Gating processes of channels induced by colicin A,its C-terminal fragment and colicin E1 in planar lipid bilayers

The dependence on pH and membrane potential of the pore formed by colicin A and its C-terminal 20 kDa fragment has been measured using planar lipid bilayers. The single channel conductance of the pore formed by both colicin A and the fragment increases with pH with an apparent pK of 6.0. At pH 5.0 the gating by membrane potential of the channels formed by either colicin A or its fragment is identical. At the same pH, quite similar pore properties were found when using the related bacteriocin, colicin E₁. In agreement with previous studies, these data indicate that the protein structure containing the lumen of the pore resides in the 20 kDa C-terminal part of the colicin A and favours the recently proposed model, based on protein sequence analysis, which proposes that colicin A, E₁ and I_B C-terminal domains are folded in the same three-dimensional structure. However, it is also shown that colicin A and not its C-terminal fragment undergoes a pH dependent transition between an acidic and a basic form of the pore with an apparent pK of 5.3. The two forms of the pore differ by their gating charge but not by the channel size. These results suggest that there is a pH dependent association between the C-terminal domain carrying the lumen of the pore and another domain of the molecule which affect the pore sensitivity to membrane potential.     

The influence of zinc and sulphur deficiency on oil-filling in peanut (Arachis hypogaea L.) kernels

In the developing peanut (Arachis hypogaea L.) kernels, the period between 15 and 35 days after podding (DAP) was identified as the active period of oil-filling. The period of active oil-filling was associated with a decrease in the starch, soluble sugars and proteins so as to make available the energy and carbon skeleton for the synthesis of oil. The oil content in the mature kernels decreased by 11, 12 and 25 per cent with Zn, S and Zn+S deficiency, respectively. In addition, proteins and starch content decreased significantly while that of soluble sugars increased slightly. The activity of malate dehydrogenase and glucose-6-phosphate dehydrogenase also decreased due to Zn as well as S deficiency. The deficiency treatments resulted in a decrease in phospholipids, free fatty acids and triacylglycerols in mature kernels. Further the proportion of 16∶0 and 18∶2 decreased while that of 18∶1 increased in developing kernels.     

Lipid peroxidation in plumbagin administered rats

Plumbagin was administered to rats at a concentration of 1,2,4,8 and 16 mg per kg body weight. After 24 h lipid peroxide levels were found to decrease in subcellular fractions of liver. Plumbagin inhibited ascorbate and nicotinafde adenine dinucleotide phosphate (reduced) dependent lipid peroxidation but was without any effect on cumene hydroperoxide dependent lipid peroxidation. Injection of 16 mg of plumbagin per kg body weight was found to decrease liver total reduced glutathione and also fcrosomal glucose-6-phosphatase. The results are discussed with reference to the anti- and prooxidant properties of plumbagin.     

Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin

The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca. -100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multistate pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2–1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.Abbreviations BLM Black lipid membranes - CCCP carbonyl cyanide m-chlorophenylhydrazone - DOPC dioleoyl phosphatidylcholine - PS phosphatidylserine - TPP⁺ tetraphenylphosphonium cation     

Correlation of bacterial viability with uptake of [14C] acetate into phenolic glycolipid-1 ofMycobacterium leprae within Schwannoma cells

The viability ofMycobacterium leprae, maintained within 33B Schwannoma cells, was estimated in terms of incorporation of [¹⁴C] acetate into its specific phenolic glycolipid-1. This measure of viability was correlated with two other assays,viz., fluorescein diacetate/ethidium bromide staining and mouse footpad growth. Observation of a 2-fold increase in the number of intracellularMycobacterium leprae over an experimental period of 12 days also corroborated this contention. Furthermore, on addition of anti-leprosy drugs to these intracellularMycobacterium leprae there was significant decrease in phenolic glycolipid-1 synthesis indicative of loss of viability of the organisms. This study also established the importance of the host cell for active bacillary metabolism, asMycobacterium leprae maintained in cell-free conditions showed no incorporation into phenolic glycolipid-1. Moreover, compromising the host’s protein synthesis capacity with cycloheximide, also led to reduction in bacillary metabolism. As this system measures the metabolic synthesis of a uniqueMycobacterium leprae component, it would be useful for development and screening of compounds acting against specific bacillary targets.     

Cholesterol binding reserve of hyperlipemic rat serum lipoproteins in chronic ethanol administration

Chronic administration of ethanol in rats caused the reduction of serum cholesterol binding reserve. The very low density and high density lipoproteins, main serum cholesterol binding reserves, were slightly increased with corresponding increases in their lipid and protein components during initial stage of alcohol consumption. However, these capacities get deminished during reversal of hyperlipemia induced by prolonged action of ethanol. This situation may be an early indicator for the initiation of hepatic damage and a variety of secondary effects of ethanol.     

Patterns of energy storage in Pseudoboeckella poppei (Crustacea,copepoda) from two contrasting lakes on Signy Island,Antarctica

The copepod Pseudoboeckella poppei (Daday) (Calanoida, Centropagidae) was sampled from Sombre and Heywood Lakes on Signy Island, Antarctica (60° S, 45° W) between January 1984 and March 1985. Sombre Lake is clear and oligotrophic with little phytoplankton and a bottom sediment low in organic content. By contrast Heywood Lake is turbid and mesotrophic; a substantial phytoplankton develops in summer and the bottom sediments are comparatively rich in organics. Both lakes freeze over for much of the year, forcing the copepods to adopt a benthic feeding strategy over winter. Adult Pseudoboeckella feed on phytoplankton when this is available, but also on detritus, diatoms and short algal filaments stirred up from the sediment. In Heywood Lake, male copepods show a smooth seasonal trend in lipid content with lipid being synthesised in early summer and utilised in late summer and winter. The summer increase in lipid content is associated with an increase in dry weight. Female lipid contents show evidence of two peaks of egg production. In Sombre Lake both male and female copepods increase in size during summer and show a wider range of lipid contents than in Heywood Lake; it is likely that this is due to the poorer winter feeding conditions which necessitate the synthesis of a much larger store of reserves during the summer. In contrast to marine calanoid copepods, lipid stores are exclusively triacylglycerol with no trace of wax ester.     

Biological effects of synthetic AKH in Manduca sexta and estimates of the amount of AKH in corpora cardiaca

Dose-response curves were measured with synthetic Manduca adipokinetic hormone (AKH) for glycogen phosphorylase activation in larvae and for lipid mobilization in adults. Both responses are known hormonal functions in Manduca sexta. In ligated larvae, full activation of glycogen phosphorylase was achieved with 0.1 pmol and half-maximal activation with 0.03-0.04 pmol. Maximal lipid mobilization in adults required 10 pmol and half-maximal mobilization 0.15 to 0.2 pmol, respectively. An estimate of AKH content of corpora cardiaca from M. sexta was gained by comparing the dose-response curves for synthetic Manduca AKH with curves from gland extracts. Corpora cardiaca extracts were also quantitated by high performance liquid chromatography. According to both estimates corpora cardiaca of adults contain 10-20 pmol AKH per pair, while a pair of larval corpora cardiaca contains 0.7-2 pmol.     

Temperature-dependent osmotic permeability in glycoproteion containing liposomes

The osmotic water outflow of large multilamellar liposomes containing ₁-acid glycoprotein was measured at a temperature near the lipid's phase transition temperature. The liposomes were formed from a mixture of DPPC, cholesterol and glycoprotein in molar ratios 100:20:1, by continuous sucrose density gradient centrifugation. These liposomes captured 35% of the radiolabeled glycoprotein. The temperature-dependent experiments showed that near phase transition temperature the initial rate of water outflow increased drastically in comparison with glycoprotein free liposomes incubated in buffer containing glycoprotein. We suggested that eventual a channel mechanism may be involved due to spontaneous incorporation of glycoprotein into the bilayer.     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1