Inactivation of endothelial derived relaxing factor by oxidized lipoproteins.

Endothelial cell derived relaxing factor (EDRF) mediated relaxation of blood vessels is impaired in vessels exposed to lipoproteins in vitro and in arteries of hyperlipidemic humans and animals. To investigate the mechanism by which lipoproteins impair the effects of EDRF, which is likely nitric oxide (NO) or a related molecule, we have bioassayed EDRF/NO activity by measuring its ability to increase cGMP accumulation in rat fetal lung cultured fibroblasts (RFL-6 cells). Low density lipoprotein modified by oxidation (ox-LDL) induced a concentration-dependent inhibition of EDRF activity that had been released from bovine aortic endothelial cells (BAEC) stimulated with bradykinin or the calcium ionophore A23187. In addition, lipoproteins directly impaired authentic NO-induced stimulation of cGMP accumulation in the detector cells; stimulation by sodium nitroprusside was unaffected. Ox-LDL or oxidized HDL3 were highly potent in blocking NO-stimulated cGMP accumulation with EC50's of approximately 1 microgram/ml. Lipid extracted from ox-LDL blocked NO-stimulated cGMP accumulation to about the same extent as intact ox-LDL, while the protein component of ox-LDL did not inhibit the cGMP response. These results suggest that the lipid component of oxidized lipoproteins inactivate EDRF after its release from endothelial cells.     

In vitro activity of Ro 23-6240, a new fluorinated 4-quinolone.

The in vitro activity of Ro 23-6240 (AM833), 6,8-difluoro-1-(2-fluoroethyl)-1,4-dihydro-4-oxo-7(4-methyl-1-piper azinyl) quinolone-3-carboxylic acid, was compared with those of norfloxacin, enoxacin, ofloxacin, and ciprofloxacin. Ro 23-6240 inhibited the majority of Enterobacteriaceae isolates at a concentration of less than or equal to 0.5 microgram/ml. It was especially active against Shigella sp., Salmonella sp., Escherichia coli, and Yersinia enterocolitica, with an MIC for 90% of the strains of less than or equal to 0.12 microgram/ml. The MIC for 90% of the strains was 1 microgram/ml for Serratia marcescens and 8 micrograms/ml for Pseudomonas aeruginosa. Staphylococcus aureus isolates, including methicillin-resistant strains, were inhibited by less than or equal to 1 microgram/ml. Streptococcal and anaerobic species were inhibited by 8 to 16 micrograms/ml. Ro 23-6240 inhibited beta-lactamase-producing bacteria resistant to broad-spectrum cephalosporins. The overall activity of Ro 23-6240 was similar to those of enoxacin, norfloxacin, and ofloxacin, but less than that of ciprofloxacin. The frequency of spontaneous resistance was low, although resistant bacteria could be isolated by repeated subculture. The activity of Ro 23-6240 was decreased in the presence of magnesium at concentrations similar to those present in urine.     

Hypoxanthine-guanine phosphoribosyltransferase. Genetic evidence for identical mutations in two partially deficient subjects.

In past reports of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency a marked degree of molecular heterogeneity has been noted. We have previously described two apparently unrelated subjects with partial HPRT deficiency, G.S. and D.B., who have a mutant form of HPRT with remarkably similar alterations in physical and kinetic properties. The mutation in G.S. is a serine to leucine substitution at amino acid 110 as determined by amino acid sequence analysis. This mutant enzyme has been designated HPRTLondon. We have examined HPRT cDNA from D.B. using two different methods to determine if the similar properties of mutant HPRT from these two subjects are the result of a common mutation. HPRT cDNA clones were obtained by routine cloning techniques and by polymerase chain reaction amplification of single-stranded cDNA reverse transcribed from mRNA derived from subject D.B. Dideoxynucleotide sequencing revealed a single mutation, a C to T transition at bp 329 in clones generated by both methods. This mutation in D.B. predicts the identical amino acid substitution described in HPRTLondon. A C to T nucleotide transition at 329 in D.B. creates an Hpa I site in exon 4 of the HPRT gene. Southern blot analysis of genomic DNA isolated from lymphoblasts derived from G.S. and D.B. revealed that both have this additional Hpa I site, indicating that the similarly altered protein sequence is due to the identical transition in the HPRT gene.     

Tigemonam, an oral monobactam.

Tigemonam is an orally administered monobactam. At less than or equal to 1 microgram/ml it inhibited the majority of strains of Escherichia coli, Klebsiella spp., Enterobacter aerogenes, Citrobacter diversus, Proteus spp., Providencia spp., Aeromonas hydrophila, Salmonella spp., Shigella spp., Serratia marcescens, and Yersinia enterocolitica. At less than or equal to 0.25 microgram/ml it inhibited Haemophilus spp., Neisseria spp., and Branhamella catarrhalis. It did not inhibit Pseudomonas spp. or Acinetobacter spp. Tigemonam was more active than cephalexin and amoxicillin-clavulanate and inhibited many members of the family Enterobacteriaceae resistant to trimethoprim-sulfamethoxazole and gentamicin. Some Enterobacter cloacae and Citrobacter freundii strains resistant to aminothiazole iminomethoxy cephalosporins and aztreonam were resistant to tigemonam. The MIC for 90% of hemolytic streptococci of groups A, B, and C and for Streptococcus pneumoniae was 16 micrograms/ml, but the MIC for 90% of enterococci, Listeria spp., Bacteroides spp., and viridans group streptococci was greater than 64 micrograms/ml. Tigemonam was not hydrolyzed by the common plasmid beta-lactamases such as TEM-1 and SHV-1 or by the chromosomal beta-lactamases of Enterobacter, Morganella, Pseudomonas, and Bacteroides spp. Tigemonam inhibited beta-lactamases of E. cloacae and Pseudomonas aeruginosa but did not induce beta-lactamases. The growth medium had a minimal effect on the in vitro activity of tigemonam, and there was a close agreement between the MICs and MBCs.     

Increased hypercholesterolemia and atherosclerosis in mice lacking both ApoE and leptin receptor

Diabetes mellitus is one of the major risk factors associated with atherosclerosis and coronary heart disease but the mechanistic links between the disease and atherosclerosis are not well understood. In this study, we investigated the effect of the deletion of the long-form leptin receptor on the progression of atherosclerosis in ApoE-/- mouse. ApoE-/-;db/db double knockout mice as well as ApoE-/-;db/+ and ApoE-/- littermates were generated by crossing ApoE-/- and db/+ mice. On a regular chow diet, ApoE-/-;db/db mice at 20 weeks of age exhibited features typical of type 2 diabetes: obesity, hyperglycemia, hyperinsulinemia and dyslipidemia and had significantly accelerated atherosclerosis compared with their age-matched ApoE-/- littermates as assessed by either the percentage of the aorta bearing lesion (5.3+/-0.9% for ApoE-/-;db/db versus 1.5+/-0.5% for ApoE-/-) or by aortic lipid content ( approximately 1.5-2-fold increase in free cholesterol and approximately 3-4-fold increase in cholesteryl ester). The atherosclerosis in these ApoE-/-;db/db mice was further accelerated by feeding mice with a Western diet and markedly inhibited by fenofibrate with a 2.5- and 5.3-fold reduction of the lesion in male and female mice, respectively. The results from this study demonstrate that type 2 diabetes can accelerate atherogenesis in mice. This mouse model may provide insight into the mechanistic link between type 2 diabetes and atherosclerosis as well as serve as a valuable tool for evaluating therapeutics.     

Genes that can be mutated to unmask hidden antigenic determinants in the cuticle of the nematode Caenorhabditis elegans.

Rabbit antisera directed against a mixture of proteins solubilized from the wild-type adult Caenorhabditis elegans cuticle were used to isolate mutants, induced by ethyl methanesulfonate treatment, that exhibit alterations in surface antigenicity by immunofluorescence. Genetic mapping and complementation data for four such mutations define two genes, srf-2(I) and srf-3(IV). The mutant phenotypes observed by immunofluorescence appear to result from unmasking of antigenic determinants that are normally hidden in the wild-type cuticle. In support of this hypothesis, surface radioiodination experiments indicate that components labeled on the wild-type surface are missing or less readily labeled on the surface of srf-2 and srf-3 mutants.     

Regulation of angiotensinogen gene expression by estrogen.

OBJECTIVE: Clarification of the role of estrogen in the regulation of angiotensinogen gene expression in multiple tissues. DESIGN: The effect of 17 beta-estradiol (E2; 10 micrograms/100 mg body weight) administration in ovariectomized (OVX) rats upon angiotensinogen messenger RNA (mRNA) levels in multiple tissues was assessed. Confounding ovarian factors were thus removed by studying the animals in the castrate state. Controls consisted of OVX and intact female rats. METHODS: Adult female Sprague-Dawley rats were ovariectomized and experiments begun 21 days postsurgery. Animals were injected with E2 and studied after 0, 1, 4, and 24 h of treatment. Levels of angiotensinogen mRNA were determined by Northern blot analysis using beta-actin mRNA as an internal standard. RESULTS: A single angiotensinogen mRNA species with molecular size of approximately 1800 bp was observed in rat liver, aorta, kidney, cardiac atria, hypothalamus and whole brain. Little or no angiotensinogen mRNA was identified in the pituitary gland. Angiotensinogen mRNA was most abundant in rat liver, hypothalamus, aorta and progressively less abundant in whole brain, cardiac atria and kidney. A twofold induction of hepatic angiotensinogen mRNA levels in E2-OVX rats was observed by 4h. The angiotensinogen mRNA levels in kidney were threefold higher by 4 h compared with OVX control animals. In aorta, the angiotensinogen mRNA level was also threefold higher by 1 h after E2 treatment. No significant effect of estradiol treatment was observed in cardiac atria although the level of angiotensinogen mRNA was higher in intact female rats compared with OVX controls. CONCLUSION: These results suggest that estrogen modulates angiotensinogen gene expression in a tissue-specific manner.     

Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.