Cytotoxic T lymphocyte unresponsiveness induced by prolonged treatment with immobilized anti-CD3 antibody. Association of impairment of cytolytic activity with temporary depletion of intracellular protein kinase C

In our study we investigated the effect of pretreatment of bulk CTL and CTL clones with immobilized anti-CD3 antibody (Ab) or PMA. Primary CTL and CTL clones were cultured in dishes coated with anti-CD3 Ab or in medium containing PMA (5 nM) and assayed for Ag-specific or Ag-nonspecific "redirected" cytolysis using FcR+ P815 cells as targets. Cytotoxic activity of bulk CTL and five of six CTL clones tested in this study were inhibited by prolonged (longer than 6 h) pretreatment with immobilized anti-CD3 Ab or PMA, whereas proliferation of CTL clones or expression of surface CD3 molecules were not. The intracellular granule enzyme (N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester esterase) activity of CTL clones was not reduced under these suppressive conditions, indicating that the incompetence of CTL is not merely due to depletion of cytolytic granules by chronic stimulation. The suppressed cytotoxicity could be recovered by culturing CTL without perturbation of CD3 molecules for 24 h. In one exceptional clone, BM10-37, pretreatment with immobilized anti-CD3 Ab or PMA did not suppress the cytotoxic activity. Immunostaining of intracellular protein kinase C (PKC) revealed that PKC was depleted after prolonged treatment with immobilized anti-CD3 Ab or PMA in those susceptible CTL clones but not in the resistant BM10-37. These findings lead us to conclude that prolonged stimulation of CD3 of CTL results in depletion of PKC and that PKC may be essential for signal transduction to deliver a lethal hit to the target cells.     

Thymic stroma-derived T cell growth factor (TSTGF). IV. Capacity of TSTGF to promote the growth of L3T4- Lyt-2- thymocytes by synergy with phorbol myristate acetate or various IL

The present study investigates the role of thymic stroma-derived T cell growth factor (TSTGF) in promoting the growth of L3T4- Lyt2- (double-negative) thymocytes. Partially purified TSTGF samples were prepared from the culture supernatant of a newly established thymic stromal cell line, MRL104.8a. The TSTGF alone induced only marginal proliferation of double-negative thymocytes, whereas this factor exerted a potent growth-promoting effect on these cells in combination with PMA. Because such an enhanced proliferation was not inhibited by anti-IL-4 or anti-IL-2R antibody, this was not due to the stimulation of an autocrine mechanism involving the production and utilization of IL-4 or IL-2. In scrutinizing PMA-equivalent physiologic substance(s), IL-1 was revealed to be capable of replacing the role of PMA in the above co-stimulation cultures and including enhanced proliferation of double-negative thymocytes in combination with TSTGF. Although TSTGF plus IL-2 or IL-4 also exhibited an appreciable or moderate synergistic effect on the growth of double-negative thymocytes, its magnitude was weaker compared with that obtained by TSTGF plus IL-1. More important, the strikingly enhanced proliferation was induced in the combinations of TSTGF, IL-1, and IL-2 or IL-4 under conditions in which the proliferation induced by IL-1 plus IL-4 or IL-1 plus IL-2 was marginal or slight. Furthermore, such strongly enhanced proliferation was also observed in the double-negative thymocyte population which was additionally depleted of T3+ cells (namely, the L3T4- Lyt-2- T3- or dull population). These results indicate the crucial role of TSTGF in the proliferation of immature thymocytes by synergy with various cytokines.     

Effects of Oxygen Depletion on Norepinephrine- and Carbachol-Stimulated Phosphoinositide Turnover in Rat Brain Slices

We examined the effects of in vitro anoxia and in vivo hypoxia (8% O2/92% N2) on norepinephrine (NE)- and carbachol-stimulated phosphoinositide (PI) turnover in rat brain slices. The formation of 3H-labeled polyPI in cortical slices was impaired by in vitro anoxia and fully restored by reoxygenation. Accumulation of 3H-labeled myo-inositol phosphates (3H-IPs) stimulated by 10(-5) M NE was significantly reduced by anoxia (control at 60 min, 1,217 +/- 86 cpm/mg of protein; anoxia for 60 min, 651 +/- 82 cpm/mg; mean +/- SEM; n = 5; p less than 0.01), and reoxygenation following anoxia resulted in overshooting of the accumulation (control at 120 min, 1,302 +/- 70 cpm/mg; anoxia for 50 min plus oxygenation for 70 min, 1,790 +/- 126 cpm/mg; n = 5; p less than 0.01). The underlying mechanisms for the two phenomena--the decrease caused by anoxia and the overshooting caused by reoxygenation following anoxia--seemed to be completely different because of the following observations. (a) Although the suppression of NE-stimulated accumulation at low O2 tensions was also observed in Ca2+-free medium, the overshooting in response to reoxygenation was not. (b) Carbachol-stimulated accumulation was significantly reduced by anoxia and was restored by reoxygenation only to control levels. Thus, the postanoxic overshooting in accumulation of 3H-IPs seems to be a specific response to NE. (c) The decrease observed at low O2 tensions was due to a decrease in Emax value, whereas the postanoxic overshooting was due to a decrease in EC50 value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of muscle force,muscle endurance,and electromyogram activity during an expedition at high altitude

Handgrip force (HF), maximal pinch force (MF), muscle endurance (ME), and the median power frequency (MdPF) of the activity shown in the electromyogram (EMG) were studied at various altitudes in eight normal healthy subjects. MF and ME were measured between the index finger and thumb, and all measurements were obtained at altitudes ranging from 610 to 4860 m during an expedition in the Qinghai Plateau in China. With the change in altitude HF, ME, and MF showed no significant change. Compared to the MdPF at 2260 m on ascent, the MdPF at other altitudes showed a significant decrease (P<0.01). Thus, we conclude that muscle performance (HF, MF, and ME) was not affected by the environment at high altitude. However, MdPF was affected and the mean MdPF at 610 m after the expedition did not recover to initial values of MdPF. We suggest these results may have been affected by fatigue and chronic exposure to the hypobaric hypoxic environment, since the members of the expedition party expressed feelings of sluggishness and fatigue after the expedition.     

The Thermostable Direct Hemolysin Gene (tdh) of Vibrio hollisae Is Dissimilar in Prevalence to and Phylogenetically Distant from the tdh Genes of Other Vibrios: Implications in the Horizontal Transfer of the tdh Gene

Vibrio hollisae strains isolated recently from patients in various locations were examined for the presence of the thermostable direct hemolysin gene (tdh) using nucleic acid hybridization and polymerase chain reaction assays. The results were consistent with the previous finding that all strains of V. hollisae carry the tdh gene. In contrast, the tdh gene has been detected in a minority of strains for other Vibrio species (V. parahaemolyticus, V. cholerae non-O1, and V. mimicus). Detailed phylogenetic analysis showed that the tdh genes of the non-V. hollisae species were very closely related to each other and that the tdh gene of V. hollisae was distantly related to the tdh genes of the non-V. hollisae species. These results and the proposed insertion sequence-mediated tdh transfer mechanism suggest that the tdh gene may have been maintained stably in V. hollisae and that the tdh genes of the non-V. hollisae species may have been involved in recent horizontal transfer.     

Suppression of gravitropic response of primary roots by submergence

Primary roots of six plant species were placed horizontally either in humid air or under water, and their growth and gravitropic responses were examined. In air, all the roots showed a normal gravitropic curvature. Under water without aeration, roots of rice (Oryza sativa L.), oat (Avena sativa L.), azuki bean (Vigna angularis Ohwi et Ohashi), and cress (Lepidium sativum L.) curved downward at almost same rate as in air, whereas the curvature of roots of maize (Zea mays L.) and pea (Pisum sativum L.) was strongly suppressed. Submergence did not cause a decrease in growth rate of these roots. When roots of maize and pea were placed horizontally under water without aeration and then rotated in three dimensions on a clinostat in air, they showed a significant curvature, suggesting that the step suppressed by submergence is not graviperception but the subsequent signal transmission or differential growth process. Constant bubbling of air through the water partly restored the gravitropic curvature of maize roots and completely restored that of pea roots. The curvature of pea roots was also partly restored by the addition of an inhibitor of ethylene biosynthesis, aminooxyacetic acid. In air, ethylene suppressed the gravitropic curvature of roots of maize and pea. Furthermore, the level of ethylene in the intercellular space of the roots was increased by submergence. These results suggest that the accumulation of ethylene in the tissue is at least partly involved in suppression of transmission of the gravity signal or of differential growth in maize and pea roots under conditions of submergence.Abbreviations AOA aminooxyacetic acid - 3-D three-dimensional Dedicated to Professor Andreas Sievers on the occasion of his retirementWe thank Professor H. Suge and Drs. H. Takahashi and H. Kataoka, Tohoku University and Dr. T. Suzuki, Yamagata University, for helpful suggestions. The present study was supported in part by a Grant for Basic Research in Space Station Utilization from the Institute of Space and Astronautical Science, Japan.     

Cloning and Characterization of Two Novel Human cDNAs (NELL1 and NELL2) Encoding Proteins with Six EGF-like Repeats

From a human fetal-brain cDNA library we isolated two novel genes encoding peptides containing six EGF-like repeats. Both showed significant homologies with nel, a gene strongly expressed in neural tissues of chicken. The cDNAs, designated NELL1 (nel-like, type 1) and NELL2 (nel-like, type 2), contained open reading frames encoding 810 and 816 amino acids, respectively. NELL2 is strongly expressed in brain of adult and fetus but only weakly in fetal kidney. NELL1 and NELL2 were mapped by FISH to chromosomal bands 11p15.1–p15.2 and 12q13.11–q13.12, respectively.