Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Fc epsilon RI-mediated hydrolysis of phosphoinositides in ghosts derived from rat basophilic leukemia cells.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on mast cells by a polyvalent Ag leads to hydrolysis of phosphoinositides (PI) catalyzed by phospholipase C (PI-PLC). To understand this phenomenon in molecular terms, it is important to obtain active, cell-free preparations. In extensive preliminary studies, we could not demonstrate Fc epsilon RI-mediated activation of PI-PLC in plasma membranes prepared by conventional methods from rat basophilic leukemia cells. We now report a stepwise approach involving preparation of cytoplasts from such cells and then hypotonic lysis of the cytoplasts to obtain active membrane vesicles. These membranes, best described as "ghosts," appear to reseal after losing greater than 90% of their soluble, cytoplasmic components and contain receptors that when aggregated, activate PI-PLC to hydrolyze endogenous phospholipids. Per unit of plasma membrane, the ghosts retain approximately 25% of Fc epsilon RI-mediated stimulation of PI-PLC relative to the cells. This activity requires ATP, magnesium, phosphoenolpyruvate, and, to a limited degree, calcium. Although an adequate amount of phosphatidylinositol biphosphate is present, the predicted spike of (1,4,5)-inositol trisphosphate is not seen, and the predominant inositol phosphate isomer is (1,4)-inositol bisphosphate. This is the first report of Fc epsilon RI-mediated activation of PI-PLC in a cytoplasm-depleted system that demonstrates activation of endogenous enzyme acting on endogenous substrate. In addition, it is the first such report for any receptor of the Ig superfamily.     

Myosin light chain 2 modulates calcium-sensitive cross-bridge transitions in vertebrate skeletal muscle.

We investigated the mechanism of the Ca2+ sensitivity of cross-bridge transitions that limit the rate of force development in vertebrate skeletal muscle. The rate of force development increases with Ca2+ concentration in the physiological range. We show here that at low concentrations of Ca2+ the rate of force development increases after partial extraction of the 20-kD light chain 2 subunit of myosin, whereas reconstitution with light chain 2 fully restores native sensitivity to Ca2+ in skinned single skeletal fibers. Furthermore, elevated free Mg2+ concentration reduces Ca2+ sensitivity, an effect that is reversed by extraction of the light chain but not by disruption of thin-filament activation by partial removal of troponin C, the Ca2+ binding protein of the thin filament. Our findings indicate that the Ca2+ sensitivity of the rate of force development in vertebrate skeletal muscle is mediated in part by the light chain 2 subunit of the myosin cross-bridge.     

Release of substance P-like immunoreactive material from the stomach of the rainbow trout

Summary The release of substance P-like immunoreactive material (SPLI) from the vascularly perfused stomach of the rainbow trout, Oncorhynchus mykiss, was studied. In most cases, SPLI was detected in the collected vascular perfusate during experimental resting conditions. Distensions of the stomach, accomplished by a water-filled intragastric balloon, produced an initial rapid relaxation of the stomach, followed by a slow further relaxation and a stimulation of contractile activity. The amount of SPLI in the vascular perfusate was significantly elevated during the distension period. Tetrodotoxin had no effect on the response to distension or on the release of SPLI during distension, indicating release from tetrodotoxin-insensitive neurons or endocrine cells. The results suggest that a substance P-like peptide may be involved in the contractile response and/or in the maintenance of muscular tone during gastric distensions in the rainbow trout. Infusion of capsaicin had no effect on the release of SPLI. However, capsaicin caused an increase in vascular flow, an effect that could be repeated on a second infusion of capsaicin, indicating that the action may not be specific to sensory neurons.Abbreviations 5-HT 5-Hydroxytryptamine - RIA radioimmunoassay - SP substance P - SPLI substance P-like immunoreactive material - TTX tetrodotoxin     

Chronopharmacokinetics and Cardiovascular Effects of Nifedipine

Orcadian phase dependency in pharmacokinetics and hemodynamic effects on blood pressure and heart rate of different galenic formulations of nifedipine (immediate-release, sustained-release, and i.v. solution) were studied in healthy subjects or in hypertensive patients. Pharmacokinetics of immediate-release but not sustained-release and i.v. nifedipine were dependent on time of day: immediate-release nifedipine had higher C_max (peak concentration) and shorter t_max (time-to-peak concentration) after morning than evening application, and bioavailibility in the evening was reduced by about 40%. Orcadian rhythm in estimated hepatic blood flow as determined by indocyanine green kinetics may contribute to these chronokinetics. A circadian time dependency was also found in nifedipine-induced effects on blood pressure and heart rate as monitored by 24-h ambulatory blood pressure measurements. In conclusion, the dose response relationship of oral nifedipine is influenced by the circadian organization of the cardiovascular system as well as by the galenic drug formulation.     

Role of Endogenous Growth Regulators in Seed Dormancy of Avena fatua: I. Short Chain Fatty Acids

The hypothesis that endogenous short chain fatty acids (C 6-C 10) are important in maintaining seeds of wild oat (Avena fatua L.) in the dormant state by acting as natural germination inhibitors (Berrie, Buller, Don, Parker, 1979 Plant Physiol 63: 758-764) was investigated. When germination of nondormant seeds was inhibited by treatment with short chain fatty acids, the seeds did not revert to a similar biochemical and physiological state as exhibited by dormant seeds. First, nonanoic acid-induced inhibition of seed germination was not reversed by hormone treatments which normally break dormancy in wild oat seeds. Second, nondormant seeds treated with short chain fatty acids maintained similar relative proportions of the pentose phosphate pathway and the Embden-Meyerhoff-Parnas pathway for respiratory glucose metabolism as that found in the nondormant controls. Seeds imbibed in the presence of nonanoic acid lost more amino acids and proteins into the imbibition solution than did the untreated controls, suggesting membrane damage had occurred. Inasmuch as increasing concentrations of nonanoic acid also progressively reduced the growth of the coleoptile and roots of intact seedlings until all growth ceased and no germination occurred, the inhibition of seed germination could be due to a nonspecific inhibition of growth of the embryo, perhaps because of disruption of membrane structure and function. Finally, no correlation between endogenous levels of short chain fatty acids in seeds or isolated embryonic axes and seed dormancy could be demonstrated.     

Identification of Strychnine Binding Sites in the Rat Retina

Abstract: [³H]Strychnine specifically binds to membrane fractions isolated from rat retinae. The binding is saturable, with an apparent dissociation constant, K _D, of 14.3 × 10⁻⁹ M and 205 fmol bound/mg protein. Specific binding is time-dependent and proportional to protein concentration. Glycine and taurine are equally potent inhibitors of [³H]strychnine binding ( K _i= 4 × 10⁻⁵ M); no other amino acids endogenously present in the retina inhibited [³H]strychnine binding.     

Nucleoside uptake by rat retina cells

Adenosine and guanosine uptake have been studied in the rat retina. Both nucleosides are taken up in a time- and temperature-dependent manner by dispersed rat retinal cells. The uptake of both nucleosides is Na⁺-dependent and Ca⁺⁺-independent. Initial rate studies of guanosine and adenosine uptake demonstrate a single uptake process for each nucleoside with K_D values of 2.1 and 2.9 uM, and maximal rates of 24 and 17 pmol/mg protein/min, respectively. Guanosine uptake was inhibited by adenosine with a K_I of 12.1 uM whereas guanosine inhibited adenosine uptake with a K_I value greater than 10^?3 M. LN⁶-phenylisopropyladenosine, a nucleoside analog, was the most potent inhibitor of adenosine and guanosine uptake with K_I values of 25 and 8 uM, respectively. Phosphodiesterase inhibitors (isobutylmethylxanthine and theophylline) and biogenic amines (dopamine, norepinephrine, and histamine) had no significant effect on the uptake of guanosine or adenosine at concentrations up to 100 uM.