One-electron-transfer reactions in biochemical systems V. Difference in the mechanism of quinone reduction by the NADH dehydrogenase and the NAD(P)H dehydrogenase (DT-diaphorase)

The mitochondrial NADH dehydrogenase catalyzes a one-electron reduction of quinones. Semiquinones thus formed have the hyperfine structures of their free anion radicals and are suggested to be detached from the enzyme. In the presence of suitable electron acceptors electron transfer occurs from the semiquinone to the acceptor. The mechanism of quinone reduction by spinach ferredoxin-NADP reductase is the same as that by the NADH dehydrogenase.

On the other hand, the NAD(P)H dehydrogenase (DT-diaphorase) prepared from liver soluble fraction catalyzes a typical two-electron reduction of quinones such as p-benzoquinone and 2-methyl-1,4-naphthoquinone. The mechanisms of one-electron and two-electron reduction of quinones are readily distinguishable by the use of an electron spin resonance spectrometer equipped with a flow apparatus and also by the use of an appropriate set of electron acceptors.

It is concluded that the reduction of quinones and oxygen by flavoproteins falls into three mechanistic categories: one-electron, two-electron and mixed-type reactions.     

Urinary thromboxane A2/prostacyclin balance reflects the pathological state of a diabetic

Levels of the stable urinary metabolites of thromboxane A2 and prostacyclin, 11-dehydro-thromboxane B2 (11-dehydro-TXB2) and 2,3-dinor-6-keto-prostaglandin F1alpha (2,3-dinor-6-keto-PGF1alpha) were measured in diabetics to elucidate the relation between the thromboxane A2/prostacyclin (TX/PGI) balance and pathological states of diabetes mellitus. 11-Dehydro-TXB2 and 2,3-dinor-6-keto-PGF1alpha were derivatized to methyl ester-propylamide-dimethylisopropylsilyl ether and methyl ester-methoxime-dimethylisopropylsilyl ether derivatives, respectively, and applied to a gas chromatography/selected ion monitoring. The TX/PGI ratios of diabetics were higher than those of healthy volunteers, suggesting the hypercoagulative states of this disease. The ratios showed positive correlations with the levels of blood glucose. The levels of hemoglobin A1c and triglyceride were correlated weakly with the ratio. Some of the patients who had relatively low levels of blood glucose also showed high TX/PGI ratios. Furthermore, the ratio increased in the order of the groups 1, 2, and 3; group 1 contained patients who did not take medicine for diabetes, group 2 contained those who took oral hypoglycemic agents, and group 3 contained those who received insulin therapy. These observations indicate that the TX/PGI ratio reflects the pathological conditions of diabetes and is a useful marker, having few different features from other markers that are presently used.     

Correlation between germ tube production, phospholipase activity and serotype distribution in Candida albicans

One-hundred and thirteen Candida albicans strains isolated from patients infected by the human immunodeficiency virus (HIV) and twenty five from HIV-negative individuals were studied. The C. albicans strains isolated from different sites of the body were tested for germ-tube (GT), phospholipase production and serotype. The results obtained indicate that the serotype A was predominant in all the groups except for the vaginal strains. No correlation was observed between phospholipase activity and serotype distribution. Germ tube (GT) production was higher among the serotype B strains. A positive correlation between GT induction and phospholipase activity was observed only for the isolates from the oral cavity. It is possible that the correlation between phospholipase activity and high GT production in C. albicans strains can facilitate the penetration through the mucosa.     

Genomic Organization Around the Centromeric End of the HLA Class I Region: Large-Scale Sequence Analysis

We previously sequenced two regions around the centromeric end of HLA class I and the boundary between class I and class III. In this paper we analyze the two regions of about 385 kb and confirm, giving a new line of evidence, that the following two pairs of the genomic segments were duplicated in evolution: (i) a 43-kb genomic segment including the HLA-B gene showing the highest polymorphism among the classical HLA class I loci (class Ia) and a 40-kb segment including the HLA-C locus showing the lowest polymorphism and (ii) a 52-kb segment including the MIC (MHC class I chain related gene) B and a 35-kb segment including MICA. We also found that repetitive elements such as SINEs, LINEs, and LTRs occupy as much as 47% of nucleotides in this 385-kb region. This unusually high content of repetitive elements indicates that repeat-mediated rearrangements have frequently occurred in the evolutionary history of the HLA class Ia region. Analysis of LINE compositions within the two pairs of duplicated segments revealed that (i) LINEs in these regions had been dispersed prior to both the duplication of the HLA-B and -C loci and the duplication of the MICB and MICA loci, and (ii) the divergence of the HLA-B and -C loci occurred prior to the duplication of the MICA and MICB loci. To find novel genes responsible for HLA class I-associated or other diseases, we performed computer analysis applying GenScan and GRAIL to GenBank's dbEST. As a result, at least five as yet uncharacterized genes were newly mapped on the HLA class I centromeric region studied. These novel genes should be analyzed further to determine their relationships to diseases associated with this region. Received: 16 June 1998 / Accepted: 18 August 1998     

Depletion of Bacillus subtilis histone-like protein, HBsu, causes defective protein translocation and induces upregulation of small cytoplasmic RNA

Small cytoplasmic RNA (scRNA) is a metabolically stable homologue of mammalian SRP RNA that contains an Alu-like domain. The Bacillus subtilis histone-like protein HBsu can bind this domain. We demonstrate here that repressing the level of HBsu results in slow growth and the accumulation of precursor of beta-lactamase fusion proteins having the signal sequence of alkaline protease, penicillin binding protein 5* (PBP5*) or CGTase. The degree of the translocation defect varied among the various signal sequences tested. A pulse-chase experiment showed that processing the alpha-amylase signal sequence is significantly inhibited in HBsu-depleted cells. Northern blot analysis indicated that repressing the HBsu gene induces scRNA upregulation, indicating that the defective translocation of presecretory proteins is not due to a reduced scRNA level. The data presented here suggest that HBsu plays a pivotal role in SRP function rather than simply stabilizing the other SRP components such as scRNA.     

Kinetic studies on the successive reaction of neuronal nitric oxide synthase from L-arginine to nitric oxide and L-citrulline

Rat neuronal nitric oxide synthase (nNOS) was heterologously expressed in Escherichia coliand purified. The conversion of L-arginine to N(omega)-hydroxy-L-arginine and further to L-citrulline in one cycle of the reaction of the purified nNOS was measured with the reaction rapid quenching method using (3)H-L-arginine as the substrate. It was found that most of the produced (3)H-N(omega)-hydroxy-L-arginine was successively hydroxylated to (3)H-L-citrulline without leaving the enzyme. From the analysis of time courses, the rate constants for each reaction step, and also for the dissociation of the intermediate, were estimated at various temperature in which the rates for the first and the second reactions were not much different each other but the rate for the dissociation of (3)H-N(omega)-hydroxy-L-arginine from the enzyme was significantly slow. Under the steady-state reaction condition, almost all of the nNOS was estimated to be active from the amount of burst formation of L-citrulline in the pre-steady state. The rate constant for the dissociation of the product L-citrulline from nNOS was calculated from the combination of results of the rapid quenching experiments and the metabolism of L-arginine in the presence of an excess amount of substrate, which was the smallest among all the rate constants in one cycle of the nNOS reaction. The activation energies for all the reaction steps were determined from the temperature dependence of the rate constants, which revealed that the rate-determining step of the nNOS reaction in the steady state was the dissociation of the product L-citrulline from the enzyme.     

NMR observation of selected segments in a larger protein: central-segment isotope labeling through intein-mediated ligation

Peptide segments in a protein, which can include an active site of interest or be a series of parts constituting the entire structure, are now selectively observed by nuclear magnetic resonance (NMR) spectroscopy using samples prepared by the intein-mediated ligation method. Two separate inteins were used to ligate NMR-transparent segments to both the ends of an NMR-visible segment, producing a partly visible intact protein molecule. The (15)N-(1)H correlation spectrum of a 370-residue maltose binding protein labeled with (15)N at a continuous segment comprising residues Gly(101)-Ser(238) showed the essential elimination of signal overlapping, the signals being at the same positions as for the uniformly labeled sample. This method will allow structural analysis by NMR of over 50-kDa proteins in combination with contemporary NMR techniques suppressing the signal decays of larger proteins.     

A possible balance of magnesium accumulations among bone, cartilage, artery, and vein in single human individuals

It is known that a large quantity of magnesium contains bones, and the magnesium contents in spongy bones decrease gradually with advancing age. To elucidate the relationships between a decrease of mineral contents in human bones and an accumulation of minerals in the other human tissues, the content of magnesium was analyzed by inductively coupled plasma-atomic emission spectrometry among human bones, arteries, veins, and cartilages in 27 subjects (17 men and 10 women). These were resected from the subjects who died in the age range 40–98 yr. Calcanei were chosen for analysis of magnesium contents in contrast with femoral, popliteal, and common carotid arteries, internal jugular and femoral veins, superior and inferior venae cavae, and pubic symphyses. The magnesium contents in the calcanei decreased gradually with aging, whereas they increased progressively in the arteries, veins, and pubic symphyses with aging. It was found that as the magnesium contents decreased in the calcanei, they increased in the arteries, such as the femoral, popliteal, and common carotid arteries, whereas they decreased inversely in the veins, such as the internal jugular and femoral veins and superior and inferior venae cavae. Furthermore, as the magnesium contents decreased in the calcanei, they hardly changed in the pubic symphyses. These suggest that magnesium released from bones is accompanied by accumulation of magnesium in the arteries.     

Age-independent constancy of mineral contents in human ribs

On age relationships of mineral contents in human bones, the contents of the sixth rib and a piece of its compact bone were determined by inductively coupled plasma atomic emission spectrometry (ICPS). The ribs were resected from 21 subjects (14 men and 7 women) who died in age ranging from 65 to 93 yr. There were no age-dependent decreases in Ca and P contents of the ribs in the age range on ICPS. It was found that there were no age-dependent decreases in Ca and P in compact bones of ribs.