Resistance of Mycobacterium chelonei-like organisms to formaldehyde.

Mycobacterium chelonei-like organisms have been isolated from patients in two outbreaks of peritonitis involving chronic peritoneal dialysis machines routinely disinfected with 2 to 3% formaldehyde. Susceptibility studies revealed that water-adapted M. chelonei-like organism strains could survive 2 h of exposure to 10% formaldehyde.     

Methyl β-lactoside: 600-MHz H- and 75-MHz C-n.m.r. studies of H- and C-enriched compounds

Using UDP-d-galactose : 2-acetamido-2-deoxy-d-glucose 4-β-d-galactosyltransferase (EC 2.4.1.22), several methyl β-lactosides have been prepared with ²H- and/or ¹³C-enrichment at specific sites to facilitate study by ¹³C (75 MHz) and ¹H (600 MHz) n.m.r. spectroscopy. ¹³C-Chemical shift assignments were verified and the ¹H-spectrum of β-lactoside was fully assigned. Sites of enrichment were selected to permit all of the potential three-bond C-C and C-H couplings through the glycosidic bond to be obtained. Replacement of H-3 of the d-glucose residue of methyl β-lactoside with ²H allowed resolution of C-1–H-4′ coupling in the 600-MHz ¹H-spectrum. Single or multiple ¹³C-enrichment at C-1, C-2, C-3, C-1′, C-3′, and/or C-4′ in the disaccharide allowed observation of intra- and inter-residue couplings. ¹³C-Spin-lattice relaxation-times (T₁) are interpreted in terms of molecular motion in solution. The data suggest that methyl β-lactoside has an extended conformation with little rotation about the glycosidic bond. Inter-residue couplings are best explained by tortion angles of φ ~ 40° and ψ ~ 15°, indicating that the conformations of β-lactoside in solution and in the crystal are similar.     

Low density lipoprotein from humans supplemented with n-3 fatty acids depresses both LDL receptor activity and LDLr mRNA abundance in HepG2 cells.

Fish oil supplementation in humans is often associated with an expanded low density lipoprotein (LDL) pool that is not thought to reflect increased production. Since data on clearance of LDL after fish oil supplementation (FO-LDL) are equivocal, normal volunteers (four men and three women) received ten capsules containing 3.6 g eicosapentaenoic acid and 2.9 g docosahexaenoic acid (approximately 2.5% total calories as methyl esters) for 2 weeks. Total plasma cholesterol was unchanged, but triglycerides decreased 30%. Low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were unchanged. Analysis of the LDL particles revealed that increased esterified cholesterol caused the FO-LDL core/surface ratio to be greater than baseline LDL (BL-LDL), resulting in a shift in mean LDL density from 1.060 to 1.056. N-3 fatty acids in FO-LDL were also increased greater than 40% at the expense of n-6 and n-9 fatty acids. Human hepatoma HepG2 cells were used to study the effects of FO-LDL on LDL receptor activity and mRNA abundance for the LDL receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and various apolipoproteins associated with cholesterol metabolism. In this system FO-LDL reduced LDL receptor activity compared to BL-LDL. Scatchard analysis revealed that LDL receptor number (Bmax) was reduced to one-third normal (P less than 0.001) whereas particle binding affinity was unchanged. The mRNA abundance for the LDL receptor and apoA-I were also depressed, even by low concentrations (10 micrograms/ml and 20 micrograms/ml LDL protein) of FO-LDL as compared to BL-LDL. HepG2 cells incubated with FO-LDL had decreased cellular free cholesterol but increased cholesteryl esters. Thus, moderate supplementation with fish oil n-3 fatty acids in normal humans enriches their LDL particles in cholesteryl esters and n-3 fatty acids. These particles depress both LDL receptor activity and LDL receptor mRNA abundance in HepG2 cells.     

Different relative abundance of neurotensin and neuromedin N in bovine ocular tissues.

Neurotensin (NT) and neuromedin N (NN) are regulatory peptides encoded by the same gene and located in tandem within a common precursor. Using specific radioimmunoassays for both peptides, their relative abundance in extracts of bovine ocular tissues has been examined. Within the retina, the molar concentration of NN was significantly higher (P less than 0.001) than that of NT. In contrast, within both choroid/sclera and iris/ciliary bodies, the molar concentration of NT was significantly higher (P less than 0.001) than that of NN. These data demonstrate that the theoretical molar ratio of 1:1, which would result from complete processing of both peptides from the common precursor, does not occur in any of the ocular tissues examined. Reverse phase HPLC of extracts of each ocular tissue confirmed the differential abundance of NT and NN. These data would suggest that the common NT/NN precursor is differentially-processed within bovine ocular tissues, a finding which may be of physiological significance.     

Mapping of carbohydrate sites on the human insulin receptor

Prior to investigating the role of individual glycosylation sites in insulin receptor function, we are mapping the sites of glycosylation in the receptor. We report here a generally applicable methodology for the isolation and identification of glycosylation sites in cell surface glycoproteins. Human insulin receptors were labeled with [3H]-sugars using a CHO cell line transfected with the human receptor cDNA. Labelled receptors were mixed with receptors purified from human placental membranes and tryptic peptides prepared. Peptides were fractionated by gel filtration chromatography to limit the number of non-glycopeptides present. Peptides were then separated by reverse phase HPLC and glycopeptides identified by scintillation counting. Using this technique we have shown the insulin receptor to be glycosylated at Asn 397 and Asn 881. This increase the known number of occupied glycosylation sites to five.     

Murine retrovirus-induced spongiform encephalopathy: productive infection of microglia and cerebellar neurons in accelerated CNS disease.

We have examined the pathological lesions and sites of infection in mice inoculated with a highly neurovirulent recombinant wild mouse ecotropic retrovirus (FrCasE). The spongiform lesions appeared initially as swollen postsynaptic neuronal processes, progressing to swelling in neuronal cell bodies, all in the absence of detectable gliosis. Infection of neurons in regions of vacuolation was not detected. However, high level infection of cerebellar granule neurons was observed in the absence of cytopathology, wherein viral protein was found associated with both axons and dendrites. Infection of ramified and amoeboid microglial cells was associated with cytopathology in the brain stem, and endothelial cell-pericyte infection was found throughout the CNS. No evidence of defective retroviral expression was observed. These results are consistent with an indirect mechanism of retrovirus-induced neuropathology.     

Esterification reactions of lipase in reverse micelles

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.