Redox-controlled,in vivo and in vitro phosphorylation of the α subunit of the light-harvesting complex I in Rhodobacter capsulatus

Labelling of Rhodobacter capsulatus cells with (³²P)Pi in a phototrophic culture results in phosphorylation of a membrane-bound polypeptide identified as the subunit of the LHI antenna complex of the photosynthetic apparatus. Phosphorylation of the same polypeptide was also observed by incubation of chromatophores with (³²P)ATP or under conditions of photophosphorylation with ADP and (³²P)Pi. The identity of the phosphorylated LHI- subunit was demonstrated by N-terminal protein sequencing of the phosphorylated polypeptide and by failure of labelling in LHI-defective mutants. Pre-aeration of the samples or addition of the oxidant potassium ferrcyanide stimulated the kinase activity whereas the presence of soluble cytoplasmic proteins impaired phosphorylation in an in vitro assay. No effect resulted from addition of reductants to the assay medium. The results indicate the presence of a membrane-bound protein kinase in R. capsulatus that phosphorylates the subunit of the LHI antenna complex under redox control.Abbreviations Pi inorganic phosphate - SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis     

Hydroxyl radical formation as a result of the interaction between primaquine and reduced pyridine nucleotides. Catalysis by hemoglobin and microsomes

Kinetic, circular dichroism, and NADH and NADPH fluorescence quenching studies indicate that these compounds interact with the antimalarial drug primaquine (PQ). The affinity of both pyridine nucleotides for PQ is similar. The data are in contrast with a previous report (Thornalley et al. (1983) Biochem. Pharmacol. 32, 3571-3575) suggesting specificity for the interaction with NADPH. The complex was seen to facilitate electron transfer from NAD(P)H to oxygen, generating oxygen-free radicals which were detected by the spin-trapping technique and to flavin nucleotides, giving rise to flavin semiquinone radicals which were demonstrated by direct ESR spectroscopy under anaerobic conditions. A twofold increase in oxygen uptake and hydroxyl radical generation by the NAD(P)H-PQ complex was observed in the presence of hemoglobin. This effect was independent of heme concentration (in the range 1 X 10(-5)-1 X 10(-4) M) and oxidation state of the iron. Under anaerobic conditions, the NAD(P)H-PQ complex reduces Fe-III to Fe-II hemoglobin, and under aerobic conditions about 65% of the heme chromophore is irreversibly destroyed. Superoxide dismutase inhibits hydroxyl radical generation by the NAD(P)H-PQ pair; this effect is not observed in the presence of hemoglobin. In the presence of microsomes there is a 10-fold increase in both oxygen consumption and hydroxyl radical generation by the NAD(P)H-PQ pair. The fact that both pyridine nucleotides are active, and the inability of SKF 525A in decreasing hydroxyl radical generation, suggests that microsomal reductases are involved in the catalysis.     

Chemotherapy of Aspergillus fumigatus keratitis: An experimental study

An experimental Keratitis study of Aspergillus fumigatus was performed in 130 rabbits divided into 12 groups of ten animals each. Three antifungal drugs (myconazole, amphotericin B and pimaricin) were tested using two procedures (topical drops and subconjunctival injections) and two different concentrations (500 and 10 000 times the MIC). In each case, the drugs were applied every 3 h starting 14 h after inoculation. Miconazole was useful at 10 mg/ ml concentration by topical drops and subconjunctival injections, but was less useful at 5 mg/ ml. Amphotericin B was useful at 5 mg/ ml concentration by topical drops and less useful at 2 mg/ ml.No differences were found between the two concentrations by subconjunctival administration. Pimaricin was useful by topical drops at 50 mg/ ml concentration and less useful at 10 mg/ ml as well as by subconjunctival injections.     

Occurrence of oscillations in ATP synthesis and hydrolysis in chromatophores of Rhodospirillum rubrum

The conditions under which an oscillatory behaviour is observed during net hydrolysis or synthesis of ATP in chromatophores of Rhodospirillum rubrum FR1 are described. In the case of ATPase the oscillations are observed at low temperature (ca. 11°C) in the dark after an initial transient behaviour. These oscillations are attenuated or disappear by the addition of an uncoupler.Oscillations are also observed during ATP synthesis. At 3°C the oscillations appear spontaneously if photophosphorylation is measured during a sufficiently long time. At 30°C the mere intercalation of a dark period also at 30°C is sufficient to trigger the oscillations in the following light period.Abbreviations Bchl Bacteriochlorophyll - FCCP carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - PMS phenazine methosulfate - TMPD, N,N,N,N tetramethyl-1,4-phenylenediamine Dedicated to Prof. Dr. Gerhart Drews as a homage for his permanent example as hard worker and careful scientist and also for his remarkable human quality     

Flow through a Collapsible Tube: Experimental Analysis and Mathematical Model

Flow through thin-wall axisymmetric tubes has long been of interest to physiologists. Analysis is complicated by the fact that such tubes will collapse when the transmural pressure (internal minus external pressure) is near zero. Because of the absence of any body of related knowledge in other sciences or engineering, previous workers have directed their efforts towards experimental studies of flow in collapsible tubes. More recently, some attention has been given towards analytical studies. Results of an extensive series of experiments show that the significant system parameter is transmural pressure. The cross-sectional area of the tube depends upon the transmural pressure, and changes in cross-section in turn affect the flow geometry. Based on experimental studies, a lumped parameter system model is proposed for the collapsible tube. The mathematical model is simulated on a hybrid computer. Experimental data were used to define the functional relationship between cross-sectional area and transmural pressure as well as the relation between the energy loss coefficient and cross-sectional area. Computer results confirm the validity of the model for both steady and transient flow conditions.     

Effects of plant size on photosynthesis and water relations in the desert shrub Prosopis glandulosa (Fabaceae)

The Jornada del Muerto basin of the Chihuahuan Desert of southern New Mexico, USA, has undergone a marked transition of plant communities. Shrubs such as mesquite (Prosopis glandulosa) have greatly increased or now dominate in areas that were previously dominated by perennial grasses. The replacement of grasses by shrubs requires an establishment phase where small shrubs must compete directly with similar-sized grass plants. This is followed by a phase in which large, established shrubs sequester nutrients and water within their biomass and alter soil resources directly under their canopy, creating “islands” of fertility. We hypothesized that these two phases were associated with shrubs having different physiological response capacities related to their age or size and the resource structure of the environment. As a corollary, we hypothesized that responses of small shrubs would be more tightly coupled to variation in soil moisture availability compared to large shrubs. To test these hypotheses, we studied gas exchange and water relations of small (establishing) and large (established) shrubs growing in the Jornada del Muerto as a function of varying soil moisture during the season. The small shrubs had greater net assimilation, stomatal conductance, transpiration, and xylem water potential than large shrubs following high summer rainfall in July, and highest seasonal soil moisture at 0.3 m. High rates of carbon assimilation and water use would be an advantage for small shrubs competing with grasses when shallow soil moisture was plentiful. Large shrubs had greater net assimilation and water-use efficiency, and lower xylem water potential than small shrubs following a dry period in September, when soil moisture at 0.3 m was lowest. Low xylem water potentials and high water-use efficiency would allow large shrubs to continue acquiring and conserving water as soil moisture is depleted. Although the study provides evidence of differences in physiological responses of different-sized shrubs, there was not support for the hypothesis that small shrubs are more closely coupled to variation in soil moisture availability than large shrubs. Small shrubs may actually be less coupled to soil moisture than large shrubs, and thus avoid conditions when continued transpiration could not be matched by equivalent water uptake.     

Reduction in Glial Fibrillary Acidic Protein mRNA Abundance Induced by (—)-Deprenyl and Other Monoamine Oxidase B Inhibitors in C6 Glioma Cells

Abstract: Gliosis is commonly observed in the CNS following tissue damage, and it also occurs in aging and in many neurodegen-erative diseases. Glial fibrillary acidic protein (GFAP) accumulation is a prominent feature of astrocytic gliosis. An inhibition or delay in GFAP synthesis could mitigate scar formation and thus reduce the formation of a physical barrier. The consequence of this would be to allow neurons and oligodendrocytes to reestablish a functional environment. (—)-Deprenyl, a specific monoamine oxidase (MAO) B inhibitor, has been used as an effective antipar-kinsonian drug, and it has been reported to possess neuroprotective and neurorescue properties. Using northern and slot blots to detect GFAP mRNA in C6 glioma cells, we have demonstrated that (—)-deprenyl decreases the abundance of GFAP mRNA in a time- and dose-dependent manner. The effect seems to be specific to MA0 B inhibitors because (+)-deprenyl and clorgyline exhibit no effect. This study indicates therefore that (-)-deprenyl may be useful for regulating astrogliosis following CNS injury as well as in some neurodegenerative diseases.     

New method for peptide purification based on selective removal of truncation peptide impurities after SPPS with orthogonal capping

Peptide purification by high-performance liquid chromatography (HPLC) is associated with high solvent consumption, relatively large effort and lack of efficient parallelization. As an alternative, many catch-and-release (c&r) purification methods have been developed over the last decades to enable the efficient parallel purification of peptides originating from solid-phase peptide synthesis (SPPS). However, with one exception, none of the c&r systems has been widely established in industry and academia until today. Herein, we present an entirely new chromatography-free purification concept for peptides synthesized on a solid support, termed reactive capping purification (RCP). The RCP method relies on the capping of truncation peptides arising from incomplete coupling of amino acids during SPPS with a reactive tag. The reactive tag contains a masked functionality that, upon liberation during cleavage from the resin, enables straightforward purification of the peptide by incubation with a resin-bound reactive moiety. In this work, two different reactive tags based on masked thiols were developed. Capping with these reactive tags during SPPS led to effective modification of truncated sequences and subsequent removal of the latter by chemoselective reaction with a maleimide-functionalized solid support. By introducing a suitable protecting group strategy, the thiol-based RCP method described here could also be successfully applied to a thiol-containing peptide. Finally, the purification of a 15-meric peptide by the RCP method was demonstrated. The developed method has low solvent consumption, has the potential for efficient parallelization, uses readily available reagents, and is experimentally simple to perform.