D1-arginine257 mutants (R257E, K, and Q) of Chlamydomonas reinhardtii have a lowered QB redox potential: analysis of thermoluminescence and fluorescence measurements

Arginine257 (R257), in the de-helix that caps the Q(B) site of the D1 protein, has been shown by mutational studies to play a key role in the sensitivity of Photosystem II (PS II) to bicarbonate-reversible binding of the formate anion. In this article, the role of this residue has been further investigated through D1 mutations (R257E, R257Q, and R257K) in Chlamydomonas reinhardtii. We have investigated the activity of the Q(B) site by studying differences from wild type on the steady-state turnover of PS II, as assayed through chlorophyll (Chl) a fluorescence yield decay after flash excitation. The effects of p-benzoquinone (BQ, which oxidizes reduced Q(B), Q(B)(-) ) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, which blocks electron flow from Q(A)(-) to Q(B)) were measured. The equilibrium constants of the two-electron gate were obtained through thermoluminescence measurements. The thermoluminescence properties were changed in the mutants, especially when observed after pretreatment with 100 microM BQ. A theoretical analysis of the thermoluminescence data, based mainly on the recombination pathways model of Rappaport et al. (2005), led to the conclusion that the free-energy difference for the recombination of Q(B)(-) with S(2) was reduced by 20-40 mV in the three mutants (D1-R257K, D1-R257Q, and D1-R257E); this was interpreted to be due to a lowering of the redox potential of Q(B)/Q(B)(-). Further, since the recombination of Q(A)(-) with S(2) was unaffected, we suggest that no significant change in redox potential of Q(A)/Q(A)(-) occurred in these three mutants. The maximum variable Chl a fluorescence yield is lowered in the mutants, in the order R257K > R257Q > R257E, compared to wild type. Our analysis of the binary oscillations in Chl a fluorescence following pretreatment of cells with BQ showed that turnover of the Q(B) site was relatively unaffected in the three mutants. The mutant D1-R257E had the lowest growth rate and steady-state activity and showed the weakest binary oscillations. We conclude that the size and the charge of the amino acid at the position D1-257 play a role in PS II function by modulating the effective redox potential of the Q(B)/Q(B)(-) pair. We discuss an indirect mechanism mediated through electrostatic and/or surface charge effects and the possibility of more pleiotropic effects arising from decreased stability of the D1/D2 and D1/CP47 interfaces.     

Chemical anatomy of hydra nervous system using antibodies against hydra neuropeptides: a review

Polyclonal antibodies against hydra neuropeptides allow us to visualize the hydra nerve net. Chemical anatomy of the hydra nerve net was archived by means of immunocytochemistry with various antibodies to hydra neuropeptides. Our goal is to describe the hydra nerve net both qualitatively and quantitatively. The present chemical anatomy results indicate (1) differences in peptide expression between ganglion cells and sensory cells, (2) large differences in neuropeptide expression between ectodermal nerve cells and endodermal nerve cells, and (3) the usefulness of quantitative chemical anatomy to analyze the entire nervous system of hydra.     

Vitamin C depletion increases superoxide generation in brains of SMP30/GNL knockout mice

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(−)]. At 4 and 8 weeks, VC levels in brains from VC(−) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(−) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.     

Isolation of stable (alphabeta)4-Tetraprotomer from Na+/K+-ATPase solubilized in the presence of short-chain fatty acids

Previously, it was demonstrated that acetate anions increase the higher oligomer (H), consuming (alphabeta) 2-diprotomer (D) and alphabeta-protomer (P) of solubilized dog kidney Na (+)/K (+)-ATPase [ Kobayashi, T. et al. (2007) J. Biochem. 142, 157-173 ]. Presently, short-chain fatty acids, such as propionate (Prop) and butyrate, have been substituted effectively for acetate. The molecular weight of 6.01 x 10 (5) for H and quantitative Na (+)/K (+)-dependent interconversion among H, D, and P showed that H was an (alphabeta) 4-tetraprotomer (T). T was optimally isolated from the enzyme solubilized in aqueous 40 mM K (+)Prop at pH 5.6 by gel chromatography performed at 0 degrees C with elution buffer containing synthetic dioleoyl phosphatidylserine (PS). K 0.5 values of K (+)-congeners constituting K (+)Prop for the maximal amount of T were NH 4 (+) > Rb (+) congruent with K (+) > Tl (+), while Na (+) had no effect. The oligomers of T, D, and P were simultaneously assayed for ATPase upon elution from the gel column, resulting in a specific activity ratio of 1:2:2. The activity of the chromatographically isolated T increased with an increasing dioleoyl PS, giving a saturated activity of 2.38 units/mg at pH 5.6 and 25 degrees C, and the active enzyme chromatography of T showed 34% dissociation into D by exposing it at 25 degrees C. On the basis of these data, the specific ATPase activities of T, D, and P were concluded to be 32, 65, and 65 units/mg, respectively, under the conventionally optimal conditions of pH 7.3 and 37 degrees C, suggesting an equivalence to a fully active enzyme for D and P but half activity for T. The physiological significance of the stable form of T remains to be investigated.     

Does Waist Circumference Add to the Predictive Power of the Body Mass Index for Coronary Risk?

Objectives: To examine the power of the combined measurements of body mass index (BMI) and waist circumference (WC) for the prediction of abnormality in coronary heart disease risk factors and to determine whether the additional measurement of WC is predictive in older men and women. Research Methods and Procedures: 1190 men and 751 women of the Baltimore Longitudinal Study of Aging were dichotomized into younger (<65 years) and older (65+ years) age groups. Coronary risk factors in the realms of glucose/insulin metabolism, blood pressure, and plasma lipids were assessed. The relationship of BMI and WC, singly and combined, to 10 risk factors for coronary heart disease was examined. Results: In younger and older men and women, BMI and WC are highly correlated (0.84 to 0.88). BMI and WC are also significantly correlated to all 10 coronary risk factors in younger men and women and to 8 of the 10 in the older men and women. Both partial correlation and logistic regression analyses revealed a modest but significant improvement in the prediction of coronary risk in younger men and women by WC after controlling for the level of BMI. There was no improvement in the older subjects. Discussion: WC adds only modestly to the prediction of coronary risk in younger subjects once BMI is known, and adds nothing to the production of risk in older subjects.     

Effect of mutations at Glu160 and Val198 on the thermostability of lactate oxidase.

We have obtained two types of thermostable mutant lactate oxidase - one that exhibited an E-to-G point mutation at position 160 (E160G) through error-prone PCR-based random mutagenesis, and another that exhibited an E-to-G mutation at position 160 and a V-to-I mutation at position 198 (E160G/V198I) through DNA shuffling-based random mutagenesis - both of which we have previously reported. Our molecular modeling of lactate oxidase suggests that the substitution of G for E at position 160 reduces the electrostatic repulsion between the negative charges of E160 and E130 in the (beta/alpha)8 barrel structure, but a thermal-inactivation experiment on the five kinds of single-mutant lactate oxidase at position 160 (E160A, E160Q, E160H, E160R, and E160K) showed that the side-chain volume of the amino acid at position 160 mainly contributes to the thermostability of lactate oxidase. We also produced V198I single-mutant lactate oxidase through site-directed mutagenesis, and analysed the thermostability of wild-type, V198I, E160G, and E160G/V198I lactate oxidase enzymes. The half-life of E160G/V198I lactate oxidase at 70 degrees C was about three times longer than that of E160G lactate oxidase, and was about 20 times longer than that of wild-type lactate oxidase. In contrast, the thermostability of the V198I lactate oxidase was almost identical to that of wild-type lactate oxidase. This indicates that the V198I mutation alone does not affect lactate oxidase thermostability, but does affect it when combined with the E160G mutation.     

Tumour necrosis factor and the lysosomal enzymes of macrophages or macrophage-like cell line

Summary The relationship between tumour necrosis factor (TNF) and macrophages or macrophage-like cell line, especially the lysosomal enzymes was investigated. The serum lysosomal enzymes and LDH activities were increased in proportion to the TNF production even in different strains of mice. Lysosomal enzymes and TNF activity were released into the supernatant of the culture medium of macrophage-enriched peritoneal exudate cells (PEC) or spleen cells derived from Propionibacterium acnes-primed mice after addition of lipopolysaccharide (LPS). After passage through a Sephadex G-10 column, TNF activity could not be detected in the supernatant of these spleen cells after addition of LPS. Also TNF activity could not be detected in the supernatant following destruction of PEC. These results suggest that TNF producibility is strongly related to the degree of activation of macrophages, especially the lysosomal enzymes. The murine macrophage-like cell line, J 774, also released TNF activity and lysosomal enzymes after addition of LPS.     

The effect of the DNA-suppressing factor (DSF) on host DNA synthesis in synchronized cell cultures

Purified host DNA-suppressing factor (DSF) produced into culture fluid of HeLa C-9 cells infected with measles virus inhibited cellular DNA synthesis in HeLa cells. When purified DSF was added into cultures of synchronous HeLa cells at the early G1-phase, cellular DNA synthesis was irreversibly inhibited. However, DSF did not affect the stability of native double-stranded DNA nor the chain-elongation of single-stranded DNA in cells of the S-phase.