The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes

We have examined the effects of extracellular and intracellular Ca2+ concentrations upon basal and insulin-stimulated 2-deoxyglucose uptake in isolated rat adipocytes. In the absence of extracellular Ca2+, both basal and insulin-stimulated glucose uptake were significantly reduced. Insulin-stimulated glucose transport was optimal at 1 and 2 mM Ca2+. Further increases in extracellular Ca2+ concentration (3 mM) significantly diminished insulin-stimulated glucose uptake. When intracellular Ca2+ concentrations were augmented by ionomycin (1 microM), insulin-stimulated glucose uptake was significantly reduced at extracellular Ca2+ concentrations of 2 and 3 mM. The levels of intracellular free Ca2+ concentrations were then measured with Ca2+ indicator fura-2. The correlation between the levels of intracellular free Ca2+ and the magnitude of insulin-stimulated glucose uptake revealed that the optimal effect of insulin is observed at Ca2+ levels between 140 and 370 nM. At both extremes outside of this window, both low and high levels of intracellular Ca2+ result in diminished cellular responsiveness to insulin. These data suggest that intracellular calcium concentrations may exert a dual role in the regulation of cellular sensitivity to insulin. First, there must exist a minimal concentration of intracellular calcium to promote insulin action. Second, increased levels of intracellular calcium may provide a critical signal for diminution of insulin action.     

Cardiac Fibroblasts Contribute to Myocardial Dysfunction in Mice with Sepsis: The Role of NLRP3 Inflammasome Activation

Myocardial contractile dysfunction in sepsis is associated with the increased morbidity and mortality. Although the underlying mechanisms of the cardiac depression have not been fully elucidated, an exaggerated inflammatory response is believed to be responsible. Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome is an intracellular platform that is involved in the maturation and release of interleukin (IL)-1β. The aim of the present study is to evaluate whether sepsis activates NLRP3 inflammasome/caspase-1/IL-1β pathway in cardiac fibroblasts (CFs) and whether this cytokine can subsequently impact the function of cardiomyocytes (cardiac fibroblast-myocyte cross-talk). We show that treatment of CFs with lipopolysaccharide (LPS) induces upregulation of NLRP3, activation of caspase-1, as well as the maturation (activation) and release of IL-1β. In addition, the genetic (small interfering ribonucleic acid [siRNA]) and pharmacological (glyburide) inhibition of the NLRP3 inflammasome in CFs can block this signaling pathway. Furthermore, the inhibition of the NLRP3 inflammasome in cardiac fibroblasts ameliorated the ability of LPS-chalenged CFs to impact cardiomyocyte function as assessed by intracellular cyclic adenosine monophosphate (cAMP) responses in cardiomyocytes. Salient features of this the NLP3 inflammasome/ caspase-1 pathway were confirmed in in vivo models of endotoxemia/sepsis. We found that inhibition of the NLRP3 inflammasome attenuated myocardial dysfunction in mice with LPS and increased the survival rate in mice with feces-induced peritonitis. Our results indicate that the activation of the NLRP3 inflammasome in cardiac fibroblasts is pivotal in the induction of myocardial dysfunction in sepsis.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor’s abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced – indicating viral protein-induced alterations in the cofactors’ binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.     

Cultured circular smooth muscle from the rabbit colon

Summary Although cultured vascular smooth muscle cells have been extensively characterized and investigated, there are very few studies of cultured intestinal smooth muscle cells. The aim of this study was to culture colonic smooth muscle (CSM) cells from the rabbit colon. Freshly isolated CSM cells from the circular muscle layer of the distal colon were prepared by collagenase digestion. In primary culture, CSM cells attached to the culture vessels by 48 to 72 h, proliferated by 3 to 7 d, and reached confluency by 14 to 17 d with a “hill-and-valley” pattern. Spontaneous contractions were not observed at any time at 21° or 37° C. Confluent primary cultures were greater than 95% CSM cells, as identified by intensely positive immunofluorescent staining to smooth muscle actin-specific CGA7 and muscle-specific HHF-35 monoclonal antibodies. Transmission electron microscopy of freshly isolated and proliferating CSM cells revealed ultrastructural features consistent with smooth muscle cells. We successfully cultured CSM cells of the rabbit from freshly isolated cells and validated these CSM cells by electron microscopy and immunocytochemical staining. These highly pure primary cultures may be used to investigate numerous aspects of CSM cell metabolism and physiology. These studies were supported by the National Institutes of Health grant to the Inflammatory Bowel Disease Center (Bethesda, MD) P30-AM-32200 and R01-DK-31147. Dr. Kao is the recipient of a Research Career Development Award from the National Foundation for Ileitis and Colitis, Inc. A preliminary report of this work was presented at the American Motility Society Meeting, Houston, TX, in October 1986, and appeared in abstract form inGastroenterology 91: 1057; 1986.     

Similar C-phycocyanins from two strains of thermotolerant cyanophyte Mastigocladus laminousus.

C-phycocyanin from two strains of the thermotolerant blue-green alga, Mastigocladus laminosus (NZ-DB2-m and I-30-m), that grow within different temperature ranges have been characterized with respect to aggregation, immunologic properties, subunit composition, and thermodenaturation. The critical thermal-denaturation temperature for phycocyanin from both strains of M. laminosus phycocyanin is 60 degrees C which is higher than that for mesophilic phyococyanin. Immunodiffusion studied have shown that these two strains of M. laminosus exhibit no antigenic differences and are closely related to the mesophilic Plectonema calothricoides and the thermophilic Synechococcus lividus (strains 3). Neither phenol nor alpha-naphthol has any effect on phycocyanin aggregation in these two strains of M. laminosus. There is also no enhancement of formation of large aggregates at their elevated temperature of cultivation. Furthermore, the phycocyanin of both strains of M. laminosus does not demonstrate any large amount of 19S or higher aggregates at any pH value. These observations suggest that the mode of adaptation of M. laminosus phycocyanin to high temperature is differnet from the previously encountered. It is also important to note that phycocyanin is essentially unchanged whether it is extracted from the same strain, M. laminosus (NZ-DBS-m), grown at either 50 degrees C or 37 degrees C.     

Mechanism of neurotensin depolarization of rabbit colonic smooth muscle

The aims of this study were (1) to measure the effect of neurotensin on the membrane potential of circular muscle of the distal colon of the rabbit and (2) to determine the mechanism by which neurotensin affects the membrane potential of this tissue. The membrane potential was measured with microelectrodes placed intracellularly and the double sucrose gap. Neurotensin (10(-11) M to 10(-7) M) dose-dependently decreased the membrane potential. The maximum decrease in membrane potential occurred with 10(-9) M neurotensin. The ED50 of neurotensin depolarization of the membrane potential was 0.87 +/- 0.33 X 10(-10) M. The frequency of the slow waves was unchanged after neurotensin. The voltage response to a constant current pulse decreased as the concentration of neurotensin increased. The amplitude of the voltage response after a 0.6 microA current pulse decreased by 6 +/- 0.5 mV after neurotensin (10(-7) M) compared to the Krebs control (P less than 0.05). Decreasing the [Na+]o to 0-23 mM did not affect the decrease in membrane potential after neurotensin. However, perfusion with a test solution containing no added Ca2+ or verapamil (10(-5) M) inhibited neurotensin depolarization of the tissue. Evidence was found that neurotensin depolarizes colonic circular smooth muscle, and the decrease in membrane potential is associated with an increase in conductance which is dependent on influx of Ca2+.     

Length, mass, and denaturation of double-stranded RNA molecules compared with DNA

The contour lengths of linear, double-stranded (ds) RNAs from mycovirus PcV and Pseudomonas bacteriophage ø6 have been measured with samples prepared for the electron microscope from 0.05 to 0.5 M NH₄Cl solutions. A linear dependence of contour length on the logarithm of ionic strength was found and compared with that of dsDNA (pBR322, linearized and open-circular forms). Conditions for molecular weight determinations of any natural dsRNA by electron microscopy have been established, and the method has been calibrated with ø6 dsRNA of known nucleotide sequence. The results imply that dsRNA in 0.20 M NH₄Cl solution has a rise per basepair of 0.271 nm, which is shorter than that in the A-conformation (4%) and in the A′-conformation (10%). The thermal behavior of dsRNA in terms of melting temperature and exhibition of fine structure of melting curves was found to be generally similar to that of dsDNA, as expected from the literature. Folding of dsRNA in ethanolic solution was similar to that of dsDNA. However, in contrast to dsDNA, coiled coils could not be induced by ethanol, which is consistent with dsRNA being stiffer than dsDNA. Concerning dsDNA, the results show that a contraction in rise per basepair by 0.1 nm is coupled with an increase in the winding angle between basepairs by 0.47°, as qualitatively predicted by polyelectrolyte theory.     

Common expression of a tumor necrosis factor resistance mechanism among gynecological malignancies

Summary The efficacy of tumor necrosis factor (TNF) as an anticancer agent is limited. This limitation might be related to the expression of a protein-synthesis-dependent resistance mechanism that prevents the lysis of tumor cells by TNF. To test this possibility eight randomly selected human cell lines, three derived from ovarian carcinomas and five derived from cervical carcinomas, were tested for their in vitro sensitivity to TNF-mediated lysis. The results of this analysis showed that all eight cell lines are normally resistant to lysis by TNF. However, in the presence of inhibitors of protein synthesis, seven of them showed a significant increase in TNF-mediated lysis. Measurement of protein synthesis showed that there is a linear correlation between the level of inhibition of protein synthesis and the level of TNF-mediated lysis. The fact that seven of eight randomly selected cell lines are resistant to TNF because they express a protein-synthesis-dependent resistance mechanism suggests that this mechanism of resistance may be common among gynecological cancers. The results also suggest that a therapy involving TNF and inhibitors of protein synthesis might be useful for the treatment of gynecological malignancies.