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61.
Smooth muscle myosin heavy chains [SM1, approximately 205 kilodaltons (kDa), and SM2, approximately 200 kDa] were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Peptide maps of the two heavy chains showed unique patterns. Limited proteolytic cleavage of purified swine stomach myosin was performed by using a variety of proteases to produce the major myosin fragments which were resolved on SDS gels. A single band was obtained for heavy meromyosin in the soluble fraction following chymotrypsin digestion. However, a variable number of bands were observed for light meromyosin fragments in the insoluble fraction after chymotrypsin digestion. Peptide mapping indicated that the two bands observed after short digestion times with chymotrypsin had relative mobility and solubility properties consistent with approximately 100- and 95-kDa light meromyosin (LMM) fragments. These results indicate that the region of difference between SM1 and SM2 lies in the LMM fragment. 相似文献
62.
Fluorinated derivatives of the chelator o-aminophenol-N,N,O-triacetic acid (APTRA) have been developed, synthesized, and analyzed for use as 19F NMR indicators of free cytosolic magnesium concentration. Magnesium dissociation constants for the 4-fluoro, 5-fluoro, and 4-methyl-5-fluoro species were determined to be 3.1, 0.9, and 0.6 mM, respectively, on the basis of UV absorption measurements at 37 degrees C in 115 mM KCl and 20 mM NaCl, pH 7.1, buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-tris-(hydroxymethyl)aminomethane. The corresponding pK values, which reflect protonation of the nitrogen atom, were determined by 19F NMR to be 4.15, 5.45, and 5.55, respectively, so that the chelators are insensitive to pH variations near the normal physiological range. The dissociation constants of these chelators for calcium ions are lower than those for magnesium but roughly 2-3 orders of magnitude above typical basal cytosolic free calcium levels, so that calcium ions will not interfere with the determinations of magnesium levels. 19F NMR studies carried out at 339.7 MHz indicate that magnesium ions are in slow exchange with the 5-fluoro and 4-methyl-5-fluoro APTRA derivatives and in fast exchange with the 4-fluoro APTRA derivative. In contrast, calcium ions were found to be in intermediate to fast exchange with all chelators. The apparent anomaly of higher thermodynamic stability of the APTRA complexes for calcium relative to magnesium but lower kinetic stability (higher k-1 values) for the calcium complexes reflects the very different association rates for the two ions. Thus, the magnesium association rates are 3 orders of magnitude slower than those for calcium ions.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
63.
The nucleolus organizer locus of Xenopus laevis lampbrush chromosomes was identified by in situ hybridization of a 3H-labelled probe complementary to 18S + 28S rDNA. The nucleolus organizer is an axial granule on chromosome III that lies four-fifths the way down this chromosome reading from its larger (left) telomere, just within an exploded region that extends to its right end, where the lateral loops are exceptionally long. By in situ hybridization of 3H-labelled oocyte and somatic 5S spacer cRNA probes to similarly RNase-treated and denatured lampbrush chromosomes, the multiple sites of oocyte and somatic 5S gene families were identified. Oocyte 5S genes lie at the larger telomeres of the 15 chromosomes that possess these structures; that is, all but chromosomes X, XVII and XVIII. There are a further four sites, all peripheral, and in three of these, on chromosomes VII, X and XI, the sequences lie on lateral loops that are resolvable with the light microscope. By contrast all of the somatic 5S gene clusters occupy peripheral sites. There are two sites on chromosome III, one of which may be shared with oocyte 5S sequences; one on chromosome VII, which is very likely shared with oocyte 5S sequences; one terminal site on chromosome X; one site on chromosome XI that lies on a single pair of long loops which are inserted in a conspicuous and recognizable axial granule, loops which certainly carry oocyte 5S sequences too; two nearly terminal sites alongside the larger telomeres on chromosomes XII and XIV; and single interstitial sites on all three of the sphere-bearing chromosomes, VIII, IX and XVI. We suggest that 5S sequences on resolvable loops are transcribed by readthrough from upstream promoters, probably by polymerase II. 相似文献
64.
Conserved epitopes on the hemagglutinin-neuraminidase proteins of human and bovine parainfluenza type 3 viruses: nucleotide sequence analysis of variants selected with monoclonal antibodies. 总被引:16,自引:10,他引:6 下载免费PDF全文
K J Coelingh C C Winter B R Murphy J M Rice P C Kimball R A Olmsted P L Collins 《Journal of virology》1986,60(1):90-96
We have previously identified 11 epitopes located in two topologically nonoverlapping antigenic sites (A and B) and a third bridging site (C) on the human type 3 parainfluenza virus (PIV3) hemagglutinin-neuraminidase (HN) glycoprotein by using monoclonal antibodies (MAbs) which inhibit hemagglutination and virus infectivity (K. L. Coelingh, C. C. Winter, and B. R. Murphy, Virology 143:569-582, 1985). We have identified three additional antigenic sites (D, E, and F) on the HN molecule by competitive-binding assays of anti-HN MAbs which have no known biological activity. Epitopes in sites A, D, and F are conserved on the bovine PIV3 HN glycoprotein and also among a wide range of human isolates. The dideoxy method was used to identify nucleotide substitutions in the HN genes of antigenic variants selected with neutralizing MAbs representing epitopes in site A which are shared by human and bovine PIV3. The deduced amino acid substitutions in the variants were located in separate hydrophilic stretches of HN residues which are conserved in the primary structures of the HN proteins of both human and bovine PIV3 strains. 相似文献
65.
Characterization of a gene coding for M proteins which is involved in host range restriction of an avian influenza A virus in monkeys. 总被引:8,自引:4,他引:4 下载免费PDF全文
The nucleotide sequence of the region of RNA segment 7 coding for the M1 and M2 proteins of avian influenza A/Mallard/New York/6750/78 was determined, and the deduced amino acid sequences were compared to other avian and human M protein sequences. The M2 proteins of the avian and human viruses have diverged much more than the M1 proteins, although amino acids specific for avian and human viruses were found in both M1 and M2 proteins. 相似文献
66.
67.
R J Baldessarini N S Kula D Francoeur S P Finklestein F Murphy J L Neumeyer 《Life sciences》1986,39(19):1765-1777
The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can induce degeneration of dopamine (DA) and other central monoamine neurons, leading to Parkinson's disease-like effects in man, monkey, and mouse. MPTP and other substituted phenylpiperidines related to synthetic analgesics including alphaprodine and meperidine were evaluated for potency vs. uptake of 0.1 microM tritiated DA, norepinephrine (NE), or serotonin (5HT) in synaptosomal preparations of mouse striatum or cerebral cortex. The most potent inhibitor of the uptake of 3H-DA was N-methyl-4-phenylpyridinium ion (MPP+; IC50 = 1 microM, Ki = 0.4 microM), a metabolite of MPTP; its effect was competitive and reversible. Other analogs of MPTP: the N-ethylindole AHR-1709, N,N-dimethyl-MPTP, and N-methyl-4-phenylpiperidine were all more potent than MPTP against 3H-DA uptake. N-dealkylation and N-propyl substitution, as well as pyridine ring substitution, decreased affinity for DA uptake while 3',4'-dihydroxyphenyl substitution increased potency and selectivity for catecholamine uptake, and quarternarization of the pyridine ring also increased potency against DA uptake. Active compounds showed higher potency against the uptake of NE than of DA. MPP+ was also more potent than MPTP in releasing endogenous DA from striatal synaptosomes (EC50 = 3 vs. 30 microM), but did not release the cytoplasmic markers tyrosine hydroxylase and lactate dehydrogenase (LDH). In contrast to MPTP, synthetic phenylpiperidine analgesics, their potential metabolites and the experimental neuroleptic agent AHR-1709 all failed to deplete striatal DA in vivo, even if active in vitro against DA uptake. 相似文献
68.
Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells 总被引:14,自引:0,他引:14
We explored the hypothesis that discrepancies in the literature concerning the nature of myosin expression in cultured smooth muscle cells are due to the appearance of a new form of myosin heavy chain (MHC) in vitro. Previously, we used a very porous sodium dodecyl sulfate gel electrophoresis system to detect two MHCs in intact smooth muscles (SM1 and SM2) which differ by less than 2% in molecular weight (Rovner, A. S., Thompson, M. M., and Murphy, R. A. (1986) Am. J. Physiol. 250, C861-C870). Myosin-containing homogenates of rat aorta cells in primary culture were electrophoresed on this gel system, and Western blots were performed using smooth muscle-specific and nonmuscle-specific myosin antibodies. Subconfluent, rapidly proliferating cultures contained a form of heavy chain not found in rat aorta cells in vivo (NM) with electrophoretic mobility and antigenicity identical to the single unique heavy chain seen in nonmuscle cells. Moreover, these cultures expressed almost none of the smooth muscle heavy chains. In contrast, postconfluent growth-arrested cultures expressed increased levels of the two smooth muscle heavy chains, along with large amounts of NM. Analysis of cultures pulsed with [35S] methionine indicated that subconfluent cells were synthesizing almost exclusively NM, whereas postconfluent cells synthesized SM1 and SM2 as well as larger amounts of NM. Similar patterns of MHC content and synthesis were found in subconfluent and postconfluent passaged cells. These results show that cultured vascular smooth muscle cells undergo differential expression of smooth muscle- and nonmuscle-specific MHC forms with changes in their growth state, which appear to parallel changes in expression of the smooth muscle and nonmuscle forms of actin (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol. 102, 343-352). The reappearance of the smooth muscle MHCs in postconfluent cells suggests that density-related growth arrest promotes cytodifferentiation, but the continued expression of the nonmuscle MHC form in these smooth muscle cells indicates that other factors are required to induce the fully differentiated state while in culture. 相似文献
69.
Independence of exercise hyperpnea and acidosis during high-intensity exercise in ponies 总被引:1,自引:0,他引:1
Pan L. G.; Forster H. V.; Bisgard G. E.; Murphy C. L.; Lowry T. F. 《Journal of applied physiology》1986,60(3):1016-1024
We investigated arterial PCO2 (PaCO2) and pH (pHa) responses in ponies during 6-min periods of high-intensity treadmill exercise. Seven normal, seven carotid body-denervated (2 wk-4 yr) (CBD), and five chronic (1-2 yr) lung (hilar nerve)-denervated (HND) ponies were studied during three levels of constant load exercise (7 mph-11%, 7 mph-16%, and 7 mph-22% grade). Mean pHa for each group of ponies became alkaline in the first 60 s (between 7.45 and 7.52) (P less than 0.05) at all work loads. At 6 min pHa was at or above rest at 7 mph-11%, moderately acidic at 7 mph-16% (7.32-7.35), and markedly acidic at 7 mph-22% (7.20-7.27) for all groups of ponies. Yet with no arterial acidosis at 7 mph 11%, normal ponies decreased PaCO2 below rest (delta PaCO2) by 5.9 Torr at 90 s and 7.8 Torr by 6 min of exercise (P less than 0.05). With a progressively more acid pHa at the two higher work loads in normal ponies, delta PaCO2 was 7.3 and 7.8 Torr by 90 s and 9.9 and 11.4 Torr by 6 min, respectively (P less than 0.05). CBD ponies became more hypocapnic than the normal group at 90 s (P less than 0.01) and tended to have greater delta PaCO2 at 6 min. The delta PaCO2 responses in normal and HND ponies were not significantly different (P greater than 0.1).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
70.
Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen 总被引:12,自引:0,他引:12
A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices. 相似文献