RESUMEN

Epoxyeicosanoids, including the epoxyeicosatrienoic acids are signaling molecules which appear to help ameliorate the effects of a wide variety of pathological conditions. The enzyme soluble epoxide hydrolase (sEH) metabolizes these molecules by converting them to their corresponding vicinal diols. Inhibition of sEH either by knockout or chemical inhibitors increases epoxyeicosanoid levels in vivo and provides significant organ protection in models of brain, cardiac, and renal injury. sEH also appears to be involved in modulating inflammation, pain pathways, pulmonary function, hypertension, and diabetes. Potent sEH inhibitors have been developed in academic, pharmaceutical, and biotech laboratories and described in the patent and scientific literature. Most of the inhibitor scaffolds employ a urea or amide which functions as an active-site transition state mimic. Arête Therapeutics compound AR9281 successfully completed phase Ia and 1b studies. A phase IIa proof of concept trial for treatment of impaired glucose tolerance has been completed, but the results are not yet reported.

Asunto(s)

Inhibidores Enzimáticos/química , Epóxido Hidrolasas/antagonistas & inhibidores , Eicosanoides/química , Eicosanoides/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Epóxido Hidrolasas/metabolismo , Pirazoles/química , Pirazoles/farmacología , Sulfonas/química , Sulfonas/farmacología

RESUMEN

Signaling by some TNF receptor family members, including CD40, is mediated by TNF receptor-associated factors (TRAFs) that interact with receptor cytoplasmic domains following ligand-induced receptor oligomerization. Here we have defined the oligomeric structure of recombinant TRAF domains that directly interact with CD40 and quantitated the affinities of TRAF2 and TRAF3 for CD40. Biochemical and biophysical analyses demonstrated that TRAF domains of TRAF1, TRAF2, TRAF3, and TRAF6 formed homo-trimers in solution. N-terminal deletions of TRAF2 and TRAF3 defined minimal amino acid sequences necessary for trimer formation and indicated that the coiled coil TRAF-N region is required for trimerization. Consistent with the idea that TRAF trimerization is required for high-affinity interactions with CD40, monomeric TRAF-C domains bound to CD40 significantly weaker than trimeric TRAFs. In surface plasmon resonance studies, a hierarchy of affinity of trimeric TRAFs for trimeric CD40 was found to be TRAF2 > TRAF3 >> TRAF1 and TRAF6. CD40 trimerization was demonstrated to be sufficient for optimal NF-kappaB and p38 mitogen activated protein kinase activation through wild-type CD40. In contrast, a higher degree of CD40 multimerization was necessary for maximal signaling in a cell line expressing a mutated CD40 (T254A) that signaled only through TRAF6. The affinities of TRAF proteins for oligomerized receptors as well as different requirements for degree of receptor multimerization appear to contribute to the selectivity of TRAF recruitment to receptor cytoplasmic domains.

Asunto(s)

Antígenos CD40/metabolismo , Proteínas/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Animales , Antígenos CD40/química , Antígenos CD40/fisiología , Humanos , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas/química , Proteínas/genética , Receptores del Factor de Necrosis Tumoral/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Transducción de Señal , Resonancia por Plasmón de Superficie , Factor 1 Asociado a Receptor de TNF , Factor 2 Asociado a Receptor de TNF , Factor 3 Asociado a Receptor de TNF , Factor 6 Asociado a Receptor de TNF

Asunto(s)

Proteínas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Humanos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Proteínas/metabolismo , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Factor 1 Asociado a Receptor de TNF , Factor 2 Asociado a Receptor de TNF , Factor 3 Asociado a Receptor de TNF

RESUMEN

One of the key steps involved in T-cell activation is binding of the tyrosine kinase ZAP-70 via its two SH2 domains to peptide segments termed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZAP-70 SH2 domains complexed to phosphopeptide revealed that the amino-terminal phosphotyrosine-binding pocket is formed at the interface between the two SH2 domains. This study was designed to further characterize the binding between TCR zeta ITAM1 and the ZAP-70 SH2 domains as well as to assess the change in conformation of SH2 domain structure upon zeta ITAM1 binding. BIAcore analysis of wild type and nonfunctional single-point mutants of ZAP-70 SH2 domains demonstrated that the amino-terminal SH2 domain can bind phosphopeptide in the absence of a functional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH2 domain prefers the RREEpYDVLDK sequence of zeta chain ITAM1 over the GQNQLpYNELNL sequence. To assess changes in protein conformation upon ITAM binding to ZAP-70 SH2 domains, fluorescence spectroscopy and analytical ultracentrifugation experiments were performed. A significant blue shift in the tryptophan emission spectrum of the SH2 domains was observed in the presence of saturating amounts of phosphopeptide, indicating a loss in solvent exposure for the tryptophan residues in the protein-phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein structure. Finally, thermal denaturation experiments showed an increase in stability and cooperativity in unfolding for the protein-phosphopeptide complex relative to the protein alone.

Asunto(s)

Proteínas de la Membrana/genética , Proteínas Tirosina Quinasas/genética , Receptores de Antígenos de Linfocitos T/genética , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Vectores Genéticos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70

RESUMEN

The development of a sensitive fluorescence binding assay for evaluating the binding of phosphotyrosyl (pY) peptides to the recombinant SH2 domain of lck in solution is described. Several fluorescent peptides containing the consensus sequence of the viral hamster polyoma middle T antigen (pYEEI) were characterized. The peptides contained either the acetamido-anilino-naphthyl sulfonic acid (AANS), acrylodan, or dansyl groups as fluorophores. The spectral features of these probes were characterized in the presence and absence of the lck SH2 domain. The binding affinities (Kd) for the fluorescent peptides studied ranged from 40 to 500 nM. The fluorescent peptide containing the sequence FTATEC(AANS)QpYEEIP exhibited the highest binding affinity (Kd = 3.98 x 10(-8) M) and largest change in emission intensity (approximately 8.7-fold) upon binding the SH2 domain. This probe was subsequently used in competitive binding assays to study the interaction of the lck SH2 domain with a series of phosphopeptides related to the pYEEIP and pYQPQP (the pY505 C-terminal) consensus sequences. The effects of peptide length and substitutions of residues within the pYEEIP sequence are discussed in terms of binding affinities. Comparison between the two peptide series revealed that the contributions of individual substitutions to binding affinity are context-dependent. The data also led to the conclusion that the presence of P at +2 results in a functional "truncation" of the binding sequence; i.e., residues at positions higher than +2 do not participate significantly in binding. This implicit truncation may actually be a desired property for the autoregulatory nature of the pYQPQP sequence, since it retains specificity for the SH2 domain while adjusting the Kd to a value appropriate for maintaining the delicate balance of receptor-ligand interactions that are involved in signal transduction events.

Asunto(s)

Péptidos/química , Familia-src Quinasas/química , Secuencia de Aminoácidos , Animales , Unión Competitiva , Secuencia de Consenso , Cricetinae , Colorantes Fluorescentes , Técnicas In Vitro , Cinética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/métodos , Espectrometría de Fluorescencia/estadística & datos numéricos , Dominios Homologos src , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo

RESUMEN

The zeta chains of the T cell receptor complex play a critical role in the initiation of proximal signaling events upon T cell activation. Three pairs of potential tyrosine phosphorylation sites are located within the cytoplasmic domains of the zeta chains. Subsequent to engagement of the T cell receptor, one or more of these tyrosine residues is phosphorylated. The phosphotyrosine residues, along with flanking amino acids, form an activation motif (and are shared by signaling subunits in the TCR, B cell receptor, and FcgammaRI) termed tyrosine-based activation motifs (ITAMs). ITAMs serve as binding sites for SH2 domain-containing proteins. Recent evidence suggests that the zeta chains provide docking space for several key signal transduction molecules such as ZAP-70, p56lck, and Shc. To determine if ZAP-70, p56lck, and Shc bind to particular zeta chain ITAM sequences, quantitative free-solution measurements of binding affinities (Kd) were obtained by use of surface plasmon resonance technology. The results indicate that binding affinities of distinct SH2 domains to individual and paired phosphorylation sites greatly differ, and may dictate the sequence of signal transduction events.

Asunto(s)

Proteínas de la Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Dominios Homologos src , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Glutatión Transferasa/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Datos de Secuencia Molecular , Fosforilación , Proteínas Recombinantes de Fusión/metabolismo , Proteína Tirosina Quinasa ZAP-70

RESUMEN

Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B<==>AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists.

Asunto(s)

Péptidos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Unión Competitiva , Cinética , Ligandos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Datos de Secuencia Molecular , Péptidos/química , Fosfotirosina , Unión Proteica , Proteínas Tirosina Quinasas/química , Análisis Espectral/métodos , Tirosina/química , Tirosina/metabolismo

RESUMEN

Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activity of reverse transcriptase of human immunodeficiency virus type-1. Nevirapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 181 and 188 of the p66 subunit of reverse transcriptase. The chromatography which was utilized to explore this binding site is described. BI-RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of nevirapine, are each crosslinked to reverse transcriptase. The use of several HPLC-based techniques employing different modes of detection makes it possible to demonstrate a dramatic difference between the two azido analogues in crosslinking behavior. In particular, by comparing HPLC tryptic peptide maps of the photoadducts formed between reverse transcriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and hence exploitable for the identification of the specifically bonded amino acid residue(s). In addition, comparison of the tryptic maps also makes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectroscopy to identify the crosslinked amino acid residue.

Asunto(s)

Marcadores de Afinidad , Cromatografía Líquida de Alta Presión/métodos , Reactivos de Enlaces Cruzados , VIH-1/enzimología , Piridinas/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Azepinas/metabolismo , Benzodiazepinonas/metabolismo , Transcriptasa Inversa del VIH , Humanos , Nevirapina , Mapeo Peptídico , Fotoquímica , Piridinas/farmacología , Inhibidores de la Transcriptasa Inversa , Análisis de Secuencia , Espectrofotometría Ultravioleta , Tripsina

RESUMEN

Rapid downregulation of L-selectin expression occurs in response to leukocyte activation, and it has been speculated to be an integral process in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is proteolytically cleaved from the cell surface; however, the nature of the cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled phytohemagglutinin-stimulated lymphoblasts and peripheral blood neutrophils. In addition, the anti-cytoplasmic domain serum, but not the antiectodomain serum, immunoprecipitate a 6-kD species from PMA activated lymphoblasts and formylmethionylleucylphenylalanine-activated neutrophils. Conversely, the antiectodomain serum but not the anti-cytoplasmic domain serum immunoprecipitate a 68-kD soluble form of L-selectin from the supernatant of PMA-activated lymphoblasts. The appearance of the 6-kD species on activated cells correlated with the disappearance of the intact form of L-selectin and the appearance of the soluble form of L-selectin. A third polyclonal serum generated against the membrane proximal region of the ectodomain also reacted with the 6-kD species, indicating that this is a transmembrane peptide of L-selectin. That the 6-kD species is derived from L-selectin was confirmed by immunoprecipitation of the 6-kD species from L-selectin transfectants but not from mock transfectants. Radiochemical sequence analysis defined a cleavage site between Lys321 and Ser322, which would predict a transmembrane fragment consistent in size with the observed 6-kD fragment. A Ser-Phe-Ser motif adjacent to the cleavage site is conserved between human, mouse, and rat L-selectin, and a related motif is found proximal to transmembrane domains of other downregulated proteins, such as ACE, CD16-II, and TNF-RII, suggesting the possibility of a common recognition motif.

Asunto(s)

Moléculas de Adhesión Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Western Blotting , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/inmunología , Línea Celular , Membrana Celular/metabolismo , Humanos , Selectina L , Linfocitos/citología , Linfocitos/inmunología , Linfocitos/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/inmunología , Ratones , Datos de Secuencia Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/citología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fitohemaglutininas , Ratas , Homología de Secuencia de Aminoácido , Acetato de Tetradecanoilforbol , Transfección

RESUMEN

Nevirapine is a highly potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) polymerase, but is inactive against HIV-2 and other polymerase. Previous studies demonstrated that residues 176-190 of HIV-1 reverse transcriptase (RT) can confer nevirapine sensitivity to HIV-2 RT. To better characterize the role of this sequence in HIV-1 RT, we have progressively substituted residues 176-190 of HIV-2 RT for those of HIV-1 RT and monitored the impact on the kinetic properties; inhibitory activity of nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[2,3-b:2',3'-e] [1,4]diazepin-6-one), E-BPU (5-ethyl-1-benzyloxymethyl-6-(phenylthio)-uracil), and TIBO-R82150 ((+)-S-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-j k] [1,4]benzodiazepin-2(1H)-thione); and inhibitor-induced fluorescence changes of the mutant enzymes. The study revealed that in addition to Try-181 and Tyr-188, a new amino acid residue (Gly-190) plays an important role in determining susceptibility to nevirapine and E-BPU, but not to TIBO-R82150. These data argue that these non-nucleoside inhibitors fit differently, even though they share a common binding pocket. Nevirapine was seen to exert inhibitory activity by altering the interaction of the enzyme with the template-primer. Kinetic parameters were modulated by the template (DNA versus RNA) as well as by some of the mutations.

Asunto(s)

Antivirales/farmacología , VIH-1/enzimología , VIH-2/enzimología , Piridinas/farmacología , ADN Polimerasa Dirigida por ARN/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Transcriptasa Inversa del VIH , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Nevirapina , Inhibidores de la Transcriptasa Inversa , Espectrometría de Fluorescencia

RESUMEN

The primary 2A/2B cleavage within cardiovirus polyprotein was examined by construction of cDNA plasmids which linked fragments from the P2 region of encephalomyocarditis virus (EMCV) and Mengovirus genomes to the EMCV 5' nontranslated region. When RNA transcripts from these clones were tested in reticulocyte extracts, the synthesized proteins were cotranslationally processed at the 2A/2B site. No viral segments outside of the P2 region were required for this activity. Engineered deletions which removed the amino-terminal two-thirds of protein 2A or the carboxyl half of protein 2B had no effect on this scission, nor did insertions into a Ser-Ala-Phe sequence (SAF) within 2B, which is conserved in most cardio- and aphthoviruses. In contrast, mutations which disrupted a conserved Asn-Pro-Gly-Pro (NPGP) sequence abolished primary scission. Precursors thus inactivated were unable to serve as substrate when simultaneously expressed with active (wild-type) 2AB sequences. Microsequencing placed the EMCV primary cleavage site between the Gly/Pro pair within the NPGP sequence. It was also determined that endogenous viral protease 3C is the previously unidentified agent responsible for cardiovirus 1D/2A scission, a cleavage that is part of the primary processing reaction in poliovirus.

Asunto(s)

Cápside/metabolismo , Virus de la Encefalomiocarditis/metabolismo , Mengovirus/metabolismo , Proteínas/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cápside/química , Cápside/genética , Electroforesis , Virus de la Encefalomiocarditis/genética , Mengovirus/genética , Datos de Secuencia Molecular , Mutagénesis/genética , Plásmidos/genética , Proteínas/química , Proteínas/genética , Proteínas Virales/química , Proteínas Virales/genética

RESUMEN

Milk production, rectal temperature, live weight gain, reproductive performance, and weather data were obtained on 150 Holstein cows managed under two cooling systems on a large dairy farm in Saudi Arabia during the summer months. Cows were paired at the onset of the trial according to days postpartum, lactation number, and current milk production. Females were then allocated either to a system that forced air, precooled by evaporative cooling, over the cows or to a system that alternately showered a fine mist onto the surface of the cows and then forced air at ambient temperature over them. The cows receiving evaporative cooling and those with spray and fan cooling were on sand and on slatted concrete floor, respectively, during the periods of cooling. The onset of estrus was observed during the night when the cows preferred the unshaded corral. For the 120-d trial period, 84% (62 of 75) of the cows receiving evaporative cooling and 60% (44 of 75) of the cows receiving spray and fan cooling became pregnant. In the evaporative cooling system, the pregnancy rate per insemination was 35.2% (179 inseminations) versus 23.2% (194 inseminations) for spray and fan cooling. The mean postpartum interval to pregnancy was 117.6 d for the evaporative cooling cows and 146.7 d for spray and fan cooling cows. The evaporative cooling system, with its open shades and sand bedding, enhanced reproductive performance and milk production compared with that of cows cooled with a spray and fan system with slatted flooring in this hot climate.

Asunto(s)

Aire Acondicionado , Bovinos/fisiología , Industria Lechera/métodos , Lactancia , Reproducción , Animales , Temperatura Corporal , Industria Lechera/economía , Clima Desértico , Estudios de Evaluación como Asunto , Femenino , Humedad , Inseminación Artificial/veterinaria , Embarazo , Arabia Saudita , Estaciones del Año , Temperatura , Aumento de Peso

RESUMEN

Nevirapine, a dipyridodiazepinone, is a highly specific inhibitor of HIV-1 reverse transcriptase (RT) which exhibits an IC50 = 84nM in enzyme assays and IC50 = 40nM against HIV-1 replication in cell culture. This nonnucleoside inhibitor acts noncompetitively with respect to nucleoside triphosphates, template and primer suggesting that nevirapine does not bind to the active site of RT. Studies employing an azido analogue of nevirapine as a photoaffinity probe indicated that one molecule of inhibitor is sufficient to inactivate one molecule of heterodimeric enzyme and demonstrated that only the p66 subunit of p66/p51 heterodimeric RT is covalently labeled by this probe. When subjected to trypic mapping, Tyr 181 and Tyr 188 were labeled with probe and consequently these aromatic residues are apparently near or actually within the RT binding site for nevirapine. The extent to which Tyr 181 and Tyr 188 participate/contribute to nevirapine binding was determined by making amino acid substitutions at these positions using the corresponding residues from HIV-2 RT which is not sensitive to nevirapine. A change at either position dramatically decreased the enzymes' sensitivity to nevirapine, as well as to TIBO derivative and Merck L-693,593, indicating that both Tyr 181 and 188 are crucial for inhibitor-enzyme interaction. Cell culture selection in the continued presence of nevirapine results in the appearance of resistant HIV-1, Tyr 181 to Cys, raising the concern that combination drug therapy will be required in the clinic.

Asunto(s)

Azepinas/farmacología , VIH-1/efectos de los fármacos , Piridinas/farmacología , Inhibidores de la Transcriptasa Inversa , Secuencia de Aminoácidos , Animales , Azepinas/inmunología , Sitios de Unión , Farmacorresistencia Microbiana , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Nevirapina , Piridinas/inmunología , Relación Estructura-Actividad , Especificidad por Sustrato

RESUMEN

Nevirapine (BI-RG-587) is a potent and specific non-nucleoside inhibitor of human immunodeficiency virus type-1 reverse transcriptase. The compound is non-competitive with respect to template, primer, and nucleoside triphosphates indicating that BI-RG-587 does not act directly at the catalytic site. The binding site for this inhibitor was investigated by employing an azido photoaffinity analogue, BI-RJ-70, to covalently label the enzyme. The resulting photoadduct was subjected to enzymatic digestion by trypsin and endoproteinase lys-C and a single, highly labeled peptide was identified as residues 174-199. Sequencing of this peptide identified Tyr-181 and Tyr-188 as labeled residues.

Asunto(s)

Azepinas/metabolismo , VIH-1/enzimología , Piridinas/metabolismo , Inhibidores de la Transcriptasa Inversa , Marcadores de Afinidad , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Nevirapina , Mapeo Peptídico , Tripsina

RESUMEN

We have proposed earlier that caldesmon inhibits the actin-activated ATPase activity of smooth muscle heavy meromyosin (HMM) by inhibiting the binding of the HMM.ATP complex to the productive site of actin (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1868-1885). This has been difficult to prove directly because caldesmon also binds to HMM and it is difficult to distinguish the actin-caldesmon-HMM complex from the actin-caldesmon complex in binding studies. We have eliminated the interaction between caldesmon and smooth HMM by digestion of caldesmon with chymotrypsin. This cleaved caldesmon inhibits the actin-activated ATPase rate of smooth HMM and this inhibition is correlated with a decrease in the binding of HMM.ATP to actin. Therefore, caldesmon functions by inhibiting the binding of the myosin-ATP complex to actin regardless of the source of myosin. We have also isolated the myosin-binding region of caldesmon and have performed a partial sequence. Comparison of this sequence with the derived sequence of caldesmon demonstrates, unequivocally, that the myosin-binding region of caldesmon begins at the amino terminus and extends beyond the first Cys residue.

Asunto(s)

Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Unión a Calmodulina/farmacología , Subfragmentos de Miosina/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Unión a Calmodulina/metabolismo , Pollos , Quimotripsina/farmacología , Molleja de las Aves/metabolismo , Cinética , Datos de Secuencia Molecular , Músculo Liso/metabolismo , Fragmentos de Péptidos/farmacología , Unión Proteica , Conejos

RESUMEN

Retroviral capsid proteins and replication enzymes are synthesized as polyproteins that are proteolytically processed to the mature products by a virus-encoded proteinase. We have purified the proteinase of human immunodeficiency virus (HIV), expressed in Escherichia coli, to approximately 90% purity. The purified enzyme at a concentration of approximately 20 nM gave rapid, efficient, and specific cleavage of an in vitro synthesized gag precursor protein. Purified HIV proteinase also induced specific cleavage of five decapeptide substrates whose amino acid sequences corresponded to cleavage sites in the HIV polyprotein but not of a peptide corresponding to a cleavage site in another retrovirus. Competition experiments with different peptides allowed a ranking of cleavage sites. Inhibition studies indicated that the HIV proteinase was inhibited by pepstatin A with an IC50 of 0.7 microM.

Asunto(s)

VIH/enzimología , Péptido Hidrolasas/metabolismo , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , VIH/genética , Cinética , Péptido Hidrolasas/genética , Péptido Hidrolasas/aislamiento & purificación , Plásmidos , Biosíntesis de Proteínas , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato , Transcripción Genética , Proteínas Virales

RESUMEN

Rabbit and bovine cardiac troponin (Tn) subunits and complexes were labeled with iodo[14C]acetamide in the presence and absence of Ca2+ to determine the effect of tertiary and quaternary structure on exposure of Cys SH groups. This procedure serves both to map regions of subunit interaction and the effects of Ca2+-induced conformational change and to indicate which Cys residues should be useful attachment sites for spectroscopic or cross-linking probes. After being labeled, Tn subunits were purified by using reversed-phase HPLC and subjected to tryptic cleavage with or without prior citraconylation. Cys-containing fragments were isolated by RP-HPLC, and the percent labeling was determined. Cys-75 and -92 of TnI were completely accessible to iodoacetamide both when TnI was labeled alone or when in the TnC-TnI complex. Both residues were largely inaccessible when Tn or the TnI-TnT complex was labeled, suggesting burial in the TnI-TnT interface. In contrast, the Cys from the N-terminal region of bovine TnT was stoichiometrically labeled when TnT was labeled alone, in native Tn or in a troponin-tropomyosin complex. Cys-35 and -84 of TnC are located in the nonfunctional Ca2+ binding loop I of cardiac TnC and helix D, respectively. For TnC alone, the percent labelings of Cys-35 and -84 were 11% and 26%, respectively (minus Ca2+), and 16% and 63%, respectively (plus Ca2+). For TnC labeled within Tn, the percent labelings of Cys-35 and -84 were 20% and 52%, respectively (minus Ca2+), and 20% and 78%, respectively (plus Ca2+).(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Miocardio/metabolismo , Troponina/metabolismo , Animales , Sitios de Unión , Calcio/farmacología , Bovinos , Cromatografía Líquida de Alta Presión , Cisteína , Técnicas In Vitro , Yodoacetamida , Fragmentos de Péptidos/aislamiento & purificación , Conformación Proteica/efectos de los fármacos , Conejos

RESUMEN

Metabolic profiles have been used in efforts to predict periparturient problems and fertility, to diagnose metabolic disease, and to assess nutritional status. Results have been varied. Until knowledge and technology provide improved blood constituent panels, the metabolic profile should not be the first step in the diagnostic process. Rather, such profiles should follow an assessment of management practices and an evaluation of diet. However, these profiles may help to confirm the diagnosis, to convince dairy farmers that management changes are desirable, or to monitor improvement in herd animals. At this point, their major contribution has been to increase our understanding of the factors contributing to changes in blood constituent concentrations, which, in turn, has led to more efficient means of diagnosis. Except in cases of gross mismanagement, these profiles do not offer a "quick fix." In many of the reported cases in which diagnosis of herd problems was attributed to the metabolic profile, the clinician should have been able to identify the problem before the profile was conducted. Profiles are to be recommended when the cause of an existing problem is still not identified or resolved after a complete evaluation. The profile may aid in identifying a factor that has been overlooked. Profiles are not for clinicians who do not have an interest in upgrading their understanding of the factors involved, or who do not have a source of knowledgeable advice.

Asunto(s)

Enfermedades de los Bovinos/diagnóstico , Enfermedades Metabólicas/veterinaria , Animales , Análisis Químico de la Sangre/veterinaria , Bovinos , Enfermedades de los Bovinos/sangre , Enfermedades de los Bovinos/metabolismo , Índices de Eritrocitos/veterinaria , Enfermedades Metabólicas/sangre , Enfermedades Metabólicas/diagnóstico

RESUMEN

Two hundred and four Holstein cows and heifers were randomly assigned to mineral supplement groups 30 d prior to expected calving. Supplement treatment groups were Cu, Mg, Cu plus Mg, and no mineral supplement. The total diet of supplemented groups averaged 15 mg/kg of Cu and .30% of Mg. Pastures consisted of bermudagrass, bahiagrass, and millet during the summer and oats and ryegrass mixture during the winter. Corn and sorghum silage were also fed. Blood samples were taken just prior to initiation of mineral supplementation and at wk 1, 2, 4, 6, and 8 postpartum. Hemoglobin and packed cell volume were determined and plasma was assayed for Cu and Mg. First service conception rates were 57% for the Cu plus Mg treatment and 27, 38, and 33% for treatments 1, 2, and 4, respectively. Ninety-two percent of the cows in the Cu plus Mg-group conceived by 210 d postpartum as opposed to an average of 75% for the other groups. Plasma Mg was different among cows grouped on a fertility basis and hemoglobin was correlated with days to conception. Plasma Mg was correlated with hemoglobin. Both were inversely related to postcalving infection and uterine involution. In summary, cows supplemented with both Cu and Mg showed improved fertility, whereas those supplemented with Cu or Mg alone did not.

Asunto(s)

Enfermedades de los Bovinos/tratamiento farmacológico , Cobre/uso terapéutico , Infertilidad Femenina/veterinaria , Lactancia/efectos de los fármacos , Óxido de Magnesio/uso terapéutico , Animales , Bovinos , Enfermedades de los Bovinos/sangre , Cobre/sangre , Cobre/farmacología , Sulfato de Cobre , Femenino , Hematócrito/veterinaria , Hemoglobinas/análisis , Infertilidad Femenina/tratamiento farmacológico , Magnesio/sangre , Óxido de Magnesio/farmacología , Embarazo

RESUMEN

Heifer, bull, fetal calf sera, and colostral whey were used to evaluate the influence of protein concentrations on percent progressive motility, head-to-head agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence of bovine spermatozoa using ejaculates from 10 bulls. In the first experiment, 10% (vol/vol) addition of undiluted colostral whey resulted in the highest head-to-head agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence. Ten percent (vol/vol) addition of whey diluted to a protein concentration equivalent to fetal calf serum produced significantly lower agglutination, acrosomal integrity, and immunoglobulin G immunofluorescence. Fetal calf serum was unable to produce agglutination and immunoglobulin G immunofluorescence of bovine spermatozoa. Heifer and bull sera produced similar responses for all seminal measurements. In Experiment 2, unheated whey and heifer serum resulted in higher response for all variables than heat inactivated whey and heifer serum. Whey treatment produced greater spermatozoal motility, agglutination, acrosomal integrity; and immunoglobulin G immunofluorescence than treatment with heifer serum. Spermatozoal immunofluorescence indicated antibodies in normal whey, bull, and heifer serum bound to spermatozoal membranes at the acrosomal region. Colostral whey was an effective source of agglutinin factor. Normal unheated whey and heifer serum did not cause sperm damage or immobilization.

Asunto(s)

Proteínas Sanguíneas , Bovinos/fisiología , Calostro/fisiología , Inmunoglobulina G/análisis , Semen/análisis , Espermatozoides/análisis , Animales , Femenino , Técnica del Anticuerpo Fluorescente , Masculino , Embarazo , Espermatozoides/inmunología