Feeding melatonin enhances the phase shifting response to triazolam in both young and old golden hamsters

Aging alters many aspects of circadian rhythmicity, including responsivity to phase-shifting stimuli and the amplitude of the rhythm of melatonin secretion. As melatonin is both an output from and an input to the circadian clock, we hypothesized that the decreased melatonin levels exhibited by old hamsters may adversely impact the circadian system as a whole. We enhanced the diurnal rhythm of melatonin by feeding melatonin to young and old hamsters. Animals of both age groups on the melatonin diet showed larger phase shifts than control-fed animals in response to an injection with the benzodiazepine triazolam at a circadian time known to induce phase advances in the activity rhythm of young animals. Thus melatonin treatment can increase the sensitivity of the circadian timing system of young animals to a nonphotic stimulus, and the ability to increase this sensitivity persists into old age, indicating exogenous melatonin might be useful in reversing at least some age-related changes in circadian clock function.     

Mevastatin,an inhibitor of HMG-CoA reductase,induces apoptosis,differentiation and Rap1 expression in HL-60 cells

It has been reported that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase suppress cell proliferation and induce apoptosis. One inhibitor which induces apoptosis is mevastatin. However, the molecular mechanism of apoptosis induction is not well understood so the effects of mevastatin on various functions of HL-60 cells were investigated. We confirmed that mevastatin activated caspase-3 by release of cytochrome c (Cyt. c) from mitochondria through a membrane permeability transition mechanism and also induced typical fragmentation and ladder formation of DNA in HL-60 cells. These effects were inhibited by mevalonate, a metabolic intermediate of cholesterol biosynthesis. Mevalonate and geranylgeraniol (GGOH) inhibited DNA fragmentation whereas farnesol (FOH) did not. Mevastatin also induced cell differentiation to monocytic cells via a mevalonate inhibitable mechanism. Furthermore, mevastatin decreased the amount of an isoprenylated membrane bound Rap1 small GTPase concomitant with an increase in cytosolic Rap1 which occurred before apoptosis and differentiation. On the contrary, both mevastatin and geranylgeranylacetone (GGA), which competes with geranylgeranyl pyrophosphate, induced membrane depolarization of isolated mitochondria without swelling and Cyt. c release. These results suggest that mevastatin-induced apoptosis of HL-60 cells might be caused indirectly by activation of the caspase cascade through the modulation of mitochondrial functions and that some relationship between a certain small GTPase molecule, such as Rap1, and mevastatin-induced apoptosis may exist.     

Loss of ypk1 function causes rapamycin sensitivity,inhibition of translation initiation and synthetic lethality in 14-3-3-deficient yeast

14-3-3 proteins bind to phosphorylated proteins and regulate a variety of cellular activities as effectors of serine/threonine phosphorylation. To define processes requiring 14-3-3 function in yeast, mutants with increased sensitivity to reduced 14-3-3 protein levels were identified by synthetic lethal screening. One mutation was found to be allelic to YPK1, which encodes a Ser/Thr protein kinase. Loss of Ypk function causes hypersensitivity to rapamycin, similar to 14-3-3 mutations and other mutations affecting the TOR signaling pathway in yeast. Similar to treatment with rapamycin, loss of Ypk function disrupted translation, at least in part by causing depletion of eIF4G, a central adaptor protein required for cap-dependent mRNA translation initiation. In addition, Ypk1 as well as eIF4G protein levels were rapidly depleted upon nitrogen starvation, but not during glucose starvation, even though both conditions inhibit translation initiation. These results suggest that Ypk regulates translation initiation in response to nutrient signals, either through the TOR pathway or in a functionally related pathway parallel to TOR.     

Effects of cytotoxic T lymphocytes (CTL) directed against a single simian immunodeficiency virus (SIV) Gag CTL epitope on the course of SIVmac239 infection

Vaccine-induced cytotoxic T lymphocytes (CTL) have been implicated in the control of virus replication in simian immunodeficiency virus (SIV)-challenged and simian-human immunodeficiency virus-challenged macaques. Therefore, we wanted to test the impact that vaccine-induced CTL responses against an immunodominant Gag epitope might have in the absence of other immune responses. By themselves, these strong CTL responses failed to control SIVmac239 replication.     

Ectomycorrhizae between <Emphasis Type="Italic">Alnus acuminata</Emphasis> H.B.K. and <Emphasis Type="Italic">Naucoria escharoides</Emphasis> (Fr.:Fr.) Kummer from Argentina

Field ectomycorrhizae of Naucoria escharoides on Alnus acuminata ("andean alder", "aliso del cerro") are described in detail for the first time. Naturally occurring ectomycorrhizal roots were sampled beneath sporocarps of N. escharoides. The samples were taken from four natural forest plots at two homogeneous A. acuminata sites (Tucumán and Catamarca Provinces, Argentina). The ectomycorrhizae were characterized morphologically and compared by means of PCR/RFLP analysis of the internal transcribed spacer region of the nuclear rDNA. The most important morphological features of the ectomycorrhizae are a white to pale yellow mantle, simple to monopodial branches, hyaline emanating hyphae, abundant hyphal bundles emerging more or less perpendicularly from a plectenchymatous mantle, and an acute or rounded apex with or without a mantle. N. escharoides fruitbodies have white basal mycelium with emanating hyphae similar to those of andean alder ectomycorrhizae. The RFLP profiles of sporocarps and mycorrhizae were the same.     

Involvement of DNA polymerase beta in protection against the cytotoxicity of oxidative DNA damage

We had shown previously that DNA polymerase beta (beta-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H2O2). This is surprising because beta-pol is thought to be involved in BER of oxidative as well as methylated DNA damage. We confirm these findings here in early-passage cells. However, with time in culture, beta-pol null cells become hypersensitive to H2O2 and other reactive oxygen species-generating agents. Analysis of in vitro BER reveals a strong deficiency in single-nucleotide BER of 8-oxoguanine (8-oxoG) by both early- and late-passage beta-pol null cell extracts. Therefore, in early-passage wild-type and beta-pol null cells, the capacity for single-nucleotide BER of 8-oxoG does not correlate with cellular sensitivity to H2O2. Expression of beta-pol protein in the late-passage null cells almost completely reverses the H2O2-hypersensitivity phenotype. Methoxyamine (MX) treatment sensitizes late-passage wild-type cells to H2O2 as expected for beta-pol-mediated single-nucleotide BER; however in beta-pol null cells, MX has no effect. The data indicate a role(s) of beta-pol-dependent repair in protection against the cytotoxicity of oxidative DNA damage in wild-type cells.     

No effect of menstrual cycle phase on glucose kinetics and fuel oxidation during moderate-intensity exercise

Resting and exercise fuel metabolism was assessed in three different phases of the menstrual cycle, characterized by different levels of estrogen relative to progesterone: early follicular (EF, low estrogen and progesterone), midfollicular (MF, elevated estrogen, low progesterone), and midluteal (ML, elevated estrogen and progesterone). It was hypothesized that exercise glucose utilization and whole body carbohydrate oxidation would decrease sequentially from the EF to the MF to the ML phase. Normal-weight healthy females, experiencing a regular menstrual cycle, were recruited. Subjects were moderately active but not highly trained. Testing occurred after 3 days of diet control and after an overnight fast (12-13 h). Resting (2 h) and exercise (50% maximal O(2) uptake, 90 min) measurements of whole body substrate oxidation, tracer-determined glucose flux, and substrate and hormone concentrations were made. No significant difference was observed in whole body fuel oxidation during exercise in the three phases (nonprotein respiratory exchange ratio: EF 0.84 +/- 0.01, MF 0.85 +/- 0.01, ML 0.85 +/- 0.01) or in rates of glucose appearance or disappearance. There were, however, significantly higher glucose (P < 0.05) and insulin (P < 0.001) concentrations during the first 45 min of exercise in the ML phase vs. EF and MF phases. In conclusion, whole body substrate oxidation and glucose utilization did not vary significantly across the menstrual cycle in moderately active women, either at rest or during 90 min of moderate-intensity exercise. During the ML phase, however, this similar pattern of substrate utilization was associated with greater glucose and insulin concentrations. Both estrogen and progesterone are elevated during the ML phase of the menstrual cycle, suggesting that one or both of these sex steroids may play a role in this response.     

Postprandial leg uptake of triglyceride is greater in women than in men

The postprandial excursion of plasma triglyceride (TG) concentration is greater in men than in women. In this study, the disposition of dietary fat was examined in lean healthy men and women (n = 8/group) in either the overnight-fasted or fed (4.5 h after breakfast) states. A [14C]oleate tracer was incorporated into a test meal, providing 30% of total daily energy requirements. After ingestion of the test meal, measures of arteriovenous differences in TG and 14C across the leg were combined with needle biopsies of skeletal muscle and adipose tissue and respiratory gas collections to define the role of skeletal muscle in the clearance of dietary fat. The postprandial plasma TG and 14C tracer excursions were lower (P = 0.04) in women than in men in the overnight-fasted and fed states. Women, however, had significantly greater limb uptake of total TG compared with men on both the fasted (3,849 +/- 846 vs. 528 +/- 221 total micro mol over 6 h) and fed (4,847 +/- 979 vs. 1,571 +/- 334 total micromol over 6 h) days. This was also true for meal-derived 14C lipid uptake. 14C content of skeletal muscle tissue (micro Ci/g tissue) was significantly greater in women than in men 6 h after ingestion of the test meal. In contrast, 14C content of adipose tissue was not significantly different between men and women at 6 h. The main effect of nutritional state, fed vs. fasted, was to increase the postmeal glucose (P = 0.01) excursion (increase from baseline) and decrease the postmeal TG excursion (P = 0.02). These results support the notion that enhanced skeletal muscle clearance of lipoprotein TG in women contributes to their reduced postprandial TG excursion. Questions remain as to the mechanisms causing these sex-based differences in skeletal muscle TG uptake and metabolism. Furthermore, nutritional state can significantly impact postprandial metabolism in both men and women.     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.