Surface-enhanced resonance Raman scattering spectroscopy of bacterial photosynthetic membranes. The carotenoid of Rhodospirillum rubrum

Resonance Raman scattering by the carotenoid, spirilloxanthin (Spx), in a suspension of chromatophores (cytoplasmic side out) isolated from the photosynthetic bacterium, Rhodospirillum rubrum, is greatly enhanced when the membranes are adsorbed onto the surface of an anodized Ag electrode. The phenomenon is the basis for surface-enhanced resonance Raman scattering (SERRS) spectroscopy. The Spx SERRS peaks observed were at 1505-1510, 1150-1155, and 1000-1005 cm-1 with laser excitation wavelengths ranging between 457.9 and 568.2 nm. Similar peaks were not observed with spheroplasts (periplasmic side out) isolated from the same species. The difference in signal detected in chromatophores and spheroplasts is not due to differences in membrane surface charge, presence of residual cell wall on the spheroplast surface, lack of adhesion of spheroplasts to metals, or large differences in pigment content per unit membrane area. Instead, the results indicate an asymmetric distribution of Spx in vivo across the membrane (i.e., it is located on the cytoplasmic side of the membrane). The results also demonstrate that the SERRS effect is extremely distance sensitive, and the thickness of a single bacterial membrane (separating the Ag electrode from the carotenoid) is sufficient to prevent detection of Spx spectra. Studies of chromatophores from the F24 strain (a reaction centerless mutant) have pin-pointed B880 antenna complex as the source of the Spx SERRS spectra, and a schematic model of the minimal structural unit of B880 is presented. This work demonstrates the potential of the SERRS technique as a probe for surface topology of pigmented membranes.     

Biochemical genetics of methylglyoxal dehydrogenases in the laboratory rat (Rattus norvegicus)

A genetic locus controlling the electrophoretic mobility of a methylglyoxal dehydrogenase (EC 1.2.1.23) in the rat is described. The locus, designatedMgd1, is expressed in liver and kidney. Inbred rat strains have fixed either alleleMgd1 ^a or alleleMgd1 ^b . Codominant expression is observed in heterozygotes, providing evidence for a tetrameric enzyme structure. Backcross progenies showed the expected 1:1 segregation ratio, and there is evidence thatMgd1 is linked toPep3 andFh1 on chromosome 13. There is also evidence for two additional methylglyoxal dehydrogenases:Mgd2, present in liver and kidney, andMgd3, present only in heart.Supported by the Deutsche Forschungsgemeinschaft (Grant Be 352/18-1).     

Surface-enhanced resonance raman scattering spectroscopy of bacterial photosynthetic membranes: orientation of the carotenoids of Rhodobacter sphaeroides 2.4.1

Surface-enhanced resonance Raman scattering (SERRS) spectra were obtained from carotenoids, in the all-trans configuration, located on the antenna complexes of Rhodobacter sphaeroides 2.4.1 membranes. Since resonance Raman (RR) spectra are barely detectable at the concentration that SERRS signals saturate, SERRS represents a very sensitive means of detecting pigments in biological systems. Prominent SERRS spectra of sphaeroidenone were detected in chromatophores (cytoplasmic side out) but not in spheroplast-derived vesicles (periplasmic side out), demonstrating that the carotenoid is asymmetrically located on the cytoplasmic side of the cell membrane. Comparison of peak frequencies from SERRS and RR spectral data suggests that the carotenoids are oriented into the membrane with the methoxy end of the isoprenoid chains located closest to the cytoplasmic side of the intracytoplasmic membrane. This work not only shows that SERRS spectroscopy can provide information on the location of a chromophore in a biological membrane but also for the first time demonstrates that SERRS data can be used to ascertain the orientation of a chromophore within the membrane. This observation greatly increases the potential of this technique for structural analysis of intact membranes at the molecular level.     

Contribution of Double-Stranded RNA and CPSF30 Binding Domains of Influenza Virus NS1 to the Inhibition of Type I Interferon Production and Activation of Human Dendritic Cells

The influenza virus nonstructural protein 1 (NS1) inhibits innate immunity by multiple mechanisms. We previously reported that NS1 is able to inhibit the production of type I interferon (IFN) and proinflammatory cytokines in human primary dendritic cells (DCs). Here, we used recombinant viruses expressing mutant NS1 from the A/Texas/36/91 and A/Puerto Rico/08/34 strains in order to analyze the contribution of different NS1 domains to its antagonist functions. We show that the polyadenylation stimulating factor 30 (CPSF30) binding function of the NS1 protein from A/Texas/36/91 influenza virus, which is absent in the A/Puerto Rico/08/34 strain, is essential for counteracting these innate immune events in DCs. However, the double-stranded RNA (dsRNA) binding domain, present in both strains, specifically inhibits the induction of type I IFN genes in infected DCs, while it is essential only for inhibition of type I IFN proteins and proinflammatory cytokine production in cells infected with influenza viruses lacking a functional CPSF30 binding domain, such as A/Puerto Rico/08/34.     

Selection and optimization of a suitable pretreatment method for miscanthus and poplar raw material

Miscanthus and poplar are very promising second‐generation feedstocks due to the high growth rates and low nutrient demand. The aim of the study was to develop a systematic approach for choosing suitable pretreatment methods evaluated with the modified severity factor (log ). Optimal pretreatment results in a high delignification grade, low cellulose solubilization and increased accessibility for enzymatic hydrolysis while revealing minimal log values. In order to do so, several reaction approaches were compared. Acid‐catalyzed organosolv processing carried out for miscanthus and poplar revealed the highest delignification grade leading to a relatively high glucose yield after enzymatic saccharification. In both cases, a design of experiments approach was used to study the influence of relevant parameters. Modeling the data resulted in the identification of optimum pretreatment conditions for miscanthus with concentrations of 0.16% H₂SO₄ and 50% EtOH at 185°C for a retention time of 60 min. Experimental validation of these conditions revealed an even higher delignification degree (88%) and glucose yield (85%) than predicted. 0.19% H₂SO₄ and 50% EtOH were determined as optimum concentrations, 182°C and 48 min identified as optimum pretreatment conditions for poplar; the delignification degree was 84% and the resulting glucose yield 70%.     

Blocking of electron donation by Mn(II) to Y(Z*) following incubation of Mn-depleted photosystem II membranes with Fe(II) in the light

The donation of electrons by Mn(II) and Fe(II) to Y(Z*) through the high-affinity (HA(Z)) site in Mn-depleted photosystem II (PSII) membranes has been studied by flash-probe fluorescence yield measurements. Mn(II) and Fe(II) donate electrons to Y(Z*) with about the same efficiency, saturating this reaction at the same concentration (ca. 5 microM). However, following a short incubation of the membranes with 5 microM Fe(II), but not with Mn(II) in room light, added Mn(II) or Fe(II) can no longer be photooxidized by Y(Z)(*). This blocking effect is caused by specifically bound, photooxidized Fe [> or =Fe(III)] and is accompanied by a delay in the fluorescence yield decay kinetics attributed to the slowing down of the charge recombination rate between Q(a-) and Y(Z*). Exogenously added Fe(III), on the other hand, does not donate electrons to Y(Z*), does not block the donation of electrons by added Mn(II) and Fe(II), and does not change the kinetics of the decay of the fluorescence yield. These results demonstrate that the light-dependent oxidation of Fe(II) by Y(Z*) creates an Fe species that binds at the HA(Z) site and causes the blocking effect. The pH dependence of Mn(II) electron donation to Y(Z*) via the HA(Z) site and of the Fe-blocking effect is different. These results, together with sequence homologies between the C-terminal ends of the D1 and D2 polypeptides of the PSII reaction center and several diiron-oxo enzymes, suggest the involvement of two or perhaps more (to an upper limit of four to five) bound iron cations per reaction center of PSII in the blocking effect. Similarities in the interaction of Fe(II) and Mn(II) with the HA(Z) Mn site of PSII during the initial steps of the photoactivation process are discussed. The Fe-blocking effect was also used to investigate the relationship between the HA(Z) Mn site and the HA sites on PSII for diphenylcarbazide (DPC) and NH2OH oxidation. Blocking of the HA(Z) site with specifically bound Fe leads to the total inhibition of electron donation to Y(Z*) by DPC. Since DPC and Mn(II) donation to PSII is noncompetitive [Preston, C., and Seibert, M. (1991) Biochemistry 30, 9615-9624], the Fe bound to the HA(Z) site can also block the DPC donation site. On the other hand, electron donation by NH2OH to PSII still occurs in Fe-blocked membranes. Since hydroxylamine does not reduce the Fe [> or =Fe(III)] specifically bound to the HA(Z) site, NH2OH must donate to Y(Z*) through its own site or directly to P680+.     

Microalgae: a green source of renewable H(2)

This article summarizes recent advances in the field of algal hydrogen production. Two fundamental approaches are being developed. One involves the temporal separation of the usually incompatible reactions of O₂ and H₂ production in green algae, and the second involves the use of classical genetics to increase the O₂ tolerance of the reversible hydrogenase enzyme. The economic and environmental impact of a renewable source of H₂ are also discussed.     

Routine prophylactic antibiotic use in the management of snakebite

Background  

Routine antibiotic prophylaxis following snakebite is not recommended but evidence suggests that it may be common practice in Zimbabwe. This study set out to determine and describe the extent of this practice at Parirenyatwa Hospital, a large teaching hospital in Zimbabwe     

Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii

The work describes a novel approach for sustained photobiological production of H(2) gas via the reversible hydrogenase pathway in the green alga Chlamydomonas reinhardtii. This single-organism, two-stage H(2) production method circumvents the severe O(2) sensitivity of the reversible hydrogenase by temporally separating photosynthetic O(2) evolution and carbon accumulation (stage 1) from the consumption of cellular metabolites and concomitant H(2) production (stage 2). A transition from stage 1 to stage 2 was effected upon S deprivation of the culture, which reversibly inactivated photosystem II (PSII) and O(2) evolution. Under these conditions, oxidative respiration by the cells in the light depleted O(2) and caused anaerobiosis in the culture, which was necessary and sufficient for the induction of the reversible hydrogenase. Subsequently, sustained cellular H(2) gas production was observed in the light but not in the dark. The mechanism of H(2) production entailed protein consumption and electron transport from endogenous substrate to the cytochrome b(6)-f and PSI complexes in the chloroplast thylakoids. Light absorption by PSI was required for H(2) evolution, suggesting that photoreduction of ferredoxin is followed by electron donation to the reversible hydrogenase. The latter catalyzes the reduction of protons to molecular H(2) in the chloroplast stroma.