Mn-porphyrins in a four-helix bundle participate in photo-induced electron transfer with a bacterial reaction center.

Hybrid complexes incorporating synthetic Mn-porphyrins into an artificial four-helix bundle domain of bacterial reaction centers created a system to investigate new electron transfer pathways. The reactions were initiated by illumination of the bacterial reaction centers, whose primary photochemistry involves electron transfer from the bacteriochlorophyll dimer through a series of electron acceptors to the quinone electron acceptors. Porphyrins with diphenyl, dimesityl, or fluorinated substituents were synthesized containing either Mn or Zn. Electrochemical measurements revealed potentials for Mn(III)/Mn(II) transitions that are ~ 0.4 V higher for the fluorinated Mn-porphyrins than the diphenyl and dimesityl Mn-porphyrins. The synthetic porphyrins were introduced into the proteins by binding to a four-helix bundle domain that was genetically fused to the reaction center. Light excitation of the bacteriochlorophyll dimer of the reaction center resulted in new derivative signals, in the 400 to 450 nm region of light-minus-dark spectra, that are consistent with oxidation of the fluorinated Mn(II) porphyrins and reduction of the diphenyl and dimesityl Mn(III) porphyrins. These features recovered in the dark and were not observed in the Zn(II) porphyrins. The amplitudes of the signals were dependent upon the oxidation/reduction midpoint potentials of the bacteriochlorophyll dimer. These results are interpreted as photo-induced charge-separation processes resulting in redox changes of the Mn-porphyrins, demonstrating the utility of the hybrid artificial reaction center system to establish design guidelines for novel electron transfer reactions.

Occurrence and greater intensity of estrus in recipient lactating dairy cows improve pregnancy per embryo transfer.

The aim of this study was to determine the association between occurrence and intensity of estrous expression with pregnancy success in recipient lactating dairy cows subjected to embryo transfer (ET). Two observational studies were conducted. Holstein cows were synchronized using the same timed ET protocol, based on estradiol and progesterone in both experiments. At 9 d after the end of the timed ET protocol only animals that had ovulated were implanted with a 7-d embryo [experiment 1 (Exp. 1); n = 1,401 ET events from 1,045 cows, and experiment 2 (Exp. 2); n = 1,147 ET events from 657 cows]. Embryos were produced in vivo (Exp. 1 and Exp. 2) and in vitro (only Exp. 2), then transferred to recipient cows as fresh or frozen-thawed. Pregnancy was confirmed at 29 and 58 d after the end of timed ET protocol. In Exp. 1, animals had their estrous expression monitored through a tail chalk applied on the tail head of the cows and evaluated daily for chalk removal (no estrus: 100% of chalk remaining; estrus: <50% of chalk remaining). In Exp. 2, cows were continuously monitored by a leg-mounted automated activity monitor. Estrous expression was quantified using the relative increase in physical activity at estrus in relation to the days before estrus. Estrous expression was classified as no estrus [<100% relative increase in activity (RI)], weak intensity (100-299% RI), and strong intensity (≥300% RI). Data were analyzed by analysis of variance using mixed linear regression models (GLIMMIX) in SAS (SAS Institute Inc.). A total of 65.2% (914/1,401) and 89.2% (1,019/1,142) of cows from Exp. 1 and Exp. 2, respectively, displayed estrus at the end of the ovulation synchronization protocol. In Exp. 1, cows expressing estrus before to ET had greater pregnancy per ET than those that did not [41.0 ± 2.3% (381/914) vs. 31.5 ± 2.9% (151/487), respectively]. Similarly, in Exp. 2, cows classified in the strong intensity group had greater pregnancy per ET compared with cows in the weak intensity and no estrus groups [41.3 ± 2.2% (213/571) vs. 32.7 ± 2.7% (115/353) vs. 11.3 ± 3.5% (26/218), respectively]. There was no effect of ET type on pregnancy per ET in Exp. 1. However, in Exp. 2, cows that received an in vivo-produced embryo, either fresh or frozen, had greater pregnancy per ET compared with cows that received in vitro-produced embryo. Cows receiving embryos in the early blastocyst and blastocyst stage had greater fertility compared with cows receiving embryos in the morula stage. There was an interaction between the occurrence of estrus and the stage of embryo development on pregnancy per ET, cows which displayed estrus and received a morula or early blastocyst had greater pregnancy per ET than cows that did not display estrus. In conclusion, the occurrence and the intensity of estrous expression improved pregnancy per ET in recipient lactating dairy cows and thus could be used as a tool to assist in the decision making of reproduction strategies in dairy farms.

Ultrafast energy transfer and excited state coupling in an artificial photosynthetic antenna.

We have studied the energy transfer dynamics in an artificial light-harvesting dyad composed of a phthalocyanine (Pc) covalently linked to a carotenoid (Car). The combination of high temporal resolution transient absorption spectroscopy with global and target analysis allowed us to quantify the efficiency of the energy transfer from the S2 excited state of the Car to the Pc at 37%, close to values observed in some natural light-harvesting complexes. In addition, following selective excitation of the Pc, we have identified the spectral signatures of the S1 excited state of the Car which appear within the ≈30 fs time resolution of our measurement. This strongly indicates excited state coupling between the S1 state of Car and the Qx state of Pc, with important implications for the regulation of photosynthetic activity.

Interaction of purified alternative oxidase from thermogenic Arum maculatum with pyruvate.

Plant alternative oxidase (AOX) activity in isolated mitochondria is regulated by carboxylic acids, but reaction and regulatory mechanisms remain unclear. We show that activity of AOX protein purified from thermogenic Arum maculatum spadices is sensitive to pyruvate and glyoxylate but not succinate. Rapid, irreversible AOX inactivation occurs in the absence of pyruvate, whether or not duroquinol oxidation has been initiated, and is insensitive to duroquinone. Our data indicate that pyruvate stabilises an active conformation of AOX, increasing the population of active protein in a manner independent of reducing substrate and product, and are thus consistent with an exclusive effect of pyruvate on the enzyme's apparent V(max).

Aortic dissection: a review of the diagnosis and initial management.

Aortic dissection is a medical emergency which carries a high mortality, particularly if undiagnosed and untreated. The diagnosis may be overlooked because the 'classical' history, examination and imaging signs are present in only a minority of cases. After reflecting on the underlying pathophysiology, this review examines aspects of the history and examination which should alert Acute Physicians to a possible diagnosis of aortic dissection. The relative benefits of different imaging techniques are summarised, along with the prognosis and treatment for different types of aortic dissection. Finally a case of aortic dissection which went undiagnosed for a month is presented to illustrate some of the key teaching points raised in this article.

Osteonecrosis of the knee in a patient receiving antiretroviral therapy.

A case of avascular osteonecrosis of the right knee is described in a patient with HIV infection. The patient had been receiving highly active antiretroviral therapy for two years prior to presentation. Osteonecrosis is an uncommon albeit serious complication of HIV infection and is associated with use of antiretroviral agents.

The design and synthesis of artificial photosynthetic antennas, reaction centres and membranes.

Artificial antenna systems and reaction centres synthesized in our laboratory are used to illustrate that structural and thermodynamic factors controlling energy and electron transfer in these constructs can be modified to optimize performance. Artificial reaction centres have been incorporated into liposomal membranes where they convert light energy to vectorial redox potential. This redox potential drives a Mitchellian, quinone-based, proton-transporting redox loop that generates a Deltamu H(+) of ca. 4.4 kcal mol(-1) comprising DeltapH ca. 2.1 and Deltapsi ca. 70 mV. In liposomes containing CF(0)F(1)-ATP synthase, this system drives ATP synthesis against an ATP chemical potential similar to that observed in natural systems.

Bias-induced forces in conducting atomic force microscopy and contact charging of organic monolayers.

Contact electrification, a surface property of bulk dielectric materials, has now been observed at the molecular scale using conducting atomic force microscopy (AFM). Conducting AFM measures the electrical properties of an organic film sandwiched between a conducting probe and a conducting substrate. This paper describes physical changes in the film caused by the application of a bias. Contact of the probe leads to direct mechanical stress and the applied electric field results in both Maxwell stresses and electrostriction. Additional forces arise from charge injection (contact charging). Electrostriction and contact charging act oppositely from the normal long-range Coulomb attraction and dominate when a charged tip touches an insulating film, causing the tip to deflect away from the film at high bias. A bias-induced repulsion observed in spin-coated PMMA films may be accounted for by either mechanism. In self-assembled monolayers, however, tunnel current signals show that the repulsion is dominated by contact charging.

Reproducible measurement of single-molecule conductivity.

A reliable method has been developed for making through-bond electrical contacts to molecules. Current-voltage curves are quantized as integer multiples of one fundamental curve, an observation used to identify single-molecule contacts. The resistance of a single octanedithiol molecule was 900 +/- 50 megohms, based on measurements on more than 1000 single molecules. In contrast, nonbonded contacts to octanethiol monolayers were at least four orders of magnitude more resistive, less reproducible, and had a different voltage dependence, demonstrating that the measurement of intrinsic molecular properties requires chemically bonded contacts.

Reduced bone mineral density in HIV-positive individuals.

A total of 105 HIV-positive patients underwent dual-energy X-ray absorbtiometry (DEXA) scan to assess bone mineral density (BMD). The prevalence of reduced BMD was found to be 71% and was higher in patients who had ever been treated with protease inhibitors (PI). Our results suggest a possible association between PI and reduced BMD, and further complicate the debate regarding when to commence treatment of HIV and with what agents to start.

Photoswitched singlet energy transfer in a porphyrin-spiropyran dyad.

A photochromic nitrospiropyran moiety (Sp) has been covalently linked to a zinc (PZn) and to a free-base (P(H2)) porphyrin. In the resulting dyads (P(Zn)-Sp(c) and P(H2)-Sp(c)), the porphyrin first excited singlet states are unperturbed by the closed form of the attached spiropyran. Excitation of the spiropyran moiety of either dyad in the near-UV region results in ring opening to a merocyanine form (P-Sp(o)) that absorbs at 600 nm. The open form re-closes thermally in 2-methyltetrahydrofuran with a time constant of 20 s, or following irradiation into the 600 nm band. Excitation of the zinc porphyrin moiety in the merocyanine form of the dyad yields 1PZn-Sp(o). The lifetime of the zinc porphyrin excited state is reduced from its usual value of 1.8 ns to 130 ps by singlet-singlet energy transfer to the merocyanine moiety to give PZn-1Sp(o). The quantum yield of energy transfer is 0.93. Quenching is also observed in the free base dyad, where 1P(H2)-Sp(o) and P(H2)-1Sp(o) exchange singlet excitation energy. This photoswitchable quenching phenomenon provides light-activated control of the porphyrin excited states, and consequently control of any subsequent energy or electron-transfer processes that might be initiated by these excited states in more complex molecular photonic or optoelectronic devices.

Purinergic-independent calcium signaling mediates recovery from hepatocellular swelling: implications for volume regulation.

Swelling of hepatocytes and other epithelia activates volume-sensitive ion channels that facilitate fluid and electrolyte efflux to restore cell volume, but the responsible signaling pathways are incompletely defined. Previous work in model HTC rat hepatoma cells has indicated that swelling elicits ATP release, which stimulates P2 receptors and activates Cl(-) channels, and that this mechanism is essential for hepatocellular volume recovery. Since P2 receptors are generally coupled to Ca(2+) signaling pathways, we determined whether hepatocellular swelling affected cytosolic [Ca(2+)], and if this involved a purinergic mechanism. Exposure of HTC cells to hypotonic media evoked an increase in cytosolic [Ca(2+)], which was followed by activation of K(+) and Cl(-) currents. Maneuvers that interfered with swelling-induced increases in cytosolic [Ca(2+)], including extracellular Ca(2+) removal and intracellular Ca(2+) store depletion with thapsigargin, inhibited activation of membrane currents and volume recovery. However, the swelling-induced increases in cytosolic [Ca(2+)] were unaffected by either extracellular ATP depletion with apyrase or blockade of P2 receptors with suramin. These findings indicate that swelling elicits an increase in hepatocellular Ca(2+), which is essential for ion channel activation and volume recovery, but that this increase does not stem from activation of volume-sensitive P2 receptors. Collectively, these observations imply that regulatory responses to hepatocellular swelling involve a dual requirement for a purinergic-independent Ca(2+) signaling cascade and a Ca(2+)-independent purinergic signaling pathway.

New insights into the regulation of plant succinate dehydrogenase. On the role of the protonmotive force.

Regulation of succinate dehydrogenase was investigated using tightly coupled potato tuber mitochondria in a novel fashion by simultaneously measuring the oxygen uptake rate and the ubiquinone (Q) reduction level. We found that the activation level of the enzyme is unambiguously reflected by the kinetic dependence of the succinate oxidation rate upon the Q-redox poise. Kinetic results indicated that succinate dehydrogenase is activated by both ATP (K(1/2) approximately 3 microm) and ADP. The carboxyatractyloside insensitivity of these stimulatory effects indicated that they occur at the cytoplasmic side of the mitochondrial inner membrane. Importantly, our novel approach revealed that the enzyme is also activated by oligomycin (K(1/2) approximately 16 nm). Time-resolved kinetic measurements of succinate dehydrogenase activation by succinate furthermore revealed that the activity of the enzyme is negatively affected by potassium. The succinate-induced activation (+/-K(+)) is prevented by the presence of an uncoupler. Together these results demonstrate that in vitro activity of succinate dehydrogenase is modulated by the protonmotive force. We speculate that the widely recognized activation of the enzyme by adenine nucleotides in plants is mediated in this manner. A mechanism that could account for such regulation is suggested and ramifications for its in vivo relevance are discussed.

Control of plant mitochondrial respiration.

Plant mitochondria are characterised by the presence of both phosphorylating (cytochrome) and non-phosphorylating (alternative) respiratory pathways, the relative activities of which directly affect the efficiency of mitochondrial energy conservation. Different approaches to study the regulation of the partitioning of reducing equivalents between these routes are critically reviewed. Furthermore, an updated view is provided regarding the understanding of plant mitochondrial respiration in terms of metabolic control. We emphasise the extent to which kinetic modelling and 'top-down' metabolic control analysis improve the insight in phenomena related to plant mitochondrial respiration. This is illustrated with an example regarding the affinity of the plant alternative oxidase for oxygen.

Gender differences in virologic response to treatment in an HIV-positive population: a cohort study.

OBJECTIVE: To establish whether a gender difference in virologic response to highly active antiretroviral treatment (HAART) exists. METHODS: A cohort of HIV-positive individuals was examined. OUTCOMES: Achievement of viral load <500 copies/ml and "failure" (failure to suppress viral load <500 copies/ml after 24 weeks or two consecutive measurements above this level after having suppressed below it). Hazard ratios (HRs) comparing the rate in women to that in men were derived using the Cox model. RESULTS: Of 366 male subjects, 79% were white and 82% were homosexual. Sixty-three percent of the 91 female subjects were African and 87% were heterosexual. The median follow-up after HAART was 94 weeks. The baseline CD4 count was higher in men (228 x 106 per liter) than in women (171 x 106 per liter) (p =.01), but the viral load was similar (p =.88). The median time to <500 copies/ml was 16 weeks. Women achieved a viral load of <500 copies/ml at a faster rate than men, with an adjusted HR of 1.46 (95% confidence interval [CI]: 0.99-2.16; p =.06). Some 261 patients failed treatment (58% of men and 53% of women) with an HR of 0.78 (95% CI: 0.51-1.21; p =.27). CONCLUSIONS: Women may achieve virologic suppression at a faster rate than men and have a more durable response. Further research should examine these responses in conjunction with clinical outcomes, because gender differences in virologic response may ultimately be of little relevance if clinical outcomes are similar.

Cloning and functional expression of a liver isoform of the small conductance Ca2+-activated K+ channel SK3.

Small conductance Ca2+-activated K+ (SK) channels have been cloned from mammalian brain, but little is known about the molecular characteristics of SK channels in nonexcitable tissues. Here, we report the isolation from rat liver of an isoform of SK3. The sequence of the rat liver isoform differs from rat brain SK3 in five amino acid residues in the NH3 terminus, where it more closely resembles human brain SK3. SK3 immunoreactivity was detectable in hepatocytes in rat liver and in HTC rat hepatoma cells. Human embryonic kidney (HEK-293) cells transfected with liver SK3 expressed 10 pS K+ channels that were Ca2+ dependent (EC(50) 630 nM) and were blocked by the SK channel inhibitor apamin (IC(50) 0.6 nM); whole cell SK3 currents inactivated at membrane potentials more positive than -40 mV. Notably, the Ca2+ dependence, apamin sensitivity, and voltage-dependent inactivation of SK3 are strikingly similar to the properties of hepatocellular and biliary epithelial SK channels evoked by metabolic stress. These observations raise the possibility that SK3 channels influence membrane K+ permeability in hepatobiliary cells during liver injury.

Mimicking photosynthetic solar energy transduction.

Increased understanding of photosynthetic energy conversion and advances in chemical synthesis and instrumentation have made it possible to create artificial nanoscale devices and semibiological hybrids that carry out many of the functions of the natural process. Artificial light-harvesting antennas can be synthesized and linked to artificial reaction centers that convert excitation energy to chemical potential in the form of long-lived charge separation. Artificial reaction centers can form the basis for molecular-level optoelectronic devices. In addition, they may be incorporated into the lipid bilayer membranes of artificial vesicles, where they function as components of light-driven proton pumps that generate transmembrane proton motive force. The proton gradient may be used to synthesize adenosine triphosphate via an ATP synthase enzyme. The overall energy transduction process in the liposomal system mimics the solar energy conversion system of a photosynthetic bacterium. The results of this research illustrate the advantages of designing functional nanoscale devices based on biological paradigms.

Incidence rate and spatio-temporal clustering of type 1 diabetes in Santiago, Chile, from 1997 to 1998

Objetibo: Estimar a taxa de incidência de diabetes tipo 1 na área urbana de Santiago, Chile, entre os dias 21 de março de 1997 e 20 de março 1998, assim como a avaliação do agrupamento espaço-temporal dos casos incidentes no período. Métodos: Foram localizados 61 casos incidentes no tempo (dia do diagnóstico) e no espaço (lugar de residência) na área do estudo. O método de Knox foi usado para avaliar o agrupamento dos casos no espaço e no tempo. Resultados: A taxa de diabetes tipo 1 foi estimada em 4,11 casos por 100.000 menores de 15 anos por ano (Intervalo de confiança 95 por cento: 3,06 ó 5,14). Essa taxa de incidência parece ter aumentado desde a última estimativa realizada na região metropolitana de Santiago, nos anos 1986-1992. Foram construídas diferentes combinações de intervalos de espaço e tempo para determinar a agregação espaço-temporal dos casos incidentes. A quase significativa agregação de casos foi detectada usando as distâncias críticas de 750 m e 60 dias (valor p não corrigido = 0,048). Conclusões: Embora o estudo só mostre resultados preliminares do agrupamento espaço-temporal dos casos de diabetes tipo 1, em Santiago, a análise dos dados sugere uma possível agregação de casos incidentes nas coordenadas de espaço e tempo

Maesaquinone: a novel inhibitor of plant mitochondrial respiratory enzymes that react with ubiquinone.

The effect of maesaquinone, 2-(14-nonadecenyl)-3,6-dihydroxy-5-methyl-1,4-benzoquinone, on plant mitochondrial respiration has been investigated. In mitochondria isolated from thermogenic Arum maculatum spadices, this compound inhibits both cytochrome and alternative pathway activities. Kinetic analyses reveal that this inhibition is the result of potent effects of maesaquinone on the alternative oxidase (ID50 < 0.3 microM) and complex III (ID50 < 5 microM). Succinate dehydrogenase and external NADH dehydrogenase are also inhibited, albeit to a lesser extent (approximately 30% at 1 microM). These data suggest that maesaquinone specifically affects the interaction of the respective enzymes with ubiquinone.

Mitochondrial electron transfer in the wheat pathogenic fungus Septoria tritici: on the role of alternative respiratory enzymes in fungicide resistance.

Certain phytopathogenic fungi are able to express alternative NADH- and quinol-oxidising enzymes that are insensitive to inhibitors of the mitochondrial respiratory Complexes I and III. To assess the extent to which such enzymes confer tolerance to respiration-targeted fungicides, an understanding of mitochondrial electron transfer in these species is required. An isolation procedure has been developed which results in intact, active and coupled mitochondria from the wheat pathogen Septoria tritici, as evidenced by morphological and kinetic data. Exogenous NADH, succinate and malate/glutamate are readily oxidised, the latter activity being only partly (approx. 70%) sensitive to rotenone. Of particular importance was the finding that azoxystrobin (a strobilurin fungicide) potently inhibits fungal respiration at the level of Complex III. In some S. tritici strains investigated, a small but significant part of the respiratory activity (approx. 10%) is insensitive to antimycin A and azoxystrobin. Such resistant activity is sensitive to octyl gallate, a specific inhibitor of the plant alternative oxidase. This enzyme, however, could not be detected immunologically. On the basis of the above findings, a conceptual mitochondrial electron transfer chain is presented. Data are discussed in terms of developmental and environmental regulation of the composition of this chain.