Evidence that cyclic AMP may regulate Ca2+-mobilization and phospholipases in thrombin-stimulated human platelets

The regulation of human platelet responses by cyclic AMP (cAMP) has been investigated by measuring thrombin-stimulated serotonin release, Ca²⁺ uptake and phospholipase activity. Thrombin-induced 1,2-diacylglycerol (DG) formation as a result of phospholipase C activation was inhibited by pretreatment with dibutyryl cAMP (dbcAMP) in a dose-dependent manner. Subsequent failure to produce phosphatidic acid (PA), which is converted from 1,2-DG by phosphorylation and would serve as intracellular Ca²⁺ ionophore, appeared to parallel the decrease in Ca²⁺ uptake activity. Phospholipase A₂ activity, monitored by the production of [³H]lysophosphatidylcholine and [³H]lysophosphatidylethanolamine, was also suppressed by dbcAMP. These data indicate that the intracellular cAMP level may be closely associated with Ca²⁺ uptake and phospholipases activation. In addition, it is suggested that alteration of intracellular cAMP regulates phospholipase activation and consequently platelet responses, perhaps by controlling available Ca²⁺ content.     

Glucose catabolism during aging and differentiation in hypocotyls ofPhaseolus mungo seedlings

Changes in activities of the glycolytic and pentose phosphate (PP) pathways in glucose catabolism in various parts of the hypocotyls obtained from 4-day-old etiolatedPhaseolus mungo seedlings were investigated by measuring the inhibition rates of respiration by iodoacetate and malonate, and the release of¹⁴CO₂ from [1-¹⁴C]- and [6-¹⁴C]glucose. The relative activity of the PP pathway in glucose catabolism was higher in the immature part (Part I) and the aged part (Part V) of the hypocotyls than in the intermediary one (Part III), while the activity of the glycolytic pathway decreased with aging. On a fresh weight basis, the enzyme activities of the glycolytic and PP pathways were higher in Part I than in Parts III and V. On a protein content basis, however, activities of the enzymes of the PP pathway increased with aging and differentiation of the hypocotyls whereas those of the glycolytic pathway decreased. Levels of nicotinamide adenine nucleotides were found to be in the following order: Part I>Part III> Part V for NAD⁺+NADH; Part I>Part V>Part III for NADP⁺+NADPH. The stimulative effect of methylene blue on decreasing the C₆/C₁ ratio was greater in Part III than in Part I, and No effect was observed in Part V. These data suggest that a decrease in the activity of the glycolytic pathway with aging and differentiation may be due to the decreasing glycolytic enzyme activities and NAD(H) content. The higher activity of the PP pathway in the immature part is attributable to larger amounts of NADP(H) and enzymes of the PP pathway. The greater contribution of the PP pathway to glucose catabolism in the aged part than in the intermediary part seems to results from a more active turnover of NADP and the relatively higher activity of the enzymes of the PP pathway than those of the glycolytic pathway.     

Establishment of five human myeloma cell lines

Summary Five human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12- PE, and KMS-12-BM, have been established at Kawasaki Medical School since 1980. As the KMS-12-PE and KMS-12-BM lines were obtained from the same patient, these five cell lines have been derived from four patients with multiple myeloma. The five myeloma cell lines are stably growing at present in RPMI 1640 medium supplemented with 10% fetal bovine serum. They can also grow in a defined culture medium without serum. That these cell lines were, human myeloma cells was confirmed by the following findings. Ultranstructually, all five cell lines showed features characteristic of plasma cells. KMM-1 and KMS-11 cells secreted lambda and kappa chains into the culture medium, respectively, but the other cell lines produced no immunoglobulins. KMM-1 expressed cytoplasmic lambda antigen, KMS-5 showed cytoplasmic delta, and KMS-11 expressed surface kappa, whereas KMS-12-PE and KMS-12-BM cells showed no surface or cytoplasmic immunoglobulins. Regarding reaction with a monoclonal plasma cell antibody (PCA-1), four of the five lines were positive, the exception being KMS-5. Another monoclonal antibody (CD38), which also recognizes plasma cells, reponded to KMM-1, KMS-12-PE, and KSM-12-BM. KMS-5 cells expressed acute lymphoblastic leukemia antigens (CALLA). These data suggest that such lines as KMM-1, KMS-11, KMS-12-PE, and KMS-12-BM represent later stages of B-cell differentiation, and that KMS-5 represents a relatively early stage of B-cell differentiation. All the cell lines lacked Epstein-Barr virus nuclear antigen, showed abnormal karyotypes of human origin, and differed from each other in the isozyme patterns examined. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.     

Isolation of Glutamyltaurine from Bovine Brains and Proof of Its γ-Linkage by the B/E Linked Scan SIMS Technique

We isolated a glutamyltaurine from bovine brains and determined its structure as gamma-glutamyltaurine (gamma-Glu-Tau; glutaurine) by use of a new mass spectrometric technique [B/E linked scan sputtered ion mass spectrometry (SIMS)], which we have recently shown to be useful for distinguishing the gamma- from the alpha-isomer of glutamyl-dipeptides. Neither the alpha-isomer of glutamyltaurine nor any aspartyltaurines could be detected in bovine brain.     

Evidence that Glyceraldehyde-3-Phosphate Dehydrogenase Is Involved in Age-Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.     

Proliferation of chick primordial germ cells cultured on stroma cells from the germinal ridge.

Fluorescent reagent-labelled PGCs isolated from the blood of 2-day-old chick embryos were cultured on stroma cells derived from 5-day-old germinal ridge in Medium 199 supplemented with 10% FBS, human IGF-1, bovine FGF-b, and murine LIF. In 7 experiments, the number of MCs increased by an average of 4.8 fold in 4 days. Intrinsic PGCs in the 5-day embryonic germinal ridge were observed loosely attached to the stroma cells, and they also increased 3.8 fold during culture for 4 days. These results indicate the possibility of applying this culture method to the production of transgenic chickens.