Diacylglycerol kinase inhibitor R59022 attenuates conjugated linoleic acid-mediated inflammation in human adipocytes

Diacylglycerol kinases (DGK) convert diacylglycerol to phosphatidic acid, which has been reported to stimulate calcium release from the endoplasmic reticulum. Based on our published data showing that trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA)-mediated intracellular calcium accumulation is linked to inflammation and insulin resistance, we hypothesized that inhibiting DGKs with {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022 would prevent t10,c12 CLA-mediated inflammatory signaling and insulin resistance in human adipocytes. Consistent with our hypothesis, {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022 attenuated t10,c12 CLA-mediated i) increased gene expression and protein secretion of interleukin (IL)-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1); ii) increased activation of extracellular signal-related kinase (ERK), cJun-NH2-terminal kinase (JNK), and cJun; iii) increased intracellular calcium levels; iv) suppressed mRNA or protein levels of peroxisome proliferator activated receptor γ, adiponectin, and insulin-dependent glucose transporter 4; and v) decreased fatty acid and glucose uptake and triglyceride content. DGKη was targeted for investigation based on our findings that i) DGKη was highly expressed in primary human adipocytes and time-dependently induced by t10,c12 CLA and that ii) t10,c12 CLA-induced DGKη expression was dose-dependently decreased with {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022. Small interfering RNA (siRNA) targeting DGKη decreased t10,c12 CLA-induced DGKη, IL-8, and MCP-1 gene expression, as well as activation of JNK and cJun. Taken together, these data suggest that DGKs mediate, in part, t10,c12 CLA-induced inflammatory signaling in primary human adipocytes.     

Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate

With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.     

Effects of T-activin on the morphology of the thymus gland in experimental injury of the lower limbs in mice (anti-stress effect of T-activin)

The effect of T-activin on thymic involution under the experimental trauma of femur was studied. T-activin in a dose of 1.0 micrograms/mouse was injected into young male (CBA X C57BL)F1 mice weighing 17.5-19.0 g before (I injection) or immediately after the fracture of the femur during 3 days. Morphometric analysis of the thymus was made 1, 5, 10 and 15 days after the trauma. It was found that T-activin suppressed the involution of the thymus, induced by the trauma, during the first 5 days and accelerate the process of its regeneration. It is suggested, that T-activin displays protective anti-stress effect on the mouse thymic involution.     

Oral 18F-fluoro-2-deoxyglucose for primate PET studies without behavioral restraint: Demonstration of principle

We describe a method of orally administering ¹⁸F-fluoro-2-deoxyglucose (FDG) for positron emission tomography (PET) scans to determine local cerebral metabolic rates for glucose (LCMRGlc), normalized to that of whole brain, in fully conscious, non-restrained primates. Oral FDG-PET studies were performed in both non-restrained and chaired monkeys, and in one human where results could be compared with traditional intravenous FDG administration. The oral route of FDG administration gave images and whole brain-normalized PET LCMRGlc results comparable to those obtained by the intravenous route. This oral FDG-PET method may provide a useful means by which to obtain measures of LCMRGlcs for brain structures, relative to each other, in non-restrained, non-druggd primates in field and laboratory studies. This method might also have clinical applications for PET studies of children. Am. J. Primatol. 42:215–224, 1997. © 1997 Wiley-Liss, Inc.     

Permeabilization of rat hepatocytes with Staphylococcus aureus alpha- toxin

Pathogenic staphylococci secrete a number of exotoxins, including alpha-toxin. alpha-Toxin induces lysis of erythrocytes and liposomes when its 3S protein monomers associate with the lipid bilayer and form a hexomeric transmembrane channel 3 nm in diameter. We have used alpha-toxin to render rat hepatocytes 93-100% permeable to trypan blue with a lactate dehydrogenase leakage less than or equal to 22%. Treatment conditions included incubation for 5-10 min at 37 degrees C and pH 7.0 with an alpha-toxin concentration of 4-35 human hemolytic U/ml and a cell concentration of 13-21 mg dry wt/ml. Scanning electron microscopy revealed signs of swelling in the treated hepatocytes, but there were no large lesions or gross damage to the cell surface. Transmission electron microscopy indicated that the nucleus, mitochondria, and cytoplasm were similar in control and treated cells and both had large regions of well-defined lamellar rough endoplasmic reticulum. Comparisons of the mannose-6-phosphatase and glucose-6-phosphatase activities demonstrated that 5-10 U/ml alpha-toxin rendered cells freely permeable to glucose-6-phosphate, while substantially preserving the selective permeability of the membranes of the endoplasmic reticulum and the functionality of the glucose-6-phosphatase system. Thus, alpha-toxin appears to have significant potential as a means to induce selective permeability to small ions. It should make possible the study of a variety of cellular functions in situ.     

Quantitative aspects of relationship between glucose 6-phosphate transport and hydrolysis for liver microsomal glucose-6-phosphatase system. Selective thermal inactivation of catalytic component in situ at acid pH.

Studies of the thermal stability of rat liver glucose-6-phosphatase (EC 3.1.3.9) were carried out to further elevate the proposal that the enzymic activity is the result of the coupling of a glucose-6-P-specific translocase and a nonspecific phosphohydrolase-phosphotransferase. Inactivation was observed when micorsomes were incubated at mild temperatures between pH 6.2 and 5.6. The rate of inactivation increased either with increasing hydrogen ion concentration or temperature. However, no inactivation was seen below 15 degrees in media as low as pH 5 or at neutral pH up to 37 degrees. The thermal stability of the enzyme may be controlled by the physical state of the membrane lipids and the degree of protonation of specific residues in the enzyme protein. Microsomes were exposed to inactivating conditions, and kinetic analyses were made of the glucose-6-P phosphohydrolase activities before and after supplementation to 0.4% sodium taurocholate. The results support the postulate and the kinetic characteristics of a given preparation of intact microsomes are determined by the relative capacities of the transport and catalytic components. Before detergent treatment, inactivation (i.e. a decrease in Vmax) was accompanied by a decrease in Km and a reduction in the fraction of latent activity, whereas only Vmax was depressed in disrupted preparations. The possibility that the inactivating treatments caused concurrent disruption of the microsomal membrane was ruled out. It is concluded that exposures to mild heat in acidic media selectively inactivate the catalytic component of the glucose-6-phosphatase system while preserving an intact permeability barrier and a functional glucose-6-P transport system. Analyses of kinetic data obtained in the present and earlier studies revealed several fundamental mathematical relationships among the kinetic constants describing the glucose-6-P phosphohydrolase activities of intact (i.e. the "system") and disrupted microsomes (i.e. the catalytic component). The quantitative relationships appear to provide a means to calculate a velocity constant (VT) and a half-saturation constant (KT) for glucose-6-P influx. The well documented, differential responses of the rat liver glucose-6-phosphatase system induced by starvation, experimental diabetes, or cortisol administration were analyzed in terms of these relationships. The possible influences of cisternal inorganic phosphate on the apparent kinetic constants of the intact system are discussed.     

Synthesis, crystal structure and pH dependent cytotoxicity of (SP-4-2)-bis(2-aminoethanolato-κN,O)platinum(II) - a representative of novel pH sensitive anticancer platinum complexes

Solid tumors are often hypoxic and consequently the pH in the tumoral tissue is decreasing with increasing tumor size (pH 5.5-7.4 in solid tumors versus pH 7.4 in normal tissues). This marked difference in pH value is a problem for weak base organic drugs and could advantageously be used for the introduction of pH sensitive anticancer platinum drugs. Synthesis and structure determination of (SP-4-2)-bis(2-aminoethanolato-κ²N,O)platinum(II), its binding behavior to 5^′-GMP and its cytotoxicity against cisplatin sensitive cell lines under standard pH screening conditions (pH 7.4) as well as in acidified cell culture medium (pH 6.0) mimicking the conditions in a number of solid tumors is presented. There is evidence that this concept in anticancer platinum therapy, namely administration of rather unreactive drugs and activation under acidic pH conditions, can be realized.     

Antiobesity mechanisms of action of conjugated linoleic acid

Conjugated linoleic acid (CLA), a family of fatty acids found in beef, dairy foods and dietary supplements, reduces adiposity in several animal models of obesity and some human studies. However, the isomer-specific antiobesity mechanisms of action of CLA are unclear, and its use in humans is controversial. This review will summarize in vivo and in vitro findings from the literature regarding potential mechanisms by which CLA reduces adiposity, including its impact on (a) energy metabolism, (b) adipogenesis, (c) inflammation, (d) lipid metabolism and (e) apoptosis.     

Effects of taktivin on antibody-forming splenic cells in rats

The paper studied the effect of Taktivin on antibody forming cells of the spleen of Wistar rats which were injected in doses 0.5 Hg and 1.0 Hg. Infusions of Taktivin were made in 24 h and 72 h after antigen immunization.