摘要:

AbstractMore than half the Pima Indians over 35 years of age have non‐insulin dependent diabetes mellitus (NIDDM). They have been the focus of prospective epidemiologic and metabolic studies for over two decades and the data collected during these studies are now providing invaluable in efforts to find genetic markers for NIDDM in humans. The Pima Indian model of this disease affords two major advantages. The population is genetically homogeneous compared to Caucasian populations, and therefore the causes of NIDDM are less heterogeneous, simplifying genetic linkage studies. Equally important, based on results from metabolic studies, two pre‐diabetic phenotypes have been identified in the Pimas: insulin resistance and a low metabolic rate. Use of these phenotypes in genetic linkage analyses should greatly improve chances of finding genetic markers for NIDDM since these phenotypes may be more closely related to the putative abnormal gene products, and actual disease genes, than is the hyperglycemia of the fully developed phenotype of NI

ISSN:0730-2312    

DOI:10.1002/jcb.240480402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe hypothesis that activation of apoptosis and DNA fragmentation is involved in TNF‐mediated cytolysis of U937 tumor cells was investigated. Morphological, biochemical, and kinetic criteria established that TNF activates apoptosis as opposed to necrosis. Within 2–3 h of exposure to TNF, U937 underwent the morphological alterations characteristic of apoptosis. This was accompanied by cleavage of DNA into multiples of nucleosome size fragments. Both of these events occurred 1–2 h prior to cell death as defined by trypan blue exclusion of51Cr release. DNA fragmentation was not a non‐specific result of cell death since U937 cells lysed under hypotonic conditions did not release DNA fragments. The percentage of cells undergoing apoptosis depended on the concentration of TNF and was augmented by the addition of cycloheximide. A TNF‐resistant variant derived from U937 did not undergo apoptosis in response to TNF, even in the presence of cycloheximide. Furthermore, TNF could still activate NFkB in this variant, suggesting that this pathway is not involved in TNF‐mediated cytotoxicity. Two agents known to inhibit TNF‐mediated cytotoxicity, ZnSo4, and 3‐aminobenzamide, were shown to inhibit TNF‐induced apoptosis. Taken altogether, these data support the hypothesis that activation of apoptosis is at least one essential step in the TNFlyticpathway in the

ISSN:0730-2312    

DOI:10.1002/jcb.240480403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe vanadate‐sensitive Mg2+‐dependent ATPase activity of the human erythrocyte ghost is believed to be involved in the shape change events that convert echinocytic ghosts to smoothed forms (biconcave discs and stomatocytes). At physiological salt concentration, pH 7.4, 2 mMATP, 5 mM Mg2+and 1 mM EGTA, the Mg2+‐ATPase activity of ghosts was inhibited strongly by millimolar concentrations of sodium fluoride: I50= 1.31 ± 0.2, mM (mean ± S.D., n = 12). The addition of aluminium chloride to 15 μM reduced the concentration of NaF required for 50% inhibition to 0.76 ± 0.21 mM (n = 10). Aluminium alone had only a small inhibitory effect of the ATPase activity (13 ± 9 %; n = 10). Desferrioxamine, a strong chalator of tervalent aluminium ion, failed to reverse the inbibition by fluoride and reversed the inhibition in the presence of aluminium and fluoride back to those values obtained with fluoride alone. Of several metal salts tested only beryllium sulfate was able to replace aluminium as an effective inhibitor in the presence of fluoride.Inhibition of the Mg2+‐ATPase activity by fluoride and the aluminofluoride complexes correlated with an inhibition of the rate of MgATP‐dependent change in red cell ghost shape from echinocytes to smoothed forms. All gross morphological changes of the smoothing process were affected, including the production of discocytes, stomatocytes and endoc

ISSN:0730-2312    

DOI:10.1002/jcb.240480404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe components of the polymorphonuclear leukocyte (PMNL) receptor for leukotriene B4(LTB4) were examined by Sephacryl S‐300 exclusion chromatography of PMNL membrane proteins, which were solubilized before and after the binding of [3H] LTB4. When the PMNL membranes were solubilized in 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propanesulfonate (CHAPS) and filtered on Sephacryl S‐300 prior to addition of [3H]LTB4, the binding activity was associated with a 65 kD protein. In contrast, the radioactivity of [3H] LTB4bound to PMNL membranes prior to solubilization was recovered predominately with a 140 kD protein. When PMNL membranes had been pretreated with pertussis toxin, but not cholera toxin, before the addition of LTB4and subsequent solubilization, radioactivity was recovered predominately with the 65 kD protein. The addition of guanylylimidodiphosphate (GMP‐PNP), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMNL membrane receptors bearing [3H] LTB4either prior to or after CHAPS solubilization reduced the yield of the 140 kD presumed LTB4receptor protein‐G protein complex. That the maximum specific binding of [35S] guanosine‐5′‐0‐3‐thiotriphosphate (GTP‐gammaS) to LTB4‐binding proteins in the Sephacryl S‐300 effluent corresponded to the 140 kD protein supported the presence of a G pr

ISSN:0730-2312    

DOI:10.1002/jcb.240480405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractBenign prostatic hypertrophy and posterior urethral valves present at both extremes of the age spectrum. Both disease processes can obstruct the urinary stream and ultimately have pathophysiological effects on detrusor structure and function. The mechanisms regulating the structural reorganization of the detrusor to a mechanical outflow obstruction are not known. In an attempt to identify maturational differences in myoctyte ultrastructure and consequent effects these might have in modifying the response of the destrusor to mechanical stimulus, we studied differences in dyanmic nuclear‐cytoskeletal interactions in detrusor tissue in an animal model. Using a drug which spcifically severs actin, cytochalasin D (CD), as an intracellular mechanical stimulus, we measured changes in nuclear area and the rate of DNA synthesis in detrusor myocytes from young (2–3 wee) and old (8–12 mon) guinea pgis. We found that there were age specific differences to intracellular mechanical stimuli in detrusor muscle. Nuclei of myocytes from young animals showed elastic recoil on severing the cell actin matrix and the tissue from young animals increased replicative DNA synthesis with an intracelluar stimulus. In contrast, nuclear shape changes in myocytes from old animals suggested less elasticity, and there was no increase in DNA synthesis with disruption of the cell actin matrix. Anti‐α‐smooth muscle actin antibody and rhodamine phalloidin staining of actin in cytochalasin D treated primary explants of detrusor mycocytes showed dose dependent disruption of the actin component of the cytoskeleton.These results suggest that there are fundamental modifications in detrusor myocyte ultrastructure with age. These maturational changes might result in differences in the pathophysiological and structural reorganization of the destrusor in response to outflow obstruction in infancy and adulthood. Furthurmore, they suggest that (1) a tensile equilibrium exists between the myocyte nucleus and cytoskeleton; (2) there appears to be a decrease in myocyte nuclear elasticity with ageing; (3)release of nuclear template restrictions increases activity of DNA polymerase α in young, but not old, detrusor myocytes; and (4) mechanico‐chemical signal transduction in detrusor myocytes may be mediated via the cytoskeleton. In addition, based on previous reports of actin within the nucleus, the results suggest that (1) nuclear actin may have a homeostatic structural role, maintaining the tensile equilibrium between nucleus and cytoskeleton, and (2) integrity of nuclear actin may function to maintain the spatial template restriction of DNA polymera

ISSN:0730-2312    

DOI:10.1002/jcb.240480406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractIn this report, conditions have been established for utilizing monoclonal antibodies and fluorescence activated flow cytometry in studying antigen expression by primary porcine stromal‐vascular cells cultured under various conditions. Single cells were isolated from cultures maintained in DME/F12 medium containing 10% fetal bovine serum, 2% pig serum, and containing 2% pig serum and 10 nM dexamethasone supplemented with growth hormone (GH), tumor necrosis factor‐alpha (TNF‐α), and transforming growth factor‐beta (TGF‐β). Flow cytometric analyses revealed that the proportion of cells expressing detectable levels of the AD‐1 cell surface antigen was greater in cultures supplemented with 2% pig serum and 10nM dexamethasone than in other media. In cultures, GH, TNF‐α and TGF‐β each inhibited lipid deposition, whereas TNF‐α and TGF‐β, but not GH, inhibited AD‐1 antigen expression. Inhibition of lipid deposition as well as antigen expression by TNF‐α and TGF‐β was reversible, but inhibition of cluster formation by GH was not reversed upon removal from cultures. In summary, differential effects of factors on surface antigen expression by preadipocytes are detectable by flow cytometry. Flow cytometric analysis using monoclonal antibodies produced against key developmentally regulated cell surface antigens is potentially a powerful analytical approach to t

ISSN:0730-2312    

DOI:10.1002/jcb.240480407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractRecent clinical studies have shown that calcium channel blockers can retard and possibly reduce the angiographic progression of coronary artery disease. Calcium channel blockers also inhibit dietary‐induced atherosclerosis in animal models of this disease. In this study, we delineate potential cellular and molecular mechanisms by which nicardipine, a dihydropyridine calcium antagonist, may alter lipoprotein and cholesterol trafficking, affect the regulatory signal transduction pathways involved in accelerating cholesteryl ester (CE) catabolism in vascular smooth muscle cells, and modulate cell‐cell interactions of vascular and inflammatory cells. We demonstrate in arterial smooth muscle cells that nicardipine increases (1) LDL binding, uptake, and degradation, (2) RNA transcript levels for the LDL receptor, (3) CE catabolic activity, (4) PGI2release, and (5) RNA transcript levels for cyclooxygenase. Furthermore, nicardipine blocked cytokine‐induced monocyte adhesion to endothelial cells and smooth muscle cells. Taken together, these findings support the hypothesis that nicardipine may function as an anti‐atherosclerotic agent by promoting CE catabolism and cholesterol clearance and by reducing monocyte adhesion to the activated endo

ISSN:0730-2312    

DOI:10.1002/jcb.240480408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractGallium(III) is a new therapeutic agent for hypercalcemia. Ga3+reduces osteoclast action, but how it inhibits the cell's physiology is unknown. In vivo, 7–12 μM Ga(III) reduces calcium release from bone, but surprisingly, 10–100 μM Ga3+; added to isolated avian osteoclasts did not reduce their degradation of L‐(5‐3H)‐proline bone.3H‐proline labels bone collagen specifically, and collagenolysis is an excellent indicator of bone dissolution because collagen is the least soluble component of bone. Ga(III)>100 μM inhibited osteoclasts in vitro, but also killed the cells. To resolve this apparent conflict, we measured67Ga distribution between bone, cells, and media. Gallium binds avidly but slowly to bone fragments. One hundred micrograms of bone clears 60% of 1 μM gallium from 500 μI of tissue culture medium, with steady state at>24 h. Osteoclasts on bone inhibited gallium binding capacity ∼ 40%, indicating a difference in available binding area and suggesting that osteoclasts protect their substrate from Ga binding. Less gallium binds to bone in serum‐containing medium than in phosphate‐buffered saline; 30% reduction of the affinity constant suggests that the serum containing medium competes with bone binding. Consequently, the effect of [Ga] on bone degradation was studied using accurately controlled amounts of Ga(III) pre‐bound to the bone. Under these conditions, gallium sensitivity of osteoclasts is striking. At 2 days, 100 μg of bone pre‐incubated with 1 ml of 1 μM Ga3+, with 10 pmoles Ga3+/μg bone, was degraded at 50% the rate of control bone; over 50 pM Ga3+/μg bone, resorption was essentially zero. In contrast, pre‐treatment of bone with [Ga3+] as high as 15 μM had no significant effect on bone resorption rate beyond 3 days, indicating that gallium below ∼150 pg/μg bone acts for a limited time and does not permanently damage the cells. We conclude that bone‐bound Ga(III) from medium concentrations<15 μM inhibits osteoclasts reversibly, while irreversible t

ISSN:0730-2312    

DOI:10.1002/jcb.240480409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractA radiometric assay has been developed for the detection of proteolytic activity capable of releasing transforming growth factor alpha (TGFα) from its membrane bound precursor. The assay is dependent upon the separation by thin layer chromatography of hydrolytic products of a nonapeptide substrate containing a radioactive iodinated tyrosine residue as a reporting group N‐terminal to an octapeptide which is cognate to the N‐terminal cleavage sequence of TGFα. We describe the selectivity of the peptidase assay with commercially purified proteases and with cell‐associated peptidases, its exquisite sensitivity, and its applicability to defining peptidase activity, which may be responsible for the processing of the membrane‐bound prepro TGFα. The activity of two different elastases had different profiles which thus may be of use in characterizing them. The characteristics of the intact and extracted HeLa cell assay with respect to time, cell density, and peptidase concentration are defined, as are conditions needed to remove endogenous, confounding, proteolytic activity from the serum used to support cell culture. Intact HeLa cell cultures exhibit both exo‐ and endo‐peptidase activity at approximately equal levels in both sparse and dense monolayer culture without relationship to cell density, and at a level equal to 1–2% of total cell activity of thes

ISSN:0730-2312    

DOI:10.1002/jcb.240480410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractMC3T3‐G2/PA6 (PA6) cells established from newborn mouse calvaria are preadipocytic stromal cells, which differentiate into adipocytes in response to glucocorticoids. We examined the effects of 1α,25‐dihydroxyvitamin D3[1α,25(OH)2D3] on adipogenesis in PA6 cells were cultured with 10−8M dexamethasone, adipocytes containing oil red O‐positive droplets first appeared on day 7 (3 days after confluence was attained) and the maximal synthesis of neutral lipids occurred on day 12. Simultaneous addition of 1α,25(OH)2D3at 10−9M completely blocked this dexamethasone‐induced neutral lipid synthesis throughout the 14‐day culture period. Dose‐response studies of vitamin D3derivatives showed that 1α,25(OH)2D3was the most potent in inhibiting neutral lipid synthesis in PA6 cells, followed by 1α‐hydroxyvitamin D3, 25‐hydroxyvitamin D3and 24R,25‐dihydroxyvitamin D3, in that order. Dexamethasone greatly enhanced incorporation of [14C]‐acetic acid into triacylglycerol in PA6 cells. The incorporation was markedly inhibited by the addition of 10−9M 1α,25(OH)2D3Instead, 1α,25(QH)2D3greatly increased incorporation of [14C]‐acetic acid into phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, irrespective of the presence or absence of dexamethasone. These results suggest that 1α,25(OH)2D3modulation of lipid metabolism in bone marrow

ISSN:0730-2312    

DOI:10.1002/jcb.240480411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY