An improved malachite green assay of phosphate: mechanism and application

The classical malachite green (MLG) assay of phosphate, which added MLG after molybdate to the acidified reaction solutions of phosphate, tolerated interference from papaverine, sildenafil, and some similar hydrophobic amines. Resonance Rayleigh scattering signals, the alleviation of interference by poly(vinyl alcohol), and the precipitation of some yellow complexes supported that the irreversible aggregation of the complexes of a hydrophobic amine of interference and phosphomolybdate reduced the amounts of phosphomolybdate accessible to MLG and caused the interference. By adding MLG before molybdate to the acidified reaction solutions of phosphate, the complexes of phosphomolybdate and MLG were preferentially formed before the complexes of phosphomolybdate and such a hydrophobic amine effectively aggregated; thereby, an improved MLG assay of phosphate with the resistance to common hydrophobic amines was developed. Using the improved MLG assay of phosphate and a phosphatase to release phosphate from AMP, a spectrometric method successfully estimated the half-inhibition concentrations of papaverine on the recombinant human cyclic nucleotide phosphodiesterase (PDE) isozyme 4 and the mixture of PDE isozymes from rabbit brain. Therefore, the improved MLG assay of phosphate was a favorable and universal technique for developing spectrometric methods for characterizing and screening inhibitors of enzymes that release phosphate during their actions.     

On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis

Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the NAD(P)⁺-coupled oxidation of succinic semialdehyde (SSA) to succinate, the last step of the γ-aminobutyrate shunt. Mycobacterium tuberculosis encodes two paralogous SSADHs (gabD1 and gabD2). Here, we describe the first mechanistic characterization of GabD1, using steady-state kinetics, pH-rate profiles, ¹H NMR, and kinetic isotope effects. Our results confirmed SSA and NADP⁺ as substrates and demonstrated that a divalent metal, such as Mg²⁺, linearizes the time course. pH-rate studies failed to identify any ionizable groups with pK_a between 5.5 and 10 involved in substrate binding or rate-limiting chemistry. Primary deuterium, solvent and multiple kinetic isotope effects revealed that nucleophilic addition to SSA is very fast, followed by a modestly rate-limiting hydride transfer and fast thioester hydrolysis. Proton inventory studies revealed that a single proton is associated with the solvent-sensitive rate-limiting step. Together, these results suggest that product dissociation and/or conformational changes linked to it are rate-limiting. Using structural information for the human homolog enzyme and ¹H NMR, we further established that nucleophilic attack takes place at the Si face of SSA, generating a thiohemiacetal with S stereochemistry. Deuteride transfer to the Pro-R position in NADP⁺ generates the thioester intermediate and [4A-²H, 4B-¹H] NADPH. A chemical mechanism based on these data and the structural information available is proposed.     

Complete genome sequence of Japanese erwinia strain ejp617, a bacterial shoot blight pathogen of pear

The Japanese Erwinia strain Ejp617 is a plant pathogen that causes bacterial shoot blight of pear in Japan. Here, we report the complete genome sequence of strain Ejp617 isolated from Nashi pears in Japan to provide further valuable insight among related Erwinia species.     

Complete genome sequence of Vibrio vulnificus MO6-24/O

Vibrio vulnificus is the causative agent of life-threatening septicemia and severe wound infections. Here, we announce the complete annotated genome sequence of V. vulnificus MO6-24/O, isolated from a patient with septicemia. When it is compared with previously known V. vulnificus genomes, the genome of this bacterium shows a unique genetic makeup, including phagelike elements, carbohydrate metabolism-related genes, and the superintegron.     

Genome sequence of Lactobacillus salivarius GJ-24, a probiotic strain isolated from healthy adult intestine

The draft genome sequence of Lactobacillus salivarius GJ-24 isolated from the feces of healthy adults was determined. Its properties, including milk fermentation activity and bacteriocin production, suggest its potential uses as a probiotic lactic acid bacterium and start culture for dairy products.     

Genome sequence of Lactobacillus johnsonii PF01, isolated from piglet feces

Lactobacillus johnsonii PF01, an autochthonous bacterium of the gastrointestinal tract, was isolated from a fecal sample from a piglet. The strain adhered specifically to the duodenal and jejunal epithelial cells of the piglet and had high bile resistance activity. Here we report the genomic sequence of L. johnsonii PF01.     

Genome sequence of Lactobacillus ruminis SPM0211, isolated from a fecal sample from a healthy Korean

Lactobacillus ruminis SPM0211 is a potential probiotic strain that shows antimicrobial activity against emerging pathogens. Here we present the draft genomic sequence of L. ruminis SPM0211, isolated from a fecal sample from a healthy Korean and describe both the common and unique features of this strain.     

Tolerance of ectomycorrhizal fungus mycelium to low temperature and freezing-thawing

Very little is known about the tolerance of ectomycorrhizal fungi to the freezing and repetitive freezing-thawing in northern coniferous forests. Isolates of Cortinarius multiformis , Russula densifolia , Suillus granulatus , and Lactarius deliciosus were exposed to a series of temperatures between +4 and -40 °C. The relative electrolyte leakage test indicated that the lethal temperature for 50% of samples was between -7.6 and -13.7 °C. Resume growth experiments showed that the 4 species of ectomycorrhizal fungi had a relatively high tolerance to the low temperatures, with L. deliciosus having the highest tolerance and C. multiformis the lowest. The repeated freezing-thawing delayed the growth of mycelium, which decreased with an increase in the number of freeze-thaw cycles.     

Defects in apoptosis increase memory CD8+ T cells following infection of Bim-/-Faslpr/lpr mice

During many infections, large numbers of effector CD8⁺ T cells are generated. After pathogen clearance, the majority of these cells undergo apoptosis, while the survivors differentiate into memory CD8⁺ T cells. Although loss of both Bim and Fas function dramatically increased antigen-specific CD8⁺ T cells in the lymph nodes following acute lymphocytic choriomeningitis virus (LCMV) infection, it was unclear whether they were pardoned effector or true memory CD8⁺ T cells. In this study, we demonstrate they are bona fide memory T cells as characterized by surface marker expression, cytokine production, homeostatic proliferation, and ability to clear a secondary challenge of pathogen. Loss of both Bim and Fas also increased the number of virus-specific CD4⁺ T cells found in the lymph nodes compared to the parental genotypes or wildtype mice. These studies illustrate that decreasing apoptosis increases the number of memory T cells and therefore could increase the efficacy of vaccines.     

Gene flow from herbicide-tolerant GM rice and the heterosis of GM rice-weed F2 progeny

Gene flow from genetically modified (GM) crops to non-GM cultivars or weedy relatives may lead to the development of more aggressive weeds. We quantified the amount of gene flow from herbicide-tolerant GM rice (Protox GM, derived from the cultivar Dongjin) to three cultivars (Dongjin, Aranghyangchal and Hwaseong) and a weedy rice line. Gene flow frequency generally decreased with increasing distance from the pollen donor. At the shortest distance (0.5 m), we observed a maximum frequency (0.039%) of gene flow. We found that the cultivar Dongjin received the greatest amount of gene flow, with the second being weedy rice. Heterosis of F2 inbred progeny was also examined between Protox GM and weedy rice. We compared growth and reproduction between F2 progeny (homozygous or hemizygous for the Protox gene) and parental rice lines (GM and weedy rice). Here, transgene-homozygous F2 progeny was significantly taller and produced more seeds than the transgene-hemizygous F2 progeny and parental lines. Although the gene flow frequency was generally low, our results suggest that F2 progeny between GM and weedy relatives may exhibit heterosis.