Effect of precursor feeding on alkaloid accumulation by a strictosidine synthase over-expressing transgenic cell line S1 of Catharanthus roseus

The transgenic S1 cell line of Catharanthus roseus (L.) G. Don has been used to study possible rate limiting steps in the terpenoid indole alkaloid (TIA) biosynthesis. Line S1 carries a recombinant, over-expressed version of the endogenous Str gene which encodes strictosidine synthase (STR; EC 4.3.3.2). STR catalyzes the stereospecific condensation of tryptamine and secologanin to strictosidine. Various concentrations and combinations of biosynthetic indole precursors L-tryptophan, tryptamine, and iridoid precursors loganin and secologanin were added to the cell suspension cultures of line S1. The largest TIA accumulation occurred when the precursor was supplied at the time of inoculation of the cells into the production medium. Line S1 could supply tryptamine endogenously up to 0.8 mM loganin feeding. The enhancement of the accumulation of TIAs by addition of loganin indicates a limitation in the terpenoid pathway. Supplying tryptamine or tryptophan along with the iridoid precursors resulted in even further increase of alkaloid accumulation. Under optimal conditions, cultures of line S1 accumulated about 600 mol l^–1 of TIAs. Also, the conversion of strictosidine into other TIAs further down the pathway seems to be a limiting step. Considering the mass balance of the intermediates fed and TIAs recovered, several yet unknown pathways must be involved in channeling away intermediates from the TIA pathway and in the breakdown of the TIAs. Our results suggest that high rates of tryptamine synthesis can still take place under conditions of low TDC activity and the flux towards tryptamine is induced by loganin feeding. However, accumulation of tryptamine seems to reduce the flux through feedback inhibition.     

Carbohydrate cycling in micro-organisms: what can (13)C-NMR tell us?

The extension of (13)C-nuclear magnetic resonance (NMR) techniques to study cellular metabolism over recent years has provided valuable data supporting the occurrence, diversity and extent of carbon cycling in the carbohydrate metabolism of micro-organisms. The occurrence of such cycles, resulting from the simultaneous operation of different and sometimes opposite individual steps, is inherently related to the network organisation of cellular metabolism. These cycles are tentatively classified here as 'reversibility', 'metabolic' and 'substrate' cycles on the basis of their balance in carbon and cofactors. Current hypotheses concerning the physiological relevance of carbohydrate cycles are discussed in light of the (13)C-NMR data. They most likely represent system-level mechanisms for coherent and timely partitioning of carbon resources to fit with the various biosynthetic, energetic or redox needs of cells and/or additional strategies in the adaptive capacity of micro-organisms to face variation in environmental conditions.     

Lesion of caudate-putamen interneurons with kainic acid alters dopamine and serotonin metabolism in the olfactory tubercle of the rat

1. The existence of functional interrelationships between dorsal and ventral regions of the rat striatum was investigated. Kainic acid (KA) was employed to induce neuronal lesions in the more dorsal striatum, the caudate-putamen (CP). Only one CP (one side) received KA. KA-induced neurotoxicity at the site of injection (CP) was evidenced by reductions in choline-acetyltransferase activity and in GABA levels, and by increases in the ratios metabolite/monoamine for dopamine (DA) and serotonin (5-HT).2. In addition to the well-known local effects, direct stereotaxic injection of KA into the CP produced distant effects in the ipsilateral olfactory tubercle (OT). A dose-dependent increase in the levels of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) and decreases in DA and 5-HT concentrations were observed in the OT ipsilateral to the CP injected with KA. With 1, 2, 3, and 4 g of KA, the ratio DOPAC+HVA/DA in the OT was 30, 79, 140, and 173% higher, respectively, than control levels. With 2, 3, and 4 g of KA, the levels of 5-HIAA were approximately 30, 60, and 120% higher than control values, and the changes in 5-HIAA were associated with significant reductions in 5-HT concentrations.3. Our results suggest that the dorsal part of the striatum exerts important regulatory functions over the most ventral striatal region, the OT. Destruction of CP interneurons by KA leads to disinhibition of DA and 5-HT activities to the OT. The functional interactions between dorsal and ventral striatal regions may play a role in the integration of fundamental life-preserving, motivational, and goal-directed olfactory motor behaviors of rodents.     

Initial net CO2 uptake responses and root growth for a CAM community placed in a closed environment

To help understand carbon balance between shoots and developing roots, 41 bare-root crassulacean acid metabolism (CAM) plants native to the Sonoran Desert were studied in a glass-panelled sealable room at day/night air temperatures of 25/15 degrees C. Net CO(2) uptake by the community of Agave schottii, Carnegia gigantea, Cylindropuntia versicolor, Ferocactus wislizenii and Opuntia engelmannii occurred 3 weeks after watering. At 4 weeks, the net CO(2) uptake rate measured for south-east-facing younger parts of the shoots averaged 1.94 micro mol m(-2) s(-1) at night, considerably higher than the community-level nocturnal net CO(2) uptake averaged over the total shoot surface, primarily reflecting the influences of surface orientation on radiation interception (predicted net CO(2) uptake is twice as high for south-east-facing surfaces compared with all compass directions). Estimated growth plus maintenance respiration of the roots averaged 0.10 micro mol m(-2) s(-1) over the 13-week period, when the community had a net carbon gain from the atmosphere of 4 mol C while the structural C incorporated into the roots was 23 mol. Thus, these five CAM species diverted all net C uptake over the 13-week period plus some existing shoot C to newly developing roots. Only after sufficient roots develop to support shoot water and nutrient requirements will the plant community have net above-ground biomass gains.     

Single nucleotide polymorphisms of thrifty genes for energy metabolism: evolutionary origins and prospects for intervention to prevent obesity-related diseases

The "thrifty" genotype and phenotype that save energy are detrimental to the health of people living in affluent societies. Individual differences in energy metabolism are caused primarily by single nucleotide polymorphisms (SNPs), some of which promote the development of obesity/type 2 diabetes mellitus. In this review, four major questions are addressed: (1) Why did regional differences in energy metabolism develop during evolution? (2) How do genes respond to starvation and affluence? (3) Which SNPs correspond to the hypothetical "thrifty genes"? (4) How can we cope with disease susceptibility caused by the "thrifty" SNPs? We examined mtDNA and genes for energy metabolism in people who live in several parts of Asia and the Pacific islands. We included 14 genes, and the SNP frequencies of PPAR gamma 2, LEPR, and UCP3-p and some other genes differ significantly between Mongoloids and Caucasoids. These differences in SNPs may have been caused by natural selection depending on the types of agriculture practiced in different regions. Interventions to counteract the adverse effects of "thrifty" SNPs have been partially effective.     

Characterization of a novel testicular form of human hormone-sensitive lipase

Hormone-sensitive lipase (HSL) is an esterase and lipase, which are essential for spermatogenesis. Two HSL mRNAs are expressed in human testis. A long form is encoded by a testis-specific exon and nine exons common to testis and adipocyte HSL. Here we show that the short-form 3.3-kb mRNA possesses a unique 5' end that is transcribed from a novel testis-specific exon. The corresponding protein is similar to the 775-amino-acid-long adipocyte HSL. Immunohistochemistry experiments on human testis sections revealed that the long form is strictly expressed in haploid germ cells whereas the short form is expressed in interstitial and tubular somatic cells as well as premeiotic germ cells.     

Disaccharide Nucleosides and Oligonucleotides on Their Basis. New Tools for the Study of Enzymes of Nucleic Acid Metabolism

The main structural features of an important group of natural compounds, disaccharide nucleosides, are reviewed. The synthesis and properties of modified oligonucleotides on their basis as well as the methods of introduction of reactive aldehyde groups are described. The last part is devoted to the application of these compounds for studies of enzymes of nucleic acid metabolism.     

Modulation of THP-1 macrophage and cholesterol-loaded foam cell apolipoprotein E levels by glycosphingolipids

Macrophages synthesize and secrete apolipoprotein E (apoE) constitutively. This process is upregulated under conditions of cholesterol loading. The response to cholesterol is antiatherogenic as it is believed to promote cholesterol efflux from the artery wall. The concentration of lactosyl ceramide (LacCer), a glycosphingolipid recently discovered to regulate cellular signaling, proliferation, and expression of adhesion molecules, is also increased in atherosclerotic tissues. Here we have investigated the effect of exogenous LacCer on macrophage apoE levels. We show that increasing macrophage LacCer levels sevenfold led to reductions in cellular and secreted apoE (15 and 30%, respectively, over a 24-h period) as determined by enzyme-linked immunosorbent assay. A similar effect was also induced by glucosyl ceramide (GlcCer) but not by ganglioside species. When macrophages were converted to cholesterol-loaded foam cells by incubation with acetylated LDL, the resulting increase in cellular apoE levels was inhibited by 26% when the cells were subsequently enriched with LacCer. After metabolic labeling of cellular glycosphingolipids with [14C]palmitate, we also discovered that high-density lipoprotein (HDL) stimulates the efflux of glycosphingolipids from foam cells. These data imply that LacCer and GlcCer may be proatherogenic due to the suppression of macrophage apoE production. Furthermore, the efflux of glycosphingolipids from macrophage foam cells to HDL could indicate a potential pathway for their removal from the artery wall and subsequent delivery to the liver.     

Studies on the mechanism of the osmoresistance of spores of Bacillus subtilis

AIMS: To determine the reason that spores of Bacillus species, in particular Bacillus subtilis, are able to form colonies with high efficiency on media with very high salt concentrations. METHODS AND RESULTS: Spores of various Bacillus species have a significantly higher plating efficiency on media with high salt concentration (termed osmoresistance) than do log or stationary phase cells. This spore osmoresistance is higher on richer media. Bacillus subtilis spores lacking various small, acid-soluble spore proteins (SASP) were generally significantly less osmoresistant than were wild-type spores, as shown previously (Ruzal et al. 1994). Other results included: (a) spore osmoresistance varied significantly between species; (b) the osmoresistance of spores lacking SASP was not restored well by amino acid osmolytes added to plating media, but was completely restored by glucose; (c) the osmoresistance of spores lacking SASP was restored upon brief germination in the absence of salt in a process that did not require protein synthesis; (d) significant amounts of amino acids generated by SASP degradation were retained within spores upon germination in a medium with high but not low salt; (e) slowing but not abolishing SASP degradation by loss of the SASP-specific germination protease (GPR) did not affect spore osmoresistance; (f) sporulation at higher temperatures produced less osmoresistant spores; and (g) spore osmoresistance was not decreased markedly by the absence of the stress sigma factor for RNA polymerase, sigmaB. CONCLUSIONS: Spore osmoresistance appears as a result of three major factors: (1) specific characteristics of spores and cells of individual species; (2) the precise sporulation conditions that produce the spores; and (3) sufficient energy generation by the germinating and outgrowing spore to allow the spore to adapt to conditions of high osmotic strength; the substrates for this energy generation can come from either the endogenous generation of amino acids by SASP degradation or from the spore's environment, in the form of a readily taken up and metabolized energy source such as glucose. SIGNFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of spore osmoresistance, a spore property that can be of major applied significance given the use of high osmotic strength with or without high salt as a means of food preservation.