Exercise-induced translocation of protein kinase C and production of diacylglycerol and phosphatidic acid in rat skeletal muscle in vivo. Relationship to changes in glucose transport

Contraction-induced translocation of protein kinase C (Richter E.A., Cleland, P.J.F., Rattigan, S., and Clark, M.G. (1987) FEBS Lett. 217, 232-236) implies a role for this enzyme in muscle contraction or the associated metabolic adjustments. In the present study, this role is further examined particularly in relation to changes in glucose transport. Electrical stimulation of the sciatic nerve of the anesthetized rat in vivo led to a time-dependent translocation of protein kinase C and a 2-fold increase in the concentrations of both diacylglycerol and phosphatidic acid. Maximum values for the latter were reached at 2 min and preceded the maximum translocation of protein kinase C (10 min). Stimulation of muscles in vitro increased the rate of glucose transport, but this required 20 min to reach maximum. There was no reversal of translocation or decrease in the concentrations of diacylglycerol and phosphatidic acid even after 30 min of rest following a 5-min period of stimulation in vivo. Translocation was not influenced by variations in applied load at maximal fiber recruitment but was dependent on the frequency of nontetanic stimuli, reaching a maximum at 4 Hz. The relationship between protein kinase C and glucose transport was also explored by varying the number of tetanic stimuli. Whereas only one train of stimuli (200 ms, 100 Hz) was required for maximal effects on protein kinase C, diacylglycerol, and phosphatidic acid, more than 35 trains of stimuli were required to activate glucose transport. It is concluded that the production of diacylglycerol and the translocation of protein kinase C may be causally related. However, if the translocated protein kinase C is involved in the activation of glucose transport during muscle contractions, an accumulated exposure to Ca2+, resulting from multiple contractions, would appear to be necessary.     

Cytomegalovirus infection stimulates expression of monocyte-associated mediator genes

Monocytes and tissue macrophages play important roles in host defense against virus infections and, in the case of human cytomegalovirus (HCMV) and HIV, may also be the reservoir for latent disease. Because these cells can also rapidly respond to most infections by secretion of inflammatory mediators, we were interested in determining if HCMV infection could have a direct activating effect on macrophage cytokine production. To do this, we primarily investigated the influence of HCMV infection on IL-1 beta-mRNA expression in peripheral blood monocytes and the promyelocytic cell line, ML-3 as well as the inflammatory response genes TNF-alpha, MAD-9, MAD-6, and MAD-2 in the promyelocytic ML-3 cell line. Exposure of ML-3 cells to the virus prior to induction of differentiation had little influence on mediator gene expression. However, induction of the macrophage phenotype by pretreatment of ML-3 cells with the phorbol ester, PMA, followed by HCMV challenge, resulted in a greatly extended period of expression of IL-1 beta, TNF-alpha, MAD-9, and CSF-1 but not MAD-6 and MAD-2. Constitutively expressed genes such as lysozyme and actin were not similarly modulated. Both RNA dot-blot and in situ hybridization studies demonstrated that infection of human peripheral blood monocytes with HCMV leads to sustained expression of IL-1 beta mRNA for up to 96 h, which contrasted markedly with mock-infected or LPS-stimulated monocytes. Flow cytometric analysis of the intracellular levels of IL-1 beta protein in ML-3 cells indicated that not only was there more protein produced in infected cells, but that the majority of the cells had responded. Enhanced levels of the intracellular form of IL-1 beta in monocytes was confirmed by Western blot analysis. Cotransfection experiments were performed using IL-1 beta-CAT chimeric plasmids together with plasmids encoding HCMV-immediate-early gene region products. Transactivation of the IL-1 beta gene by region 2 of the immediate-early gene was observed in ML-3 cells that had been induced to differentiate prior to transfection. No stimulation of IL-1 beta promoter activity was observed in ML-3 cells that were undifferentiated prior to transfection. In summary, HCMV infection, although not leading to productive infection, nonetheless may contribute to the pathology of the infection through enhancement of monocyte inflammatory mediator gene expression with subsequent stimulation of protein synthesis.     

High-pressure, high-temperature bioreactor for comparing effects of hyperbaric and hydrostatic pressure on bacterial growth.

We describe a high-pressure reactor system suitable for simultaneous hyperbaric and hydrostatic pressurization of bacterial cultures at elevated temperatures. For the deep-sea thermophile ES4, the growth rate at 500 atm (1 atm = 101.29 kPa) and 95 degrees C under hydrostatic pressure was ca. three times the growth rate under hyperbaric pressure and ca. 40% higher than the growth rate at 35 atm.     

A new method for study of normal lens developmentin vitro using pulsatile delivery of PDGF or EGF in HL-1 serum-free medium

Summary A new culture method was used to study increases in wet and dry weight and soluble protein during normal development of the transparent lens. Seven different media with more than ten different additives were tested for their effects on cultured lens transparency.In vivo, rat lenses increased 53% in soluble protein content between 3 and 5.5 days of age. Only HL-1 serum-free medium containing 15 μg/ml insulin plus 1–2 ng/ml BB platelet-derived growth factor (PDGF), or 5–7 ng/ml epidermal growth factor (EGF) allowed similar growthin vitro, during the same time period. Normal lens grwoth occurred in culture when fresh medium was delivered to lenses as a pulse every 4–6 hours. Lenses decreased in dry weight and soluble protein content, and became opaque when the same medium was delivered continuously. Lenses increased only 26% and 32% in soluble protein content when delivered pulses of HL-1 medium containing BB PDGF or EGF in the absence of insulin. We suggest that pulsatile delivery of medium containing insulin and PDGF or EGF stimulates lens growth during developmentin vitro. This pulsatile organ culture system is presented as a new approach for studying the effects of growth factors on cell proliferation, differentiation, and receptor regulation in a developing tissue. This work was supported by grants from EY-07031 and EY-04542 from the National Eye Institute and a grant from the Oculon Corporation. Editor's statement This report documents an in vitro system that may mimic lens development and response to growth regulators and hormones. The system may be useful for application to other organs and provide a foundation for cell and molecular level analysis.     

Investigating causation in cancer clusters

Questions concerning clusters of cancer cases frequently arise in public health practice. The process of investigating any such cluster requires awareness that such case groupings can easily occur by chance and that any search for biologically meaningful causes will be severely constrained by various methodologic difficulties. These include (1) the long and probably variable latent periods between causative events and cancer diagnosis, (2) the limited numbers of cases available for study in any given cluster situation, and (3) the clinical non-specificity of cancer cases whereby no readily available means are at hand to identify the specific causes for any particular case. Evaluation of any given cluster should involve careful consideration of such limitations, together with a preliminary assessment of the specific cases involved and their community or workplace setting, before more intensive study is undertaken. Received: 22 February 1996 / Accepted in revised form: 13 June 1996     

Lateral diffusion in planar lipid bilayers: a fluorescence recovery after photobleaching investigation of its modulation by lipid composition, cholesterol, or alamethicin content and divalent cations.

In spite of the fact that planar lipid bilayers are still the best-suited artificial membrane system for the study of reconstituted ion channels and receptors, data dealing with their physical characterization, especially as regards dynamics, are scanty. A combined electrical and optical chamber was designed and allowed fluorescence recovery after photobleaching recovery curves to be recorded from stable virtually solvent-free bilayers. D, the lateral diffusion coefficient of N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, was found to be relatively insensitive to the phospholipid composition (headgroup, chain unsaturation, etc.), whereas inclusion of 33-50% cholesterol in the membrane reduced D by a factor of 2. Divalent cations significantly reduced D of negatively charged bilayers. These results compare well with data gathered on other model and natural systems. In addition, the incorporation of the voltage-dependent pore-former alamethicin did slightly reduce lipid lateral mobility. This study demonstrates the feasibility of such experiments with planar bilayers, which are amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.     

Identification of VCR, a repeated sequence associated with a locus encoding a hemagglutinin in Vibrio cholerae O1.

We have determined the nucleotide sequence of a 6.3-kb BamHI fragment of the chromosome of Vibrio cholerae 569B that includes the sequence of the mannose-fucose-resistant hemagglutinin reported previously (V.L. Franzon, A. Barker, and P. A. Manning, Infect. Immun. 61:3032-3037, 1993). This region contains nine copies of a 124-bp direct repeat, here named VCR, of imperfect dyad symmetry, that are shown by Southern hybridization to occur at least 60 to 100 times in the V. cholerae O1 chromosome. Large-scale chromosomal mapping suggests that the repeats are confined to about 10% of the chromosome. Related sequences are also found in non-O1 V. cholerae but not in other members of the family Vibrionaceae. However, VCR is unrelated to other previously described repetitive sequences.     

Site-directed mutagenesis of the yeast PRP20/1SRM1 gene reveals distinct activity domains in the protein product

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.