Energy-linked nicotinamide-nucleotide transhydrogenase. Characterization of reconstituted ATP-driven transhydrogenase from beef heart mitochondria

The interaction between pure transhydrogenase and ATPase (Complex V) from beef heart mitochondria was investigated with transhydrogenase-ATPase vesicles in which the two proteins were co-reconstituted by dialysis or dilution procedures. In addition to phosphatidylcholine and phosphatidylethanolamine, reconstitution required phosphatidylserine and lysophosphatidylcholine. Transhydrogenase-ATPase vesicles catalyzed a 20-30-fold stimulation of the reduction of NADP+ or thio-NADP+ by NADH and a 70-fold shift of the apparent equilibrium expressed as the nicotinamide nucleotide ratio [NADPH][NAD+]/[NADP+][NADH]. In both of these respects, the transhydrogenase-ATPase vesicles were severalfold more efficient than beef heart submitochondrial particles. By measuring the ATP-driven transhydrogenase and the oligomycin-sensitive ATPase activities simultaneously and under the same conditions at low ATP concentrations, i.e. below 15 microM, the ATP-driven transhydrogenase/oligomycin-sensitive ATPase activity ratio was found to be about 3. This value is consistent with the stoichiometries of three protons translocated per ATP hydrolyzed and one proton translocated per NADPH formed and with a mechanism where the two enzymes interact through a delocalized proton-motive force.     

Single potassium channels in membranes of isolated mesophyll barley vacuoles

Summary Voltage-dependent K channels could be identified in on-cell and excised patch-clamp records on membranes of isolated plant cell vacuoles. The current through a membrane patch is dominated by a channel population with a conductance of about 121 pS in symmetrical 250mm KCl solution. The single channel adopts at least two conducting levels the 121-pS state being most frequently observed. The channel shows outward rectification, representing a cation flux into the vacuoles. The rectification appears to be caused by a vanishing open probability and a short channel lifetime at hyperpolarizing voltages. A selectivity ratio of potassium over sodium of about 6 was derived as an estimate. Occasionally, an additional population of K channels with a single-channel conductance of approximately 18 pS is observed. This channel type exhibits outward rectification as well.     

A phylogenetic study of the antenna cleaner in Formicidae,Mutillidae, and Tiphiidae (Insecta,Hymenoptera)

Summary The morphology of the tibio-tarsal antenna cleaner (strigilis) of 30 species of Formicidae, 14 species of Mutillidae and 9 species of Tiphiidae was investigated and is described comparatively. In Formicidae, there is a common type of strigilis. The spur has a posterior-dorsal comb and its anterior side is covered with squamae, which usually form a brush. Posteriorly, the basitarsal notch bears a comb and anteriorly specialized paddle-shaped hairs. These characters may be apomorphic for Formicidae. In several Ponerinae and in Myrmecia there is also a velum on the spur. In two species of ants which are strongly parasitic (Teleutomyrmex schneideri and Anergates atratulus ) there are reduced antenna cleaners. Mutillidae (except Myrmosa) have another common type of strigilis: the spur bears a velum with a smooth rim and a clear apex with two rows of teeth. The notch in the basitarsus is usually deeper than that of ants; there is a comb, but no paddle-shaped hairs. The strigilis of Myrmosa females has no velum but there are two prominent rows of teeth on the spur. In the male, the velum is reduced to a slender strip. In Tiphiidae the antenna cleaners show considerable diversity. In Methocha the spur bears a comb but no velum; the spur of Tiphia has a velum with a serrated rim; the spur of Myzinum is equipped with a velum with a smooth rim; in Thynnus the surface of the velum is wrinkled or undulating. An apex (without teeth) is present in all investigated Tiphiidae. The notch of the basitarsus bears a comb except in female Myzinum, where the teeth seem to have fused, thus forming a rim. It is suggested that a velum with a serrated or toothed edge and an apex with two rows of teeth are plesiomorphic for aculeate Hymenoptera. The antenna cleaners in Mutillidae are remarkably similar to those in some bees. This fact is interpreted as partly due to convergence and as partly symplesiomorphic. In several species of ants, there is a specialized cuticular area (bande poreuse) anterior to the comb of the notch, which is characterized by fissures or holes. These are presumably openings of an excretory gland.     

Naturally occurring monoterpenoids in needles of Picea abies (L.) Karst

Summary An analysis was made of the effects of different sampling and extraction techniques on the amounts and pattern of monoterpenoids isolated from needles of Norway spruce. The following isolation and analysis procedure was finally adopted: liquid nitrogen-cooled needles were pulverized by a microdismembrator, extracted with pentane overnight at 2°–3°C and concentrated to a volume not less than 3 ml/g fresh weight on a Vigreux column. The crude extract was injected splitless (with solvent split) onto a cold programmed temperature vaporized (PTV) precolumn of a gas chromatograph and the vaporizable compounds heated to a capillary column. This method was tested for production of artefacts and quantitative extraction and applied to needles of eleven 80-year-old spruce trees.     

Concentration gradients of free sterols,steryl esters and lipid phosphorus in the trunkwood of Scot's pine (Pinus sylvestris L.)

The amounts of free sterols, steryl esters and lipid phosphorus were determined in the sapwood and heartwood of mature, and in the outer and inner sapwood of young Pinus sylvestris trees. In the mature trees (up to 70 years old) the heartwood contains significantly higher amounts of free sterols than the sapwood. No radial gradient can be demonstrated in the amounts of steryl esters. Lipids extracted from the sapwood contain higher amounts of phosphorus than those from the heartwood. Stems of young Pinus sylvestris trees (up to 13 years old) show in the inner sapwood higher amounts of both free sterols and steryl esters than the peripheral younger wood zone. The inner sapwood of the young stems shows slightly higher amounts of lipid phosphorus than the outer sapwood. The results indicate that Pinus sylvestris accumulates both free sterols and steryl esters in the stems at a very early stage of the life cycle. Sterol accumulation in the innermost parts of the stems seems not to depend on heartwood formation.     

Immunohistochemical procedures for the demonstration of peptide- and tyrosine hydroxylase-containing nerve fibers in cryostat sections of unfixed rapidly frozen tissue stored for long periods of time

Summary Traditional protocols for the immunohistochemical localization of peptides and tyrosine hydroxylase (TH) in nerve fibers in cryostat sections require the tissue to be thoroughly fixed and rinsed and to be processed for the cryostat sectioning and the immunohistochemical staining more or less directly after freezing. In the present study it was tested whether also unfixed, rapidly frozen tissue, conforming to guinea pig and bovine heart specimens, can be used for the visualization of neuropeptides [neuropeptide Y (NPY) and substance P (S P)] and TH in cryostat sections. The following observations were made: 1) NPY-immunoreactive (IR) and S P-IR nerve fibers could be clearly identified in both fixed and unfixed sections of this type of tissue. 2) TH-IR nerve fibers could be detected in unfixed tissue if the sections were post-fixed with aldehydes by the use of a two-step fixation process related to a sudden change of pH. However, the outlines of the nerve fibers were sometimes diffuse. 3) Storage of unfixed tissue for periods of up to 2.5 yeart at –80° C did not lead to a decrease in immunoreactivity. 4) Somewhat higher concentrations of primary antibodies had to be used for sections of unfixed tissue than for sections of fixed tissue when the FITC method was used. This waste of antibodies was partly overcome by use of the biotin-streptavidin method. The glyoxylic acid induced catecholamine(CA)-fluorescence method for demonstration of sympathetic nerve fibers was also applied and was found to give optimal results after storage of tissue for up to 2.5 years. The study shows that the use of unfixed rapidly frozen tissue represents a fast and realistic method for the demonstration of neuropeptide immunoreactivity, that it to some extent can be used for the visualization of TH-containing nerve fibers and that it is a suitable method to maintain longterm neuropeptide and TH immunoreactivity as well as long-term CA-fluorescence reaction.     

Cytochrome P-450 dependent ethanol oxidation. Kinetic isotope effects and absence of stereoselectivity

Deuterium isotope effects [D(V/K)] and stereoselectivity of ethanol oxidation in cytochrome P-450 containing systems and in the xanthine-xanthine oxidase system were compared with those of yeast alcohol dehydrogenase. The isotope effects were determined by using both a noncompetitive method, including incubation of unlabeled or [1,1-2H2]ethanol at various concentrations, and a competitive method, where 1:1 mixtures of [1-13C]- and [2H6]ethanol or [2,2,2-2H3]- and [1,1-2H2]ethanol were incubated and the acetaldehyde formed was analyzed by gas chromatography/mass spectrometry. The D(V/K) isotope effects of the cytochrome P-450 dependent ethanol oxidation were about 4 with liver microsomes from imidazole-, phenobarbital- or acetone-treated rabbits or with microsomes from acetone- or ethanol-treated rats. Similar isotope effects were reached with reconstituted membranes containing the rabbit ethanol-inducible cytochrome P-450 (LMeb), whereas control rat microsomes and membranes containing rabbit phenobarbital-inducible P-450 LM2 oxidized the alcohol with D(V/K) of about 2.8 and 1.8, respectively. Addition of FeIIIEDTA either to microsomes from phenobarbital-treated rabbits or to membranes containing P-450 LMeb significantly lowered the isotope effect, which approached that of the xanthine-xanthine oxidase system (1.4), whereas desferrioxamine had no significant effect. Incubations of all cytochrome P-450 containing systems or the xanthine-xanthine oxidase systems with (1R)- and (1S)-[1-2H]ethanol, revealed, taking the isotope effects into account, that 44-66% of the ethanol oxidized had lost the 1-pro-R hydrogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic variability in mitochondrial DNA in a regional population of the great tit (Parus major)

Genetic variation of mitochondrial DNA (mtDNA) in 18 great tits (Parus major) from three neighboring localities in Sweden was investigated with eight tetranucleotide restriction endonucleases. The 18 individuals could be separated into 13 different maternal lineages. The high number of female lineages present in this regional population contrasts with a low level of sequence divergence between the different mtDNA clones, with a mean of 0.19% sequence divergence between all individuals. There was no obvious spatial structuring of mtDNA clones among the three localities. The presence of a high number of different clones with a low degree of sequence divergence could be explained by the effects of a large long-term effective population size, with the mtDNA clones having diverged about 25,000–200,000 years ago.This study was supported by the Swedish Natural Science Research Council, the Erik Philip-Sörensen Foundation, and the Nilsson-Ehle Foundation.     

Inhibition of follicle-stimulating hormone-induced ovulation by indomethacin in the perfused rat ovary

In isolated, perfused ovaries of rats treated with pregnant mare's serum gonadotropin (PMSG), purified preparations of ovine follicle-stimulating hormone (FSH) (oFSH-211B) and rat FSH (rFSH-I-6), 100 ng/ml, were found to induce ovulations (4.8 +/- 0.9, n = 4, and 6.4 +/- 2.0, n = 5, ovulations per ovary, respectively). Indomethacin (5 micrograms/ml) added to the perfusate inhibited this ovulatory effect and exogenous prostaglandin F2 alpha (PGF2 alpha) (1 microgram/ml), or prostaglandin E2 (PGE2) (0.5 microgram/ml), reversed the blockade. Ovine FSH and rFSH had only a weak stimulatory effect on estradiol release, and only rFSH caused a significant increase in progesterone accumulation. Indomethacin reduced the stimulatory effect of rFSH on progesterone release, and this effect was reversed by PGE2 but not by PGF2 alpha. In a 6-h incubation experiment with preovulatory rat follicles, we tested the biological activity of gonadotropins used to induce oocyte maturation. The concentration of FSH used in the perfusion experiments induced oocyte maturation in more than 88% of the oocytes studied. The data confirm earlier findings that FSH can induce ovulations and show that prostaglandins are involved in this process. The data also indicate that prostaglandins might be involved in the FSH-induced increase of progesterone levels.     

Interaction specificity of violamycin BI and BII with DNA using the hyperchromic spectra method

Interaction specificity of the anthracycline antibiotics violamycin BI and violamycin BII in respect to A.T and G.C pairs was investigated. For comparison denaturation of complexes with A.T and G.C specific ligands distamycin A and actinomycin D are presented. Making use of the least squares hyperchromic spectra measured in the course of thermal denaturation were partitioned into the components corresponding to the melting of A.T and G.C base pairs and dissociation of ligand. The mutual dependence of AT and GC denaturation allows one to draw conclusions about specificity of interaction. In case of both violamycins only slight preference of interaction with AT-rich regions was detected. The dissociation of violamycin BII in the latest stage of thermal denaturation was found to be cooperative.