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1.
The mixed lymphocyte culture (MLC) and antigen-mediated proliferative response represent important correlates to the in vivo phenomena of allograft rejection and delayed hypersensitivity. This study defines an in vitro model to measure mouse lymphocyte responsiveness to allogeneic cells, antigen (tuberculoprotein), and nonspecific mitogens. Results describe optimal cells concentration, time and conditions of culture. Optimal conditions include the use of high cell concentration, flat-bottomed vials, RPMI-1640 medium, and fresh human serum. Peripheral blood lymphocytes demonstrated greater proliferation than lymph node lymphocytes, which in turn demonstrated greater activity than splenic lymphocytes. Significant proliferation occurred in serum-free media, dialyzed against fresh serum and supplemented with hydrocortisone and carrier protein. The MLC response in the mouse appears dependent on multiple subpopulations of cells and on soluble substances produced by them. 相似文献
2.
Influence of myosin isoforms on contractile properties of intact muscle fibers from Rana pipiens 总被引:3,自引:0,他引:3
Lutz GJ Sirsi SR Shapard-Palmer SA Bremner SN Lieber RL 《American journal of physiology. Cell physiology》2002,282(4):C835-C844
The myosin heavy chain (MHC) andmyosin light chain (MLC) isoforms in skeletal muscle of Ranapipiens have been well characterized. We measured theforce-velocity (F-V) properties of single intact fast-twitchfibers from R. pipiens that contained MHC types 1 or 2 (MHC1or MHC2) or coexpressed MHC1 and MHC2 isoforms. Velocities weremeasured between two surface markers that spanned most of the fiberlength. MHC and MLC isoform content was quantified after mechanicsanalysis by SDS-PAGE. Maximal shortening velocity(Vmax) and velocity at half-maximal tension(VP 50) increased with percentage of MHC1(%MHC1). Maximal specific tension (Po/CSA, wherePo is isometric tension and CSA is fiber cross-sectional area) and maximal mechanical power (Wmax) alsoincreased with %MHC1. MHC concentration was not significantlycorrelated with %MHC1, indicating that the influence of %MHC1 onPo/CSA and Wmax was due to intrinsicdifferences between MHC isoforms and not to concentration. TheMLC3-to-MLC1 ratio was not significantly correlated withVmax, VP 50,Po/CSA, or Wmax. These data demonstrate the powerful relationship between MHC isoforms and F-V properties of the two most common R. pipiensfiber types. 相似文献
3.
Knowledge of the embryonic morphogenetic movements of any groupof vertebrates must be explored by experimental means in orderto answer the questions and correct the errors left over fromsimply watching development or studying a series of stages inserial sections. The holostean fishes are among numerous importantgroups of vertebrates that have not previously been exploredby the newer cell-marking techniques. Because of superficialresemblances of the epibolic blastodisc and yolk plug of theAmia embryo to those of amphibia, earlier reports usually postulatedfor this species an invagination of cells over a dorsal lip.Vital staining of the surface cells calls this into question,showing that most if not all of the mesoderm and endoderm isformed from originally internal cells without contribution fromthe outer layers that form the general ectoderm and the nervoussystem. The prostomal thickening, a cluster of cells under theso-called dorsal lip, was earlier considered to arise from varioussources and seen as the agent which formed more or less of theendoderm by a process called invagination. Evidence is presentedthat the prostomal thickening is not a constant feature in Amiaembryos, when present has not been derived from the embryo'sexternal epithelial covering, and has little if anything todo with formation of the endoderm. 相似文献
4.
In rye the total phosphorus, potassium, and iron content eachincreases linearly with increase in seedling weight up to tenweeks from germination. The rate of increase of mineral withweight varies between genotypes. In F1 families showing hybridvigour and, also, in faster growing inbred families the rateof increase of mineral with increasing weight is low comparedwith that in less vigorous families. It is suggested that amore efficient utilization of mineral material accounts in partfor the greater vigour of some F1 and inbred genotypes. 相似文献
5.
M A Bean M Akiyama Y Kodera B Dupont J A Hansen 《Journal of immunology (Baltimore, Md. : 1950)》1979,123(4):1610-1614
A nontransfused male patient with recurrent bladder carcinomata has been demonstrated to have blood T lymphocytes that suppress the MLC responsiveness of lymphocytes only from normal individuals positive for HLA-B14. Unexpectedly, this T-suppressor cell differs from three other reported blood T-suppressor cells arising in man in that it apparently does not require HLA-D locus compatibility between the suppressor cell and the lymphocyte being suppressed. 相似文献
6.
Four inbred (Wf9, Hy2, B10, and 3811) plus three hybrid(Wf9 x 38.11, US13, and B10 x Hy2) corn (Zea mays) seedlingstrains (minus seed coat and endosperm) were assayed for totaland free myoinositol content, using a mutant inositol-less straino Neurospora crassa. Over a 120-h germination period, it wasfound that the concentration of total myoinositol decreasedin both of the respective varieties. The inbred corn seedling strains, assayed in this study, appearto demonstrate a lower decrease in total myoinositol than dothe hybrids, over the same 120-h germination period. Concomitantly,the hybrid strains have a higher percentage increase in freemyoinositol than do the inbred varieties, over the same germinationtime period. The hybrid corn seedling strains appear to havea greater capacity to mediate the metabolism of total to freemyoinositol, during the early stages of germination, comparedto the inbred varieties. 相似文献
7.
CS7BL/6 mice were sensitized with an ip injection of allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the tumor allosensitized mice were cultured and tested for their responsiveness to mitogens and alloantigens, and for their ability to generate cytotoxic cells in vitro. The results indicate that 15 day tumor-sensitized spleen cells are hypo-responsive in mixed lymphocyte culture (MLC) with DBA/2 or AKR as stimulating spleen cells. The cells which are hypo-responsive in MLC can proliferate in response to mitogens and they also can generate cytotoxic cells in vitro. MLC reactivity recovers in about 2–3 months which is months after the mice have rejected their tumors. The mechanism of MLC hypo-responsiveness was investigated. The results suggest the presence of a suppressor cell which does not appear to be a macrophage or a B-cell. The suppressor cell can be separated from the cytotoxic cell and therefore appears to be a noncytotoxic T-cell. 相似文献
8.
Depolarization induces Rho-Rho kinase-mediated myosin light chain phosphorylation in kidney tubular cells 总被引:2,自引:0,他引:2
Szászi K Sirokmány G Di Ciano-Oliveira C Rotstein OD Kapus A 《American journal of physiology. Cell physiology》2005,289(3):C673-C685
Myosin-based contractility plays important roles in the regulation of epithelial functions, particularly paracellular permeability. However, the triggering factors and the signaling pathways that control epithelial myosin light chain (MLC) phosphorylation have not been elucidated. Herein we show that plasma membrane depolarization provoked by distinct means, including high extracellular K+, the lipophilic cation tetraphenylphosphonium, or the ionophore nystatin, induced strong diphosphorylation of MLC in kidney epithelial cells. In sharp contrast to smooth muscle, depolarization of epithelial cells did not provoke a Ca2+ signal, and removal of external Ca2+ promoted rather than inhibited MLC phosphorylation. Moreover, elevation of intracellular Ca2+ did not induce significant MLC phosphorylation, and the myosin light chain kinase (MLCK) inhibitor ML-7 did not prevent the depolarization-induced MLC response, suggesting that MLCK is not a regulated element in this process. Instead, the Rho-Rho kinase (ROK) pathway is the key mediator because 1) depolarization stimulated Rho and induced its peripheral translocation, 2) inhibition of Rho by Clostridium difficile toxin B or C3 transferase abolished MLC phosphorylation, and 3) the ROK inhibitor Y-27632 suppressed the effect. Importantly, physiological depolarizing stimuli were able to activate the same pathway: L-alanine, the substrate of the electrogenic Na+-alanine cotransporter, stimulated Rho and induced Y-27632-sensitive MLC phosphorylation in a Na+-dependent manner. Together, our results define a novel mode of the regulation of MLC phosphorylation in epithelial cells, which is depolarization triggered and Rho-ROK-mediated but Ca2+ signal independent. This pathway may be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and thereby regulate paracellular transport. membrane potential; Na+-alanine cotransport; epithelium; phosphatidylinositol 3-kinase; LLC-PK1 cells 相似文献
9.
Human peripheral blood leukocytes were exposed to either PWM or Con A mitogens. Cells activated by both these mitogens were able to depress proliferation in an MLC, and to inhibit the generation of spontaneous killer cell (SK) and induced T-cell cytotoxic activity. PWM-activated cells incubated in media for 48 hr were able to elaborate a soluble factor in vitro. This factor suppressed cytotoxicity, and was active only when present at the initiation of MLC cultures. In contrast, cells exposed to Con A were able to suppress immune responsiveness, but this population did not release a soluble factor which could inhibit cytotoxicity. PWM induction appears to be dependent on phagocytic cells, while Con A activation is less dependent on this adherent population. An enriched adherent cell population, stimulated with PWM, was able to suppress cytotoxicity. Thus, the PWM-stimulated system of suppression is mediated through a soluble factor and is dependent on adherent cells. 相似文献
10.
Effects of unweighting and clenbuterol on myosin light and heavy chains in fast and slow muscles of rat 总被引:3,自引:0,他引:3
Stevens L Firinga C Gohlsch B Bastide B Mounier Y Pette D 《American journal of physiology. Cell physiology》2000,279(5):C1558-C1563
To investigate the plasticityof slow and fast muscles undergoing slow-to-fast transition, rat soleus(SOL), gastrocnemius (GAS), and extensor digitorum longus (EDL) muscleswere exposed for 14 days to 1) unweighting by hindlimbsuspension (HU), or 2) treatment with the2-adrenergic agonist clenbuterol (CB), or 3)a combination of both (HU-CB). In general, HU elicited atrophy, CBinduced hypertrophy, and HU-CB partially counteracted the HU-induced atrophy. Analyses of myosin heavy (MHC) and light chain (MLC) isoformsrevealed HU- and CB-induced slow-to-fast transitions in SOL (increasesof MHCIIa with small amounts of MHCIId and MHCIIb) and theupregulation of the slow MHCIa isoform. The HU- and CB-induced changesin GAS consisted of increases in MHCIId and MHCIIb("fast-to-faster transitions"). Changes in the MLC composition ofSOL and GAS consisted of slow-to-fast transitions and mainlyencompassed an exchange of MLC1s with MLC1f. In addition, MLC3f waselevated whenever MHCIId and MHCIIb isoforms were increased. Becausethe EDL is predominantly composed of type IID and IIB fibers, HU, CB,and HU-CB had no significant effect on the MHC and MLC patterns. 相似文献
11.
CBA spleen T lymphocytes were stimulated by the T mitogens concanavalin-A (Con-A), phytohemagglutinin (PHA), and leukoagglutinin (LA). On the 2nd to 3rd culture day the activated cells (blasts) were separated from the nonactivated cells (lymphocytes) by 1g velocity sedimentation. The lymphocytes which were not activated during the primary culture (lymphocyte fraction from the velocity sedimentation) were then stimulated by the same mitogens or in one-way MLC to DBA/2 m, and tested for relevant target lysis after MLC stimulation. Primary stimulation with Con-A abolished the responses to Con-A, to PHA, and to LA, whereas primary stimulation with PHA or with LA abolished the responses to these mitogens but left behind a considerable Con-A response. Stimulation with any one of the listed T mitogens did not significantly affect the MLC responses. While primary stimulation with Con-A abolished the relevant target cell lysis after MLC stimulation, primary stimulation with PHA or with LA reduced it only slightly. Assuming that the various mitogens stimulate separate subpopulations of T cells, the results seem to indicate that the Con-A-responsive population includes the PHA- and LA-responsive populations but not the MLC-responsive population. It also appears that the T cells generated to killer cells during MLC are mainly confined to the concanavalin-responsive population. 相似文献
12.
The role of gibberellic acid (GA3) in controlling the secretion(across the plasma membrane) and release (through the cell wall)of acid phosphatase (E.C. 3.1.3.2
[EC]
.) from Avena aleurone layershas been investigated. Evidence from this comparative studywith intact aleurone layers and isolated aleurone protoplastsreveals that the secretion of acid phosphatase is under GA3control. The mechanism underlying secretion and release of theenzyme from aleurone cells is discussed. Key words: Avena fatua, Acid phosphatase, Aleurone protoplasts, Gibberellic acid, Secretion 相似文献
13.
Noll T Schäfer M Schavier-Schmitz U Piper HM 《American journal of physiology. Cell physiology》2000,279(3):C717-C723
In cultured porcine aortic endothelial monolayers, theeffect of ATP on myosin light chain (MLC) phosphorylation, whichcontrols the endothelial contractile machinery, was studied. ATP (10 µM) reduced MLC phosphorylation but increased cytosolicCa2+ concentration ([Ca2+]i).Inhibition of the ATP-evoked [Ca2+]i rise byxestospongin C (10 µM), an inhibitor of the inositol trisphosphate-dependent Ca2+ release from endoplasmicreticulum, did not affect the ATP-induced dephosphorylation of MLC. MLCdephosphorylation was prevented in the presence of calyculin A (10 nM),an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activatesMLC dephosphorylation in a Ca2+-independent manner. In thepresence of calyculin A, MLC phosphorylation was incremented afteraddition of ATP, an effect that could be abolished when cellswere loaded with the Ca2+ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester (10 µM). Thus ATP also activates aCa2+-dependent kinase acting on MLC. In summary, ATPsimultaneously stimulates a functional antagonism toward bothphosphorylation and dephosphorylation of MLC in which thedephosphorylation prevails. In endothelial cells, ATP is the firstphysiological mediator identified to activate MLC dephosphorylation bya Ca2+-independent mechanism. 相似文献
14.
The inbred strain STS/A exhibits a higher proliferative response in the mixed lymphocyte culture (MLC) to stimulator cells
of all 11 tested inbred mouse strains with 10 different major histocompatibility complex (MHC) haplotypes, as well as to stimulation
with IL-2 than does the strain BALB/cHeA. However, alloantigen-stimulated BALB/c cells produce more IL-2 than STS/A cells.
To study the genetic basis of these differences, we used 20 recombinant congenic strains (RCS) of the CcS/Dem series. Each
of these CcS/Dem RC strains contains a different subset of about 12.5% of genes from the STS/A strain and the remaining approximately
87.5% of BALB/c origin genes. As a result the multiple non-linked genes responsible for phenotypic differences between BALB/c
and STS/A became separated into different CcS/Dem strains. The strain distribution pattern (SDP) of high or low MLC response
of individual CcS/Dem strains to stimulator cells of four different strains was almost identical, indicating that differences
in responsiveness, rather than the alloantigenic difference itself, determine the magnitude of the response, and that the
responsiveness to different alloantigens is largely controlled by the same genes. The SDP of IL-2 stimulation was different
from that of MLC responsiveness. The differences in the proliferative responses observed among individual CcS/Dem strains
were not due to differences in numbers of CD3+, CD4+ or CD8+ cells or to the observed differences in IL-2 production, and hence they likely reflect genetically determined intrinsic properties
of T cells. These results show that a set of non-linked genes controls proliferative responses in MLC irrespective of the
MHC haplotype of the stimulator cells, and that stimulation with IL-2 and production of IL-2 are controlled by different subsets
of genes. Since the genomes of all RCS are extensively characterized by microsatellite markers, they can be used to map the
genes controlling proliferative responsiveness to stimulation with alloantigens and IL-2. 相似文献
15.
Previous studies have demonstrated defective monocyte leukotaxis in human sarcoidosis and tuberculosis, diseases associated with chronic cellular immune activation. In the present study, we sought to determine if acute cellular immune activation might also alter this monocyte function, using a primate model of skin allograft rejection. A profound, short-lived depression of monocyte leukotactic responsiveness occurred during the time of allograft rejection. The leukotactic defect was mediated by a cell-directed inhibitor in plasma, a heat-stable protein of 230,000 daltons molecular weight. Neither monocyte leukotactic responsiveness nor plasma inhibitory activity was affected by autografting or nonspecific cutaneous inflammation. A physicochemically and functionally similar inhibitor was produced in vitro during the course of one-way primed mixed lymphocyte cultures. The cell-directed inhibitor appears to be a lymphocyte product which is clearly distinguishable from MIF and leukotactic lymphokines. 相似文献
16.
The water status and water content of embryos of Quercus roburhave been determined during development. Using psychrometers,changes in osmotic potential at full imbibition and the proportionof water at zero reciprocal water potential, R0, were estimatedfrom curves of embryo relative water content and reciprocalwater potential. The specific moisture content of embryos decreasedduring development, with a concomitant increase in the osmoticpotential. However, R0 also increased such that, during thelast four weeks of development, there was little net changein the content of osmoticum per dry weight. Evidence is presentedthat starch stored in the cotyledon may be the major site ofmatrix-bound water, R0. Isolated embryonic axes had a slightly higher osmotic potentialthan the whole embryo but the R0 was much lower. The differencesbetween whole embryo and axis water relations parameters emphasizethe need to take account of R0 when discussing seed water statusand survival. The results are considered in relation to currentmodels of seed water status and recalcitrant behaviour. Key words: Quercus robur L., embryo water relations, development, starch 相似文献
17.
Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway 总被引:1,自引:0,他引:1
Reiss Gilles; Heesen Stephen te; Zimmerman Jenet; Robbins Phillips; Aebi Markus 《Glycobiology》1996,6(5):493-498
N-Linked protein glycosylation in most eukaryotic cells initiateswith the transfer of the oligosaccharide Glc3Man9GlcNAc2 fromthe lipid carrier dolichyl pyrophosphate to selected asparagineresidues. In the yeast Saccharomyces cerevisiae, alg mutationswhich affect the assembly of the lipid-linked oligosaccharideat the membrane of the endoplasmic reticulum result in the accumulationof lipid-linked oligosaccharide intermediates and a hypoglycosylationof proteins. Exploiting the synthetic growth defect of alg mutationsin combination with mutations affecting oligosaccharyl transferaseactivity (Stagljar et al., 1994), we have isolated the ALG6locus. alg6 mutants accumulate lipid-linked Man9GlcNAc2, suggestingthat this locus encodes an endoplasmic glucosyltransferase.Alg6p has sequence similarity to Alg8p, a protein required forglucosylation of Glc1Man9GlcNAc2. Saccharomyces cerevisiae endoplasmic reticulum glycosyltransferase dolichol 相似文献
18.
Evidence has been obtained that starving leaves of Kleinia radicansin the summer condition produce a volatile substance, whichreacts with potassium permanganate, and which accumulates andpoisons the leaves when they are enclosed, as in a Dixon respirometer.A microrespirometer has been designed for use with continuousair-currents. Using this, the course of oxygen intake by leavesin darkness resembles that of CO2 output (previous results)and shows an initial phase of diurnal fluctuation of O2 intake.Humidity has also some effect, for an early increase in O2 intakeoccurs under dry conditions. 相似文献
19.
A diphosphopyridine nucleotide specific isocitric dehydrogenasehas been isolated from pea-seedling mitochondria and purified10-fold. In the presence of manganese the enzyme catalyses theoxidative decarboxylation of d-isocitrate to -oxoglutarate andCO2, but under similar conditions does not catalyse the reversereaction. Evidence is presented indicating that -SH groups ofthe enzyme are essential for activity. The properties of theenzyme are described. 相似文献
20.
A J Norin S L Kamholz K L Pinsker E E Emeson F J Veith 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(2):332-337
Alloreactivity of intragraft and peripheral blood lymphocytes from tolerant canine lung allograft recipients was examined. Tolerance was induced by variable periods of treatment with cyclosporine. Analysis of effector cells from lung allografts (obtained by bronchoalveolar lavage) revealed the absence of specific cytolytic T lymphocyte (CTL) activity and the presence of a low level of cytolytic activity detected in a lectin-dependent cell-mediated cytotoxicity assay. In contrast, high levels of specific CTL activity and lectin-dependent activity were detected in cell preparations from lung allografts undergoing rejection. Tolerant recipients retained normal ability to generate specific CTL activity to third party alloantigens in mixed lymphocyte cultures (MLC) but had diminished ability to generate CTL to donor alloantigens in recipient X donor MLC. Addition of exogenous interleukin 2 to these MLC was unable to restore donor-specific CTL activity. Lymphocytes from tolerant recipients were, however, capable of generating proliferative responses and lectin-dependent cytotoxicity on exposure to donor alloantigens in MLC. Evidence presented in this report suggests that the lectin-dependent cytolytic activity generated in these MLC is mediated by lymphokine-activated killer cells. Such cells are likely to be activated by interleukin 2 released in the proliferative response. The results support the proposal that the cyclosporine-induced tolerant state is characterized by the relative inability to respond against major histocompatibility complex class I antigens in contrast to class II antigens and/or minor histocompatibility antigens since MLC-induced CTL are directed, for the most part, against class I molecules. 相似文献