Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances

Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2^−/−) mice extended the life span of Tk2^−/− mice from 3 weeks to at least 20 months. The Dm-dNK^+/−Tk2^−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK^+/−Tk2^−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK^+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues.     

Cytotoxicity and antioxidant activity of 5-(2,4-dimethylbenzyl)pyrrolidin-2-one extracted from marine Streptomyces VITSVK5 spp.

The aim of the present study was to evaluate the cytotoxicity and antioxidant activity of 5-(2,4-dimethylbenzyl)pyrrolidin-2-one (DMBPO) extracted from marine Streptomyces VITSVK5 spp. The strain was isolated from sediment samples collected at the Marakkanam coast of Bay of Bengal, India. Systematic screening of isolates for anti-Aspergillus activity resulted in the identification of Streptomyces species designated as Streptomyces VITSVK5 spp. Bioactivity guided extraction and purification yielded a compound 5-(2,4-dimethylbenzyl)pyrrolidin-2-one (DMBPO) and was tested for cytotoxicity and antioxidant activity. The structure of the extracted compound was established by spectroscopic studies and identified as 5-(2,4-dimethylbenzyl)pyrrolidin-2-one (DMBPO). DMBPO exhibited cytotoxic activity on HEP 2 and Hep G2 cell lines with the IC₅₀ value of 2.8 μg/ml and 8.3 μg/ml, respectively, as compared to Vero cell line (22.6). DMBPO showed the hemolytic EC₅₀ value of 288 μg/ml on human erythrocytes. DMBPO treatment showed fewer (31.7%) aberrations, gaps and chromatid breaks as compared to untreated controls (27.8%) of human chromosomes. DMBPO also exhibited significant (44.13% at 5 μg/ml DMBPO) DPPH radical scavenging activity and total antioxidant activity (50.10% at 5 μg/ml DMBPO). The results of this study showed that DMBPO is cytotoxic to cancer cells and possesses antioxidant property.     

nodD alleles of Sinorhizobium fredii USDA191 differentially influence soybean nodulation, nodC expression, and production of exopolysaccharides

All Rhizobium strains examined to date have one or multiple alleles of nodD. At least one copy of nodD and the presence of flavonoid exudates are required for nod gene induction and nodulation. Sinorhizobium fredii USDA191 has two copies of nodD. In this study, we demonstrate that inactivation of either copy of nodD caused a reduction in basal levels of expression of nodC. Extra copies of nodD1 had no effect on the expression of nodC when compared with the wild type, but extra copies of nodD2 abolished the inducer requirement, thereby rendering nodC constitutive. A nodD1 mutant was unable to nodulate soybean cultivars 'Peking' and 'McCall'. Inactivation of nodD2 or addition of extra copies of nodD1 or nodD2 caused delayed nodulation on Peking, and reduced the number of nodules on McCall. Both nodD alleles of S. fredii USDA191 appear to be involved in regulation of exopolysaccharide production; however, nodD2 appears to be more important in this respect than nodD1.     

Pyruvate decarboxylase: a key enzyme for the oxidative metabolism of lactic acid by Acetobacter pasteurianus

Acetobacter pasteurianus, an obligately oxidative bacterium, is the first organism shown to utilize pyruvate decarboxylase (PDC) as a central enzyme for oxidative metabolism. In plants, yeast, and other bacteria, PDC functions solely as part of the fermentative ethanol pathway. During the growth of A. pasteurianus on lactic acid, the central intermediate pyruvate is cleaved to acetaldehyde and CO(2) by PDC. Acetaldehyde is subsequently oxidized to its final product, acetic acid. The presence of the PDC enzyme in A. pasteurianus was confirmed by zymograms stained for acetaldehyde production, enzyme assays using alcohol dehydrogenase as the coupling enzyme, and by cloning and characterization of the pdc operon. A. pasteurianus pdc was also expressed in recombinant Escherichia coli. The level of PDC activity was regulated in response to growth substrate, highest with lactic acid and absent with mannitol. The translated PDC sequence (548 amino acids) was most similar to that of Zymomonas mobilis, an obligately fermentative bacterium. A second operon ( aldA) was also found which is transcribed divergently from pdc. This operon encodes a putative aldehyde dehydrogenase (ALD2; 357 amino acids) related to class III alcohol dehydrogenases and most similar to glutathione-dependent formaldehyde dehydrogenases from alpha-Proteobacteria and Anabeana azollae.     

Tumour eradication and induction of memory against murine mesothelioma by combined immunotherapy

Numerous immunotherapy treatments for cancer are undergoing clinical trials or are already approved for use. One particular area of interest is targeting mechanisms of immune tolerance. Using a murine model of mesothelioma, we investigated the roles of regulatory T-cells, intratumoural transforming growth factor (TGF)-β and the negative regulator molecule cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in immune tolerance to tumours. It was found that treatments targeting a single negative regulator molecule mechanism were not as effective against tumours as targeting multiple mechanisms simultaneously. Most importantly, it was found that a combined triple treatment of anti-CD25 monoclonal antibody (mAb), anti-CTLA-4 mAb and TGF-β soluble receptor resulted in long-term clearance of tumours and memory against tumour rechallenge. These data suggest that clinical application of immunotherapies against tumours may be improved by simultaneously targeting multiple mechanisms of immune suppression.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Starch Accumulation

The investigation dealt with starch accumulation in four species of Cuscuta (Cuscuta campestris, C. indecora, C. planiflora and C. reflexa), a leafy mistletoe (Dendrophthoe falcata) and a chlorophyll-lacking root parasite (Orobanche aegyptiaca). The highest content of starch occurred In O. aegyptiaca, with a maximum of 45 per cent of dry weight Starch in Cuscuta filaments and mistletoe leaves showed a maximum of about 10 per cent of dry weight. The starch content varied along the length of the Cuscuta vine, with a maximum in the apical region. Orobanche had a higher starch content when it was still submerged than it was fully developed. Cuscuta vines did not show any marked diurnal alteration in the starch content. The content of ethanol-soluble carbohydrate was only a tenth of the starch in Orobanche, but was relatively higher in the other parasites. the neutral sugars in Cuscuta filaments were sucrose and glucose, whereas fructose was also present in mistletoe and Orobanche. Raffinose and stachyose were absent or present only ill traces in parasite tissue. Starch granules from Cuscuta and Orobanche bad ADPG/UDPG-starch synthetase activity and homogenates starch phosphorylase activity. The former enzyme appeared to be responsible for synthesis of starch and the latter for utilization. The four different species of Cuscnta, growing on alfalfa, had more or less the same activity of starch synthetase and also of phosphorylase activity. Hosts infected by Cuscuta had significantly less starch per plant than the controls. A characteristic feature of invasion by Cuscuta and Orobanche was increased phosphorylase activity in the host tissues. The protein content of the tissues of Cuscuta and Orobanche was of a lower level than that of the host shoot system or foliage, indicating that the parasite differed from the host in having a higher carbon (of starch) to nitrogen (of protein) ratio.     

Threonine-insensitive Homoserine Dehydrogenase from Soybean: GENOMIC ORGANIZATION, KINETIC MECHANISM, AND IN VIVO ACTIVITY*

Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (K_i = 160–240 mm) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (K_i∼150 μm). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content.     

Dynamics of epileptiform activity in mouse hippocampal slices

Increase of the extracellular K⁺ concentration mediates seizure-like synchronized activities in vitro and was proposed to be one of the main factors underlying epileptogenesis in some types of seizures in vivo. While underlying biophysical mechanisms clearly involve cell depolarization and overall increase in excitability, it remains unknown what qualitative changes of the spatio-temporal network dynamics occur after extracellular K⁺ increase. In this study, we used multi-electrode recordings from mouse hippocampal slices to explore changes of the network activity during progressive increase of the extracellular K⁺ concentration. Our analysis revealed complex spatio-temporal evolution of epileptiform activity and demonstrated a sequence of state transitions from relatively simple network bursts into complex bursting, with multiple synchronized events within each burst. We describe these transitions as qualitative changes of the state attractors, constructed from experimental data, mediated by elevation of extracellular K⁺ concentration.     

Estradiol inhibits glucocorticoid receptor expression and induces glucocorticoid resistance in MCF-7 human breast cancer cells

Our study has shown that treatment of MCF-7 human breast cancer cells with 17-beta estradiol (E(2)) produced significant decreases in glucocorticoid receptor (GR) concentrations and GR mRNA levels. E(2) pre-treatment of MCF-7 cells stably transfected with the GR responsive pMTV-CAT reporter (MCF-7-MTV cells), caused significant attenuation of dexamethasone (DEX)-induced chloramphenicol acetyl transferase (CAT). In MCF-7 cells transiently transfected with [(GRE)(3)-Luc] reporter plasmid, E(2) pre-treatment significantly suppressed DEX-induced luciferase, which was abolished by the estrogen receptor antagonist ICI 182,780. We examined the effect of chronic E(2) treatment as well as E(2) withdrawal on GR function and abundance. MCF-7-MTV cells were treated with vehicle (control) or E(2) for up to 16 days. A third group received E(2) for 5 days followed by E(2) withdrawal from day 6 to 16. Chronic E(2) treatment almost totally abrogated DEX-induced CAT and reduced GR to very low levels. Interestingly, in the group subjected to E(2) withdrawal, neither the DEX response nor GR abundance recovered and reached control values suggesting that the estrogen mediated suppression is long lasting and could not be easily reversed. The E(2) induced resistance to glucocorticoid action may be of potential clinical significance in a number of settings including breast cancer, neuroendocrine response to stress and osteoporosis and could possibly contribute to the differences in glucocorticoid responsiveness among patients.