Characterization of Solt, a novel SoxLZ/Sox6 binding protein expressed in adult mouse testis.
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SoxLZ/Sox6, a member of the Sox protein family, contains a leucine zipper motif in addition to an HMG box, which is its DNA binding domain. Here we have identified a novel SoxLZ/Sox6 binding protein, termed Solt, which we obtained independently using both a far-Western blot and a yeast two-hybrid screen. Like SoxLZ/Sox6 mRNA, Solt mRNA was exclusively expressed in the testis in mouse. Solt contains an unusual leucine zipper, which bound to the leucine zipper region of SoxLZ/Sox6 in vitro. In transient transfection assays in CHO cells with SoxLZ/Sox6 containing the transactivational region of herpes simplex virus VP16, expression of a reporter gene that carries a cis binding region for Sox proteins was significantly enhanced by the co-expression of Solt and Ca(2+)/calmodulin-dependent protein kinase IV. (+info)
Heterologous expression of CYP2K1 and identification of the expressed protein (BV-CYP2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase.
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LMC2 is the most abundant constitutively expressed hepatic cytochrome P450 found in sexually immature rainbow trout (Onchorynchus mykiss) and is also the isozyme that activates the carcinogen aflatoxin B1 (AFB1). This P450 has been cloned, sequenced, and designated as CYP2K1. The present report describes the heterologous expression of enzymatically active CYP2K1 (BV-CYP2K1) in baculovirus Spodoptera frugiperda (Sf9) insect cells and its catalytic and immunoreactivity characterization in comparison with that of the previously purified LMC2 P450. Homogenates of Sf9 cells expressing the CYP2K1 enzyme and LMC2 both catalyzed the hydroxylation of lauric acid and the epoxidation of AFB1 in the presence of rat NADPH-cytochrome P450 reductase. Both LMC2 and BV-CYP2K1 catalyzed the oxidation of lauric acid primarily at the (omega-1) position plus small amounts at the (omega-2) position. Formation of AFB1 epoxide was shown indirectly by the appearance of an AFB1 epoxide-glutathione conjugate when P450 incubation mixtures contained AFB1, glutathione (GSH) together with mouse liver cytosol or purified rat GSH-transferase. When the AFB1 epoxide-GSH conjugate produced by BV-CYP2K1 and purified LMC2 was analyzed by HPLC using a chiral column, it had a retention time identical to that produced by CYP3A4, a human P450 known to form exclusively the AFB1 exo-epoxide. These results, therefore, confirm that the cDNA-expressed CYP2K1 protein is catalytically and immunologically identical to purified trout LMC2 and that these two enzymes produce primarily the highly carcinogenic stereoisomeric exo-epoxide form of AFB1. (+info)
Ligand-independent dimerization and activation of the oncogenic Xmrk receptor by two mutations in the extracellular domain.
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Overexpression of the oncogenic receptor tyrosine kinase ONC-Xmrk is the first step in the development of hereditary malignant melanoma in the fish Xiphophorus. However, overexpression of its proto-oncogene counterpart (INV-Xmrk) is not sufficient for the oncogenic function of the receptor. Compared with INV-Xmrk, the ONC-Xmrk receptor displays 14 amino acid changes, suggesting the presence of activating mutations. To identify such activating mutations, a series of chimeric and mutant receptors were studied. None of the mutations present in the intracellular domain was found to be involved in receptor activation. In the extracellular domain, we found two mutations responsible for activation of the receptor. One is the substitution of a conserved cysteine (C578S) involved in intramolecular disulfide bonding. The other is a glycine to arginine exchange (G359R) in subdomain III. Either mutation leads to constitutive dimer formation and thereby to activation of the ONC-Xmrk receptor. Besides, the presence of these mutations slows down the processing of the Xmrk receptor in the endoplasmic reticulum, which is apparent as an incomplete glycosylation. (+info)
A purine-sensitive pathway regulates multiple genes involved in axon regeneration in goldfish retinal ganglion cells.
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In lower vertebrates, retinal ganglion cells (RGCs) can regenerate their axons and reestablish functional connections after optic nerve injury. We show here that in goldfish RGCs, the effects of several trophic factors converge on a purine-sensitive signaling mechanism that controls axonal outgrowth and the expression of multiple growth-associated proteins. In culture, goldfish RGCs regenerate their axons in response to two molecules secreted by optic nerve glia, axogenesis factor-1 (AF-1) and AF-2, along with ciliary neurotrophic factor. The purine analog 6-thioguanine (6-TG) blocked outgrowth induced by each of these factors. Previous studies in PC12 cells have shown that the effects of 6-TG on neurite outgrowth may be mediated via inhibition of a 47 kDa protein kinase. Growth factor-induced axogenesis in RGCs was accompanied by many of the molecular changes that characterize regenerative growth in vivo, e.g. , increased expression of GAP-43 and certain cell surface glycoproteins. 6-TG inhibited all of these changes but not those associated with axotomy per se, e.g., induction of jun family transcription factors, nor did it affect cell survival. Additional studies using RGCs from transgenic zebrafish showed that expression of Talpha-1 tubulin is likewise stimulated by AF-1 and blocked by 6-TG. The purine nucleoside inosine had effects opposite to those of 6-TG. Inosine stimulated outgrowth and the characteristic pattern of molecular changes in RGCs and competitively reversed the inhibitory effects of 6-TG. We conclude that axon regeneration and the underlying program of gene expression in goldfish RGCs are mediated via a common, purine-sensitive pathway. (+info)
Hepatic fatty acid-binding proteins of a teleost, Lateolabrax japonicus. The primary structures and location of a disulfide bond.
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Two fatty acid-binding proteins (FABP), FABP-1 and FABP-2, were purified from the liver cytosol of the teleost, Lateolabrax japonicus (Japan sea bass), and characterized. The complete primary structure of FABP-2 was determined by protein analysis to be the following: MDFSGTWQVY AQENYEEFLR AMELPADVIK MAKDIKPITE IKQSGNDFVV TSKTPGKTVT NSFTIGKEAD ITTMDGKKIR CVVNLEGGKL VCNTGKFCHI QELRGGEMVE TLTMGSTTLI RKSKKM. Partial peptide sequences of FABP-1 were also determined. Phylogenetic analysis indicates that FABP-2 is a homologue of mammalian hepatic FABP, whereas FABP-1 is most similar to the members of mammalian cardiac FABP subfamily. L. japonicus FABP-2 contains three cysteine residues, and a disulfide bond is identified between Cys-81 and Cys-92. A theoretical model of FABP-2 generated by a homology modeling method indicates close proximity of the two cysteine residues in the three-dimensional structure. This is a rather rare case of cytosolic protein having a disulfide bond under the normally reducing conditions of the cytosol, though the presence or absence of disulfide bonds does not seem to affect the ligand-binding ability. (+info)
The contribution of dendritic Kv3 K+ channels to burst threshold in a sensory neuron.
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Voltage-gated ion channels localized to dendritic membranes can shape signal processing in central neurons. This study describes the distribution and functional role of a high voltage-activating K(+) channel in the electrosensory lobe (ELL) of an apteronotid weakly electric fish. We identify a homolog of the Kv3.3 K(+) channel, AptKv3.3, that exhibits a high density of mRNA expression and immunolabel that is distributed over the entire soma-dendritic axis of ELL pyramidal cells. The kinetics and pharmacology of native K(+) channels recorded in pyramidal cell somata and apical dendrites match those of AptKv3.3 channels expressed in a heterologous expression system. The functional role of AptKv3.3 channels was assessed using focal drug ejections in somatic and dendritic regions of an in vitro slice preparation. Local blockade of AptKv3.3 channels slows the repolarization of spikes in pyramidal cell somata as well as spikes backpropagating into apical dendrites. The resulting increase in dendritic spike duration lowers the threshold for a gamma-frequency burst discharge that is driven by inward current associated with backpropagating dendritic spikes. Thus, dendritic AptKv3.3 K(+) channels influence the threshold for a form of burst discharge that has an established role in feature extraction of sensory input. (+info)
Molecular cloning and characterization of spiggin. An androgen-regulated extraorganismal adhesive with structural similarities to von Willebrand Factor-related proteins.
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One of the most definitive examples of a vertebrate extraorganismal structural protein can be found in three-spined sticklebacks (Gasterosteus aculeatus). In the breeding male the kidney hypertrophies and synthesizes an adhesive protein called "spiggin," which is secreted into the urinary bladder from where it is employed as a structural thread for nest building. This paper describes the first molecular characterization of spiggin and demonstrates that this adhesive is a protein complex assembled from a potential of three distinct subunits (alpha, beta, and gamma). These subunits arise by alternative splicing, and 11-ketoandrogens induce their expression in stickleback kidneys. Analysis of the predicted amino acid sequence of each subunit reveals a modular organization whose structural elements display a similarity to the multimerization domains found within von Willebrand Factor-related proteins. These results implicate that spiggin utilizes a conserved multimerization mechanism for the formation of a viscous agglutinate from its constituent subunits in the urinary bladders of male sticklebacks. This novel extraorganismal structural protein is therefore ideally suited to its function as an adhesive thread. (+info)
Synergy of histone-derived peptides of coho salmon with lysozyme and flounder pleurocidin.
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Recent research has identified endogenous cationic antimicrobial peptides as important factors in the innate immunity of many organisms, including fish. It is known that antimicrobial activity, as well as lysozyme activity, can be induced in coho salmon (Oncorhynchus kisutch) mucus after exposure of the fish to infectious agents. Since lysozyme alone does not have antimicrobial activity against Vibrio anguillarum and Aeromonas salmonicida, a four-step protein purification protocol was used to isolate and identify antibacterial fractions from bacterially challenged coho salmon mucus and blood. The purification consisted of extraction with hot acetic acid, extraction and concentration on a C(18) cartridge, gel filtration, and reverse-phase chromatography on a C(18) column. N-terminal amino acid sequence analyses revealed that both the blood and the mucus antimicrobial fractions demonstrated identity with the N terminus of trout H1 histone. Mass spectroscopic analysis indicated the presence of the entire histone, as well as fragments thereof, including a 26-amino-acid N-terminal segment. These fractions inhibited the growth of antibiotic-supersuscptible Salmonella enterica serovar Typhimurium, as well as A. salmonicida and V. anguillarum. Synthetic peptides identical to the N-terminally acetylated or C-terminally amidated 26-amino-acid fragment were inactive in antimicrobial assays, but they potentiated the antimicrobial activities of the flounder peptide pleurocidin, lysozyme, and crude lysozyme-containing extracts from coho salmon. The peptides bound specifically to anionic lipid monolayers. However, synergy with pleurocidin did not appear to occur at the cell membrane level. The synergistic activities of inducible histone peptides indicate that they play an important role in the first line of salmon defenses against infectious pathogens and that while some histone fragments may have direct antimicrobial effects, others improve existing defenses. (+info)