An integrated metabonomic approach to describe temporal metabolic disregulation induced in the rat by the model hepatotoxin allyl formate

The time-related metabolic events in rat liver, plasma, and urine following hepatotoxic insult with allyl formate (75 mg/kg) were studied using a combination of high-resolution liquid state and magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopic methods together with pattern recognition analysis. The metabonomics results were compared with the results of conventional plasma chemistry and histopathological assessments of liver damage. Various degrees of liver damage were observed in different animals, and this variation was reflected in all of the analyses. Furthermore, each analysis revealed a high degree of functional and structural recovery by the end of the study. The allyl formate-induced changes included hepatocellular necrosis, hepatic lipidosis, decreased liver glycogen and glucose, decreased plasma lipids, increased plasma creatine and tyrosine, increased urinary taurine and creatine, and decreased urinary TCA cycle intermediates. The observed reductions in hepatic glycogen and glucose suggest increased glucose utilization and are consistent with the expected depletion of hepatic ATP following mitochondrial impairment, assuming that there is a consequent increase in energy production from glycolysis. The increase in plasma tyrosine is consistent with impaired protein synthesis, a known consequence of ATP depletion. Partial least squares-based cross-correlation of the variation in the liver and plasma NMR profiles indicated that the allyl formate-induced increase in liver lipids correlated with the decrease in plasma lipids. This suggests disruption in lipid transport from the liver to plasma, which could arise through impaired apolipoprotein synthesis, as with ethionine.     

The role of glutathione, membrane sphingomyelin, and its metabolites in oxidative stress-induced calcium "dysregulation" in PC12 cells

Previous research showed that increasing membrane sphingomyelin (SPH) levels in rat pheochromocytoma (PC12) cells to the same extent as that seen in some brain regions with aging dramatically increases the vulnerability to oxidative stress (OS). These increases in vulnerability were determined by assessing deficits in the ability of these cells to extrude and/or sequester Ca2+ following 30 mM KCl-induced depolarization (recovery). The purpose of the present experiments was to discern whether increasing the levels of particular SPH metabolite(s), i.e., ceramide (Cer), sphingosine (Ssine), or sphingosine-1-phosphate (SPP), or indirectly increasing the concentrations of these metabolites with sphingomylinase (Sase), would interact with the cell's sensitivity to OS induced by low (5 microM) or high (nonlethal, 300 microM) H2O2. In addition, the OS vulnerability was examined as above under decreased SPH levels by exposing the cells to L-cycloserine (Lcc), which prevents SPH synthesis. Both Sase and SPP significantly decreased Ca2+ recovery of PC12 cells after H2O2 exposure. Conversely, Lcc-treated cells showed no further OS-induced decrements in recovery below those seen in controls. SPP significantly decreased glutathione levels (GSH) in the absence of OS. Repletion of GSH with 20 mM N-acetylcysteine significantly attenuated the effect of 5 microM H2O2 on recovery in SPP-treated cells and decreased sensitivity of SPP-treated cells to low doses of OS. Overall, our results suggest a critical role for GSH and SPP in the regulation of OS vulnerability, especially as it relates to Ca2+ homeostasis.     

Oligomerization characteristics of cysteine string protein

CSP function is vital to synaptic transmission, however; the precise nature of its role remains controversial. Conflicting reports support either a role for CSP: (i) in exocytosis or (ii) in the regulation of transmembrane calcium fluxes. Here we have examined the self-association of CSP to form oligomers that are stable upon SDS-PAGE. To understand the structural requirements for CSP self-association a series of CSP deletion mutants were constructed, expressed, and purified. This analysis revealed an interesting pattern of oligomerization. Amino acids between 83 and 136 were observed to be important for self-association. The recombinant CSP oligomers as well as the CSP monomers directly associate with Ni(2+)-NTA agarose. Thus CSP-CSP interactions may be an important consideration for current working models of CSP chaperone activity at the synapse.     

Change in the chemical signature of the ant leptothorax lichtensteini bondroit with time

The cuticular hydrocarbons of foraging workers of the ant Leptothorax lichtensteini have been identified by gas chromatography-mass spectrometry. Characterization of these compounds at regular intervals, by gas chromatography, has shown a change with time in the relative proportions of some of the hydrocarbons: n-hexacosane, 4-methylhexacosane, 4-methyloctacosane, 3-methylnoncosane. Some of which constitute a part of the colony signature. These changes occur in a synchronous manner, and in the same way for all the individuals of a colony tested at the same time. The changes also appear in queenless colonies; nevertheless, the presence of a queen seems to accelerate the change over the course of 1 yr. Certain hypotheses are formulated as to the mode of regulation of the synthesis of cuticular hydrocarbons responsible for these changes with time and as to the role played by the queen in this regulation. The implications of such a dynamic system for the process of nestmate recognition are discussed.     

Recombination after protoplast fusion in the yeast Candida tropicalis

Candida tropicalis protoplasts obtained by snail enzyme treatment were induced to fuse by the use of polyethylene-glycol. Heterokaryons formed by two auxotrophic strains were selected by complementation on minimal medium. These heterokaryons were unstable and readily dissociated into their nuclear components. Under appropriate conditions, the parental nuclei of an heterokaryon fused. The homokaryon so obtained was unstable and segregated into various types of auxotrophic and prototrophic recombinants.List of Abbreviations Used MM minimal medium - YEA yeast extract agar (complete medium) - YPGT yeast-peptone-glucosethiol (medium for protoplast preparation) - PTP medium for cell pretreatment (used before the action of snail enzyme) - PEG polyethylene glycol - p-FPA para-fluorophenylalanine - 5-FC 5-fluorocytosine     

Pattern of recovery of species-specific cuticular hydrocarbon mixtures by Reticulitermes santonensis and Reticulitermes lucifugus grassei after being removed from a mixed group. Is the acquisition of allospecific hydrocarbons reversible?

Each of the termite species Reticulitermes santonensis and Reticulitermes lucifugus grassei has its own particular cuticular chemical profile. When members of the two species are placed together to form artificially mixed species groups, their chemical profiles undergo changes: Each species acquires all the hydrocarbons which initially characterized the other species. When the members of a mixed group which had been kept together for 24 h were split into two homospecific groups, the cuticular profiles of the members of both groups immediately showed a sharp drop in both the homospecific and allospecific components. In R. santonensis, the homospecific hydrocarbons subsequently increased in quantity, reaching values which were higher on the 33rd day after the separation than those initially recorded in this species; whereas in R. lucifugus grassei, the homospecific hydrocarbon proportions were still lower on the 33rd day than the initial values. In both species, the allospecific hydrocarbon levels began to increase sharply on the 5th day after separation, and the homospecific products still showed no tendency to return to the initial proportions 33 days after separation. In the light of these results, some hypotheses are put forward as to what mechanisms might posibly regulate the hydrocarbon profiles of these two species. Arch. Insect Biochem. Physiol. 35:237-259, 1997.© 1997 Wiley-Liss, Inc.     

Treatments resulting in pregnancy in nonovulating, hormone-treated oocyte recipient mares

Synchronization of follicle growth between oocyte donor and recipient mares is difficult. To avoid this, recipient mares in a clinical program were used during a period of low follicular activity, and were treated with estrogen before transfer and progesterone after transfer. Five pregnancies were established after oocyte transfer to nonovulating, hormone-treated recipient mares. One pregnancy was lost before 30 d gestation, and the other 4 foals were carried to term. One foal died at birth. Establishment and maintenance of pregnancy in these mares indicates that nonovulating, hormone-treated mares may offer an alternative to cyclic recipients in oocyte transfer programs.     

Variations in chemical mimicry by the ectoparasitic mite Varroa jacobsoni according to the developmental stage of the host honey-bee Apis mellifera

The ectoparasitic mite Varroa jacobsoni poses a major threat to the survival of European honey-bee populations. Development of effective control methods is therefore much needed. Study of interspecific chemical communication between the parasite and host is a particularly promising avenue of research. Previous study has shown that the cuticular hydrocarbons of the parasite mite Varroa jacobsoni are qualitatively identical to those of its honey-bee host Apis mellifera (Nation J.L., Sanford M.T., Milne K., 1992. Cuticular hydrocarbons from Varroa jacobsoni. Experimental and Applied Acarology 16, 331-344). The purpose of the present study was to compare the cuticular hydrocarbon patterns of the two species at different stages of bee development. Cuticular components were identified by gas chromatography/mass spectrometry. The proportion of each component was calculated at three stages of bee development (larvae, pupa, emerging bee). The degree of chemical mimicry between the parasite and host was evaluated by multivariate analyses using the resulting proportions for each category of individuals. There were four main findings. The first was that the proportions of some components are different at the larval, pupal and imago stage of bee development. Second, Varroa profiles vary depending on the developmental stage of the host. Third, the cuticular profile of adult mites is more similar to that of the stage of the host than that of later and/or earlier stages except for parasites collected from emerging adult bees. Fourth, the degree of mimicry by Varroa is greater during larval and pupal stages than during the emerging adult bee stages. The role of chemical mimicry - although it is not perfect - in enabling parasites to infest bee colonies by the parasite is discussed.