Studies on phosphoglyceromutase from chicken breast muscle: chemical modification of lysyl residues.

To examine the role of lysyl residues in the activity of the enzyme, phosphoglyceromutase (PGM) from chicken breast muscle was chemically modified with trinitrobenzenesulfonate (TNBS) and pyridoxal 5'-phosphate. Trinitrophenylation resulted in modification of about nine lysines per mole of PGM with almost complete activity loss. Substrate (3-PGA) offered some protection to TNBS inactivation but cofactor (2,3-DPGA) did not. Reduction of the Schiff's base complex between pyridoxal 5'-phosphate and PGM gave irreversible inactivation of the enzyme. Inactivation was due to incorporation of 1 mol of pyridoxal 5'-phosphate per mole of PGM dimer through the epsilon-amino group of a lysyl residue. The effect of pyridoxal 5'-phosphate was specific for intact native enzyme and reaction with only one lysine per dimer was not due to induced conformational changes nor to dissociation of the reacted enzyme. 3-PGA prevented much of the reaction with pyridoxal 5'-phosphate with preservation of 70% of the activity and was a competitive inhibitor of the active site directed reagent. Cofactor (2,3-DPGA) acting noncompetitively, reduced the rate at which inactivation occurred with pyridoxal 5'-phosphate. Incorporation of 2,3-[32P]DPGA into PGM irreversibly inactivated with pyridoxal 5'-phosphate and NaBH4 was incomplete indicating hindrance to phosphorylation in the modified enzyme. The results indicate that a lysyl residue is located at or near the active site of PGM and that it is probably involved in the binding of 3-PGA.     

Details of the nucleic acid binding site of T4 gene 32 protein revealed by proteolysis and DNA Tm depression methods

The affinities and location of oligonucleotides bound to intact and truncated bacteriophage T4 gene 32 protein have been elucidated by two independent and sensitive methods. The nucleic acid binding site is located within the core domain of 32 protein, residues 22-253. Oligonucleotides protect the core domain against proteolysis catalyzed by mammalian endoproteinase Arg-C. Of the three cleavage sites, Arg111, within the internal "LAST" ((Lys/Arg)3(Ser/Thr)2) motif, is selectively protected. We have previously suggested that these LAST residues, Lys-Arg-Lys-Thr-Ser, residues 110-114, are involved in nucleic acid binding, and our results are also consistent with crystallographic studies. The inhibitory effects of oligonucleotides on the kinetics of core domain proteolysis were used to quantify binding affinities. In addition, affinities of oligonucleotides for both core domain and intact protein were obtained from their effect on the Tm-depressing activities of these proteins. For both core and intact protein, the degree of affinity increases with oligonucleotide length. The presence of a 5' terminal phosphate increases the affinity two- to fourfold. Placement of methylphosphonodiester (uncharged) linkages at alternating linkages vastly lowers binding affinity for the intact protein and core domain. We conclude that at least two and likely three adjacent phosphodiester linkages are a minimal requirement for binding, further defining the electrostatic component of the interaction. The length-dependence of binding affinity suggests that additional interactions, both ionic and non-ionic, likely occur with longer oligonucleotides.     

Fermentation of molasses using a thermotolerant yeast,Kluyveromyces marxianus IMB3: simplex optimisation of media supplements

 The use of molasses as a substrate for ethanol production by the thermotolerant yeast Kluyveromyces marxianus var. marxianus was investigated at 45°C. A maximum ethanol concentration of 7.4% (v/v) was produced from unsupplemented molasses at a concentration of 23% (v/v). The effect on ethanol production of increasing the sucrose concentration in 23% (v/v) molasses was determined. Increased sucrose concentration had a similar detrimental effect on the final ethanol produced as the increase in molasses concentration. This indicated that the effect may be due to increased osmotic activity as opposed to other components in the molasses. The optimum concentration of the supplements nitrogen, magnesium, potassium and fatty acid for maximum ethanol production rate was determined using the Nelder and Mead (Computer J 7:308–313, 1965) simplex optimisation method. The optimum concentrations of the supplements were 0.576 g l^-1 magnesium sulphate, 0.288 g l^-1 potassium dihydrogen phosphate and 0.36% (v/v) linseed oil. Added nitrogen in the form of ammonium sulphate did not affect the ethanol production rate. Received: 29 January 1996/Received revision: 23 April 1996/Accepted: 29 April 1996     

Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor

Interventions such as glycogen depletion, which limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether the novel glycogen phosphorylase inhibitor 5-Chloro-N-[(1S,2R)-3-[(3R,4S)-3,4-dihydroxy-1-pyrrolidinyl)]-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (ingliforib; CP-368,296) could reduce infarct size in both in vitro and in vivo rabbit models of ischemia-reperfusion injury (30 min of regional ischemia, followed by 120 min of reperfusion). In Langendorff-perfused hearts, constant perfusion of ingliforib started 30 min before regional ischemia and elicited a concentration-dependent reduction in infarct size; infarct size was reduced by 69% with 10 microM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg.kg(-1).h(-1) infusion) selected to achieve a free plasma concentration equivalent to an estimated EC(50) in the isolated hearts (1.2 microM, 0.55 microg/ml) significantly reduced infarct size by 52%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores were preserved in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate-pressure product in the ingliforib group, although heart rate was modestly decreased postischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection and treating diabetic patients at increased risk of cardiovascular disease.     

Microbial diversity and biomarker analysis of modern freshwater microbialites from Laguna Bacalar,Mexico

Laguna Bacalar is a sulfate‐rich freshwater lake on the Yucatan Peninsula that hosts large microbialites. High sulfate concentrations distinguish Laguna Bacalar from other freshwater microbialite sites such as Pavilion Lake and Alchichica, Mexico, as well as from other aqueous features on the Yucatan Peninsula. While cyanobacterial populations have been described here previously, this study offers a more complete characterization of the microbial populations and corresponding biogeochemical cycling using a three‐pronged geobiological approach of microscopy, high‐throughput DNA sequencing, and lipid biomarker analyses. We identify and compare diverse microbial communities of Alphaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria that vary with location along a bank‐to‐bank transect across the lake, within microbialites, and within a neighboring mangrove root agglomeration. In particular, sulfate‐reducing bacteria are extremely common and diverse, constituting 7%–19% of phylogenetic diversity within the microbialites, and are hypothesized to significantly influence carbonate precipitation. In contrast, Cyanobacteria account for less than 1% of phylogenetic diversity. The distribution of lipid biomarkers reflects these changes in microbial ecology, providing meaningful biosignatures for the microbes in this system. Polysaturated short‐chain fatty acids characteristic of cyanobacteria account for <3% of total abundance in Laguna Bacalar microbialites. By contrast, even short‐chain and monounsaturated short‐chain fatty acids attributable to both Cyanobacteria and many other organisms including types of Alphaproteobacteria and Gammaproteobacteria constitute 43%–69% and 17%–25%, respectively, of total abundance in microbialites. While cyanobacteria are the largest and most visible microbes within these microbialites and dominate the mangrove root agglomeration, it is clear that their smaller, metabolically diverse associates are responsible for significant biogeochemical cycling in this microbialite system.     

The chemokine receptor CXCR3 attenuates the control of chronic Mycobacterium tuberculosis infection in BALB/c mice

The chemokine receptor CXCR3 plays a significant role in regulating the migration of Th1 cells. Given the importance of Th1 immunity in the control of tuberculous infection, the results of the present study demonstrating that CXCR3-deficient BALB/c mice are more resistant to Mycobacterium tuberculosis, compared with wild-type mice, is surprising. This enhanced resistance manifests in the chronic but not the acute phase of infection. Remarkable differences in the cellular composition of the pulmonic granuloma of the CXCR3(-/-) and wild-type mice were found, the most striking being the increase in the number of CD4(+) T cells in the knockout strain. In the chronic phase of infection, the number of CD69-expressing CD4(+) T lymphocytes in the lungs of CXCR3(-/-) mice was higher than in wild-type mice. Additionally, at 1 mo postinfection, the number of IFN-gamma-producing CD4(+) T cells in the lungs and mediastinal lymph nodes of the CXCR3-deficient strain was elevated compared with wild-type mice. Pulmonic expression of IFN-gamma, IL-12, TNF-alpha, or NO synthase 2, the principal antimycobacterial factors, were equivalent in the two mouse strains. These results indicate that: 1) CXCR3 plays a role in modulating the cellular composition of tuberculous granuloma; 2) CXCR3 impairs antimycobacterial activity in chronic tuberculosis; and 3) in the absence of CXCR3, mice exhibit a heightened state of CD4(+) T lymphocyte activation in the chronic phase of infection that is associated with enhanced CD4(+) T cell priming. Therefore, CXCR3 can attenuate the host immune response to M. tuberculosis by adversely affecting T cell priming.     

An international parentage and identification panel for the domestic cat (Felis catus)

Seventeen commercial and research laboratories participated in two comparison tests under the auspices of the International Society for Animal Genetics to develop an internationally tested, microsatellite-based parentage and identification panel for the domestic cat (Felis catus). Genetic marker selection was based on the polymorphism information content and allele ranges from seven random-bred populations (n = 261) from the USA, Europe and Brazil and eight breeds (n = 200) from the USA. Nineteen microsatellite markers were included in the comparison test and genotyped across the samples. Based on robustness and efficiency, nine autosomal microsatellite markers were ultimately selected as a single multiplex 'core' panel for cat identification and parentage testing. Most markers contained dinucleotide repeats. In addition to the autosomal markers, the panel included two gender-specific markers, amelogenin and zinc-finger XY, which produced genotypes for both the X and Y chromosomes. This international cat parentage and identification panel has a power of exclusion comparable to panels used in other species, ranging from 90.08% to 99.79% across breeds and 99.47% to 99.87% in random-bred cat populations.     

Structure and rearrangements in the carboxy-terminal region of SpIH channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels regulate the spontaneous firing activity and electrical excitability of many cardiac and neuronal cells. The modulation of HCN channel opening by the direct binding of cAMP underlies many physiological processes such as the autonomic regulation of the heart rate. Here we use a combination of X-ray crystallography and electrophysiology to study the allosteric mechanism for cAMP modulation of HCN channels. SpIH is an invertebrate HCN channel that is activated fully by cAMP, but only partially by cGMP. We exploited the partial agonist action of cGMP on SpIH to reveal the molecular mechanism for cGMP specificity of many cyclic nucleotide-regulated enzymes. Our results also elaborate a mechanism for the allosteric conformational change in the cyclic nucleotide-binding domain and a mechanism for partial agonist action. These mechanisms will likely extend to other cyclic nucleotide-regulated channels and enzymes as well.     

Individualized survival and treatment response predictions for breast cancers using phospho-EGFR, phospho-ER, phospho-HER2/neu, phospho-IGF-IR/In, phospho-MAPK, and phospho-p70S6K proteins

The development and progression of breast cancer involves the activation of numerous protein kinases, and the change in phosphorylation is a hallmark of protein kinase activation. In this study, we identified a comprehensive profile to predict individual breast cancer patients' survival and treatment responses using the Random Committee algorithm. The profile incorporated a subset of phosphorylated signal protein expressions and several selected clinical factors of breast cancer. The parameters of our profile were identified by supervised feature selection algorithms, Gain Ratio Attribute Evaluation and Relief. The results showed that the overall accuracy of survival prediction reached 92.3% for individual breast cancer patients with the use of the expression profiles of phospho-EGFR, phospho-ER, phospho-HER2/neu, phospho-IGFIR/In, phospho-MAPK, and phospho-p70S6K plus the selected clinical factors. The results also indicated that the overall accuracy of treatment response prediction was 92.6% with the use of the level of phospho-EGFR, phospho-ER, phospho-HER2/neu, phospho-MAPK, and phospho-p70S6K plus the selected clinical information. The prediction system combines multiple signal protein activation profiles and relevant clinical information, and provides a unique guideline to aid individualized decision-making in the clinical management of breast cancer.