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101.
Summary Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells
capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14–15 wk
gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics
in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through
2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum
(differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in
the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages,
however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold;
expression of immunoreactive sarcomeric actin andα-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation
medium for 20 days before passage and expansion in growth medium. Using isopycinc centrifugation, a high-density cell fraction
was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined
immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing
fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells
proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed
by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable
levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum
withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells my provide a useful experimental system
for the study of human cardiac muscle cell biology. 相似文献
102.
Reactive oxygen species and elastase mediate lung permeability after acid aspiration. 总被引:10,自引:0,他引:10
G Goldman R Welbourn L Kobzik C R Valeri D Shepro H B Hechtman 《Journal of applied physiology》1992,73(2):571-575
Acid aspiration leads to increased neutrophil (PMN) oxidative metabolism, an event associated with lung leukosequestration and permeability increase. Neutropenia protected the vascular barrier function against acid injury. This study tests whether active oxygen species and elastase (which are presumably released by adherent PMNs) affect the microvascular barrier. Anesthetized rats underwent tracheostomy and insertion of a cannula into a lung segment. This was followed by localized instillation of 0.1 N HCl (n = 18) or saline (n = 18). Sequestration of PMNs in acid-aspirated and nonaspirated segments was 77 and 46 PMNs/high-power field (HPF), respectively, which was higher than control values of 11 and 8 PMNs/10 HPF in saline-aspirated and nonaspirated regions (P less than 0.05). Acid aspiration was associated with increased protein concentration in bronchoalveolar lavage (BAL) fluid to 3,550 and 2,900 micrograms/ml in the aspirated and nonaspirated lungs, respectively, which were higher than control values of 420 and 400 micrograms/ml (P less than 0.05). Acid aspiration also led to increased lung wet-to-dry weight ratios (W/D) of 6.6 and 5.4, which were higher than control values of 3.4 and 3.3 (P less than 0.05). Intravenous treatment of rats (n = 18) 90 min after aspiration with scavengers of reactive oxygen species, superoxide dismutase (1,500 U/kg), and catalase (5,000 U/kg), both conjugated to polyethylene glycol, did not reduce PMN sequestration but attenuated acid aspiration-induced increase in protein accumulation in BAL fluid in the aspirated and nonaspirated segments (990 and 610 micrograms/ml) as well as the increased lung W/D (4.6 and 4.0; all P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
103.
The zooplankton-phytoplankton interface in lakes of contrasting trophic status: an experimental comparison 总被引:4,自引:4,他引:0
We report here the results of an experimental study designed to compare algal responses to short-term manipulations of zooplankton
in three California lakes which encompass a broad range of productivity (ultra-oligotrophic Lake Tahoe, mesotrophic Castle
Lake, and strongly eutrophic Clear Lake). To assess the potential strength of grazing in each lake, we evaluated algal responses
to a 16-fold range of zooplankton biomass. To better compare algal responses among lakes, we determined algal responses to
grazing by a common grazer (Daphnia sp.) over a range ofDaphnia densities from 1 to 16 animals per liter. Effects of both ambient grazers andDaphnia were strong in Castle Lake. However, neither ambient zooplankton norDaphnia had much impact on phytoplankton in Clear Lake. In Lake Tahoe, no grazing impacts could be demonstrated for the ambient zooplankton
butDaphnia grazing had dramatic effects. These results indicate weak coupling between phytoplankton and zooplankton in Clear Lake and
Lake Tahoe, two lakes which lie near opposite extremes of lake trophic status for most lakes. These observations, along with
work reported by other researchers, suggest that linkages between zooplankton and phytoplankton may be weak in lakes with
either extremely low or high productivity. Biomanipulation approaches to recover hypereutrophic lakes which aim only to alter
zooplankton size structure may be less effective if algal communities are dominated by large, inedible phytoplankton taxa. 相似文献
104.
O-antigen units are nonuniformly distributed among lipid A-core molecules in lipopolysaccharide (LPS) from gram-negative bacteria, as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the actual distribution patterns are complex, multimodal, and strain specific. Although the basic biochemical steps involved in synthesis and polymerization of O-antigen monomers and their subsequent attachment to lipid A-core are known, the mechanism by which specific multimodal distribution patterns are attained in mature LPS has not been previously considered theoretically or experimentally. We have developed probability equations which completely describe O-antigen distribution among lipid A-core molecules in terms of the probability of finding a nascent polymer (O antigen linked to carrier lipid) of length k (Tk) and the probability that a nascent polymer of length k will be extended to k + 1 by polymerase (pk) or transferred to lipid A-core by ligase (qk). These equations were used to show that multimodal distribution patterns in mature LPS cannot be produced if all pk are equal to p and all qk are equal to q, conditions which indicate a lack of selectivity of polymerase and ligase, respectively, for nascent O-antigen chain lengths. A completely stochastic model (pk = p, qk = q) of O-antigen polymerization and transfer to lipid A-core was also inconsistent with observed effects of mutations which resulted in partial inhibition of O-antigen monomer synthesis, lipid A-core synthesis, or ligase activity. The simplest explanation compatible with experimental observations is that polymerase or ligase, or perhaps both, have specificity for certain O-antigen chain lengths during biosynthesis of LPS. Our mathematical model indicates selectively probably was associated with the polymerase reaction. Although one may argue for a multimodal distribution pattern based on a kinetic mechanism i.e., varying reaction parameters in space or in time during cell growth, such a model requires complex sensory and regulatory mechanisms to explain the mutant data and mechanisms for sequestering specific components of LPS biosynthesis to explain the distribution pattern in normal cells. We favor the simple alternative of enzyme specificity and present generalized equations which should be useful in analysis of other analogous biochemical systems. 相似文献
105.
Extracellular Ca2(+)-dependent inducible alkaline phosphatase from extremely halophilic archaebacterium Haloarcula marismortui. 总被引:1,自引:0,他引:1
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When starved of inorganic phosphate, the extremely halophilic archaebacterium Haloarcula marismortui produces the enzyme alkaline phosphatase and secretes it to the medium. This inducible extracellular enzyme is a glycoprotein whose subunit molecular mass is 160 kDa, as estimated by sodium dodecyl sulfate-gel electrophoresis. The native form of the enzyme is heterogeneous and composed of multiple oligomeric forms. The enzymatic activity of the halophilic alkaline phosphatase is maximal at pH 8.5, and the enzyme is inhibited by phosphate. Unlike most alkaline phosphatases, the halobacterial enzyme requires Ca2+ and not Zn2+ ions for its activity. Both calcium ions (in the millimolar range) and NaCl (in the molar range) are required for the stability of the enzyme. 相似文献
106.
B J Undem T J Torphy D Goldman F H Chilton 《The Journal of biological chemistry》1990,265(12):6750-6758
A mouse spleen-derived mast cell line (PT-18) was employed to examine the mechanisms of adenosine 3':5'-monophosphate (cAMP)-mediated inhibition of antigen-induced lipid mediator biosynthesis. Specifically, we tested the hypothesis that increasing cAMP in mast cells inhibits lipid mediator biosynthesis by a mechanism independent of effects on histamine release (degranulation) or changes in cytosolic calcium concentration. Forskolin inhibited antigen-induced prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4) production by 30-50%. In contrast, forskolin had no inhibitory effect on antigen-induced increases in cytosolic calcium concentration, as monitored by the calcium indicator fura-2, or histamine release from the cells. The combination of the phosphodiesterase inhibitor isobutylmethylxanthine with forskolin inhibited the antigen-induced production of PGD2 and LTC4 by 90-100% and histamine release by about 60%. These responses were accompanied by a virtual abolition of the antigen-induced increase in cytosolic calcium. To test further the hypothesis that increasing cAMP can lead to inhibition of lipid mediator biosynthesis in the absence of effects on cytosolic calcium, we employed the calcium ionophores A23187 and ionomycin. Forskolin alone or in combination with isobutylmethylxanthine had no effect on ionophore-induced increases in cytosolic calcium but effectively inhibited leukotriene biosynthesis. In addition, increasing cyclic AMP led to an inhibition of ionophore-induced production of platelet-activating factor and liberation of arachidonic acid. These data suggest that a relatively modest increase in cAMP-dependent protein kinase activity in mast cells leads to inhibition of the lipase-catalyzed cleavage of arachidonic acid from membrane phospholipids in the absence of measurable effects on either histamine release or changes in cytosolic calcium concentration. This effect results in a selective inhibition of the biosynthesis of lipid mediators including LTC4, LTB4, PGD2, and platelet-activating factor. 相似文献
107.
Modifications of position 12 in parathyroid hormone and parathyroid hormone related protein: toward the design of highly potent antagonists 总被引:1,自引:0,他引:1
M Chorev M E Goldman R L McKee E Roubini J J Levy C T Gay J E Reagan J E Fisher L H Caporale E E Golub 《Biochemistry》1990,29(6):1580-1586
Truncated N-terminal fragments of parathyroid hormone (PTH), [Tyr34]bovine PTH(7-34)NH2, and parathyroid hormone related protein (PTHrP), PTHrP(7-34)NH2, inhibit [Nle8,18,[125I]iodo-Tyr34]-bPTH(1-34)NH2 binding and PTH-stimulated adenylate cyclase in bone and kidney assays. However, the receptor interactions of these peptides are 2-3 orders of magnitude weaker than those of their agonist counterparts. To produce an antagonist with increased receptor-binding affinity but lacking agonist-like properties, structure-function studies were undertaken. Glycine at position 12 (present in all homologues of PTH and in PTHrP), which is predicted in both hormones to participate in a beta-turn, was examined by substituting conformational reporters, such as D- or L-Ala, Pro, and alpha-aminoisobutyric acid (Aib), in both agonist and antagonist analogues. Except for N-substituted amino acids, which substantially diminished potency, substitutions were well tolerated, indicating that this site can accept a wide latitude of modifications. To augment receptor avidity, hydrophobic residues compatible with helical secondary structure were introduced. Incorporation of the nonnatural amino acids D-Trp, D-alpha-naphthylalanine (D-alpha-Nal), or D-beta-Nal into either [Tyr34]bPTH(7-34)NH2 or [Nle8,18,Tyr34]bPTH(7-34)NH2 resulted in antagonists that were about 10-fold more active than their respective 7-34 parent compound. Similarly, [D-Trp12]PTHrP(7-34)NH2 was 6 times more potent than the unsubstituted peptide but retained partial agonistic properties, although markedly reduced, similar to PTHrP(7-34)NH2. The antagonistic potentiating effect was configurationally specific.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
108.
The uptake of morphine was significantly reduced in most regions of the brains of conscious, unrestrained rats within 10 minutes after treatment with an analog of ACTH/MSH (4–9), ORG-2766. The effect was most obvious in regions with significant densities of enkephalin receptors, namely basal ganglia, hippocampus and cortex. The results explain, in part, how some fragments and analogs of ACTH/MSH may antagonize behavioral actions of morphine, even though some of these peptides lack significant opiate receptor binding properties. We believe that this effect of ORG-2766 is related to an action on the permeability characteristics of the brain microvasculature. The underlying mechanism is unknown. 相似文献
109.
Hereditary heat-labile hexosaminidase B: its implication for recognizing Tay-Sachs genotypes 总被引:2,自引:2,他引:0
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R Navon J Nutman R Kopel L Gaber N Gadoth B Goldman M Nitzan 《American journal of human genetics》1981,33(6):907-915
Two pairs of alleles, at the two loci of hexosaminidase (HEX), were found to segregate in an Arab inbred family: the normal and the mutant Tay-Sachs (TSD) alleles of HEX A, and the normal and a mutant allele of HEX B. Since the mutant HEX B is heat labile, no reliable identification of TSD genotypes can be obtained in its presence, as long as the proportions of HEX A and B are estimated by the routinely used heat-inactivation method. The genotypes may be correctly identified in such cases by separation of the two isoenzymes on ion-exchange chromatography, estimating their individual activities, and calculating the ratio between them. Of the nine genotype combinations possible with these two pairs of alleles, five have been identified in the reported family by this procedure. 相似文献
110.
A surface polysaccharide of Escherichia coli O111 contains O-antigen and inhibits agglutination of cells by O-antiserum 总被引:18,自引:9,他引:9
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R C Goldman D White F Orskov I Orskov P D Rick M S Lewis A K Bhattacharjee L Leive 《Journal of bacteriology》1982,151(3):1210-1221
The repeating pentasaccharide of O-antigen from Escherichia coli O111 contains galactose, glucose, N-acetylglucosamine, and colitose, the latter representing the major antigenic determinant. Phenol extraction of this strain was previously shown to release two fractions (I and II) containing O-antigen carbohydrate, and both fractions were believed to be lipopolysaccharide. We have now characterized fractions I and II and conclude that only fraction II represents lipopolysaccharide. Fraction II contains phosphate, 2-keto-3-deoxyoctonate, beta-hydroxymyristic acid, and potent endotoxin activity, whereas fraction I was deficient in all of these properties of the lipid A and core oligosaccharide regions of lipopolysaccharide. Fractions I and II each represented 50% of the total cellular O-antigen, and both were present on the cell surface. Both fractions were metabolically stable, and no precursor-product relationship existed between them. Fraction II had a number-average molecular weight of 15,800, corresponding to an average of 12 O-antigen repeats per molecule. In contrast, fraction I had a number-average molecular weight of 354,000, corresponding to an average of 404 O-antigen repeats per molecule. Before heat treatment, cells of E. coli O111 are poorly agglutinated by O-serum; although this indicates the presence of a capsule, the corresponding K-antigen was never detected. We conclude that fraction I, when present on the cell surface, inhibits agglutination of unheated cultures of E. coli O111 by O-serum because: (i) a variant strain which lacks fraction I was agglutinated by O-serum without prior heating; (ii) erythrocytes coated with purified fraction I behaved like bacteria containing fraction I in showing inhibition of O-serum agglutination; and (iii) heat treatment released fraction I and rendered bacterial cells agglutinable in O-serum. 相似文献