The genome stability in Corynebacterium species due to lack of the recombinational repair system

Corynebacterium species are members of gram-positive bacteria closely related to Mycobacterium species, both of which are classified into the same taxonomic order Actinomycetales. Recently, three corynebacteria, Corynebacterium efficiens, Corynebacterium glutamicum, and Corynebacterium diphtheriae have been sequenced independently. We found that the order of orthologous genes in these species has been highly conserved though it has been disrupted in Mycobacterium species. This synteny suggests that corynebacteria have rarely undergone extensive genome rearrangements and have maintained ancestral genome structures even after the divergence of corynebacteria and mycobacteria. This is the first report that the genome structures have been conserved in free-living bacteria such as C. efficiens and C. glutamicum, although it has been reported that obligate parasites such as Mycoplasma and Chlamydia have the stable genomes. The comparison of recombinational repair systems among the three corynebacteria and Mycobacterium tuberculosis suggested that the absence of recBCD genes in corynebacteria be responsible for the suppression of genome shuffling in the species. The genome stability in Corynebacterium species will give us hints of the speciation mechanism with the non-shuffled genome, particularly the importance of horizontal gene transfer and nucleotide substitution in the genome.     

Purification and antifungal activity of recombinant chitinase from Escherichia coli carrying the family 19 chitinase gene of Streptomyces sp. J-13-3

A recombinant chitinase was purified from the cell extract of Escherichia coli JM109 transformed by plasmid pUC19 carrying the gene encoding family 19 chitinase of Streptomyces sp. J-13-3 by column chromatography on DEAE-Sepharose, CM-Sepharose, and Bio-Gel P-100. The final preparation was homogenous in polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 32,000. The recombinant chitinase hydrolyzed the trimer to hexamer of N-acetylglucosamine and had the identical N-terminal amino acid sequence of the mature protein, indicating removal of the signal sequence by E. coli signal peptidase. The fungal growth in well (200 microl of medium) of microplate by measurement of absorbance at 595 nm indicated that the chitinase (10 microg) completely and half inhibited growth of Trichoderma reesei and Aspergillus niger respectively.     

Validation of a simple gas chromatographic-mass spectrometric method for the determination of gamma-butyrolactone in human plasma

A gas chromatographic-mass spectrometric (GC-MS) method is described for the determination of human plasma levels of gamma-butyrolactone (GBL) is described. The method is sensitive and simple. The plasma sample spiked with the internal standard was extracted by dichloromethane (CH(2)Cl(2)) in acidic conditions, and the concentrated organic layer was injected into GC-MS. Because of endogenous GBL in human plasma, the method used a standard calibration curve. The calibration curve was linear from 10 to 1000 ng/ml. The method has been validated for accuracy and precision with the relative error and C.V. for intra- and inter-day within 10%. GBL-spiked plasma samples stored at -80 degrees C were stable for a 3-month period. The stability of plasma samples after three cycles of freezing and thawing and of prepared samples on an autosampler for 48 h were demonstrated. Plasma concentrations of GBL before and after administration of UFT were 24.3+/-14.2 and 84.9+/-22.4 ng/ml, respectively.     

Temperature-sensitive intracellular Mg2+ block of L-type Ca2+ channels in cardiac myocytes

We examined the concentration-dependent blocking effects of intracellular Mg2+ on L-type Ca2+ channels in cardiac myocytes using the whole cell patch-clamp technique. The increase of L-type Ca2+ channel current (I(Ca)) (due to relief of Mg2+ block) occurred in two temporal phases. The rapid phase (runup) transiently appeared early (<5 min) in dialysis of the low-Mg2+ solution; the slow phase began later in dialysis (>10 min). Runup was not blocked by intracellular GTP (GTP(i)). The late phase of the I(Ca) increase (late I(Ca)) was suppressed by GTP(i) (0.4 mM) and was observed in myocytes of the guinea pig or frog at higher (32 or 24 degrees C, respectively) rather than lower temperatures (24 or 17.5 degrees C, respectively). At pMg = 6.0, raising the temperature from 24 to 32 degrees C evoked late I(Ca) with a Q10 of 14.5. Restoring the temperature to 24 degrees C decreased I(Ca) with a Q10 of only 2.4. The marked difference in the Q10 values indicated that late I(Ca) (pMg = 5-6) is an irreversible phenomenon. Phosphorylation suppressed the intracellular [Mg2+] dependency of late I(Ca). This effect of phosphorylation together with the inhibitory action of GTP(i) on Mg2+-dependent blocking of I(Ca) are common properties of mammalian and amphibian cardiomyocytes.     

Molecular basis for exaggerated sensitivity to mexiletine in the cardiac isoform of the fast Na channel

Cardiac sodium channels have been shown to have a higher sensitivity to local anesthetic agents, such as lidocaine, than the sodium channels of other tissues. To examine if this is also true for mexiletine, we have systematically measured mexiletine sensitivity of the Na channel isoforms, rH1, (mu)1, and rBII, which were transiently expressed in human embryonic kidney (HEK) 293 cells. We confirmed that the cardiac isoform rH1 exhibited the highest sensitivity among the three tested channel isoforms. In rH1, (mu)1, and rBII, the respective IC(50) values were 62, 294, and 308 microM mexiletine, in regard to tonic block, and 18, 54, and 268 microM mexiletine, in relation to use (8 Hz)-dependent block. The relatively high drug sensitivity of rH1 was an invariant finding, irrespective of channel state or whether channels were subjected to infrequent or frequent depolarizing stimuli. Mutating specific amino acids in the skeletal muscle isoform (mu)1 (namely, (mu)1-I433V and (mu)1-S251A) to those of the cardiac isoform at putative binding sites for local anesthetic agents revealed that only one of the point mutations ((mu)1-S251A) has relevance to the high cardiac drug sensitivity, because mexiletine produced significantly more use-dependent and tonic block in (mu)1-S251A than wild-type (mu)1.     

Crystallization and preliminary X-ray crystallographic studies of Trypanosoma brucei prostaglandin F(2 alpha) synthase

Prostaglandin F(2 alpha) is a potent mediator of various physiological and pathological processes. Trypanosoma brucei prostaglandin F(2 alpha) synthase (TbPGFS) catalyzes the NADPH-dependent reduction of 9,11-endoperoxide PGH(2) to PGF(2 alpha), and could thus be involved in the elevation of the PGF(2 alpha) concentration during African trypanosomiasis. In the present report, the purification and crystallization of recombinant TbPGFS are described. The active recombinant enzyme was crystallized by the hanging-drop vapor-diffusion meth-od using ammonium sulfate as a precipitant. The crystal belonged to a tetragonal space group, P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters of a = b = 112.3 A, and c = 140.0 A. Native data up to 2.6 A resolution were collected from the crystal using our home facility.     

X-ray structure of Galdieria Rubisco complexed with one sulfate ion per active site

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the reactions of carboxylation and oxygenation of ribulose-1,5-bisphosphate. These reactions require that the active site should be closed by a flexible loop (loop 6) of the large subunit. Rubisco from a red alga, Galdieria partita, has the highest specificity for carboxylation reaction among the Rubiscos hitherto reported. The crystal structure of unactivated Galdieria Rubisco has been determined at 2.6 A resolution. The electron density map reveals that a sulfate binds only to the P1 anion-binding site of the active site and the loop 6 is closed. Galdieria Rubisco has a unique hydrogen bond between the main chain oxygen of Val332 on the loop 6 and the epsilon-amino group of Gln386 of the same large subunit. This interaction is likely to be crucial to understanding for stabilizing the loop 6 in the closed state and to making a higher affinity for anionic ligands.     

Thermal regulation of fatty acid synthetase from Brevibacterium ammoniagenes.

The fatty acid composition of Brevibacterium ammoniagenes was affected by the temperature of growth. As the growth temperature was lowered, the proportion of unsaturated fatty acids increased. The fatty acid synthetase obtained from B. ammoniagense produced oleic acid as well as saturated fatty acids. The ratio of unsaturated to saturated fatty acids synthesized by this enzyme in vitro was dependent on the temperature of the enzyme reaction but not on the growth temperature of B. ammoniagenes from which the enzyme was prepared. These results suggest that the changes of composition in cellular fatty acids reflect the temperature dependence of the fatty acid synthetase.     

Stereochemical studies of hydrogen incorporation from nucleotides with fatty acid synthetase from Brevibacterium ammoniagenes.

The biosynthesis of fatty acids from malonyl-CoA and acetyl-CoA was investigated with an enzyme preparation which was purified 100-fold from Brevibacterium ammoniagenes. Fatty acids synthesized in the presence of D2O and stereospecifically deuterated NADPH and NADH were isolated and analyzed by mass chromatography to examine the localization of deuterium in the molecule. The following results were obtained: 1) HB hydrogen of NADPH was used for beta-ketoacyl reductase. 2) HB hydrogen of NADH was used for enoyl reductase. 3) Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom) and some were also found on the odd-numbered methylene carbon atoms. 4) Hydrogen atoms from NADPH was found on the odd-numbered methylene carbon atoms (1 hydrogen per carbon). 5) Hydrogen atoms from NADH was also found on the odd-numbered methylene carbon atoms, but the number of incorporated hydrogen atoms was less than expected. The exchange of HB hydrogen of NADH with water catalyzed by enoyl reductase was suspected. 6) The exchange of methylene hydrogen atoms of malonyl-CoA with protons of water was suggested by 13C NMR analysis.     

Solubilization and characterization of leukotriene B4 receptor-GTP binding protein complex from porcine spleen

A high amount of leukotriene B4 (LTB4) binding protein was observed in the porcine spleen. It was solubilized and partially purified from spleen membrane with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Scatchard analysis indicated the presence of a single class of receptor with Kd and Bmax values of 0.26 nM and 120 fmol/mg protein, respectively. The receptor was specific for LTB4, and Ki values for 20-hydroxy- and 20-carboxy-LTB4, both inactive metabolites of LTB4, were 1.7 nM and over 1,000 nM, respectively. By the addition of 10 microM GTP gamma S, a low affinity binding site appeared with a Kd value of 390 nM. A pretreatment of the receptor-GTP binding protein complex with islet-activating protein (IAP) increased the inhibitory effect of GTP gamma S on LTB4 binding, indicating that the LTB4 receptor is coupled with an IAP-sensitive GTP-binding protein in the porcine spleen.