Intracellular expression of Vitreoscilla hemoglobin improves S-adenosylmethionine production in a recombinant Pichia pastoris

To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.     

Sex differences in expression of calcium-handling proteins and beta-adrenergic receptors in rat heart ventricle

Human studies reveal sex differences in myocardial function as well as in the incidence and manifestation of heart disease. Myocellular Ca(2+) cycling regulates normal contractile function; whereas cardiac dysfunction in heart failure has been associated with alterations in Ca(2+)-handling proteins. Beta-adrenergic receptor (beta-AR) signaling regulates activity of several Ca(2+)-handling proteins and alterations in beta-AR signaling are associated with heart disease. This study examines sex differences in expression of beta(1)-AR, beta(2)-AR, and Ca(2+)-handling proteins including: L-type calcium channel (Ca(v)1.2) , ryanodine calcium-release channels (RyR), sarcoplasmic reticular Ca(2+) ATPase (SERCA2), phospholamban (PLB) and Na(+)-Ca(2+) exchange protein (NCX) in healthy hearts from male and female Sprague-Dawley rats. Protein levels were examined using Western blot analysis. Abundance of mRNA was determined by real time RT-PCR normalized to abundance of GAPDH mRNA. Contraction parameters were measured in right ventricular papillary muscle in the presence and absence of isoproterenol. Results demonstrate that female ventricle has significantly higher levels of Ca(v)1.2, RyR, and NCX protein compared to males. Messenger RNA abundance for RyR, and NCX protein was significantly higher in females whereas Ca(v)1.2 mRNA was higher in males. No differences were detected in beta-ARs, SERCA2 or PLB. Female right papillary muscle had a faster maximal rate of force development and decline (+/- dF/dt). There were no sex differences in response to isoproterenol. Results show significant sex differences in expression of key ventricular Ca(2+)-handling proteins that are associated with small functional differences in +/- dF/dt. Further studies will determine whether differences in the abundance of these key proteins play a role in sex disparities in the incidence and manifestation of heart disease.     

Comparison of DMO and flow cytometric methods for measuring intracellular pH and the effect of hyperthermia on the transmembrane pH gradient

Intracellular pH (pHi) was measured in both unheated and heated cells by the distribution of the weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C (14C-DMO), and by the fluorescence intensity ratio (I530/I630) of the pH sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF), analyzed by flow cytometry (FCM). BCECF-loaded Chinese hamster ovary (CHO) cells were analyzed by FCM after they had incubated in fresh medium at 37 degrees C for 90 min, during which time a decrease in fluorescence ratio stabilized. After stabilization, the pHi determined for CHO cells by the FCM method at pHe values of 6.0-8.1 agreed-within 0.1 pH units with that determined by the 14C-DMO method. There is a pH gradient across the plasma membrane that is not affected by heat. In CHO cells, the gradient, determined by DMO and FCM, is less or greater than pHe by 0.30 and 0.15 pH units at pHe 7.4 and 6.3, respectively, and in NG108-15 cells, the gradient determined by DMO increases to 0.50 pH units at pHe 6.3. Both cells maintained their pH gradients for at least 4 h after heating, although 99.9% of the cells were reproductively dead (survival of 10(-3)) after heating at 45.5 degrees C either at the normal pHe of 7.4 or at a low pHe of 6.4-6.7.     

Comparison of molecular linkage maps and QTLs for morphological traits in two reciprocal backcross populations of rice

Comparison of maps and QTLs between populations may provide us with a better understanding of molecular maps and the inheritance of traits. We developed and used two reciprocal BC1F1 populations, IP/DS//IP and IP/DS//DS, for QTL analysis. DS (Dasanbyeo) is a Korean tongil-type cultivar (derived from an indica x japonica cross and similar to indica in its genetic make-up) and IP (Ilpumbyeo) is a Korean japonica cultivar. We constructed two molecular linkage maps corresponding to each backcross population using 196 markers for each map. The length of each chromosome was longer in the IP/DS//IP population than in the IP/DS//DS population, indicating that more recombinants were produced in the IP/DS//IP population. Distorted segregation was observed for 44 and 19 marker loci for the IP/DS//IP and IP/DS//DS populations, respectively; these were mostly skewed in favor of the indica alleles. A total of 36 main effect QTLs (M-QTLs) and 15 digenic epistatic interactions (E-QTLs) were detected for the seven traits investigated. The phenotypic variation explained (PVE) by M-QTLs ranged from 3.4% to 88.2%. Total PVE of the M-QTLs for each trait was significantly higher than that of the E-QTLs. The total number of M-QTLs identified in the IP/DS//IP population was higher than in the IP/DS//DS population. However, the total PVE by the M-QTLs and E-QTLs together for each trait was similar in the two populations, suggesting that the two BC1F1 populations are equally useful for QTL analysis. Maps and QTLs in the two populations were compared. Eleven new QTLs were identified for SN, SF, GL, and GW in this study, and they will be valuable in marker-assisted selection, particularly for improving grain traits in tongil-type varieties.     

The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Alcaligenes eutrophus . Incorporation of [1-¹⁴C]-acetyl-CoA into poly-3-hydroxybutyrate (PHB) by systems reconstituted from purified preparations of either 3-ketothiolase, AcAc-CoA reductase and PHB synthase, occurred only when NADPH-AcAc-CoA reductase was present. The NADH reductase was active with all of the d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C₄-C₁₀), whereas the NADPH reductase was only active with d (−)-3-hydroxyacyl-CoAs (C₄-C₆). The products of AcAc-CoA reduction by the NADH- and NADPH-linked enzymes were l (+)-3-hydroxybutyryl-CoA and d (−)-3-hydroxybutyryl-CoA, respectively. The NADH-linked enzyme had an M _r of 150,000 (containing identical M _r 30,000 sub-units) and the NADPH-linked enzyme appeared to be a tetramer ( M _r 84,000) with identical sub-units ( M _r 23,000). K _m^app values of 22 μM and 5 μM for AcAc-CoA and 13 μM (NADH) and 19 μM (NADPH) for the coenzymes were determined for the NADH- and NADPH-linked enzymes, respectively.     

The Mitochondrial Genome of Raphanus sativus and Gene Evolution of Cruciferous Mitochondrial Types

To explore the mitochondrial genes of the Cruciferae family, the mitochondrial genome of Raphanus sativus (sat) was sequenced and annotated. The circular mitochondrial genome of sat is 239,723 bp and includes 33 protein-coding genes, three rRNA genes and 17 tRNA genes. The mitochondrial genome also contains a pair of large repeat sequences 5.9 kb in length, which may mediate genome reorga-nization into two sub-genomic circles, with predicted sizes of 124.8 kb and 115.0 kb, respectively. Furthermore, gene evolution of mitochondrial genomes within the Cruciferae family was analyzed using sat mitochondrial type (mitotype), together with six other re-ported mitotypes. The cruciferous mitochondrial genomes have maintained almost the same set of functional genes. Compared with Cycas taitungensis (a representative gymnosperm), the mitochondrial genomes of the Cruciferae have lost nine protein-coding genes and seven mitochondrial-like tRNA genes, but acquired six chloroplast-like tRNAs. Among the Cruciferae, to maintain the same set of genes that are necessary for mitochondrial function, the exons of the genes have changed at the lowest rates, as indicated by the numbers of single nucleotide polymorphisms. The open reading frames (ORFs) of unknown function in the cruciferous genomes are not conserved. Evolutionary events, such as mutations, genome reorganizations and sequence insertions or deletions (indels), have resulted in the non- conserved ORFs in the cruciferous mitochondrial genomes, which is becoming significantly different among mitotypes. This work represents the first phylogenic explanation of the evolution of genes of known function in the Cruciferae family. It revealed significant variation in ORFs and the causes of such variation.     

Induction of Crassulacean Acid Metabolism in the Facultative Halophyte Mesembryanthemum crystallinum by Abscisic Acid

The facultative halophyte, Mesembryanthemum crystallinum, shifts its mode of carbon assimilation from the C₃ pathway to Crassulacean acid metabolism (CAM) in response to water stress. In this study, exogenously applied abscisic acid (ABA), at micromolar concentrations, could partially substitute for water stress in induction of CAM in this species. ABA at concentrations of 5 to 10 micromolar, when applied to leaves or to the roots in hydroponic culture or in soil, induced the expression of CAM within days (as indicated by the nocturnal accumulation of total titratable acidity and malate). After applying ABA there was also an increase in phosphoenolpyruvate carboxylase and NADP-malic enzyme activities. The degree and time course of induction by ABA were comparable to those induced by salt and water stress. Electrophoretic analyses of leaf soluble protein indicate that the increases in phosphoenolpyruvate carboxylase activity during the induction by ABA, salt, and water stress are due to an increase in the quantity of the enzyme protein. ABA may be a factor in the stress-induced expression of CAM in M. crystallinum, serving as a functional link between stress and biochemical adaptation.