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.     

Advantages and limitations of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3- sn-phosphatidylcholine as a fluorescent membrane probe

We have investigated the behavior of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn -phosphatidylcholine (DPHpPC) in synthetic, multilamellar phosphatidylcholine vesicles. This fluorescent phospholipid has photophysical properties similar to its parent fluorophore, diphenylhexatriene (DPH). DPHpPC preferentially partitioned into fluid phase lipid (Kf/s = 3.3) and reported a lower phase transition temperature as detected by fluorescence anisotropy than that observed by differential scanning calorimetry. Calorimetric measurements of the bilayer phase transition in samples having different phospholipid to probe ratios demonstrated very slight changes in membrane phase transition temperature (0.1-0.2 degree C) and showed no measurable change in transition width. Nonetheless, measurements of probe fluorescence properties suggested that DPHpPC disrupts its local environment in the membrane and may even induce perturbed probe-rich local domains below the phospholipid phase transition. Temperature profiles of steady-state fluorescence anisotropy, limiting anisotropy, differential tangent, and rotational rate were similar to those of DPH below the main lipid phase transition but indicated more restricted rotational motion above the lipid phase transition temperature. As for DPH, the fluorescence decay of DPHpPC could be described by either a single or double exponential both above and below the DPPC phase transition. The choice seemed dependent on the treatment of the sample. The intensity-weighted average lifetime of DPHpPC was roughly 1.5 ns shorter than that of DPH. In summary, the measured properties of DPHpPC and its lipid-like structure make it a powerful probe of membrane structure and dynamics.     

Morphology and phase behavior of two types of unilamellar vesicles prepared from synthetic phosphatidylcholines studied by freeze-fracture electron microscopy and calorimetry

Differential scanning calorimetry and freeze-fracture electron microscopy have been used to characterize the phase behavior and morphology of two types of unilamellar vesicles composed of synthetic phosphatidylcholines. The first type displayed an average diameter of roughly 100 nm and was formed by slow dilution and dialysis of octylglucoside-solubilized lipid. These large, unilamellar vesicles were termed dialyzed, octylglucoside vesicles and could be obtained as a fairly well defined and uniform population of vesicles. The second vesicle type was prepared by a unique procedure involving dialysis of deoxycholate-solubilized lipid at its pre-transition temperature. This procedure produced a much more heterogeneous distribution of vesicle sizes (500 to 4000 nm in diameter) and left some dilamellar and oligolamellar species which could not be conveniently separated from the giant, unilamellar vesicles constituting the major portion of the sample. Both populations of vesicles displayed phase behavior similar, but not identical to that of large, multilamellar vesicles (LMV). Fracture-face morphology of the gel phase was also observed to differ between the two unilamellar and the multilamellar species. LMV have previously been shown to have clear undulated or banded fracture-faces in the P beta phase, while octylglucoside vesicles are shown here to have facetted fracture-faces. Giant, unilamellar vesicles displayed a faint banded morphology similar to but less distinct than that of the LMV P beta phase. These results have demonstrated that bilayer apposition is not required to support the banded fracture-face morphology characteristic of the P beta phase but that a limiting curvature is necessary.     

Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease

The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.     

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.