AQR1 gene (ORF YNL065w) encodes a plasma membrane transporter of the major facilitator superfamily that confers resistance to short-chain monocarboxylic acids and quinidine in Saccharomyces cerevisiae

We report results on the functional analysis of Saccharomyces cerevisiae ORF YNL065w, predicted to code for a protein belonging to the poorly characterized major facilitator superfamily (MFS) of transporters that are involved in multidrug resistance (MDR). YNL065w is important for a moderate increase of yeast tolerance to ketoconazole and to the cationic dye crystal violet; it protects the cell against short-chain monocarboxylic acids (C(2)-C(6)), but not against highly liposoluble acids such as octanoic acid or the phenoxyacetic-acid herbicides 2,4-D and MCPA; it is also a determinant of resistance to the antiarrhytmic and antimalarial drug quinidine. The encoding ORF was, thus, denominated the AQR1 gene. Results obtained using an AQR1-lacZ fusion indicate that gene expression is very low and it is not stimulated under weak acid stress. The encoded putative transporter was localized in the plasma membrane by fluorescence microscopy observation of the overproduced Aqr1-GFP fusion protein distribution.     

Demonstration of dimethylnonanoyl-CoA thioesterase activity in rat liver peroxisomes followed by purification and molecular cloning of the thioesterase involved.

Peroxisomes play an indispensable role in cellular fatty acid oxidation in higher eukaryotes by catalyzing the chain shortening of a distinct set of fatty acids and fatty acid derivatives including pristanic acid (2,6,10,14-tetramethylpentadecanoic acid). Earlier studies have shown that pristanic acid undergoes three cycles of beta-oxidation in peroxisomes to produce 4,8-dimethylnonanoyl-CoA (DMN-CoA) which is then transported to the mitochondria for full oxidation to CO(2) and H(2)O. In principle, this can be done via two different mechanisms in which DMN-CoA is either converted into the corresponding carnitine ester or hydrolyzed to 4,8-dimethylnonanoic acid plus CoASH. The latter pathway can only be operational if peroxisomes contain 4,8-dimethylnonanoyl-CoA thioesterase activity. In this paper we show that rat liver peroxisomes indeed contain 4,8-dimethylnonanoyl-CoA thioesterase activity. We have partially purified the enzyme involved from peroxisomes and identified the protein as the rat ortholog of a known human thioesterase using MALDI-TOF mass spectrometry in combination with the rat EST database. Heterologous expression studies in Escherichia coli established that the enzyme hydrolyzes not only DMN-CoA but also other branched-chain acyl-CoAs as well as straight-chain acyl-CoA-esters. Our data provide convincing evidence for the existence of the second pathway of acyl-CoA transport from peroxisomes to mitochondria by hydrolysis of the CoA-ester in peroxisomes followed by transport of the free acid to mitochondria, reactivation to its CoA-ester, and oxidation to CO(2) and H(2)O. (c)2002 Elsevier Science.     

Factors governing 3(10)-helix vs alpha-helix formation in peptides: percentage of C(alpha)-tetrasubstituted alpha-amino acid residues and sequence dependence

As an additional step toward the dissection of the factors responsible for the onset of 3(10)-helix vs alpha-helix in peptides, in this paper we describe the results of a three-dimensional (3D) structural analysis by x-ray diffraction of the N(alpha)-acylated heptapeptide alkylamide mBrBz-L-Iva-L-(alphaMe)Val-L-Abu-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe characterized by a single (L-Abu3) C(alpha)-trisubstituted and six C(alpha)-tetrasubstituted alpha-amino acids. We find that in the crystal state this peptide is folded in a mixed helical structure with short elements of 3(10)-helix at either terminus and a central region of alpha-helix. This finding, taken together with the published NMR and x-ray diffraction data on the all C(alpha)-methylated parent sequence and its L-Val2 analog (also the latter heptapeptide has a single C(alpha)-trisubstituted alpha-amino acid) strongly supports the view that one C(alpha)-trisubstituted alpha-amino acid inserted near the N-terminus of an N(alpha)-acylated heptapeptide alkylamide sequence may be enough to switch a regular 3(10)-helix into an essentially alpha-helical conformation. As a corollary of this work, the x-ray diffraction structure of the N(alpha)-protected, C-terminal tetrapeptide alkylamide Z-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe, also reported here, is clearly indicative of the preference of this fully C(alpha)-methylated, short peptide for the 3(10)-helix. As the same terminally blocked sequence is mixed 3(10)/alpha-helical in the L-Abu3 heptapeptide amide but regular 3(10)-helical in the tetrapeptide amide and in the parent heptapeptide amide, these results point to an evident plasticity even of a fully C(alpha)-methylated short peptide.     

Seasonal Changes of Nitrogen Storage Compounds in a Rhizomatous Grass Calamagrostis epigeios

The seasonal dynamics in content and distribution of N-rich compounds between overwintering organs of Calamagrostis epigeios were examined. Samples were taken both from plants grown in natural conditions and in containers with controlled nutrient supply. There were significant changes in content of nitrate, free amino acids and soluble protein in all investigated plant parts during the course of a year. Amino acids showed both the highest maximum and seasonal fluctuation among the all N compounds observed and, therefore, appear to have a central role in N storage. Their content rises in the autumn, remains stable during winter and declines quickly at the beginning of spring. The most abundant amino acids in the end of winter storage period - asparagine, arginine and glutamine - constituted about 90 % of N in fraction of free amino acids. The portion of N stored in soluble proteins, however, was considerably smaller compare to both amino acids and nitrate. The amount of N stored in rhizomes of C. epigeios was smaller than in roots and stubble base before the onset of spring re-growth. This indicates that roots and stubble base are particularly important for winter N storage in this species.     

Simulation on long-term operation of an anaerobic bioreactor for Korean food wastes

A mathematical model was formulated to simulate the long-term performance of an anaerobic bioreactor designed to digest Korean food wastes. The system variables of various decomposition steps were built into the model, which predicts the temporal characters of solid waste, and volatile fatty acid (VFA) in the reactor, and gas production in response to various input loadings and temperatures. The predicted values of VFA and gas production were found to be in good agreement with experimental observations in batch and repeated-input systems. Finally, long-term reactor performance was simulated with respect to the seasonal temperature changes from 5°C in winter to 25°C in summer at different food waste input loadings. The simulation results provided us with information concerning the success or failure of a process during long-term operation.     

Effects of culture conditions on the mycolic acid composition of isolates of Rhodococcus spp. from activated sludgefoams

The influence of two different carbon sources and three incubation temperatures on the mycolic acid compositions of three Rhodococcus isolates from activated sludge was examined using Selective Ion Monitoring (SIM) gas chromatography-mass spectrometry (GC-MS). Considerable qualitative and quantitative differences were detected in the mycolic acid compositions of the three very closely related isolates grown under the same conditions. Culture age also affected both the chain lengths and proportions of saturated mycolic acids detected in cell extracts, but not in the same manner for each isolate. Mycolic acids generally were of shorter chain lengths in cells grown with Tween 80 compared to glucose grown cells in strain 11R but the opposite situation occurred with strains A7 and D5. In all three, the proportion of unsaturated mycolic acids decreased with increasing growth temperatures. The taxonomic relevance of these observations and possible explanations for the observed changes in mycolic acid composition under various culture conditions are discussed.     

Modification of phospholipids fatty acid composition in reuber H35 hepatoma cells: effect on HMG-CoA reductase activity

There is controversy about the effect of saturated and polyunsaturated fats on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the main regulatory enzyme of cholesterogenic pathway. Results from dietary studies are difficult to interpret because diets normally contain a mixture of fatty acids. Therefore, we have used Reuber H35 hepatoma cells whose phospholipids were enriched in different individual fatty acids and have studied their effects on the cellular reductase activity. Lauric, myristic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids were supplemented to the culture medium coupled to bovine serum albumin. The four fatty acids were incorporated into phospholipids from cells grown in media containing whole serum or lipoprotein-poor serum (LPPS). Reductase activity of cells cultivated in a medium with LPPS was three to four times higher than those cultivated in medium with whole serum. Saturated fatty acids increased reductase activity of cells grown in medium with whole serum, whereas n-3 polyunsaturated fatty acids (PUFA) decreased it. However, both saturated and polyunsaturated fatty acids increased reductase activity when serum lipoproteins were removed. In conclusion, this is one of the first reports demonstrating that saturated and n-3 PUFA only show differential effects on HMG-CoA reductase activity in the presence of lipoproteins.     

Effects of long-term administration of N-3 polyunsaturated fatty acids (PUFA) and selective estrogen receptor modulator (SERM) derivatives in ovariectomized (OVX) mice

We studied the beneficial effects of dietary consumption of n-3 polyunsaturated fatty acids (PUFA) and two selective estrogen receptor modulator (SERM) derivatives (SERM-I and SERM-II) and their combined effect on serum lipids, skin dermis and adipose layers, bone marrow adipogenesis, and cytokine secretion in mice. Two different ovariectomized (OVX) models were studied: treatment began immediately post-OVX in one and 3 months post-OVX in the other. Our results showed that n-3 PUFA and both SERMs decreased triglyceride levels in the serum, and that SERMs also decreased serum cholesterol levels while n-3 PUFA had no similar effect. SERMs had no effect on IL-6, IL-1 beta, or IL-10 levels, but they decreased ex vivo tumor necrosis factor (TNF-alpha). N-3 PUFA decreased secretion of non-induced IL-6 and TNF-alpha from cultured BMC and IL-1 beta levels in vivo (i.e., in bone marrow plasma), but its main effect was a significant elevation in the secretion of IL-10, a known anti-inflammatory cytokine. OVX-induced B-lymphopoiesis was not affected by LY-139481 (SERM-I) while LY-353381 (SERM-II) exhibited an estrogen-antagonistic effect in sham and OVX mice and elevated the amount of B-cells in bone marrow. Fish oil consumption prevented the elevation in B-lymphopoiesis caused by OVX, but had no curative effect on established augmented B-lymphopoiesis. This activity could be mediated via the elevation of IL-10 which was shown to suppress B-lymphopoiesis. Both SERMs and n-3 PUFA inhibited the increase in adipose tissue thickness caused by OVX in mice. Our results showed that n-3 PUFA, could prevent some of the deleterious outcomes of estrogen deficiency that were not affected by SERMs. We observed no significant beneficial effects of the combined administration of SERM-I, SERM-II, and PUFA on the studied parameters.The exact mechanism by which polyunsaturated fatty acids exert their activities is still not clear, but peroxisome proliferator-activated receptors (PPARs) might be involved in processes which are modulated by n-3 PUFA.     

Fatty acid variations in symbiotic dinoflagellates from Okinawan corals

The fatty acid composition of polar lipids and triacylglycerols was determined in different morphophysiological types of symbiotic dinoflagellates (SD) isolated from the hydrocoral Millepora intricata and the scleractinian corals Pocillopora damicornis, Seriatopora caliendrum, Seriatopora hystrix and Stylophora pistillata from a fringing reef of Sesoko Island, Okinawa, Japan. The distribution of the fatty acids among the morphophysiologically distinct types of SD reported in these corals makes it possible to readily distinguish one type of SD from the other. Moreover, differences were found both in polar lipids and triacylglycerols. The polar lipids of SD from M. intricata showed a very distinctive fatty acid profile. A combination of large proportions of 18:4 (n-3), 18:5 (n-3), 22:5 (n-6), and 22:6 (n-3) and negligible amounts of 20:4 (n-6), and 20:5 (n-3) in SD from M. intricata was particularly noteworthy. The fatty acid profiles of SD from P. damicornis and SD isolated from S. caliendrum and S. hystrix differed in the proportion of 18:4 (n-3) and 22:6 (n-3). It is suggested that fatty acids might provide useful information on possible taxonomic differences among symbiotic dinoflagellates. It is assumed that biochemical differences can reflect the genetic diversity of the morphophysiological types of SD associated with several species of hermatypic corals from this region.