 and wortmannin caused a 5-fold reduction in diapedesis, while IC87114 only decreased diapedesis 2-fold. PI3K, activity was specifically required for THP-1 cell adhesion and spreading on VCAM-1, but not on ICAM-1 protein substrates. Flow cytometric analysis demonstrated that PI3K, inhibition decreased the amount of conformationally active , 1-integrins, while having no effect on the prevalence of conformationally active , 2-integrins expressed on the cell surface. In addition, PI3K, inhibition resulted in a 4-fold decrease in the activation state of Rac-1 and Cdc42. Conclusions: These results demonstrate the specific necessity of PI3K, in regulating monocytic integrin activation and the general role of PI3K signaling during diapedesis, implicating PI3K as a target for therapeutic intervention. [source]

Oxidation of specific methionine and tryptophan residues of apolipoprotein A-I in hepatocarcinogenesis

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2005
Jokin Fernández-Irigoyen
Abstract Hepatocellular carcinoma (HCC) is the fifth most common neoplasm with more than 500,000 new cases diagnosed yearly. Although major risk factors of HCC are currently known, the identification of biological targets leading to an early diagnosis of the disease is considered one of the priorities of clinical hepatology. In this work we have used a proteomic approach to identify markers of hepatocarcinogenesis in the serum of a knockout mice deficient in hepatic AdoMet synthesis (MAT1A,/,), as well as in patients with HCC. Three isoforms of apolipoprotein A-I (Apo A-I) with different pI were identified in murine serum. Isoform 1 is up-regulated in the serum of MAT1A,/, mice much earlier than any histological manifestation of liver disease. Further characterization of the differential isoform by electrospray MS/MS revealed specific oxidation of methionine 85 and 216 to methionine sulfoxide while the sequence of the analogous peptides on isoforms 2 and 3 showed the nonoxidized methionine residues. Enrichment of an acidic isoform of Apo A-I was also assessed in the serum of hepatitis B virus patients who developed HCC. Specific oxidation of methionine 112 to methionine sulfoxide and tryptophans 50 and 108 to formylkinurenine were identified selectively in the up-regulated isoform. Although it is not clear at present whether the occurrence of these modifications has a causal role or simply reflects secondary epiphenomena, this selectively oxidized Apo A-I isoform may be considered as a pathological hallmark that may help to the understanding of the molecular pathogenesis of HCC. [source]

TMPRSS2:ERG fusion transcripts in urine from prostate cancer patients correlate with a less favorable prognosis

APMIS, Issue 8 2009
KARI ROSTAD
Rostad K, Hellwinkel OJC, Haukaas SA, Halvorsen OJ, Øyan AM, Haese A, Budäus L, Albrecht H, Akslen LA, Schlomm T, Kalland K-H. TMPRSS2:ERG fusion transcripts in urine from prostate cancer patients correlate with a less favorable prognosis. APMIS 2009; 117: 575,82. The transcription factor ERG is highly upregulated in the majority of prostate cancers due to chromosomal fusion of the androgen responsive promoter of TMPRSS2 to the ERG reading frame. Our aim was to identify this gene fusion in urine samples from prostate cancer patients prior to radical treatment and to compare fusion status with clinicopathological variables. Urine fractions from 55 patients (with and without prior prostatic massage) were analyzed for the presence of TMPRSS2:ERG isoforms using real-time qPCR. Sixty-nine percent of urine samples following prostatic massage were positive for TMPRSS2:ERG isoforms a or b, five out of which were positive for both, vs 24% of samples obtained without prior massage. Isoform a seems to be most prevalent and some patients may be positive for more than one fusion variant, reflecting the multifocality of prostate cancer. Prostatic massage prior to sampling, analysis of pelleted urine material and detection of cDNA provided the highest sensitivity. Positive statistical correlations were identified between TMPRSS2:ERG fusion and high s-PSA, pathological stage and Gleason score. Our findings contribute to the increasing elucidation of the role of TMPRSS2:ERG in the development of prostate cancer. [source]

Skeletal muscle fibre diversity and the underlying mechanisms

ACTA PHYSIOLOGICA, Issue 4 2010
M. Canepari
Abstract The review first briefly summarizes how myosin isoforms have been identified as the major determinant of the functional variability among skeletal muscle fibres. The latter feature is a major characteristic of muscle fibres and a major basis of skeletal muscle heterogeneity and plasticity in vivo. Then, evidence is reported, which indicates that the properties of muscle fibres can vary with no change in the myosin isoform they express. Moreover, the physiological and pathological conditions (ageing, disuse, exercise training, muscular dystrophy) in which such myosin isoform independent change in functional properties occurs and the possible underlying mechanisms are considered. Finally, the known molecular bases of the functional differences among slow and fast isoforms are briefly dealt with. [source]

Localization and functional characterization of the human NKCC2 isoforms

ACTA PHYSIOLOGICA, Issue 3 2010
I. Carota
Abstract Aim:, Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na+/K+/2Cl, cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. Methods:, RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were performed to characterize human NKCC2 isoforms. Results:, All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant isoform in humans; similarly, isoform-specific in situ hybridization showed high expression levels of human NKCC2A along the TAL. Compared to NKCC2B and NKCC2F, human NKCC2A had the lowest Cl, affinity as determined by 86Rb+ uptake studies in oocytes. All NKCC2 isoforms were more efficiently inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the highly conserved sequence encoded by exon 4. Conclusion:, The human NKCC2 is an example of how differential splicing forms the basis for a diversification of transporter protein function. [source]

Correlation of dystrophin,glycoprotein complex and focal adhesion complex with myosin heavy chain isoforms in rat skeletal muscle

ACTA PHYSIOLOGICA, Issue 4 2009
S. Masuda
Abstract Aim:, The dystrophin,glycoprotein complex (DGC) and focal adhesion complex (FAC) are transmembrane structures in muscle fibres that link the intracellular cytoskeleton to the extracellular matrix. DGC and FAC proteins are abundant in slow-type muscles, indicating the structural reinforcement which play a pivotal role in continuous force output to maintain posture for long periods. The aim of the present study was to examine the expression of these structures across fast-type muscles containing different myosin heavy chain (MHC) isoform patterns which reflect the fatigue-resistant characteristics of skeletal muscle. Methods:, We measured the expression of dystrophin and ,1 integrin (representative proteins of DGC and FAC respectively) in plantaris, extensor digitorum longus, tibialis anterior, red and white portions of gastrocnemius, superficial portion of vastus lateralis and diaphragm, in comparison with soleus (SOL) and cardiac muscle from rats. Results:, The expression of dystrophin and ,1 integrin correlated positively with the percentage of type I, IIa and IIx MHC isoforms and negatively with that of type IIb MHC isoform in fast-type skeletal muscles, and their expression was abundant in SOL and cardiac muscle. Conclusion:, Our results support the idea that DGC and FAC are among the factors that explain the fatigue-resistant property not only of slow-type but also of fast-type skeletal muscles. [source]

Na+/H+ exchangers and the regulation of volume

ACTA PHYSIOLOGICA, Issue 1-2 2006
R. T. Alexander
Abstract The regulation of volume is fundamental to life. There exist numerous conditions that can produce perturbations of cell volume. The cell has developed mechanisms to directly counteract these perturbations so as to maintain its physiological volume. Directed influx of the major extracellular cation, sodium, serves to counteract a decreased cell volume through the subsequent osmotically coupled movement of water to the intracellular space. This process, termed regulatory volume increase is often mediated by the ubiquitous sodium/hydrogen ion exchanger, NHE1. Similarly, the maintenance of intravascular volume is essential for the maintenance of blood pressure and consequently the proper perfusion of vital organs. Numerous mechanisms exist to counterbalance alterations in intravascular volume, not the least of which is the renal absorption of sodium filtered at the glomerulus. Two-thirds of filtered sodium and water are absorbed in the renal proximal tubule, a mechanism that intimately involves the apical sodium/hydrogen ion exchanger, NHE3. This isoform is fundamental to the maintenance and regulation of intravascular volume and blood pressure. In this article, the effects of cell volume on the activity of these different isoforms, NHE1 and NHE3, will be described and the consequences of their activity on intracellular and intravascular volume will be explored. [source]

Expression of Na+/HCO3, co-transporter proteins (NBCs) in rat and human skeletal muscle

ACTA PHYSIOLOGICA, Issue 1 2004
J. M. Kristensen
Abstract Aim:, Sodium/bicarbonate co-transport (NBC) has been suggested to have a role in muscle pH regulation. We investigated the presence of NBC proteins in rat and human muscle samples and the fibre type distribution of the identified NBCs. Methods and results:, Western blotting of muscle homogenates and sarcolemmal membranes (sarcolemmal giant vesicles) were used to screen for the presence of NBCs. Immunohistochemistry was used for the subcellular localization. The functional test revealed that approximately half of the pH recovery in sarcolemmal vesicles produced from rat muscle is mediated by bicarbonate-dependent transport. This indicates that the NBCs are preserved in the vesicles. The western blotting experiments demonstrated the existence of at least two NBC proteins in skeletal muscle. One NBC protein (approximately 150 kDa) seems to be related to the kidney/pancreas/heart isoform NBC1, whereas the other protein (approximately 200 kDa) is related to the NBC4 isoform. The two NBC proteins represent the electrogenic isoforms named NBCe1 and NBCe2. Membrane fractionation and immunofluorescence techniques confirmed that the two NBCs are located in the sarcolemmal membrane as well as in some internal membranes, probably the T-tubules. The two NBCs localized in muscle have distinct fibre type distributions. Conclusions:, Skeletal muscle possesses two variants of the sodium/bicarbonate co-transporter (NBC) isoforms, which have been called NBCe1 and NBCe2. [source]

Human B cells express a CD45 isoform that is similar to murine B220 and is downregulated with acquisition of the memory B-cell marker CD27,

CYTOMETRY, Issue 1 2003
Jack J. H. Bleesing
Abstract Background Differences between human and murine B cells exist at all stages of B-cell development, including the stage of memory B-cell formation. B cells in mice are identified with the pan,B-cell,specific CD45 isoform, B220. In initial studies in humans, it appeared that B220 expression did not include all B cells. This study was performed to expand on those preliminary findings. Methods Multiparameter flow cytometric detection of B220 expression on B cells was combined with a variety of B-cell markers. Results In contrast to mice, B220 was not a pan,B-cell marker in humans but was downregulated in the majority of B cells that acquired the human memory B-cell marker, CD27, whereas a minor memory B-cell subset remained B220+, suggesting differences in differentiation. Conclusions The B220 isoform in humans is developmentally regulated in humans, tied to the acquisition of a memory phenotype, and as such can be used as a differentiation-specific CD45 isoform, akin to the use of CD45 isoforms to distinguish between naive and memory T-cell subsets. Patients with immunodeficiency disorders, associated with defective memory B-cell generation and absent or reduced CD27+ B cells, showed a corresponding lack of B220 downregulation consistent with altered differentiation of B-cell subsets. Cytometry Part B (Clin. Cytometry) 51B:1,8, 2003. Published 2002 Wiley-Liss, Inc. [source]

The actin gene family: Function follows isoform,

CYTOSKELETON, Issue 10 2010
Benjamin J. Perrin
Although actin is often thought of as a single protein, in mammals it actually consists of six different isoforms encoded by separate genes. Each isoform is remarkably similar to every other isoform, with only slight variations in amino acid sequence. Nevertheless, recent work indicates that actin isoforms carry out unique cellular functions. Here, we review evidence drawn from localization studies, mouse models, and biochemical characterization to suggest a model for how in vivo mixing of actin isoforms may influence cytoskeletal function in cells. © 2010 Wiley-Liss, Inc. [source]

A minor ,-tubulin essential for mammalian cell proliferation

CYTOSKELETON, Issue 9 2008
Rajat Bhattacharya
Abstract Mammals use tubulin from multiple genes to construct microtubules. Some genes are expressed in a tissue specific manner, while others are expressed in almost all cell types. ,5-Tubulin is a minor, ubiquitous isoform whose overexpression was recently shown to disrupt microtubules. Using inhibitory RNA, we now report that suppression of ,5 production in both human and hamster cells blocks cell proliferation. Cells depleted of ,5 either trigger the mitotic checkpoint and undergo apoptosis; or they experience a transient mitotic block, a high incidence of lagging chromosomes, and progression into G1 without cytokinesis to become large, flat cells with elevated DNA content. Microtubules appear to be normally organized in cells depleted of ,5, but they are rich in acetylated ,-tubulin indicating that they may be more stable than normal. The results provide the first evidence that a specific isoform of ,-tubulin is required for mitosis. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source]

Molecular identification and localization of cellular titin, a novel titin isoform in the fibroblast stress fiber

CYTOSKELETON, Issue 6 2007
Peter J. Cavnar
Abstract We previously discovered a large titin-like protein,c-titin,in chicken epithelial brush border and human blood platelet extracts that binds ,-actinin and organizes arrays of myosin II bipolar filaments in vitro. RT-PCR analysis of total RNA from human megakaryoblastic (CHRF-288-11) and mouse fibroblast (3T3) nonmuscle cells reveal sequences identical to known titin gene exon sequences that encode parts of the Z-line, I-band, PEVK domain, A-band, and M-line regions of striated muscle titins. In the nonmuscle cells, these sequences are differentially spliced in patterns not reported for any striated muscle titin isoform. Rabbit polyclonal antibodies raised against expressed protein fragments encoded by the Z-repeat and kinase domain regions react with the c-titin band in Western blot analysis of platelet extracts and immunoprecipitate c-titin in whole platelet extracts. Immunofluorescent localization demonstrates that the majority of the c-titin colocalizes with ,-actinin and actin in 3T3 and Indian Muntjac deer skin fibroblast stress fibers. Our results suggest that differential expression of titin gene exons in nonmuscle cells yields multiple novel isoforms of the protein c-titin that are associated with the actin stress fiber structures. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

Myosin16b: The COOH-tail region directs localization to the nucleus and overexpression delays S-phase progression

CYTOSKELETON, Issue 1 2007
Richard S. Cameron
Abstract Rat Myo16a and Myo16b comprise the founding members of class XVI myosin and are characterized by an N-terminal ankyrin repeat domain thought to mediate an association with protein phosphatase 1 catalytic subunits 1, and 1,. Myo16b is the principal isoform and reveals predominant expression in developing neural tissue. Here, we use COS-7 cells as a model system to develop an understanding of Myo16b function. We find that Myo16b displays predominant localization in the nucleus of cells transitioning through interphase, but is not associated with processes of mitosis. Using a panel of EGFP-Myo16b-expression plasmids in transient transfection studies, we identified the COOH-terminal residues 1616,1912 as necessary and solely sufficient to target Myo16b to the nucleus. We show that the Myo16b-tail region directs localization to a nuclear compartment containing profilin and polymerized actin, which appears to form a three-dimensional meshwork through the depth of the nucleus. Further, we demonstrate that this compartment localizes within euchromatic regions of the genome and contains proliferating cell nuclear antigen (PCNA) and cyclin A, both markers of S-phase of the cell cycle. Cells transiently expressing Myo16b or Myo16b-tail region show limited incorporation of BrdU, delayed progression through S-phase of the cell cycle, and curtailed cellular proliferation. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source]

Myoblast attachment and spreading are regulated by different patterns by ubiquitous calpains

CYTOSKELETON, Issue 4 2006
Germain Mazères
Abstract The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced ,- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed. Cell Motil. Cytoskeleton 63: 2006. © 2006 Wiley-Liss, Inc. [source]

Expression of constructs of the neuronal isoform of myosin-Va interferes with the distribution of melanosomes and other vesicles in melanoma cells

CYTOSKELETON, Issue 2 2002
João Carlos da Silva Bizario
Abstract Myosin-Va has been implicated in melanosome translocation, but the exact molecular mechanisms underlying this function are not known. In the dilute, S91 melanoma cells, melanosomes move to the cell periphery but do not accumulate in the tips of dendrites as occurs in wild-type B16 melanocytes; rather, they return and accumulate primarily at the pericentrosomal region in a microtubule-dependent manner. Expression of the full-length neuronal isoform of myosin-Va in S91 cells causes melanosomes to disperse, occupying a cellular area approximately twice that observed in non-transfected cells, suggesting a partial rescue of the dilute phenotype. Overexpression of the full tail domain in S91 cells is not sufficient to induce melanosome dispersion, rather it causes melanosomal clumping. Overexpression of the head and head-neck domains of myosin-Va in B16 cells does not alter the melanosome distribution. However, overexpression of the full tail domain in these cells induces melanosome aggregation and the appearance of tail-associated, aggregated particles or vesicular structures that exhibit variable degrees of staining for melanosomal and Golgi ,-COP markers, as well as colocalization with the endogenous myosin-Va. Altogether, the present data suggest that myosin-Va plays a role in regulating the direction of microtubule-dependent melanosome translocation, in addition to promoting the capture of melanosomes at the cell periphery as suggested by previous studies. These studies also reinforce the notion that myosin-V has a broader function in melanocytes by acting on vesicular targeting or intracellular protein trafficking. Cell Motil. Cytoskeleton 51:57,75, 2002. © 2002 Wiley-Liss, Inc. [source]

Neuron-specific expression of atp6v0c2 in zebrafish CNS

DEVELOPMENTAL DYNAMICS, Issue 9 2010
Ah-Young Chung
Abstract Vacuolar ATPase (V-ATPase) is a multi-subunit enzyme that plays an important role in the acidification of a variety of intracellular compartments. ATP6V0C is subunit c of the V0 domain that forms the proteolipid pore of the enzyme. In the present study, we investigated the neuron-specific expression of atp6v0c2, a novel isoform of the V-ATPase c-subunit, during the development of the zebrafish CNS. Zebrafish atp6v0c2 was isolated from a genome-wide analysis of the zebrafish mibta52b mutant designed to identify genes differentially regulated by Notch signaling. Whole-mount in situ hybridization revealed that atp6v0c2 is expressed in a subset of CNS neurons beginning several hours after the emergence of post-mitotic neurons. The ATP6V0C2 protein is co-localized with the presynaptic vesicle marker, SV2, suggesting that it is involved in neurotransmitter storage and/or secretion in neurons. In addition, the loss-of-function experiment suggests that ATP6V0C2 is involved in the control of neuronal excitability. Developmental Dynamics 239:2501,2508, 2010. © 2010 Wiley-Liss, Inc. [source]

Non-core subunit eIF3h of translation initiation factor eIF3 regulates zebrafish embryonic development

DEVELOPMENTAL DYNAMICS, Issue 6 2010
Avik Choudhuri
Abstract Eukaryotic translation initiation factor eIF3, which plays a central role in translation initiation, consists of five core subunits that are present in both the budding yeast and higher eukaryotes. However, higher eukaryotic eIF3 contains additional (non-core) subunits that are absent in the budding yeast. We investigated the role of one such non-core eIF3 subunit eIF3h, encoded by two distinct genes,eif3ha and eif3hb, as a regulator of embryonic development in zebrafish. Both eif3h genes are expressed during early embryogenesis, and display overlapping yet distinct and highly dynamic spatial expression patterns. Loss of function analysis using specific morpholino oligomers indicates that each isoform has specific as well as redundant functions during early development. The morphant phenotypes correlate with their spatial expression patterns, indicating that eif3h regulates development of the brain, heart, vasculature, and lateral line. These results indicate that the non-core subunits of eIF3 regulate specific developmental programs during vertebrate embryogenesis. Developmental Dynamics 239:1632,1644, 2010. © 2010 Wiley-Liss, Inc. [source]

Gene targeted ablation of high molecular weight fibroblast growth factor-2

DEVELOPMENTAL DYNAMICS, Issue 2 2009
Mohamad Azhar
Abstract Fibroblast growth factor-2 (FGF2) is produced as high molecular weight isoforms (HMW) and a low molecular weight isoform (LMW) by means of alternative usage of translation start sites in a single Fgf2 mRNA. Although the physiological function of FGF2 and FGF2 LMW has been investigated in myocardial capillarogenesis during normal cardiac growth, the role of FGF2 HMW has not been determined. Here, we report the generation of FGF2 HMW-deficient mice in which FGF2 HMW isoforms are ablated by the Tag-and-Exchange gene targeting technique. These mice are normal and fertile with normal fecundity, and have a normal life span. Histological, immunohistochemical, and morphometric analyses indicate normal myocardial architecture, blood vessel, and cardiac capillary density in young adult FGF2 HMW-deficient mice. These mice along with the FGF2- and FGF2 LMW-deficient mice that we have generated previously will be very useful for elucidating the differential functions of FGF2 isoforms in pathophysiology of cardiovascular diseases. Developmental Dynamics 238:351,357, 2009. © 2008 Wiley-Liss, Inc. [source]

Nucleolar colocalization of TAF1 and testis-specific TAFs during Drosophila spermatogenesis

DEVELOPMENTAL DYNAMICS, Issue 10 2007
Chad E. Metcalf
Abstract In Drosophila, testis-specific TBP-associated factors (tTAFs) predominantly localize to spermatocyte nucleoli and regulate the transcription of genes necessary for spermatocyte entry into meiosis. tTAFs are paralogs of generally expressed TAF subunits of transcription factor IID (TFIID). Our recent observation that the generally expressed TAF1 isoform TAF1-2 is greatly enriched in testes prompted us to explore the functional relationship between general TAFs and tTAFs during spermatogenesis. Analysis by immunofluorescence microscopy revealed that among the general TFIID subunits examined (TATA-box binding protein [TBP], TAF1, TAF4, TAF5, and TAF9), only TAF1 colocalized with the tTAF Mia in spermatocyte nucleoli. Nucleolar localization of TAF1, but not Mia, was disrupted in tTAF mutant flies, and TAF1 dissociated from DNA prior to Mia as spermatocytes entered meiosis. Taken together, our results suggest stepwise assembly of a testis-specific TFIID complex (tTFIID) whereby a TAF1 isoform, presumably TAF1-2, is recruited to a core subassembly of tTAFs in spermatocyte nucleoli. Developmental Dynamics 236:2836,2843, 2007. © 2007 Wiley-Liss, Inc. [source]

Xenopus Lefty requires proprotein cleavage but not N-linked glycosylation to inhibit nodal signaling

DEVELOPMENTAL DYNAMICS, Issue 8 2007
Joby J. Westmoreland
Abstract The Nodal and Nodal-related morphogens are utilized for the specification of distinct cellular identity throughout development by activating discrete target genes in a concentration-dependant manner. Lefty is a principal extracellular antagonist involved in the spatiotemporal regulation of the Nodal morphogen gradient during mesendoderm induction. The Xenopus Lefty proprotein contains a single N-linked glycosylation motif in the mature domain and two potential cleavage sites that would be expected to produce long (XleftyL) and short (XleftyS) isoforms. Here we demonstrate that both isoforms were secreted from Xenopus oocytes, but that XleftyL is the only isoform detected when embryonic tissue was analyzed. In mesoderm induction assays, XleftyL is the functional blocker of Xnr signaling. When secreted from oocytes, vertebrate Lefty molecules were N-linked glycosylated. However, glycan addition was not required to inhibit Xnr signaling and did not influence its movement through the extracellular space. These findings demonstrate that Lefty molecules undergo post-translational modifications and that some of these modifications are required for the Nodal inhibitory function. Developmental Dynamics 236:2050,2061, 2007. © 2007 Wiley-Liss, Inc. [source]

Transient expression of thyroid hormone nuclear receptor TR,2 sets S opsin patterning during cone photoreceptor genesis

DEVELOPMENTAL DYNAMICS, Issue 5 2007
M.L. Applebury
Abstract Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR,2, the nuclear thyroid hormone receptor , isoform 2, regulates dorsal repression. To determine the molecular mechanism by which TR,2 acts, we compared the spatiotemporal expression of TR,2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR,2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR,2 is expressed uniformly, but transiently, across the retina. mRNA levels are maximal by E17 at completion of cone genesis and again minimal before P5. S opsin is also transcribed by E13, but only in ventral cones. Repression in dorsal cones is established by E17, consistent with the occurrence of patterning during cone cell genesis. The uniform expression of TR,2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR,2. The mechanism by which TR,2 functions was probed in transgenic animals with TR,2 ablated, TR,2 that is DNA binding defective, and TR,2 that is ligand binding defective. These studies show that TR,2 is necessary for dorsal repression, but not ventral activation of S opsin. TR,2 must bind DNA and the ligand T3 (thyroid hormone) to repress S opsin. Once repression is established, T3 no longer regulates dorsal S opsin repression in adult animals. The transient, embryonic action of TR,2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors. Developmental Dynamics 236:1203,1212, 2007. © 2007 Wiley-Liss, Inc. [source]

Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevis

DEVELOPMENTAL DYNAMICS, Issue 3 2005
Teun P. De Boer
Abstract Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (,62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals. Developmental Dynamics 233:864,871, 2005. © 2005 Wiley-Liss, Inc. [source]

Proprotein convertase genes in Xenopus development

DEVELOPMENTAL DYNAMICS, Issue 3 2005
Sylvia Nelsen
Abstract Proprotein convertases (PCs) are a family of serine endoproteases that proteolytically activate many precursor proteins within various secretory pathway compartments. Loss-of-function studies have demonstrated a critical role for these proteases in embryonic patterning and adult homeostasis, yet little is known about how substrate selectivity is achieved. We have identified Xenopus orthologs of three PCs: furin, PC6, and PC4. In addition to previously described isoforms of PC6 and furin, four novel splice isoforms of PC6, which are predicted to encode constitutively secreted proteases, and a putative transmembrane isoform of PC4 were identified. Furin and PC6 are expressed in dynamic, tissue-specific patterns throughout embryogenesis, whereas PC4 transcripts are restricted primarily to germ cells and brain in adult frogs. Developmental Dynamics 233:1038,1044, 2005. © 2005 Wiley-Liss, Inc. [source]

The vesicular integral protein-like gene is essential for development of a mechanosensory system in zebrafish

DEVELOPMENTAL NEUROBIOLOGY, Issue 12 2008
Mabel Chong
Abstract The zebrafish hi472 mutation is caused by a retroviral insertion into the vesicular integral protein-like gene, or zVIPL, a poorly studied lectin implicated in endoplasmic reticulum (ER)-Golgi trafficking. A mutation in the shorter isoform of zVIPL (zVIPL-s) results in a reduction of mechanosensitivity and consequent loss of escape behavior. Here we show that motoneurons and hindbrain reticulospinal neurons, which normally integrate mechanosensory inputs, failed to fire in response to tactile stimuli in hi472 larvae, suggesting a perturbation in sensory function. The hi472 mutant larvae in fact suffered from a severe loss of functional neuromasts of the lateral line mechanosensory system, a reduction of zVIPL labeling in support cells, and a reduction or even a complete loss of hair cells in neuromasts. The Delta-Notch signaling pathway is implicated in cellular differentiation of neuromasts, and we observed an increase in Notch expression in neuromasts of hi472 mutant larvae. Treatment of hi472 mutant larvae with DAPT, an inhibitor of Notch signaling, or overexpression of the Notch ligand deltaB in hi472 mutant blastocysts produced partial rescue of the morphological defects and of the startle response behavior. We conclude that zVIPL-s is a necessary component of Delta-Notch signaling during neuromast development in the lateral line mechanosensory system. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008 [source]

Effects of early weaning on anxiety and prefrontal cortical and hippocampal myelination in male and female wistar rats

DEVELOPMENTAL PSYCHOBIOLOGY, Issue 4 2008
Yuka 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]