Stereoselective metabolism of pentoxifylline in vitro and in vivo in humans

Pentoxifylline increases erythrocyte flexibility, reduces blood viscosity, and inhibits platelet aggregation and is thus used in the treatment of peripheral vascular disease. It is transformed into at least seven phase I metabolites, of which two, M1 and M5, are active. The reduction of the keto group of pentoxifylline to a secondary alcohol in M1 takes place chiefly in erythrocytes, is rapidly reversible, and creates a chiral center. The aims of this study were: to develop HPLC methods to separate the enantiomers of M1, to investigate the kinetics of the reversible biotransformation of pentoxifylline to (R)- and (S)-M1 in hemolysed erythrocyte suspension, and to quantify the formation of the enantiomers of M1 (as well as M4 and M5) after intravenous and oral administration of pentoxifylline to human volunteers. (R)- and (S)-M1 could be separated preparatively on a cellobiohydrolase column, while determination in blood or plasma was by HPLC after chiral derivatization with diacetyl-L-tartaric acid anhydride. The metabolism of pentoxifylline to (R)-M1 in suspensions of hemolysed erythrocytes followed simple Michaelis-Menten kinetics (K(m) = 11 mM), while that to (S)-M1 was best described by a two-enzyme model (K(m) = 1.1 and 132 mM). Studies with inhibitors indicated that the enzymes were of the carbonyl reductase type. At a therapeutic blood concentration of pentoxifylline, the calculated rate of formation of (S)-M1 is 15 times higher than that of the (R)-enantiomer. Back-conversion of M1 to pentoxifylline was 3-4 times faster for the (S)- than for the (R)-enantiomer. In vivo, the R:S plasma concentration ratio of M1 ranged from 0.010-0.025 after intravenous infusion of 300 or 600 mg of pentoxifylline, and from 0.019-0.037 after oral administration of 600 mg. The biotransformation of pentoxifylline to M1 was thus highly stereoselective in favor of the (S)-enantiomer both in vitro and in vivo.     

Abnormal in vivo metabolism of apoB-containing lipoproteins in human apoE deficiency

The present study was undertaken to elucidate the metabolic basis for the increased remnants and lipoprotein(a) [Lp(a)] and decreased LDL apolipoprotein B (apoB) levels in human apoE deficiency. A primed constant infusion of (13)C(6)-phenylalanine was administered to a homozygous apoE-deficient subject. apoB-100 and apoB-48 were isolated, and tracer enrichments were determined by gas chromatography-mass spectrometry, then kinetic parameters were calculated by multicompartmental modeling. In the apoE-deficient subject, fractional catabolic rates (FCRs) of apoB-100 in VLDL and intermediate density lipoprotein and apoB-48 in VLDL were 3x, 12x, and 12x slower than those of controls. On the other hand, the LDL apoB-100 FCR was increased by 2.6x. The production rate of VLDL apoB-100 was decreased by 45%. In the Lp(a) kinetic study, two types of Lp(a) were isolated from plasma with apoE deficiency: buoyant and normal Lp(a). (125)I-buoyant Lp(a) was catabolized at a slower rate in the patient. However, (125)I-buoyant Lp(a) was catabolized at twice as fast as (131)I-normal Lp(a) in the control subjects. In summary, apoE deficiency results in: 1) a markedly impaired catabolism of VLDL/chylomicron and their remnants due to lack of direct removal and impaired lipolysis; 2) an increased rate of catabolism of LDL apoB-100, likely due to upregulation of LDL receptor activity; 3) reduced VLDL apoB production; and 4) a delayed catabolism of a portion of Lp(a).     

Longitudinal changes in aerobic power in older men and women.

The purpose of this study was to describe the longitudinal (10 yr) decline in aerobic power [maximal O(2) uptake (Vo(2 max))] and anaerobic threshold [ventilatory threshold (T(Ve))] of older adults living independently in the community. Ten years after initial testing, 62 subjects (34 men, mean age 73.5 +/- 6.4 yr; 28 women, 72.1 +/- 5.3 yr) achieved Vo(2 max) criteria during treadmill walking tests to the limit of tolerance, with T(Ve) determined in a subset of 45. Vo(2 max) in men showed a rate of decline of -0.43 ml.kg(-1).min(-1).yr(-1), and the decline in Vo(2 max) was consequent to a lowered maximal heart rate with no change in the maximum O(2) pulse. The women showed a slower rate of decline of Vo(2 max) of -0.19.ml.kg(-1).min(-1).yr(-1) (P < 0.05), again with a lowered HR(max) and unchanged O(2) pulse. In this sample, lean body mass was not changed over the 10-yr period. Changes in Vo(2 max) were not significantly related to physical activity scores. T(Ve) showed a nonsignificant decline in both men and women. Groupings of young-old (65-72 yr at follow-up) vs. old-old (73-90 yr at follow-up) were examined. In men, there were no differences in the rate of Vo(2 max) decline. The young-old women showed a significant decline in Vo(2 max), whereas old-old women, initially at a Vo(2 max) of 19.4 +/- 3.1 ml.kg(-1).min(-1), showed no loss in Vo(2 max). The longitudinal data, vs. cross-sectional analysis, showed a greater decline for men but similar estimates of the rates of change in women. Thus the 10-yr longitudinal study of the cohort of community-dwelling older adults who remained healthy, ambulatory, and independent showed a 14% decline in Vo(2 max) in men, and a smaller decline of 7% in women, with the oldest women showing little change over the 10-yr period.     

Single-nucleotide polymorphisms in LPA explain most of the ancestry-specific variation in Lp(a) levels in African Americans

Lipoprotein(a) (Lp(a)) is an important causal cardiovascular risk factor, with serum Lp(a) levels predicting atherosclerotic heart disease and genetic determinants of Lp(a) levels showing association with myocardial infarction. Lp(a) levels vary widely between populations, with African-derived populations having nearly 2-fold higher Lp(a) levels than European Americans. We investigated the genetic basis of this difference in 4464 African Americans from the Jackson Heart Study (JHS) using a panel of up to 1447 ancestry informative markers, allowing us to accurately estimate the African ancestry proportion of each individual at each position in the genome. In an unbiased genome-wide admixture scan for frequency-differentiated genetic determinants of Lp(a) level, we found a convincing peak (LOD = 13.6) at 6q25.3, which spans the LPA locus. Dense fine-mapping of the LPA locus identified a number of strongly associated, common biallelic SNPs, a subset of which can account for up to 7% of the variation in Lp(a) level, as well as >70% of the African-European population differences in Lp(a) level. We replicated the association of the most strongly associated SNP, rs9457951 (p = 6 × 10(-22), 27% change in Lp(a) per allele, ～5% of Lp(a) variance explained in JHS), in 1,726 African Americans from the Dallas Heart Study and found an even stronger association after adjustment for the kringle(IV) repeat copy number. Despite the strong association with Lp(a) levels, we find no association of any LPA SNP with incident coronary heart disease in 3,225 African Americans from the Atherosclerosis Risk in Communities Study.     

Mutations in oxyR resulting in peroxide resistance in Xanthomonas campestris

A spontaneous Xanthomonas campestris pv. phaseoli H(2)O(2)-resistant mutant emerged upon selection with 1 mM H(2)O(2). In this report, we show that growth of this mutant under noninducing conditions gave high levels of catalase, alkyl hydroperoxide reductase (AhpC and AhpF), and OxyR. The H(2)O(2) resistance phenotype was abolished in oxyR-minus derivatives of the mutant, suggesting that elevated levels and mutations in oxyR were responsible for the phenotype. Nucleotide sequence analysis of the oxyR mutant showed three nucleotide changes. These changes resulted in one silent mutation and two amino acid changes, one at a highly conserved location (G197 to D197) and the other at a nonconserved location (L301 to R301) in OxyR. Furthermore, these mutations in oxyR affected expression of genes in the oxyR regulon. Expression of an oxyR-regulated gene, ahpC, was used to monitor the redox state of OxyR. In the parental strain, a high level of wild-type OxyR repressed ahpC expression. By contrast, expression of oxyR5 from the X. campestris pv. phaseoli H(2)O(2)-resistant mutant and its derivative oxyR5G197D with a single-amino-acid change on expression vectors activated ahpC expression in the absence of inducer. The other single-amino-acid mutant derivative of oxyR5L301R had effects on ahpC expression similar to those of the wild-type oxyR. However, when the two single mutations were combined, as in oxyR5, these mutations had an additive effect on activation of ahpC expression.     

Erythrocyte-associated transients in PO2 revealed in capillaries of rat mesentery

Mathematical models have predicted the existence of Po(2) gradients between erythrocytes in capillaries in the usual case where plasma contributes substantial resistance to oxygen diffusion. According to theoretical predictions, these gradients could be detected as rapid Po(2) fluctuations (erythrocyte-associated transients, EATs) along the capillary. However, verification of a model and correct choice of its parameters can be made only on the basis of direct experimental measurements. We used phosphorescence quenching microscopy to measure Po(2) in 52 capillaries of rat mesentery to obtain plasma Po(2) values 100 times/s at a given point along a capillary. A 532-nm laser generated 10-micros pulses of light, concentrated by a x100 objective, onto a spot 0.9 microm in diameter. The presence of erythrocytes in the excitation region was detected on the basis of phosphorescence amplitude (PA), proportional to the amount of plasma encountered by the laser beam, and on the basis of the intensity of transmitted laser light (LT), detected by a photodiode placed under the capillary. The data revealed correlated waveforms in PA, LT, and Po(2) in capillaries. The magnitude of the Po(2) gradients between erythrocytes and plasma was correlated with average capillary Po(2). EATs in Po(2) were more readily detected in capillaries with relatively low oxygenation. The correlation coefficients between PA and Po(2) for the half of the capillaries (n = 26) below the median Po(2) (mean Po(2) = 17 mmHg; R = -0.72) was higher than that for the other half (mean Po(2) = 39 mmHg; R = -0.38). These results support the theoretical predictions of EATs and plasma Po(2) gradients in capillaries.     

NO可能作为H2O2的下游信号介导ABA诱导的蚕豆气孔关闭

ABA、H2O2和硝普钠(SNP)均能诱导蚕豆气孔关闭.NO的清除剂c-PTIO可以减轻由ABA或H2O2所诱导的蚕豆气孔关闭的程度,而过氧化氢酶(CAT)则不能减轻NO诱导的气孔关闭程度.激光共聚焦显微检测结果显示,10μmo1/L的ABA处理后,胞内H2O2的产生速率明显高于NO的产生速率;CAT几乎可完全抑制ABA所诱导的DAF的荧光增加;外源H2O2能显著诱导胞内DAF的荧光增加;c-PTIO对ABA诱导的DCF荧光略有促进作用,但外源SNP不能诱导胞内DCF荧光增加.这些结果表明,在ABA诱导气孔关闭过程中,H2O2可能在NO的上游起作用并受NO的负反馈调节.     

Regulation of glucose production in rainbow trout: role of epinephrine in vivo and in isolated hepatocytes

The rate of hepatic glucose production (R(a) glucose) of rainbow trout (Oncorhynchus mykiss) was measured in vivo by continuous infusion of [6-(3)H]glucose and in vitro on isolated hepatocytes to examine the role of epinephrine (Epi) in its regulation. By elevating Epi concentration and/or blocking beta-adrenoreceptors with propranolol (Prop), our goals were to investigate the mechanism for Epi-induced hyperglycemia to determine the possible role played by basal Epi concentration in maintaining resting R(a) glucose and to assess indirect effects of Epi in the intact animal. In vivo infusion of Epi caused hyperglycemia (3.75 +/- 0.16 to 8.75 +/- 0.54 mM) and a twofold increase in R(a) glucose (6.57 +/- 0.79 to 13.30 +/- 1.78 micromol. kg(-1). min(-1), n = 7), whereas Prop infusion decreased R(a) from 7.65 +/- 0.92 to 4.10 +/- 0.56 micromol. kg(-1). min(-1) (n = 10). Isolated hepatocytes increased glucose production when treated with Epi, and this response was abolished in the presence of Prop. We conclude that Epi-induced trout hyperglycemia is entirely caused by an increase in R(a) glucose, because the decrease in the rate of glucose disappearance normally seen in mammals does not occur in trout. Basal circulating levels of Epi are involved in maintaining resting R(a) glucose. Epi stimulates in vitro glucose production in a dose-dependent manner, and its effects are mainly mediated by beta-adrenoreceptors. Isolated trout hepatocytes produce glucose at one-half the basal rate measured in vivo, even when diet, temperature, and body size are standardized, and basal circulating Epi is responsible for part of this discrepancy. The relative increase in R(a) glucose after Epi stimulation is similar in vivo and in vitro, suggesting that indirect in vivo effects of Epi, such as changes in hepatic blood flow or in other circulating hormones, do not play an important role in the regulation of glucose production in trout.     

S-Transnitrosylation of albumin in human plasma and blood in vitro and in vivo in the rat

S-Nitrosoalbumin (SNOALB) is the most abundant physiological circulating nitric oxide (NO) carrier regulating NO-dependent biological actions in humans. The mechanisms of its formation and biological actions are still incompletely understood. Nitrosation by authentic NO and S-transnitrosylation of the single sulfhydryl group located at Cys-34 of human albumin by the physiological S-nitroso compounds S-nitrosocysteine (SNOC) and S-nitrosoglutathione (GSNO) are two possible mechanisms. On a quantitative basis, we investigated by gas chromatography-mass spectrometry the contribution of these two mechanisms to SNOALB formation in human plasma and blood in vitro. GSNO and SNOC (0-100 microM) rapidly and efficiently (recovery=35%) S-transnitrosylated albumin to form SNOALB. NO (100 microM) S-nitrosated albumin to SNOALB at a considerably lower extent (recovery=5%). The putative NO-donating drugs glyceryl trinitrate and sodium nitroprusside (each 100 microM) failed completely in S-nitrosating albumin. Bubbling NO into human plasma and blood resulted in formation of SNOALB that inhibited ADP-induced platelet aggregation. Infusion of GS(15)NO in the rat resulted in formation of S(15)NOALB, [(15)N]nitrate and [(15)N]nitrite. Our results suggest that S-transnitrosylation of albumin by SNOC and GSNO could be a more favored mechanism for the formation of SNOALB in the circulation in vivo than S-nitrosation of albumin by NO itself.     

Capacity for Methane Oxidation in Landfill Cover Soils Measured in Laboratory-Scale Soil Microcosms

Laboratory-scale soil microcosms containing different soils were permeated with CH(inf4) for up to 6 months to investigate their capacity to develop a methanotrophic community. Methane emissions were monitored continuously until steady states were established. The porous, coarse sand soil developed the greatest methanotrophic capacity (10.4 mol of CH(inf4) (middot) m(sup-2) (middot) day(sup-1)), the greatest yet reported in the literature. Vertical profiles of O(inf2), CH(inf4), and methanotrophic potential in the soils were determined at steady state. Methane oxidation potentials were greatest where the vertical profiles of O(inf2) and CH(inf4) overlapped. A significant increase in the organic matter content of the soil, presumably derived from methanotroph biomass, occurred where CH(inf4) oxidation was greatest. Methane oxidation kinetics showed that a soil community with a low methanotrophic capacity (V(infmax) of 258 nmol (middot) g of soil(sup-1) (middot) h(sup-1)) but relatively high affinity (k(infapp) of 1.6 (mu)M) remained in N(inf2)-purged control microcosms, even after 6 months without CH(inf4). We attribute this to a facultative, possibly mixotrophic, methanotrophic microbial community. When purged with CH(inf4), a different methanotrophic community developed which had a lower affinity (k(infapp) of 31.7 (mu)M) for CH(inf4) but a greater capacity (V(infmax) of 998 nmol (middot) g of soil(sup-1) (middot) h(sup-1)) for CH(inf4) oxidation, reflecting the enrichment of an active high-capacity methanotrophic community. Compared with the unamended control soil, amendment of the coarse sand with sewage sludge enhanced CH(inf4) oxidation capacity by 26%; K(inf2)HPO(inf4) amendment had no significant effect, while amendment with NH(inf4)NO(inf3) reduced the CH(inf4) oxidation capacity by 64%. In vitro experiments suggested that NH(inf4)NO(inf3) additions (10 and 71 (mu)mol (middot) g of soil(sup-1)) inhibited CH(inf4) oxidation by a nonspecific ionic effect rather than by specific inhibition by NH(inf4)(sup+).     

A role for cyclooxygenase-2 in lipopolysaccharide-induced anorexia in rats

Because nonselective cycloooxygenase (COX) inhibition attenuated anorexia after lipopolysaccharide (LPS) administration, we tested the ability of resveratrol (2.5, 10, and 40 mg/kg) and NS-398 (2.5, 10, and 40 mg/kg), selective inhibitors of the two COX isoforms COX-1 and -2, respectively, to attenuate LPS (100 microg/kg ip)-induced anorexia. NS-398 (10 and 40 mg/kg) administered with LPS at lights out attenuated LPS-induced anorexia, whereas resveratrol at all doses tested did not. Because prostaglandin (PG) E(2) is considered the major metabolite synthesized by COX, we measured plasma and cerebrospinal fluid (CSF) PGE(2) levels after LPS administration. LPS induced a time-dependent increase of PGE(2) in CSF but not in plasma. NS-398 (5, 10, and 40 mg/kg) blocked the LPS-induced increase in CSF PGE(2), whereas resveratrol (10 mg/kg) did not. These results support a role of COX-2 in mediating the anorectic response to peripheral LPS and point at PGE(2) as a potential neuromodulator involved in this response.     

Seasonal fluctuations in Vitis vinifera root respiration in the field

We studied the seasonal fluctuation of soil respiration (R(S)), and its root-dependent (R(R)) and basal (R(B)) components, in a Vitis vinifera (Chardonnay) vineyard. The R(S) components were estimated through independent field methods (y-intercept and trenching) and modeled on the basis of a Q(10) response to soil temperature, and fine and coarse root respiration coefficients. The effect of assimilate availability on R(R) was assessed through a trunk girdling treatment. The apparent Q(10) for R(R) was twice that of R(B) (3.5 vs 1.6) and increased linearly with increasing vine root biomass. The fastest R(R) of fine roots was during rapid fruit growth and the fastest R(R) of coarse roots was immediately following fruit development. R(S) was estimated at 32.6 kg ha(-1) d(-1) (69% as a result of R(R) ) for the hottest month and at 7.6 kg ha(-1) d(-1) (18% as a result of R(R)) during winter dormancy. Annual R(S) was low compared with other natural and cultivated ecosystems: 5.4 Mg ha(-1) (46% as a result of R(R)). Our estimates of annual vineyard R(S) are the first for any horticultural crop and suggest that the assumption that they are similar to those of annual crops or forest trees might lead to an overestimation.     

Group X secretory PLA2 in neutrophils plays a pathogenic role in abdominal aortic aneurysms in mice

Group X secretory PLA(2) (sPLA(2)-X) is expressed in neutrophils and plays a role in the pathogenesis of neutrophil-mediated tissue inflammation and injury. This study tested the hypothesis that sPLA(2)-X in neutrophils may contribute to the pathogenesis of abdominal aortic aneurysms (AAA) using sPLA(2)-X(-/-) mice. AAA was created by application of CaCl(2) to external surface of aorta. As a result, the aortas of sPLA(2)-X(-/-) mice had smaller diameters (percent increase from baseline; 24.8 ± 3.5% vs. 49.9 ± 9.1%, respectively; P < 0.01), a reduced grade of elastin degradation, and lower activities of elastase and gelatinase (26% and 19% lower, respectively) after CaCl(2) treatment compared with sPLA(2)-X(+/+) mice. In sPLA(2)-X(+/+) mice, immunofluorescence microscopic images showed that the immunoreactivity of sPLA(2)-X was detected only in neutrophils within aortic walls 3 days, 1, 2, and 6 wk after CaCl(2) treatment, whereas the immunoreactivity was not detected in macrophages or mast cells in aortic walls. sPLA(2)-X immunoreactivity also was colocalized in cells expressing matrix metalloproteinase (MMP)-9. Neutrophils isolated from sPLA(2)-X(-/-) mice had lower activities of elastase, gelatinase, and MMP-9 in response to stimuli compared with sPLA(2)-X(+/+) mice. The attenuated release of elastase and gelatinase from sPLA(2)-X(-/-) neutrophils was reversed by exogenous addition of mouse sPLA(2)-X protein. The adoptive transfer of sPLA(2)-X(+/+) neutrophils days 0 and 3 after CaCl(2) treatment reversed aortic diameters and elastin degradation grades in the lethally irradiated sPLA(2)-X(+/+) mice reconstituted with sPLA(2)-X(-/-) bone marrow to an extent similar to that seen in sPLA(2)-X(+/+) mice. In conclusion, sPLA(2)-X in neutrophils plays a pathogenic role in AAA in a mice model.     

Progress in the prediction of pKa values in proteins

The pK(a) -cooperative aims to provide a forum for experimental and theoretical researchers interested in protein pK(a) values and protein electrostatics in general. The first round of the pK(a) -cooperative, which challenged computational labs to carry out blind predictions against pK(a) s experimentally determined in the laboratory of Bertrand Garcia-Moreno, was completed and results discussed at the Telluride meeting (July 6-10, 2009). This article serves as an introduction to the reports submitted by the blind prediction participants that will be published in a special issue of PROTEINS: Structure, Function and Bioinformatics. Here, we briefly outline existing approaches for pK(a) calculations, emphasizing methods that were used by the participants in calculating the blind pK(a) values in the first round of the cooperative. We then point out some of the difficulties encountered by the participating groups in making their blind predictions, and finally try to provide some insights for future developments aimed at improving the accuracy of pK(a) calculations.     

Involvement of peripheral type of benzodiazepine receptor in social isolation stress-induced decrease in pentobarbital sleep in mice

Our previous studies have shown that central-type benzodiazepine (BZD) receptors (CBR) and neurosteroids capable of modulating GABA(A) receptor function are involved in the decrease of pentobarbital (PB)-induced sleep caused by social isolation stress in mice. In this study, to further clarify the mechanism underlying this decrease, we investigated the possible involvement of peripheral-type BZD receptors (PBR) which play an important role in neurosteroidogenesis in PB sleep in socially isolated mice. Socially isolated mice showed significantly shorter duration of PB-induced sleep than group-housed animals. When injected intracerebroventricularly (i.c.v.), FGIN-1-27 (FGIN, 25-100 nmol), a selective PBR agonist, and PK11195 (PK, 14-28 nmol), a PBR antagonist, and pregnenolone (PREG, 15-30 nmol), a neurosteroid precursor, dose-dependently normalized the PB sleep in isolated mice without having an effect on the group-housed animals. In contrast, pregnenolone sulfate (PS, 24 nmol), an endogenous neurosteroidal negative allosteric modulator of the GABA(A) receptor, reduced PB sleep in group-housed but not isolated mice. PS, at the same dose, significantly attenuated the effects of FGIN (100 nmol), PK (28 nmol) and PREG (30 nmol) in isolated mice, while FGIN (100 nmol), PK (28 nmol) and pregnenolone (30 nmol) significantly blocked the effect of PS (24 nmol) in group-housed mice. These results suggest that the PBR-mediated decrease in the genesis of neurosteroid(s) possessing a GABA(A) receptor agonistic profile is also partly involved in the down regulation of the GABA(A) receptor following long-term social isolation and contributes to the decrease of PB-induced sleep in isolation stressed mice.     

Ascorbate-mediated LHCII protein phosphorylation--LHCII kinase regulation in light and in darkness

A freeze-thaw cycle of isolated thylakoids in darkness in the presence of ascorbate was employed as a novel experimental system to activate the light-harvesting complex (LHC) II kinase. Under these conditions ascorbate reduces Q(A), the primary quinone electron acceptor of photosystem (PS) II, and the subsequent reduction of plastoquinone and the cytochrome (cyt) b(6)f complex results in the activation of the LHCII kinase. Using this activation system, several facets of regulation of LHCII protein phosphorylation were unravelled. (i) Myxothiazol inhibited the activation of LHCII protein phosphorylation, thus being a potent inhibitor of electron flow not only in cyt bc complexes but in darkness also in cyt b(6)f complexes. (ii) Oxygen, the only electron acceptor in darkness, was required for LHCII kinase activation demonstrating that after a full reduction of the cyt b(6)f complex, an additional plastoquinol oxidation cycle in the quinol oxidation (Qo) site is required for LHCII kinase activation. (iii) In the absence of electron flow, when the intersystem electron carriers are reduced, the activated LHCII kinase has a half-life of 40 min, whereas the fully activated LHCII kinase becomes deactivated in a time scale of seconds upon oxidation of the cyt b(6)f complex, indicating that the kinase constantly reads the redox poise of the cyt b(6)f complex. (iv) The LHCII kinase is more tightly bound to the thylakoid membrane than the PS II core protein kinase(s). It is concluded that oxidation of plastoquinol at the Qo site of the reduced cyt b(6)f complex is required for LHCII kinase activation, while rapid reoccupation of the Qo site with plastoquinol is crucial for sustenance of the active state of the LHCII kinase.     

Microgravity-induced changes in aortic stiffness and their role in orthostatic intolerance.

Microgravity (microG)-induced orthostatic intolerance (OI) in astronauts is characterized by a marked decrease in cardiac output (CO) in response to an orthostatic stress. Since CO is highly dependent on venous return, alterations in the resistance to venous return (RVR) may be important in contributing to OI. The RVR is directly dependent on arterial compliance (C(a)), where aortic compliance (C(ao)) contributes up to 60% of C(a). We tested the hypothesis that microG-induced changes in C(a) may represent a protective mechanism against OI. A retrospective analysis on hemodynamic data collected from astronauts after 5- to 18-day spaceflight missions revealed that orthostatically tolerant (OT) astronauts showed a significant decrease in C(a) after spaceflight, while OI astronauts showed a slight increase in C(a). A ground-based animal model simulating microG, hindlimb-unweighted rats, was used to explore this phenomenon. Two independent assessments of C(ao), in vivo pulse wave velocity (PWV) of the thoracic aorta and in vitro pressure-diameter squared relationship (PDSR) measurements of the excised thoracic aorta, were determined. PWV showed a significant increase in aortic stiffness compared with control, despite unchanged blood pressures. This increase in aortic stiffness was confirmed by the PDSR analysis. Thus both actual microG in humans and simulated microG in rats induces changes in C(ao). The difference in C(a) in OT and OI astronaut suggests that the microG-induced decrease in C(a) is a protective adaptation to spaceflight that reduces the RVR and allows for the maintenance of adequate CO in response to an orthostatic stress.     

Mutations in laminin alpha 1 result in complex, lens-independent ocular phenotypes in zebrafish

We report phenotypic and genetic analyses of a recessive, larval lethal zebrafish mutant, bal(a69), characterized by severe eye defects and shortened body axis. The bal(a69) mutation was mapped to chromosome 24 near the laminin alpha 1 (lama1) gene. We analyzed the lama1 gene sequence within bal(a69) embryos and two allelic mutants, bal(arl) and bal(uw1). Missense (bal(a69)), nonsense (bal(arl)), and frameshift (bal(uw1)) alterations in lama1 were found to underlie the phenotypes. Extended analysis of bal(a69) ocular features revealed disrupted lens development with subsequent lens degeneration, focal cornea dysplasia, and hyaloid vasculature defects. Within the neural retina, the ganglion cells showed axonal projection defects and ectopic photoreceptor cells were noted at inner retinal locations. To address whether ocular anomalies were secondary to defects in lens differentiation, bal(a69) mutants were compared to embryos in which the lens vesicle was surgically removed. Our analysis suggests that many of the anterior and posterior ocular defects in bal(a69) are independent of the lens degeneration. Analysis of components of focal adhesion signaling complexes suggests that reduced focal adhesion kinase activation underlies the anterior segment dysgenesis in lama1 mutants. To assess adult ocular phenotypes associated with lama1 mutations, genetic mosaics were generated by transplanting labeled bal cells into ocular-fated regions of wild-type blastulas. Adult chimeric eyes displayed a range of defects including anterior segment dysgenesis and cataracts. Our analysis provides mechanistic insights into the developmental defects and ocular pathogenesis caused by mutations in laminin subunits.     

Effect of exogenous melatonin on in vivo and in vitro prostaglandin secretion in Rasa Aragonesa ewes

The effect of exogenous melatonin on prostaglandin secretion was measured on Rasa Aragonesa ewes. Fourteen ewes received an 18 mg melatonin implant (M+) on 10 April and were compared with 13 control animals (without implants M-). Twenty days later, intravaginal pessaries were inserted in all animals to induce a synchronized oestrus (day 0). On day 14, ewes were injected, i.v., with 0.5 IU oxytocin. Plasma 15-ketodihydro-PGF(2alpha) (PGFM) concentrations were measured to assess uterine secretory responsiveness to oxytocin. After euthanasia, pieces of endometrium were collected to determine progesterone content and PGE(2) and PGF(2alpha) secretion in vitro, in the presence or absence of either 20 microg/ml recombinant ovine interferon-tau (roIFNt) or 1 nmol/l oxytocin in the medium. Endometrial progesterone content was similar in the two treatments (M+: 50.25+/-17.34 ng/mg tissue, M-: 43.08+/-11.21 ng/mg tissue). M+ ewes that responded to oxytocin had significantly higher plasma PGFM concentrations between 10 and 80 min after oxytocin administration, a higher mean PGFM peak (P<0.001), higher plasma PGFM levels after the challenge (P<0.05) and higher plasma progesterone concentrations (P<0.01) than control ewes. In the in vitro experiment, M+ and M- control samples secreted similar amounts of PGE(2). The presence of roIFNtau and oxytocin only stimulated PGE(2) production (P<0.05) in M- tissues. Control M+ tissues secreted higher amounts of PGF(2alpha) (P=0.07) and PGF(2alpha) secretion was significantly (P<0.01) stimulated by roIFNtau. Oxytocin produced this effect only in M- samples (P<0.01). In conclusion, although previous studies have demonstrated a positive effect of melatonin on lamb production, PGF(2alpha) secretion is higher in vitro and the PGE(2):PGF(2alpha) ratio is unfavourable in response to IFNtau, which could affect embryo survival. Whether or not these mechanisms are similar in pregnant ewes remains to be elucidated.     

Hydrodynamics and mass transfer in Aspergillus niger fermentations in bubble column and loop bioreactors

The influence of Aspergillus niger broth rheology, bioreactor geometry, and superficial gas velocity on the volumetric liquid phase oxygen transfer coefficient (k(L)a(L)), riser gas holdup (epsilon(GR)), and circulating liquid velocity (u(LR)) was studied in a bubble column (BC) and two external-circulation-loop airlift (ECLAL) bioreactors. The results are compared to those of previous studies on homogeneous fluids and in particular with a recent study on non-Newtonian carboxymethylcellulose (CMC) solutions conducted in the same contactors used for the A. niger fermentations. As expected from the CMC-based studies, in the heterogeneous broths of A. niger epsilon(GR), k(L)a(L), and u(LR) decreased with increasing broth apparent viscosity; epsilon(GR) and k(L)a(L) decreased with increasing downcomer-to-riser cross-sectional area ratio, A(d)/A(r), whereas u(LR) increased with increasing A(d)/A(r). Gas holdup data in the airlift fermentations of A. niger were well predicted by the CMC-based correlation. However, the CMC-based correlations produced conservative estimations of k(L)a(L) and overestimates of u(LR) compared to the observed values in the A. niger broths.