摘要:

AbstractMethods for the induction of an exudate of polymorphonuclear neutrophilic leukocytes (PMN) in the peritoneal cavity of C57BL, BALB/c, SJL and CBA mice were analysed. Peritoneal exudates in male mice were highly enriched for PMN (80‐90%) three hours after a single injection of calcium caseinate whereas eosionophils comprised less than 1% of the exudate population. Female mice were a less satisfactory source of PMN because the proportion of eosinophils in the exudate was variable. Purification of PMN from peritoneal exudate cells was performed on the basis of light scattering using a Becton‐Dickinson cell sorter or by density gradient centrifugation with graded polyvinylpyrroliodone‐coated silica particles (Percoll). Both techniques yielded approximately 97% pure PMN preparations. Electrophoretic analysis of the PMN proteins revealed an abundance of lactoferrin and actin, but several other proteins were also present in high concentrations. Proteolytic degradation of several high molecular weight proteins (>90,000) was prevented by the addition of phenylmethylsulphonyl fluoride (PMSF) and ethylene diamine tetracetic acid (EDTA). Surface iodination, using diphenyl, tetrachloroglycouril (IODO‐DEN), indicated that there were six tyrosine‐containing proteins present on the external cell membrane. The apparent molecular weights of these surface proteins ranged from 185,000 to 90,000 and the major125I‐labeled protein had an apparent molecular weight of 90,000. Neither actin nor lactoferrin was labeled with125I unless cell viability was lost during the iodination procedure. Standard conditions for labeling the cell surface only, required low iodide and IODO‐GEN concentrations. Biosynthetic labeling of PMN using35S‐methionine increased the sensitivity of detection for most of the proteins, but some of the granule storage proteins (such as lactoferrin) were not effectively labeled wit

ISSN:0021-9541    

DOI:10.1002/jcp.1041000102

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

年代:1979

数据来源: WILEY

摘要:

AbstractThe equilibrium distribution of 5,5‐dimethyloxazoladine 2,4‐dione (DMO) between intra‐ and extracellular volume was used to estimate intracellular pH (pHi) inTetrahymena pyriformis. In control experiments, DMO was found to equilibrate rapidly in response to a pH gradient. Under normal growth conditions, pHi was constant over a finite range of external pH, being maintained near pH 7.1 over the external pH range 5.25 to 7.3. This same range of external pH was also optimal for growth. pHi was monitored during the cell cycle of a synchronous population ofT. pyriformisGL. The cells were synchronized either by starvation/refeeding or heat shock. Under both conditions, there were two alkaline shifts of approximately 0.4 pH units per cell cycle. These shifts in pH retained a constant remporal relationship to S phase and were not affected by changes in the time, duration, or magnitude of cytoki

ISSN:0021-9541    

DOI:10.1002/jcp.1041000103

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

年代:1979

数据来源: WILEY

摘要:

AbstractThe potential of nanomelic chondrocytes to synthesize chondroitin sulfate was investigated by providing the mutant cells with p‐nitrophenyl‐β‐D‐xyloside, a compound which acts as an artificial acceptor for glycosaminoglycan synthesis. Under these conditions the synthesis of chondroitin sulfate in nanomelic and normal chondrocytes is comparable. The chondroitin sulfate synthesized by the mutant is indistinguishable in molecular size and composition from that synthesized by similarly treated normal chon

ISSN:0021-9541    

DOI:10.1002/jcp.1041000104

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

年代:1979

数据来源: WILEY

摘要:

AbstractClones of Chinese hamster ovary (CHO) cells were isolated by single‐step selection for resistance to killing by Concanavalin A (ConA) and certain cellular and membrane properties were examined. The ConA‐resistant isolates were only about 2‐fold more resistant than wild type cells to the selecting lectin, but exhibited pleiotropic temperature‐sensitivity for growth, markedly altered morphology and adherence, and significant differences in susceptibility to other agents such as colchicine. Two revertants to full temperature‐resistance were isolated from different ConA‐resistant mutants. One revertant clone had reacquired wild type sensitivity to ConA while the other revertant remained ConA‐resistant. The two series of wild type, ConA‐resistant, and temperature revertant clones were analyzed for altered mobility of cell surface glycoproteins using lactoperoxidase/125I and galactose oxidase/[3H]borohydride labelling procedures. The ConA‐resistant clones showed increased mobility on polyacrylamide gels of three classes of labelled proteins, in the molecular weight ranges 225,000, 200,000, and 130,000 daltons. These changes persisted in the temperature‐revertant that remained ConA‐resistant, while two of the altered protein classes were restored to wild type mobility in the revertant that regained ConA‐sensitivity. Cell hybridization experiments indicated that the temperature‐sensitive phenotypes of different ConA‐resistant isolates are recessive and noncomplementing, implying that the same gene is affected in each case. The reversions to temperature resistance appear to be recessive suppressor m

ISSN:0021-9541    

DOI:10.1002/jcp.1041000105

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

年代:1979

数据来源: WILEY

摘要:

AbstractThe binding of [3H]tuftsin to normal and in vivo stimulated mouse peritoneal macrophage populations was studied at 22°C. The [3H]tuftsin binding to thioglycollate‐stimulated macrophages was shown to be rapid and saturable, with an equilibrium dissociation constant (KD) (calculated from a Scatchard plot) of 5.3 × 10−8M. The calculated number of binding sites per macrophage amounts to approximately 72,000. Binding competition studies with unlabelled tuftsin yielded a KDof 5.0 × 10−8M. [3H] [N‐Acetyl‐Thr1]tuftsin, an inactive analog of tuftsin, failed to bind specifically to thioglycollatestimulated macrophages. [N‐Acetyl‐Thr1]tuftsin and the tripeptide [Des‐Arg4]tuftsin failed to compete for tuftsin binding sites, while [D‐Arg4]tuftsin, an analog with small tuftsin‐like activity, exhibited a low degree of inhibition of [3H]tuftsin binding. Thus a rather high degree of specificity is involved in the binding of the tetrapeptide.Normal as well as six different macrophage populations induced by stimulation with thioglycollate, concanavalin‐A, starch, mineral oil, glucan and Bacillus Calmette Guerrin (BCG), exhibited a similar degree of binding of [3H]tuftsin. Corynebacterium parvum (CP)‐stimulated macrophages, on the other hand, showed a 6‐ to 10‐fold‐lower capacity for tuftsin binding.See Note added in proof on p. 62.Under similar experimental conditions, mouse fibroblast and lymphocyte preparations revealed no detectable specific binding.Tuftsin augmented the phagocytic response of normal and stimulated macrophages assessed both for phagocytosis mediated via the Fc‐receptor and via non‐specific receptors. CP‐stimulated macrophages did not exhibit an increased phagocyti

ISSN:0021-9541    

DOI:10.1002/jcp.1041000106

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

年代:1979

数据来源: WILEY

摘要:

AbstractThe relationship between replication and the synthesis of matrix sulfated proteoglycans was investigated with fetal rat chondrocytes grown in monolayer culture. The effect of N6O2′ dibutyryl adenosine 3′, 5′ cyclic monophosphate (DBcAMP), adenosine 3′, 5′ cyclic monophosphate (cAMP), 8 Bromo adenosine 3′, 5′ cyclic monophosphate (8 Br‐cAMP), sodium butyrate and hydroxyurea was examined. Between 0.05 and 0.5 mM DBcAMP, a dose related inhibition of cell division and stimulation of [35SO 4=] incorporation into matrix proteoglycans was demonstrated. At the higher concentrations of DBcAMP, cell division was completely inhibited and the enhancement of [35SO 4=] incorporation into matrix proteoglycans ranged between 40 and 120% (P<0.01). Utilizing14C‐glucosamine and photometric determination of proteoglycans with Alcian Blue, it was demonstrated that the increase in sulfate incorporation reflected enhanced accumulation of extracellular matrix. The effects of DBcAMP were mimickled by 8 Br‐cAMP, suggesting they were mediated by the adenylyl cyclase system. cAMP (0.05‐0.5 mM), sodium butyrate (0.1‐0.5 mM) and hydroxyurea (0.5‐5 mM) partially or fully inhibited cell division, but either failed or only slightly enhanced sulfate incorporation. The enhanced sulfated proteoglycan deposition promoted by DBcAMP began 8 to 12 hours after serum stimulation, its onset occurred prior to thymidine incorporation and the effect persisted for 28 hours. Determination of cell volume demonstrated an increase in size of DBcAMP treated chondrocytes between 8 and 12 hours, coincident with the onset of increased sulfate incorporation. These results are consistent with a model where matrix sulfated proteoglycan deposition by chondrocytes is mediated by intracellular cAMP levels and occurs in

ISSN:0021-9541    

DOI:10.1002/jcp.1041000107

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

年代:1979

数据来源: WILEY

摘要:

AbstractInfection of BALB/c mice with Rauscher leukemia virus (RLV) gives rise to pronounced erythrocytopoiesis manifesting in splenomegaly and is associated with progressive development of anemia. In the spleen erythroid colony forming units (CFU‐E) increase exponentially up to 800‐fold that of normal levels by the third week of infection. In vitro these CFU‐E are dependent on erythropoietin for colony formation, their erythropoietin requirements being higher than that of CFU‐E from normal mice. Numbers of CFU‐E in spleen and degree of splenomegaly in anemic RLV infected mice were also shown to be modified by red blood cell transfusion, but progression of the disease was not stopped. Erythroid burst forming units (BFU‐E) were also responsive to erythropoietin. However, a small proportion of cells also formed BFU‐E colonies at concentrations which did not support growth of normal marrow BFU‐E.When compared to normal, CFU‐E found in RLV‐infected spleen have similar velocity sedimentation rates. However, buoyant density separation of leukemic spleen cells indicated that CFU‐E were more homogeneous (modal density 1.0695 g/cm3) than CFU‐E from normal spleen. Analysis of physical properties of CFU‐E and the nonhemoglobinized erythroblast‐like cells, which accumulate in the spleen showed that they differed mainly in their distribution of cell diameter.Our findings show that erythroid progenitor cells in RLV infected mice are responsive to erythropoietin in vitro. Also in vivo erythropoiesis appears to be under control of erythropoietin but other factors which lead to progression of RLV disease apparently exist. Most proerythroblast‐like cells, which are characteristic of this disease, apparently lack the potential to form colonies and

ISSN:0021-9541    

DOI:10.1002/jcp.1041000108

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

年代:1979

数据来源: WILEY

摘要:

AbstractBovine corneal and aortal endothelial cell cultures were established from primary explants and subcultured for at least 40 passages. With both cell, exogenous thymidine, folate or folinate markedly increased the proliferation of these cells and decreased their serum requirement in Medium 199.Medium 199 supplemented with thymidine was particularly useful for cell survival at low densities; clones were readily produced when single cells were plated as low as 0.07 cells. cm−2. In contrast to the results of others, neither fibroblast growth factor nor epidermal growth factor were necessary for cell proliferation or survival at low densitie

ISSN:0021-9541    

DOI:10.1002/jcp.1041000109

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

年代:1979

数据来源: WILEY

摘要:

AbstractWhen BHK21 cells transformed by hamster sarcoma virus aregrown in the presence of 5‐Bromodeoxyuridine (BUdr), several in vitro properties of the transformed cells such as morphology, adhesiveness, and alignment, revert towards a state close to that of untransformed cells. We have studied plasma membrane changes associated with this phenotypic reversion by several different biochemical methods. Reversion is accompanied by a reappearance of Fibronectin, an increase in a membrane‐associated protein of M. W. 195,000, a decrease in glycosylation and exposure of a glycoprotein M. W. 100,000 which is increased in transformed cells and a decrease in Con A‐agglutinability. On the other hand, several membrane changes associated with malignant transformation namely, the increase in an integral membrane protein M. W. 177,000, the higher rate of hexose uptake, the increase in high molecular weight surface glycopeptides and, to some extent, the increase in the density of intramembrnous particles, did not revert under BUdr treatment. Thus, membrane properties of transformed cells may be dissociated into two main groups by BUdr treatment. In addition, the exposure and glycosylation of a growth‐regulated membrane protein M. W. 160,000 was highly sensitive

ISSN:0021-9541    

DOI:10.1002/jcp.1041000110

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

年代:1979

数据来源: WILEY

摘要:

AbstractWe have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood.The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+‐ATPase activity were characterized. The Na+, K+‐ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by iptimal concentrations of Na+and K+(Na+, K+‐ATPase) was 1.5 nmol of Pihydrolyzed, μ g protein−1, 30 min−1(range 0.9‐2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The Kmfor K+was approximately 1.0 mM and the Kmfor Na+was approximately 15 mM.Active Na+and K+transport and ouabain‐sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+‐ATPase activity must increase also to transduce energy for the transport of Na+and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+‐ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+‐ATPase when studied at the Kmfor ATP, Na+, K+or Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action

ISSN:0021-9541    

DOI:10.1002/jcp.1041000111

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

年代:1979

数据来源: WILEY