A major myristylated substrate of protein kinase C and protein kinase C itself are differentially regulated during murine B- and T-lymphocyte development and activation.

The regulation and expression of protein kinase C (PKC) and phosphomyristin C (PMC) (a principal substrate of PKC which is the major myristylated protein in lymphocyte and glioma lines that express it) in murine B and T lymphocytes were investigated. Both PMC and PKC are differentially regulated during T-cell development. The level of PMC expression is highest in CD4-8-, intermediate in CD4+8+, and lowest in J11d-, CD4, or CD8 single-positive thymocytes. PKC is equally expressed by all three thymic populations. In striking contrast to thymocytes, resting peripheral lymph node T cells and T-cell clones express little if any PMC and reduced levels of PKC. Neither PKC nor PMC is significantly induced upon the activation of lymph node T cells: treatment with anti-CD3 antibodies or anti-CD3 and interleukin-2 fails to induce PKC, whereas PMC is not induced by anti-CD3 alone and is only slightly induced by anti-CD3 and interleukin-2. In contrast to the situation with T cells, PMC and PKC are constitutively expressed at moderate levels in mature B cells. PMC is greatly increased in B-cell blasts generated by cross-linking the antigen receptor with anti-immunoglobulin. These results demonstrate that PMC and PKC are differentially regulated during the development and activation of B and T cells, suggesting that cellular events that rely upon PKC and PMC may differ during ontogeny and activation of different lymphocyte subsets.     

Differences between wheat and rice in the enzymic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase and the relationship to photosynthetic gas exchange

The kinetic parameters of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39) in wheat (Triticum aestivum L.) and rice (Oryza sativa L.) were determined by rapidly assaying the leaf extracts. The respective K _m and V _max values for carboxylase and oxygenase activities were significantly higher for wheat than for rice. In particular, the differences in the V _max values between the two species were greater. When the net activity of CO₂ exchange was calculated at the physiological CO₂-O₂ concentration from these kinetic parameters, it was 22% greater in wheat than in rice. This difference in the in-vitro RuBP-carboxylase/oxygenase activity between the two species reflected a difference in the CO₂-assimilation rate per unit of RuBP-carboxylase protein. However, there was no apparent difference in the CO₂-assimilation rate for a given leaf-nitrogen content between the two species. When the RuBP-carboxylase/oxygenase activity was estimated at the intercellular CO₂ pressure from the enzyme content and kinetic parameters, these estimated enzyme activities in wheat and rice were similar to each other for the same rate of CO₂ assimilation. These results indicate that the difference in the kinetic parameters of RuBP carboxylase between the two species was offset by the differences in RuBP-carboxylase content and conductance for a given leaf-nitrogen content.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic - PAR photosynthetically active radiation - RuBP ribulose-1,5-bisphosphate     

Induction of gametocytogenesis in Plasmodium falciparum by the culture supernatant of hybridoma cells producing anti-P. falciparum antibody

There have been many unsuccessful attempts to induce gametocytogenesis in vitro. In the present experiment, however, we found that RPMI-CS medium and RPMI-FS medium prepared by dissolving powdered RPMI 1640 medium in the culture supernatants of hybridoma cells, hybrid line D21 and 219.5, respectively, that produce anti-P. falciparum antibody induced gametocytogenesis. Gametocytogenesis was consistently observed from 3 days after addition of these media. The culture supernatant of anti-P. falciparum antibody producing hybridoma cells did not induce gametocytogenesis in the absence of RPMI 1640 medium. RPMI-MS medium, prepared by dissolving powdered RPMI 1640 medium in the culture supernatant of myeloma cells, SP2/O-Ag 14, which was used as a control, induced a few gametocytes.     

Glucagon-stimulated phosphorylation of rat liver glycogen synthase in isolated hepatocytes

Addition of glucagon (20 nM) to the isolated hepatocytes from 24-h starved male rats results in an inactivation of glycogen synthase. The A0.5 for glucose-6-P is increased 2-fold over the control but the S0.5 for UDP-glucose is not significantly affected. The glucagon-stimulated inactivation of glycogen synthase is also accompanied by a 60-120% increase in the phosphorylation of the synthase. Glycogen synthase labeled with 32P by incubation of the hepatocytes with [32P] PO4(3-) was recovered by immunoprecipitation and the resulting immunoprecipitate was subjected to tryptic digestion. Analysis of the 32P-labeled peptides reveals that the sites corresponding to those phosphorylated by cAMP-dependent protein kinase and glycogen synthase (casein) kinase-1 (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057) are rapidly phosphorylated in response to glucagon. These results demonstrate that glucagon not only triggers the activation of cAMP-dependent protein kinase through an increase in the intracellular level of cAMP but also, by an unknown mechanism, activates a Ca2+- and cAMP-independent protein kinase.     

Changes in the Amounts of Ribulose Bisphosphate Carboxylase Synthesized and Degraded during the Life Span of Rice Leaf (Oryza sativa L.)

In situ synthesis and degradation of ribulose bisphosphate carboxylase(RuBPCase) were studied quantitatively in the 12th leaf bladeof the rice plant during the life span of the leaf. Levels ofRuBPCase protein were determined by rocket immunoelectrophoresis.The amounts of RuBPCase synthesized and degraded were estimatedusing ¹⁵N tracer. RuBPCase was scarcely recognized in the leaf when the tip ofthe leaf had just emerged from the 1 lth leaf sheath. Then itincreased rapidly and reached its maximum content a week afterthe completion of leaf expansion. At this time RuBPCase accountedfor 56% of the soluble leaf protein N (26% of the total leafN). The total amount of RuBPCase synthesized up to this timewas about 90% of the amount synthesized throughout the leaf'slife. Degradation of RuBPCase started about the time when it reachedthe maximum content and proceeded at a faster rate during senescencethan that of the remaining soluble protein. When the leaf hadsenesced completely, it contained little measurable RuBPCasealthough the total leaf N was about 30% of the maximum level.These results clearly suggest that RuBPCase is a major N componentwhich is used as remobilized N for the growth of young tissues. Influx and efflux of N and the synthesis and degradation ofRuBPCase are discussed in relation to leaf age. (Received February 18, 1983; Accepted June 16, 1983)     

Type I protein kinase C isozyme in the visual-information-processing pathway of monkey brain

Previously using PKC isozyme-specific antibodies for immunoblot analysis, we demonstrated the heterogeneous distribution of PKC isozymes in various regions of monkey and rat brains and that type I PKC was most abundant in cerebellum, hippocampus, amygdala, and cerebral cortex (Huang et al.: J Biol Chem 262:15714-15720, 1987). Using these antibodies, we have also demonstrated that type I, II, and III PKC are products of PKC genes gamma, beta, and alpha, respectively (Huang et al.: Biochem Biophys Res Commun 149:946-952, 1987). By immunocytochemical analysis, type I PKC-specific antibody showed strong reactivity in various types of neuron in hippocampal formation, amygdala, cerebellum, and neocortex. In hippocampal formation, granule cells of dentate gyrus and pyramidal cells of hippocampus were heavily stained. By immunoblot analysis, relative levels of PKC isozymes in several areas of monkey cerebral cortex involved in the visual information processing and storage were determined. Both type II and III PKCs appeared to be evenly distributed and at moderate levels, type I PKC formed a gradient of increasing concentration rostral along the cerebral cortex of occipital to temporal and then to the limbic areas. Neurobehavioral studies have demonstrated that the neocortical and limbic areas of the anterior and medial temporal regions participate more directly than the striate, prestriate, and posterior temporal regions in the storage of visual representations and that both hippocampus and amygdala are important in the memory formation. As type I PKC is present at high levels in hippocampus, amygdala, and anterior temporal lobe, we predict that the type I protein kinase C may participate in the plastic changes important for mnemonic function.     

Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2

The cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 for fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because of the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10(-7) M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10(-7) M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase.     

Glutelin accumulation and changes in the levels of its mRNA in the superior and inferior spikelets of rice ear during ripening

Glutelin accumulation in the apical spikelet of the top primary branch (superior spikelet) and the second spikelet of the lowest secondary branch (inferior spikelet) of the ear of the rice plant (Oryza sativa L.) was characterized during grain filling.In the superior spikelet, the accumulation of dry matter and nitrogen started immediately after flowering and rapidly reached the maturation level by 20 days after heading (DAH). At 7 DAH, total RNA content had already reached its maximum level and glutelin mRNA content 70% of its maximum. The increase in glutelin mRNA was followed by a rapid increase in glutelin between 7 and 16 DAH.In the inferior spikelet dry matter, nitrogen and glutelin accumulation were low immediately after flowering and increased only after grain filling of the superior spikelet was almost complete. Total RNA and glutelin mRNA increased much later at slower rates than in the superior spikelet.It is very likely that the retardation of dry matter, total nitrogen and glutelin accumulation in the inferior spikelet is due to retardation of differentiation and development of endosperm tissue, and to glutelin gene expression in endosperm cells. It is suggested that the delayed development resulted from limited partitioning of nutrients to the inferior spikelet at the early stage of ripening.     

HNF-4 increases activity of the rat Apo A1 gene.

Apolipoprotein A1 (Apo A1) is the major protein component of high density lipoprotein (HDL) particles. HDL particles mediate the removal of cholesterol from extra-hepatic tissues via a process known as reverse cholesterol transport. Augmented production of Apo A1 will likely be beneficial to those who suffer from the consequences of hypercholesterolemia. One approach to increase expression of the protein is to identify nuclear factor(s) that enhance Apo A1 promoter activity. Therefore, we have used transient transfection to study a limited portion (-474 to -7) of the gene and showed that a cis-regulatory element, site C had a permissive effect on the ability of an adjacent site B to increase promoter activity by 30-fold. The importance of element C prompted us to identify the factor(s) that interact with this site. Results showed that HNF-4, a new member of the thyroid/steroid hormone receptor superfamily interacts with site C to enhance activity of the promoter. Based on this observation and that of the known inhibitory effects of ARP-1 on site C, we postulate a model which may account for the tissue-specific expression of the rat Apo A1 gene.     

Relation between Nitrogen and Ribulose-1,5-bisphosphate Carboxylase in Rice Leaves from Emergence through Senescence

The relation between N content and ribulose-l,5-bisphosphate(RuBP) carboxylase protein was examined in the 12th leaf bladeof rice. Plants were grown under different amounts of N afterthe emergence of the 12th leaf blade. RuBP carboxylase proteinincreased with leaf N during leaf expansion. The synthesis ofRuBP carboxylase predominated during this period, and changesin the amounts of carboxylase synthesized until leaf death paralleledchanges in the N influx to the leaves. When the carboxylasereached its maximum content, the proportion of RuBP carboxylaseto leaf N was 27 to 28% irrespective of N treatment. As theleaf senesced, however, this proportion differed significantlywith the treatment. It was higher in the N-deficient leaf thanin the N-sufficient leaf. This was due to different patternsof RuBP carboxylase degradation for the treatments during senescence.RuBP carboxylase was degraded actively during the early stageof senescence in the N-sufficient leaf, whereas its degradationproceeded almost constantly in the N-deficient leaf during senescence. (Received October 17, 1983; Accepted January 27, 1984)