Saccharin exposure increases the potency and aversive property of naloxone in drug-naive rats

In naive rats previously given saccharin (0.1%) as the only drinking fluid for 14 days, naloxone produced a conditioned taste aversion at a dose (0.09 mg/kg, SC) that had little or no effect in normal controls. The magnitude of this effect of the saccharin increased between 2 and 7 days after cessation of the treatment. Under these experimental conditions, evidence of an interaction between the saccharin exposure and a naloxone response was also seen with rectal temperature measurements. Therefore, in rats having no history of morphine, previous consumption of saccharin may potentiate various actions of naloxone including its aversive property.     

Abnormal perception of food size in anorexia nervosa

The hypothesis that patients with anorexia nervosa exaggerate the perceived size of food was tested. Video recordings of five items of food and four neutral objects of a similar size were made such that the size of each object increased steadily from half to twice its normal size. Each of the nine objects was placed on a ledge inside a dummy television screen next to the video screen, the food items alternating with the neutral objects, and 20 female patients with anorexia nervosa and 20 female controls matched for age were asked to adjust the size of the video recording to that of the real object. Although there was no overall difference in perceptual accuracy between patients and controls, both groups perceived the food items as being bigger than the neutral objects, the patients exaggerating the size of the food significantly more than the controls.These results imply that patients with anorexia might start eating more easily when admitted if their food were presented as small portions on large plates.     

Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic mechanism.

The mechanism by which CD8+ T cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in acutely infected CD4+ T cells was investigated. Cytotoxicity was not involved, as the antiviral activity of the CD8+ cells did not correlate with the ability to lyse HIV-infected or uninfected CD4+ T cells. In addition, the frequency of HIV-infected CD4+ cells increased during coculture with CD8+ T cells even in the absence of detectable levels of virus replication. Moreover, separation of the CD4+ and CD8+ cells by a 0.4-micron-pore-size filter delayed HIV replication, indicating a role, at least in part, for a soluble factor. However, cell contact was required for optimal antiviral activity. These results extend further the observation on the mechanism of antiviral HIV activity by CD8+ cells from infected individuals. They support the conclusion that CD8+ cells can play a major role in preventing development of disease in HIV-infected individuals.     

Intranasal activity of the growth hormone releasing peptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 in conscious dogs

This series of experiments was conducted to evaluate the growth hormone (GH) releasing activity of intranasally administered His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6, SK&F 110679) in conscious dogs. Intranasal administration of GHRP-6 increased plasma growth hormone levels in the conscious dog in a dose-related manner. Doses of 0.25 and 0.5 mg/kg produced GH levels of 11.3 +/- 4.8 ng/ml and 28.6 +/- 8.0 ng/ml, respectively. Peak levels were observed 15 minutes after dosing and GH levels were elevated for up to 105 minutes after intranasal dosing. Intranasal administration of isotonic saline did not produce any change in basal (negligable) GH levels. When GHRP-6 was given by the intravenous route, a maximal dose of 0.5 mg/kg, produced a peak plasma GH concentration of 60.8 +/- 10.5 ng/ml. Saline had no effect on GH levels when given intravenously. Using the intravenous and intranasal GH response data (i.e., area under the time-response curves), the intranasal bioavailability of GHRP-6 was estimated to be 34.4 to 44.9%. The results of these studies suggest that significant activity and excellent bioavailability can be achieved when GHRP-6 is administered by the intranasal route to conscious dogs. Based on these results, the intranasal activity of GHRP-6 should be evaluated in man. The successful intranasal administration of this peptide in man should provide GH therapy with reduced patient discomfort and better patient compliance when compared to presently available parenterally administered remedies.     

New mutations in cloned Escherichia coli umuDC genes: novel phenotypes of strains carrying a umuC125 plasmid

The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD⁺C⁺ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD⁺C⁺ plasmids. pBR322-derived umuD⁺C⁺ plasmids inhibited the induction of the SOS response of lexA⁺ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA⁺ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA⁺ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD⁺C⁺ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.     

Effects of chilling on the biochemical and functional properties of thylakoid membranes

The mechanism of chilling resistance was investigated in 4-week-old plants of the chilling-sensitive cultivated tomato, Lycopersicon esculentum Mill. cv H722, and rooted cuttings of its chilling-resistant wild relative, L. hirsutum Humb. and Bonpl., which were chilled for 3 days at 2°C with a 14-hour photoperiod and light intensity of 250 micromoles per square meter per second. This chilling stress reduced the chlorophyll fluorescence ratio, stomatal conductance, and dry matter accumulation more in the sensitive L. esculentum than in the resistant L. hirsutum. Photosynthetic CO₂ uptake at the end of the chilling treatment was reduced more in the resistant L. hirsutum than in L. esculentum, but recovered at a faster rate when the plants were returned to 25°C. The reduction of the spin trap, Tiron, by isolated thylakoids at 750 micromoles per square meter per second light intensity was taken as a relative indication of the tendency for the thylakoids to produce activated oxygen. Thylakoids isolated from the resistant L. hirsutum with or without chilling treatment were essentially similar, whereas those from chilled leaves of L. esculentum reduced more Tiron than the nonchilled controls. Whole chain photosynthetic electron transport was measured on thylakoids isolated from chilled and control leaves of the two species at a range of assay temperatures from 5 to 25°C. In both species, electron transport of the thylakoids from chilled leaves was lower than the controls when measured at 25°C, and electron transport declined as the assay temperature was reduced. However, the temperature sensitivity of thylakoids from chilled L. esculentum was altered such that at all temperatures below 20°C, the rate of electron transport exceeded the control values. In contrast, the thylakoids from chilled L. hirsutum maintained their temperature sensitivity, and the electron transport rates were proportionately reduced at all temperatures. This sublethal chilling stress caused no significant changes in thylakoid galactolipid, phospholipid, or protein levels in either species. Nonchilled thylakoid membranes from L. hirsutum had fourfold higher levels of the fatty acid 16:1, than those from L. esculentum. Chilling caused retailoring of the acyl chains in L. hirsutum but not in L. esculentum. The chilling resistance of L. hirsutum may be related to an ability to reduce the potential for free radical production by close regulation of electron transport within the chloroplast.     

Further characterization of the magnesium chelatase in isolated developing cucumber chloroplasts : substrate specificity, regulation, intactness, and ATP requirements

Mg-chelatase catalyzes the first step unique to the chlorophyll branch of tetrapyrrole biosynthesis, namely the insertion of Mg into protoporphyrin IX (Proto). Mg-chelatase was assayed in intact chloroplasts from semi-green cucumber (Cucumis sativus, cv Sumter) cotyledons. In the presence of Proto and MgATP, enzyme activity was linear for 50 minutes. Plastid intactness was directly related to (and necessary for) Mg-chelatase activity. Uncouplers and ionophores did not inhibit Mg-Chelatase in the presence of ATP. The nonhydrolyzable ATP analogs, β,γ-methylene ATP and adenylylimidodiphosphate, could not sustain Mg-chelatase activity alone and were inhibitory in the presence of ATP (I₅₀ 10 and 3 millimolar, respectively). Mg-chelatase was also inhibited by N-ethylmaleimide (I₅₀, 50 micromolar) and the metal ion chelators 2,2′-dipyridyl and 1, 10 phenanthroline (but not to the same degree by their nonchelating analogs). In addition to Proto, the following porphyrins acted as Mg-chelatase substrates, giving comparable specific activities: deuteroporphyrin, mesoporphyrin, 2-ethyl, 4-vinyl Proto and 2-vinyl, 4-ethyl Proto. Mg-chelatase activity and freely exchangeable heme levels increased steadily with greening, reaching a maximum and leveling off after 15 hours in the light. Exogenous protochlorophyllide, chlorophyllide, heme, and Mg-Proto had no measurable effect on Mg-chelatase activity. The potent ferrochelatase inhibitors, N-methylmesoporphyrin and N-methylprotoporphyrin, inhibited Mg-chelatase at micromolar concentrations.     

Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax.

The processing of phagosomes containing Legionella pneumophila and Escherichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22 degrees C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium. T. vorax ingested L. pneumophila normally, but by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.     

Isolation of a yeast mutant deficient in pyruvate carboxylase activity

To improve our understanding of the catalytic mechanism and regulatory properties of pyruvate carboxylase (EC 6.4.1.1), an important biotin-dependent enzyme, we have sought to isolate mutants in Saccharomyces cerevisiae which are defective in pyruvate carboxylase activity. One mutant was isolated which was unable to grow on glucose minimal medium unless supplemented with aspartate. Although the enzyme had only 25% of the wild type pyruvate carboxylase activity, Western analysis and RNase protection analysis demonstrated that the mutant gene was expressed at approximately 70% of the wild type level. On the basis of genetic crosses and complementation tests, we have attributed the defect to mutations in the PYC gene encoding pyruvate carboxylase.