Growth of mixed cultures on mixtures of substitutable substrates: the operating diagram for a structured model

The growth of mixed microbial cultures on mixtures of substrates is a problem of fundamental biological interest. In the last two decades, several unstructured models of mixed-substrate growth have been studied. It is well known, however, that the growth patterns in mixed-substrate environments are dictated by the enzymes that catalyse the transport of substrates into the cell. We have shown previously that a model taking due account of transport enzymes captures and explains all the observed patterns of growth of a single species on two substitutable substrates (J. Theor. Biol. 190 (1998) 241). Here, we extend the model to study the steady states of growth of two species on two substitutable substrates. The model is analysed to determine the conditions for existence and stability of the various steady states. Simulations are performed to determine the flow rates and feed concentrations at which both species coexist. We show that if the interaction between the two species is purely competitive, then at any given flow rate, coexistence is possible only if the ratio of the two feed concentrations lies within a certain interval; excessive supply of either one of the two substrates leads to annihilation of one of the species. This result simplifies the construction of the operating diagram for purely competing species. This is because the two-dimensional surface that bounds the flow rates and feed concentrations at which both species coexist has a particularly simple geometry: It is completely determined by only two coordinates, the flow rate and the ratio of the two feed concentrations. We also study commensalistic interactions between the two species by assuming that one of the species excretes a product that can support the growth of the other species. We show that such interactions enhance the coexistence region.     

Orthotopic expression of human 15-lipoxygenase (LO)-1 in the dorsolateral prostate of normal wild-type C57BL/6 mouse causes PIN-like lesions

The lipid-peroxidating enzyme, 15-lipoxygenase (LO)-1 and its metabolite, 13-S-hydroxyoctadecadienoic acid (13-S-HODE), likely contribute to prostate tumorigenesis. Thus, this study evaluated adenovirus-mediated overexpression of 15-LO-1 on normal mouse prostate. Adenovirus expressing either human 15-LO-1 tagged with green fluorescent protein (GFP) or GFP alone was orthotopically injected into the dorsolateral prostates of C57BL/6 mice, three times over the course of 60 days. On day 90, pathological changes in prostate tissue were assessed by hematoxylin and eosin (H&E) staining. Expression of the proliferation marker Ki-67 was evaluated by immunohistochemistry and expression of angiogenesis markers were analyzed by an antibody array. Based on the latter study, immunoprecipitation analysis was used to measure the effect of 13-S-HODE, with or without conditioned media, on fibroblast growth factor-a and b (FGF-a and FGF-b) expression in human PrEC (normal prostate epithelial), PrSMC (normal prostate smooth muscle) and PrSC (normal prostate stromal) lines. Expression of viral 15-LO-1-GFP, but not GFP alone, resulted in the development of a prostate intraepithelial neoplasia (PIN)-like phenotype with increased expression of Ki-67. Aberrant 15-LO-1 expression also induced the angiogenic markers FGF-a and FGF-b. Human PrEC, PrSMC and PrSC lines demonstrated an increase in FGF-b expression upon stimulation with 13-S-HODE, which was further increased by the addition of conditioned media from the epithelial or smooth muscle cells. Using adenoviral mediated 15-LO-1 gene delivery, this study suggests that aberrant 15-LO-1 overexpression in normal prostate can trigger events leading to prostate epithelial and stromal cell proliferation. Thus, our findings demonstrate the effectiveness of this viral system for 15-LO-1 expression studies in tissues.     

Cloning and expression of chicken granulocyte-macrophage colony stimulating factor (GMCSF) gene

Granulocyte-macrophage colony stimulating factor (GMCSF), a multifunctional cytokine can enhance immune responses when administered along with DNA vaccine. Aim of the present study was to clone and express the chicken GMCSF cytokine for use as 'genetic adjuvant'. Chicken GMCSF gene 435bp was amplified using specific primers in which restriction sites of BamHI and HindIII were at forward and reverse primers respectively. The PCR product was cloned into eukaryotic expression vector pcDNA 3.1(+) and clones were confirmed by restriction digestion and nucleotide sequencing. Functional activity of recombinant GMCSF was checked by expression of GMCSF specific mRNA in transfected Vero cells by RT-PCR of total RNA isolated from transfected Vero cells. The recombinant plasmid can be used as genetic adjuvant in chicken.     

Protein structure evaluation using an all-atom energy based empirical scoring function

Arriving at the native conformation of a polypeptide chain characterized by minimum most free energy is a problem of long standing interest in protein structure prediction endeavors. Owing to the computational requirements in developing free energy estimates, scoring functions--energy based or statistical--have received considerable renewed attention in recent years for distinguishing native structures of proteins from non-native like structures. Several cleverly designed decoy sets, CASP (Critical Assessment of Techniques for Protein Structure Prediction) structures and homology based internet accessible three dimensional model builders are now available for validating the scoring functions. We describe here an all-atom energy based empirical scoring function and examine its performance on a wide series of publicly available decoys. Barring two protein sequences where native structure is ranked second and seventh, native is identified as the lowest energy structure in 67 protein sequences from among 61,659 decoys belonging to 12 different decoy sets. We further illustrate a potential application of the scoring function in bracketing native-like structures of two small mixed alpha/beta globular proteins starting from sequence and secondary structural information. The scoring function has been web enabled at www.scfbio-iitd.res.in/utility/proteomics/energy.jsp.     

Effects of amylin on feeding of goldfish: interactions with CCK

In mammals, amylin (AMY) is a peptide that is secreted from the pancreas in response to a meal. AMY inhibits food intake and may also contribute to the anorectic effects of the brain-gut peptide cholecystokinin (CCK). In this study, we assessed the role of AMY in the regulation of food intake in goldfish (Carassius auratus) and its interactions with CCK. Fish were injected intraperitoneally (i.p.) with mammalian AMY and intracerebroventricularly (i.c.v.) with mammalian AMY, alone or in combination with the sulfated octapeptide CCK-8S. We also assessed the effects of i.c.v. injections of AC187, an amylin receptor antagonist on the central actions of both AMY and CCK-8S, as well as the effects of i.c.v. injections of proglumide, a CCK receptor antagonist, on the central effects of AMY. AMY injected i.p. at 100 ng/g but not 25 or 50 ng/g or i.c.v. at 10 ng/g but not 1 ng/g significantly decreased food intake as compared to saline-treated fish. Fish co-treated i.c.v. with AMY at 1 ng/g and CCK-8S at 1 ng/g had a food intake lower than that of control fish and fish treated with either 1 ng/g CCK-8S or 1 ng/g AMY, suggesting a synergy between the two systems. Whereas low i.c.v. doses of AC187 (30 ng/g) had no effect, moderate doses (50 ng/g) induced an increase in food intake, indicating a role of endogenous AMY in satiety in goldfish. Blocking central amylin receptors with i.c.v. AC187 (30 ng/g) resulted in an inhibition of both i.c.v. AMY- and CCK-induced reduction in feeding. Blocking central CCK receptors with i.c.v. proglumide (25 ng/g) resulted in an inhibition of both i.c.v. CCK-induced and AMY-induced decrease in food intake. Our results show for the first time in fish that AMY is a potent anorexigenic factor and that its actions are interdependent with those of CCK.     

Kidney plays a major role in ammonia homeostasis after portasystemic shunting in patients with cirrhosis

The kidney plays an important role in ammonia metabolism. In this study the hypothesis was tested that the kidney can acutely diminish ammonia release after portacaval shunting. Thirteen patients with cirrhosis (6 female/7 male, age 54.4 +/- 3.3 yr) were studied. Blood was sampled prior to and 1 h after transjugular intrahepatic stent-shunt (TIPSS) insertion from the portal vein, a hepatic vein, the right renal vein, and the femoral vein, and renal and liver plasma flow were measured. Prior to TIPSS, renal ammonia release was significantly higher than ammonia release from the splanchnic region, which was not significantly different from zero. TIPSS insertion did not change arterial ammonia concentration or ammonia release from the splanchnic region but reduced renal ammonia release into the circulation (P < 0.05) to values that were not different from zero. TIPSS resulted in a tendency toward increased venous-arterial ammonia concentration differences across leg muscle. Post-TIPSS ammonia efflux via portasystemic shunts was estimated to be seven times higher than renal efflux. Kidneys have the ability to acutely diminish systemic ammonia release after portacaval shunting. Diminished renal ammonia release and enhanced muscle ammonia uptake are important mechanisms by which the cirrhotic patient maintains ammonia homeostasis after portasystemic shunting.     

Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions

Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations.     

Bhageerath: an energy based web enabled computer software suite for limiting the search space of tertiary structures of small globular proteins

We describe here an energy based computer software suite for narrowing down the search space of tertiary structures of small globular proteins. The protocol comprises eight different computational modules that form an automated pipeline. It combines physics based potentials with biophysical filters to arrive at 10 plausible candidate structures starting from sequence and secondary structure information. The methodology has been validated here on 50 small globular proteins consisting of 2-3 helices and strands with known tertiary structures. For each of these proteins, a structure within 3-6 A RMSD (root mean square deviation) of the native has been obtained in the 10 lowest energy structures. The protocol has been web enabled and is accessible at http://www.scfbio-iitd.res.in/bhageerath.     

Genetic polymorphisms and possible gene-gene interactions in metabolic and DNA repair genes: effects on DNA damage

We investigated in a central European population, the association between genetic polymorphisms in several genes coding for xenobiotic metabolizing enzymes (CYP1A1, CYP2E1, EPHX1, GSTP1, GSTM1 and GSTT1) and in DNA repair genes (XPD, XPG, XPC and XRCC1) and the levels of single-strand breaks (SSBs) and SSB endonuclease III sensitive sites (endoIII sites) in peripheral blood lymphocytes. No significant differences in the mean levels of SSBs and endoIII sites after stratification for main confounders and occupational exposure were observed in the studied population. Significantly higher levels of SSBs were observed in individuals bearing the wild-type alleles (AA) (0.75+/-0.51SSB/10(9)Da) and heterozygous (AC) genotypes (0.67+/-0.49SSB/10(9)Da) compared to those with homozygous XPD (CC) genotype (0.43+/-0.28SSB/10(9)Da, P=0.033). A moderate increase in the levels of SSBs was also found in individuals with the homozygous XPG exon 15 wild type (GG) and heterozygous (GC) genotypes in comparison to those with the homozygous (CC) genotype (P=0.066) and in individuals with low activity EPHX1 genotype in comparison to those with high activity genotype. Nevertheless, these differences were not statistically significant. No other significant association was found. When gene-gene interactions were evaluated, a combination of EPHX1 activity genotypes with that of either XPD or XPG significantly (P=0.003 and 0.016, respectively) modulated SSB levels resulting in a three-fold difference between the "protective" and the "adverse" genotype-combinations. Almost three-fold differences in SSB levels were found between the "protective" and the "adverse" genotype-combinations of EPHX1 activity genotype and GSTM1 or GSTT1 genotypes, respectively. In conclusion, our results suggest a relation between markers of genotoxicity and polymorphisms in genes coding for xenobiotic metabolizing and DNA repair enzymes as well as a modulating effect of combinations of these polymorphisms.