917 resultados para Temperance (Virtue)


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Late endosomes and the Golgi complex maintain their cellular localizations by virtue of interactions with the microtubule-based cytoskeleton. We study the transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network in vitro. We show here that this process is facilitated by microtubules and the microtubule-based motor cytoplasmic dynein; transport is inhibited by excess recombinant dynamitin or purified microtubule-associated proteins. Mapmodulin, a protein that interacts with the microtubule-associated proteins MAP2, MAP4, and tau, stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. The present study shows that mapmodulin also stimulates the initial rate with which mannose 6-phosphate receptors are transported from late endosomes to the trans-Golgi network in vitro. These findings represent the first indication that mapmodulin can stimulate a vesicle transport process, and they support a model in which the microtubule-based cytoskeleton enhances the efficiency of vesicle transport between membrane-bound compartments in mammalian cells.

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The murine ZnT3 gene was cloned by virtue of its homology to the ZnT2 gene, which encodes a membrane protein that facilitates sequestration of zinc in endosomal vesicles. ZnT-3 protein is predicted to have six transmembrane domains and shares 52% amino acid identity with ZnT-2, with the homology extending throughout the two sequences. Human ZnT-3 cDNAs were also cloned; the amino acid sequence is 86% identical to murine ZnT-3. The mouse ZnT3 gene has 8 exons and maps to chromosome 5. Northern blot and reverse transcriptase–PCR analyses demonstrate that murine ZnT-3 expression is restricted to the brain and testis. In situ hybridization reveals that within the brain, ZnT-3 mRNA is most abundant in the hippocampus and cerebral cortex. Antibodies raised against the C-terminal tail of mouse ZnT-3 react with the projections from these neurons and produce a pattern similar to that obtained with Timm’s reaction, which reveals histochemically reactive zinc within synaptic vesicles. We propose that ZnT-3 facilitates the accumulation of zinc in synaptic vesicles.

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From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate/glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (Km for growth ≈8 mM but with Vmax for generation time ≈1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak). The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme IIGlc of the PTS, which we designate ptsG-F. Exconjugants that had acquired ptsG+ from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (Vmax ≈7 h 20 min), even though they were rich in Mak activity. A mutant of E. coli also rich in Mak but unable to grow on glucose by virtue of transposon-mediated inactivations both of ptsG and of the genes specifying enzyme IIMan (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose. Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl α-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence.

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Selenium has been increasingly recognized as an essential element in biology and medicine. Its biochemistry resembles that of sulfur, yet differs from it by virtue of both redox potentials and stabilities of its oxidation states. Selenium can substitute for the more ubiquitous sulfur of cysteine and as such plays an important role in more than a dozen selenoproteins. We have chosen to examine zinc–sulfur centers as possible targets of selenium redox biochemistry. Selenium compounds release zinc from zinc/thiolate-coordination environments, thereby affecting the cellular thiol redox state and the distribution of zinc and likely of other metal ions. Aromatic selenium compounds are excellent spectroscopic probes of the otherwise relatively unstable functional selenium groups. Zinc-coordinated thiolates, e.g., metallothionein (MT), and uncoordinated thiolates, e.g., glutathione, react with benzeneseleninic acid (oxidation state +2), benzeneselenenyl chloride (oxidation state 0) and selenocystamine (oxidation state −1). Benzeneseleninic acid and benzeneselenenyl chloride react very rapidly with MT and titrate substoichiometrically and with a 1:1 stoichiometry, respectively. Selenium compounds also catalyze the release of zinc from MT in peroxidation and thiol/disulfide-interchange reactions. The selenoenzyme glutathione peroxidase catalytically oxidizes MT and releases zinc in the presence of t-butyl hydroperoxide, suggesting that this type of redox chemistry may be employed in biology for the control of metal metabolism. Moreover, selenium compounds are likely targets for zinc/thiolate coordination centers in vivo, because the reactions are only partially suppressed by excess glutathione. This specificity and the potential to undergo catalytic reactions at low concentrations suggests that zinc release is a significant aspect of the therapeutic antioxidant actions of selenium compounds in antiinflammatory and anticarcinogenic agents.

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Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid generation of nested deletions with Cre recombinase was developed and tested. A loxP site was targeted to the Notch1 gene on chromosome 2. A targeted line was cotransfected with a second loxP site and a plasmid for transient expression of Cre. Independent random integrations of the second loxP site onto the targeted chromosome in direct repeat orientation created multiple nested deletions. By virtue of targeting in an F1 hybrid embryonic stem cell line, F1(129S1×Cast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extending six or seven centiMorgans. The cytology of the deletion chromosomes were determined by fluorescent in situ hybridization. Eight deletions were cytologically normal, but the two largest deletions had additional rearrangements. Three deletions, including the largest unrearranged deletion, have been transmitted through the germ line. Several endpoints also have been cloned by plasmid rescue. These experiments illustrate the means to rapidly create and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells.

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The Molecular Biology Database Collection is an online resource listing key databases of value to the biological community. This Collection is intended to bring fellow scientists’ attention to high-quality databases that are available throughout the world, rather than just be a lengthy listing of all available databases. As such, this up-to-date listing is intended to serve as the initial point from which to find specialized databases that may be of use in biological research. The databases included in this Collection provide new value to the underlying data by virtue of curation, new data connections or other innovative approaches. Short, searchable summaries of each of the databases included in the Collection are available through the Nucleic Acids Research Web site, at http://www.nar.oupjournals.org.

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Induction of phase 2 enzymes and elevations of glutathione are major and sufficient strategies for protecting mammals and their cells against the toxic and carcinogenic effects of electrophiles and reactive forms of oxygen. Inducers belong to nine chemical classes and have few common properties except for their ability to modify sulfhydryl groups by oxidation, reduction, or alkylation. Much evidence suggests that the cellular “sensor” molecule that recognizes the inducers and signals the enhanced transcription of phase 2 genes does so by virtue of unique and highly reactive sulfhydryl functions that recognize and covalently react with the inducers. Benzylidene-alkanones and -cycloalkanones are Michael reaction acceptors whose inducer potency is profoundly increased by the presence of ortho- (but not other) hydroxyl substituent(s) on the aromatic ring(s). This enhancement correlates with more rapid reactivity of the ortho-hydroxylated derivatives with model sulfhydryl compounds. Proton NMR spectroscopy provides no evidence for increased electrophilicity of the β-vinyl carbons (the presumed site of nucleophilic attack) on the hydroxylated inducers. Surprisingly, these ortho-hydroxyl groups display a propensity for extensive intermolecular hydrogen bond formation, which may raise the reactivity and facilitate addition of mercaptans, thereby raising inducer potencies.

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Hereditary hemochromatosis (HH) is a common chronic human genetic disorder whose hallmark is systemic iron overload. Homozygosity for a mutation in the MHC class I heavy chain paralogue gene HFE has been found to be a primary cause of HH. However, many individuals homozygous for the defective allele of HFE do not develop iron overload, raising the possibility that genetic variation in modifier loci contributes to the HH phenotype. Mice deficient in the product of the β2-microglobulin (β2M) class I light chain fail to express HFE and other MHC class I family proteins, and they have been found to manifest many characteristics of the HH phenotype. To determine whether natural genetic variation plays a role in controlling iron overload, we performed classical genetic analysis of the iron-loading phenotype in β2M-deficient mice in the context of different genetic backgrounds. Strain background was found to be a major determinant in iron loading. Sex played a role that was less than that of strain background but still significant. Resistance and susceptibility to iron overload segregated as complex genetic traits in F1 and back-cross progeny. These results suggest the existence of naturally variant autosomal and Y chromosome-linked modifier loci that, in the context of mice genetically predisposed by virtue of a β2M deficiency, can profoundly influence the severity of iron loading. These results thus provide a genetic explanation for some of the variability of the HH phenotype.

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p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helix-loop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1.

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Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor.

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In the replication of human immunodeficiency virus type 1 (HIV-1), gag MA (matrix), a major structural protein of the virus, carries out opposing targeting functions. During virus assembly, gag MA is cotranslationally myristoylated, a modification required for membrane targeting of gag polyproteins. During virus infection, however, gag MA, by virtue of a nuclear targeting signal at its N terminus, facilitates the nuclear localization of viral DNA and establishment of the provirus. We now show that phosphorylation of gag MA on tyrosine and serine prior to and during virus infection facilitates its dissociation from the membrane, thus allowing it to translocate to the nucleus. Inhibition of gag MA phosphorylation either on tyrosine or on serine prevents gag MA-mediated nuclear targeting of viral nucleic acids and impairs virus infectivity. The requirement for gag MA phosphorylation in virus infection is underscored by our finding that a serine/threonine kinase is associated with virions of HIV-1. These results reveal a novel level of regulation of primate lentivirus infectivity.

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Radiolabeled antibodies have shown promise for the treatment of lymphoma and for solid tumor targeting. Campath-1H is a humanized monoclonal antibody that reacts with the CD52 antigen present on human lymphoid and myeloid cells. Campath-1H is a gamma1 (G1) isotype that induces lymphopenia via an Fc-mediated mechanism(s). Isotype switches were engineered, and the resulting antibodies were expressed in NS0 mouse myeloma cells and biosynthetically radiolabeled with [35S]methionine. The forms included G1, G4, and a G4 variant that contained alanine substitutions at (EU numbering) Leu-235, Gly-237, and Glu-318. All isotypes bound antigen equivalently as assessed by target cell binding in vitro. The G4 variant had a greatly reduced capacity to interact with Fc receptor by virtue of reduced binding to THP-1 human myeloid cells and by a 1000-fold increase in EC50 to intermediate antibody-dependent cellular cytotoxicity. The pharmacokinetics of the isotypes were compared in CD-1 (nu/nu) mice bearing an experimental antigen-expressing tumor. The plasma half-life and tumor uptake were increased for the G4 variant. The G4 variant showed significantly less spleen, liver, and bone uptake but similar uptake in the lung, kidney, and stomach and lower tissue-to-blood ratios. Immunogenicity was assessed after repeated monthly administrations of unlabeled antibody in BALB/c mice. A 50% reduction in the incidence of anti-globulin response was observed for the G4 variant. These properties suggest that antibodies with reduced Fc receptor interaction merit additional study as potential targeting vehicles relative to other isotypes for radioimmunotherapy or situations where diminished normal tissue binding contributes to efficacy.

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We have isolated the NIL1 gene, whose product is an activator of the transcription of nitrogen-regulated genes, by virtue of the homology of its zinc-finger domain to that of the previously identified activator, the product of GLN3. Disruption of the chromosomal NIL1 gene enabled us to compare the effects of Gln3p and of Nil1p on the expression of the nitrogen-regulated genes GLN1, GDH2, and GAP1, coding respectively for glutamine synthetase, NAD-linked glutamate dehydrogenase, and general amino acid permease. Our results show that the nature of GATAAG sequence that serve as the upstream activation sequence elements for these genes determines their abilities to respond to Gln3p and Nil1p. The results further indicate that Gln3p is inactivated by an increase in the intracellular concentration of glutamine and that Nil1p is inactivated by an increase in intracellular glutamate.

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Expansins are unusual proteins discovered by virtue of their ability to mediate cell wall extension in plants. We identified cDNA clones for two cucumber expansins on the basis of peptide sequences of proteins purified from cucumber hypocotyls. The expansin cDNAs encode related proteins with signal peptides predicted to direct protein secretion to the cell wall. Northern blot analysis showed moderate transcript abundance in the growing region of the hypocotyl and no detectable transcripts in the nongrowing region. Rice and Arabidopsis expansin cDNAs were identified from collections of anonymous cDNAs (expressed sequence tags). Sequence comparisons indicate at least four distinct expansin cDNAs in rice and at least six in Arabidopsis. Expansins are highly conserved in size and sequence (60-87% amino acid sequence identity and 75-95% similarity between any pairwise comparison), and phylogenetic trees indicate that this multigene family formed before the evolutionary divergence of monocotyledons and dicotyledons. Sequence and motif analyses show no similarities to known functional domains that might account for expansin action on wall extension. A series of highly conserved tryptophans may function in expansin binding to cellulose or other glycans. The high conservation of this multigene family indicates that the mechanism by which expansins promote wall extensin tolerates little variation in protein structure.

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Metazoan cyclin C was originally isolated by virtue of its ability to rescue Saccharomyces cerevisiae cells deficient in G1 cyclin function. This suggested that cyclin C might play a role in cell cycle control, but progress toward understanding the function of this cyclin has been hampered by the lack of information on a potential kinase partner. Here we report the identification of a human protein kinase, K35 [cyclin-dependent kinase 8 (CDK8)], that is likely to be a physiological partner of cyclin C. A specific interaction between K35 and cyclin C could be demonstrated after translation of CDKs and cyclins in vitro. Furthermore, cyclin C could be detected in K35 immunoprecipitates prepared from HeLa cells, indicating that the two proteins form a complex also in vivo. The K35-cyclin C complex is structurally related to SRB10-SRB11, a CDK-cyclin pair recently shown to be part of the RNA polymerase II holoenzyme of S. cerevisiae. Hence, we propose that human K35(CDK8)-cyclin C might be functionally associated with the mammalian transcription apparatus, perhaps involved in relaying growth-regulatory signals.