Insulin-Like Growth Factor-I-Enhanced Secretion Is Abolished in Protein Kinase C-Deficient Chromaffin Cells

Abstract: Previous studies have demonstrated that bovine chromaffin cells cultured in medium with 10 nM insulin-like growth factor-I (IGF-I) secrete about twofold more catecholamine when exposed to secretory stimuli than do cells cultured without IGF-I. The purpose of this study was to determine whether protein kinase C (PKC) is involved in the effect of IGF-I on secretion from these cells. PKC was down-regulated in the cells by 16–18 h of treatment with β-phorbol didecanoate (β-PDD; 100 nM). Such treatment had no effect on high-K⁺-stimulated secretion from cells cultured without IGF-I; however, secretion from cells cultured with IGF-I was reduced to a level comparable to that in cells cultured without the peptide. The inactive isomer, α-PDD (100 nM), had no effect on secretion from untreated or IGF-I-treated chromaffin cells. The effect of β-PDD was time and concentration dependent, with 100 nM β-PDD producing a maximal effect in 8–10 h. In situ PKC activity measured in permeabilized cells treated with PMA (300 nM) was decreased by～40% by 10 h and was reduced to almost basal levels by 18 h. Immunoblotting experiments demonstrated that both α-and ε-PKC were lost from the cells with time courses similar to that seen in the in situ PKC assay. Overnight treatment with the PKC inhibitor H7 (100 μM) prevented the enhanced secretion normally seen in IGF-l-treated cells, whereas HA1004 had no effect. High-K⁺-stimulated ⁴⁵Ca²⁺ uptake in IGF-I-treated cells was attenuated by long-term treatment with β-PDD (200 nM) or H7 (100 μM). Together these observations suggest that PKC is required for IGF-I-enhanced secretion from chromaffin cells.     

Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C

Abstract: Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attributable to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events downstream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N-and L-type calcium channels.     

Pentylenetetrazole-Induced Chemoshock Affects Protein Kinase C and Substrate Proteins in Mouse Brain

Abstract: Protein kinase C (PKC) activity, western blot analysis of PKCα, β, γ, ε, and ζ by isozyme-specific antibodies, and in vitro phosphorylation of endogenous substrate proteins were studied in the mice brain after pentyl-enetetrazole-induced chemoshock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified by DE-52 columns eluted with buffer A containing 100 or 200 m M KCI. This method consistently separates cytosolic and membrane proteins and various PKC isoforms. The 100 m M KCI eluates from DE-52 columns contain more PKC α and β in both cytosol and membrane than the 200 m M KCI eluates, whereas PKCγ, ε, and ζappear in equal amounts in these two eluates. The kinase activity assayed by phosphorylation of exogenous histone was increased in the chemoshocked mice in both the cytosol and membrane of 200 m M KCI eluates. In further analysis by immunoblotting, this increased activity was found to be due to the increase in content of PKC7 isozyme. As for novel-type ε and ζ isozymes, they were not altered in the chemoshocked mice. From autoradiography, the endogenous substrate 17-kDa neurogranin, which was shown below 21 kDa, was mostly eluted by 100 m M KCI from the DE-52 column, whereas 43-kDa neuromodulin, which was also demonstrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, only appeared in the 200 m M KCI eluates. The in vitro phosphorylation of neuromodulin was found to be increased in the chemoshocked mice. Therefore, the increased phosphorylation of neuromodulin and increased content of the PKCγ isoform were involved in the pentylenetetrazole-induced chemoshock.     

Insects and fungi on a C3 sedge and a C4 grass exposed to elevated atmospheric CO2 concentrations in open-top chambers in the field

The effects of elevated atmospheric CO₂ concentration on plant-fungi and plant-insect interactions were studied in an emergent marsh in the Chesapeake Bay. Stands of the C₃ sedge Scirpus olneyi Grey, and the C₄ grass Spartina patens (Ait.) Muhl. have been exposed to elevated atmospheric CO₂ concentrations during each growing season since 1987. In August 1991 the severities of fungal infections and insect infestations were quantified. Shoot nitrogen concentration ([N]) and water content (WC) were determined. In elevated concentrations of atmospheric CO₂, 32% fewer S. olneyi plants were infested by insects, and there was a 37% reduction in the severity of a pathogenic fungal infection, compared with plants grown in ambient CO₂ concentrations. S. olneyi also had reduced [N], which correlated positively with the severities of fungal infections and insect infestations. Conversely, S. patens had increased WC but unchanged [N] in elevated concentrations of atmospheric CO₂ and the severity of fungal infection increased. Elevated atmospheric CO₂ concentration increased or decreased the severity of fungal infection depending on at least two interacting factors, [N] and WC; but it did not change the number of plants that were infected with fungi. In contrast, the major results for insects were that the number of plants infected with insects decreased, and that the amount of tissue that each insect ate also decreased.     

Allozyme diversity in Chinese,Seguin and American chestnut (Castanea spp.)

Allozyme genetic variability in three chestnut (Castanea) species was investigated using 19 loci from ten enzyme systems. G-tests of heterogeneity of isozymic allele distribution showed significant differences between the three species at 15 of the 19 loci, and between the 13 C. mollissima populations at 13 of the 19 loci examined. C. mollissima was found to possess a significantly-higher value of mean gene heterozygosity (H=0.3050±0.0419), the percentage of polymorphic loci (P=84.21%) and the average number of alleles per locus (A=2.05), than any other species in the Castanea section Eucastanon. When the genetic variability of populations of C. mollissima from four regions in China was investigated, the population from the Changjiang river region showed a markedly higher mean gene heterozygosity (H=0.3480±0.0436) than populations from the other regions. Genetic relationships among the four regions were assessed by Nei's genetic identity I and standard genetic distance D. An approximately-identical distance between the population from the Changjiang river region and populations from the three other regions was observed, while populations from the latter regions showed almost the same genetic distance from each other. These data, when considered with information existing prior to this study, contribute to an understanding of the possible origin and progenitor of the chestnut species.     

Dynamic approaches to the mechanism of photosynthesis

An account of the author's life and scientific research is presented. Two main lines of research have been pursued: (1) Studies on the physiological aspect of photosynthesis started from experiments with crops under field conditions and then extended to the study of photosynthesis in nature; and (2) studies on the mechanism of photophosphorylation and related problems which began with the measurement of quantum requirement of photophosphorylation. This work led to the discovery of the high energy state of phosphorylation and many other interesting findings. In recent years, efforts have been made to study the operation and regulation of photosynthetic apparatus with a view to link the above-mentioned lines of research together.Written at the invitation of Govindjee.     

Relationships between biovolume and carbon and nitrogen content of bacterioplankton

Abstract Cell volume, carbon and nitrogen content were determined for bacteria grown in batch cultures in water samples collected at five localities in western Florida, USA. Cultures were set up by inoculating 0.2 μm filtered water with 2.5 to 7.0% of 1.0 μm filtered water. Biovolumes of the bacteria were measured by epifluorescence photomicrography. Bacterial carbon and nitrogen contents were determined with a CHN analyser. During incubations, bacterial volumes doubled from 0.070±0.037 μ m³(mean ± S.E.) to 0.153 ± 0.036 μ m³ at early stationary phase. Bacterial C:N ratios ranged between 2.8 and 10.3, with a mean of 6.5, and were inversely correlated with cell volumes. Conversion factors for volume to carbon and nitrogen content were relatively high and variable, ranging from 0.21 to 161 pg C μm⁻³ (mean: 0.72 pg C μm⁻³) and from 0.05 to 0.25 pg N μm⁻³ (mean: 0.12 pg N μm⁻³). Small cells contained more C and N per unit volume than did large cells. The data suggested that biovolume to biomass conversion factors may be higher than previously thought and may be highly variable both temporally and geographically.     

Depolarization and Neurotransmitters Increase Neuronal Protein Tyrosine Phosphorylation

Abstract: In rat hippocampal slices and in neurons in primary culture, K⁺-induced depolarization increased markedly and rapidly tyrosine phosphorylation of a 110-kDa protein (pp110) and, to a lesser degree, of a 120-kDa protein (pp120), in a calcium-dependent fashion. Qlutamate, 1-aminocyclopentane- trans -1,3-dicarboxylic acid (an agonist of metabotropic glutamate receptors), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (an agonist of ionotropic glutamate receptors) stimulated also tyrosine phosphorylation of pp110 and pp120. These effects were not observed in astrocytes in primary culture. In hippocampal slices tyrosine phosphorylation of pp110 and pp120 was stimulated by Ca²⁺-ionophores and by phorbol esters and antagonized by a chelator of intracellular Ca²⁺and by drugs that inhibit protein kinase C. Stimulation of muscarinic and α₁,-adrenergic receptors increased also tyrosine phosphorylation of pp110 and pp120. These results demonstrate that membrane depolarization and stimulation of neurotransmitter receptors activate a tyrosine phosphorylation pathway in neurons. This pathway involves an increase in intracellular Ca²⁺ concentrations and the activation of protein kinase C. It may provide a biochemical basis for some neurotrophic effects of electrical activity and neurotransmitters and may contribute to the role of tyrosine phosphorylation in long-term potentiation.     

Streptococcal erythrogenic toxin type C is not a phosphorylated protein. Description of two different purification procedures and investigation of its phosphorylation state

Abstract Erythrogenic toxin type C (ETC) from different streptococcal group A strains was successively purified by absorption on phenylsepharose, acidic dialysis of the eluate at 40% saturated ammonium sulphate solution, CM-Sepharose chromatography, finally by immunoaffinity chromatography on monoclonal antibodies. Second, after growing of bacteria in the presence of [³²P]orthophosphate to phosphorylate ETC, the ETC was purified with phenylsepharose following immunoaffinity chromatography. The occurrence of phosphoamino acids in the purified ETC was investigated by an immunoassay. No phosphoamino acids could be detected in the ETC molecule. Also after radiolabelling with ³²P it was not possible to demonstrate a radioactive signal. The treatment with alkaline phosphatase has no influence on the mitogenicity or position of ETC in isoelectric focusing. The results obtained led to the conclusion that in contrast to the literature, ETC is not a phosphorylated protein.     

Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen.

Three-dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site-directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3-dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.