Glucose transport into the extremely halophilic archaebacteria

Penetration of glucose into cells of several extremely halophilic archaebacteria of the Halobacterium and Haloferax genera (Halobacterium saccharovorum and Halobacterium salinarium, Haloferax volcanii and Haloferax mediterranei) has been studied. Some characteristics of transport systems of carbohydrate-utilizing halobacteria Halobacterium saccharovorum, Haloferax mediterranei and Haloferax volcanii (pH and temperature optima, stereospecificity, kinetic parameters) have been determined. Inability of H. salinarium cells for active glucose transport has been shown. The dependence of glucose transport on the Na⁺ ions gradient (on the whole cells and membrane vesicles) has been demonstrated. Cells or membrane vesicles of carbohydrate-utilizing halobacteria grown in media containing this sugar indicated the activation of glucose transport, whereas cells grown in media without sugars did not. This fact has allowed us to conclude that corresponding transport systems are inducible.     

The membrane proteins of the methanol-induced peroxisome of Candida boidinii. Initial characterization and generation of monoclonal antibodies

Peroxisomes are massively induced when methylotrophic yeasts are cultured on methanol as the sole carbon and energy source. An analysis of the protein composition of the peroxisomal membrane and the generation of probes against two peroxisomal membrane proteins (PMPs) have been undertaken. Peroxisomes from Candida boidinii were obtained from sucrose gradients as previously described or from a novel one-step purification of the organelle on a Percoll gradient. The protein composition of the membranes from these two preparations was virtually identical. About 10 proteins comprise nearly all of its protein mass. The most prominent proteins have molecular masses of 120, 100, 47, 31-32 (a triplet), and 20 kDa; significant amounts of alcohol oxidase and dihydroxyacetone synthase, the two abundant matrix proteins, also remain associated with the membrane. Glycosylation of the membrane proteins could not be detected. Exposure of the membrane to chaotropes shows that PMPs 100 and 20 are the most easily removable, whereas PMP 47 appears to be the most tightly associated. Mice were injected with peroxisomal membrane, and hybridoma lines were isolated that produced antibody against PMP 20, PMP 47, and dihydroxyacetone synthase. Indirect immunofluorescence with these monoclonal antibodies confirmed that all three proteins are localized to the peroxisomal cluster. Immunoblotting experiments demonstrated that peroxisomal membrane as well as matrix proteins are induced by methanol.     

Changes in HDL subfractions in patients with type I diabetes mellitus before and after metabolic control

In order to further investigate the behaviour of high density lipoproteins in diabetes mellitus, we studied HDL subclasses, HDL2 and HDL3, in 10 patients with newly detected, untreated insulin-deficient diabetes before starting insulin treatment and after getting a good metabolic control. We used the extractive method of Abell to determine HDL-cholesterol after LDL and VLDL precipitation with polyanions and HDL3-cholesterol after HDL2 precipitation with dextransulphate 15,000 m.w. After insulin therapy, we observed a significant increase in HDL-cholesterol and a decrease in serum triglycerides. Only HDL2-cholesterol, but not HDL3-cholesterol, raised; moreover, we found a significant inverse relationship between HDL-cholesterol (and also HDL2-cholesterol) and triglycerides. So, we think that an increase of lipoprotein lipase activity, owing to insulin treatment, could account for our results.     

Nystatin-induced increase in photocurrent in the system ‘bacteriorhodopsin proteoliposome/bilayer planar membrane’

Proteoliposomes were reconstituted from bacteriorhodopsin sheets, asolectin and cholesterol with or without nystatin. Bacteriorhodopsin-mediated electrogenesis was monitored using (1) a proteoliposome suspension and phenyldicarbaundecaborane (PCB^?) probe or (2) proteoliposomes associated with planar bilayer membrane and orthodox electrometer techniques. In the light, PCB^? was shown to be taken up by proteoliposomes. The PCB^? uptake was inhibited by addition of nystatin to an incubation mixture with proteoliposomes if they were reconstituted in the presence of nystatin. Extraproteoliposomal nystatin was without influence if nystatin was omitted from the reconstitution mixture. The nystatin-containing proteoliposomes were associated with a planar bilayer asolectin membrane in the presence of Ca²⁺. It was found that in such a system, bacteriorhodopsin generated a photocurrent charging the proteoliposome-containing (cis-side) compartment negatively and the trans-side compartment positively. The photoresponse was shown to be increased several-fold by addition of nystatin to the trans-side solution. Nystatin addition was ineffective if proteoliposomes were reconstituted without nystatin. Taking into account that nystatin forms ion-permeable pores in a membrane only if present on both sides of the membrane and that this membrane is bilayer, one can explain the above data assuming that (1) the intraproteoliposomal solution does not mix with the extraproteoliposomal one when proteoliposomes are attached to a planar black membrane and (2) the attached proteoliposomes are separated from the trans-side bathing solution by a bimolecular membrane. If this is the case, nystatin in the trans-side bathing solution and inside the attached proteoliposome can form pores across that part of the planar membrane which separates the proteoliposome interior from the trans-side solution. Through these pores, H⁺ (pumped by bacteriorhodopsin from the cis-side solution into the proteoliposome interior) or some other intraproteoliposomal ions can be equilibrated with those in the trans-side solution. As a result, the bacteriorhodopsin-generated photocurrent increases.     

Reconstitution of biological molecular generators of electric current. Cytochrome oxidase.

1. Direct measurement of the electric current generation by cytochrome oxidase has been carried out. To this end, two procedures were used. The simpler one consists in formation of planar artificial membrane from the mixture of decane solution of soya bean phospholipids and beef heart cytochrome oxidase. Addition of cytochrome c and ascorbate to one of the two compartments separated by the cytochrome oxidase-containing planar membrane was found to result in a transmembrane electric potential difference being formed (plus on cytochrome c side of the membrane). Maximal values of potential differences obtained by this method were about 40 mV. Much higher potentials were observed when another ("photeoliposome-planar membrane") method was applied. In this case cytochrome oxidase was reconstituted with phospholipid to form proteoliposomes which adhered to planar phospholipid membrane in the presence of Ca2+ ions. Addition of cytochrome c and ascorbate to the proteoliposome-containing compartment gives rise to generation of an electric potential difference across the planar membrane, which reached 100 mV at a current of about 1 X 10(-11) A (minus in the proteoliposome-free compartment). The electromotive force of this generator was estimated as being about 0.2 V. If ascorbate and proteoliposomes were added into different compartments, a penetrating hydrogen atom carrier (phenazine methosulfate, (PMS) or tetramethyl-p-phenylenediamine (TMPD)) was required for a membrane potential to be formed. Generation of an electric potential difference of the opposite direction (plus in the proteoliposome-free compartment) was revealed in experiments with cytochrome oxidase proteoliposome containing cytochrome c in their interior. In this case, addition of PMS or TMPD was necessary. 2. In the suspension of cytochrome oxidase proteoliposome the uptake of a cationic penetrant (tetraphenyl phosphonium cation) was found to be coupled with electron transfer via external cytochrome c. Electron transfer via intraproteoliposomal cytochrome c induced the uptake of anionic penetrants (tetraphenyl borate and phenyldicarbaundecaborane anions). 3. All the above effects were sensitive to cyanide and protonophorous uncouplers. 4. In proteoliposomes containing both cytochrome oxidase and bacteriorhodopsin, the light- and oxidation-dependent generations of membrane potential have been revealed. 5. The data obtained are in agreement with Mitchell's idea of transmembrane electron flow in the cytochrome oxidase segment of the respiratory chain.     

Reconstitution of biological molecular generators of electric current. H+-ATPase.

1. Generation of a transmembrane electric potential difference by oligomycin-sensitive ATPase complex, incorporated into spherical or planar phospholipid membrane, has been demonstrated. To this end, penetrating anion probe and direct voltmeter measurement of electric potential across phospholipid membrane were used. It was found that ATP-induced electric response is sensitive to oligomycin and protonophorous uncouplers. 2. The effect of variations in the phospholipid component of proteoliposomes on the electric generation was studied. It was revealed that the usage of mitochondrial phospholipids and phosphatidylethanolamine allows the highest values of membrane potential to be obtained in the case of ATPase proteoliposomes. In the case of cytochrome oxidase and bacteriorhodopsin proteoliposomes, phosphatidylserine was also shown to be quite suitable. Phosphatidylcholine was absolutely ineffective in all cases. 3. In proteoliposomes, containing both ATPase and bacteriorhodopsin, ATP and light induced generation of the electric field of the same direction. 4. In ATPase + cytochrome oxidase proteoliposomes, ATP hydrolysis and ascorbate oxidation was found to support electric generation of the same direction if cytochrome c was inside vesicles. Oxidation via external cytochrome c resulted in formation of electric field of the direction, opposite to that induced by ATP hydrolysis. 5. The data obtained in experiments with proteoliposomes of different types are discussed. The conclusion is made that conversion of energy of different resources into electric form is a common feature of membraneous energy transducers, which is in agreement with the Mitchellian principle of cellular energetics.     

Application of Abscisic Acid (S-ABA) to ‘Crimson Seedless’ Grape Berries in a Mediterranean Climate: Effects on Color, Chemical Characteristics, Metabolic Profile, and S-ABA Concentration

‘Crimson Seedless’ is a table grape cultivar that often fails to develop adequate red color in Mediterranean climates. Application of abscisic acid (S-ABA) may be an aid for improving color, but its potential effects on overall quality and S-ABA concentration of the berry should be also considered. We tested two concentrations (200 and 400 mg/L) and different times of application (from 1 week after veraison up to 9 days before harvest) of a commercial formulation of S-ABA (ProTone^®) to verify the effect on harvestable bunches, color, chemical characteristics, metabolic profile, and S-ABA concentration in the berry. It was found that either the application of S-ABA at 400 mg/L one week after veraison or the application of S-ABA at 400 mg/L one week and four weeks after veraison positively affected the berry skin color, shifting the hue (h°) from 20 to a more red-violet hue (h° = 11–12). In general, the application of S-ABA, with the exception of the late treatments, enhanced coloration of the berries and increased the amount of harvestable bunches at the first pick because it promoted the skin-coloring process. S-ABA did not affect berry firmness but reduced the berry detachment force. Nevertheless, the values remained sufficiently high and the general quality of the bunch was not compromised. Ripening parameters (°Brix, pH, titratable acidity) were not affected by S-ABA applications, and even the primary metabolite profile was not influenced by the treatments as ascertained by multivariate statistical analyses [principal component analyses (PCA) and partial least squares discriminant analysis (PLS-DA)] applied to nuclear magnetic resonance (NMR) data. The S-ABA concentration in the berry, when treatments were performed around veraison, was within the natural range for grape (10–400 ng/g f.w.), whereas when late treatments were applied (few days before harvest), the concentration was higher (more than 1,000 ng/g f.w.). The best results for yield, quality, and S-ABA concentration in the berry were observed for the treatments performed a few days after veraison at the dose of 400 mg/L. This study gives new information about the positive effects of S-ABA on color without any particular change in the metabolic profile of the berry.     

The history of renalase from amine oxidase to α-NAD(P)H-oxidase/anomerase

Renalase is a recently discovered secretory protein, which plays a certain (still poorly understood) role in regulation of blood pressure. The review summarizes own and literature data on structure and catalytic properties of renalase accumulated since the first publication on this protein (2005). Initial reports on FADdependent amine oxidase activity were not confirmed in independent experiments performed in different laboratories. In addition, proposed amine oxidase activity of circulating extracellular renalase requires the presence of FAD, which has not been detected either in blood or urinary renalase. Moreover, renalase excreted into urine lacks its N-terminal peptide, which is ultimately needed for accommodation of the FAD cofactor. Results of the Aliverti’s group on NAD(P)H binding by renalase and weak diaphorase activity of this enzyme stimulated further studies of renalase as NAD(P)H oxidase catalyzing reaction of catecholamine co-oxidation. However, physiological importance of such extracellular catecholamine-metabolizing activity remains unclear due to existence of much more active enzymatic systems (e.g., neutrophil NAD(P)H oxidase, xanthine oxidase/xanthine) in circulation, which can perform such co-oxidation reactions. Recently α-NAD(P)H oxidase/anomerase activity of renalase, which also promotes oxidative conversion of β-NADH isomers inhibiting activity of NAD-dependent dehydrogenases, has been described. However, its possible contribution to the antihypertensive effect of renalase remains unclear. Thus, the antihypertensive effect of renalase still remains a phenomenon with unclear biochemical mechanim(s) and functions of intracellular and extracellular (circulating) renalases obviously differ.     

Transmission of Grapevine virus A and Grapevine leafroll‐associated viruses 1 and 3 by Planococcus ficus and Planococcus citri fed on mixed‐infected plants

The Grapevine virus A (GVA) and Grapevine leafroll‐associated viruses 1 and 3 (GLRaV‐1 and GLRaV‐3) are associated with grapevine diseases that induce severe reductions in yield and berry quality. These three viruses are known to coexist in both grapevine and insect vectors, but their cotransmission has been poorly characterised so far. This study investigates the acquisition and transmission of GLRaV‐1, GLRaV‐3 and GVA by Planococcus ficus and Planococcus citri (Hemiptera: Pseudococcidae) following feeding on multiple‐infected plants. The retention and load of the three viruses in the two insect species were analysed. After feeding onto GVA, GLRaV‐1 and GLRaV‐3 mixed‐infected grapevines, nymphs of P. ficus and P. citri showed similar virus acquisition rates and retained low quantities of viruses until the third post‐acquisition day. Despite the similar acquisition patterns, the two vectors differed in transmission efficiency: P. ficus showed a higher efficiency in transmitting GVA and GLRaV‐3, whereas P. citri transmitted GLRaV‐1 more efficiently. When focusing on the virus cotransmission, it appears that GVA could be transmitted to grapevine without GLRaV‐1 and/or GLRaV‐3 and that the transmission of both GLRaVs could take place in the absence of GVA. This comparative study involving different viruses and vector species improves the current knowledge of the semi‐persistent transmission of these three viruses and contributes to the understanding of grapevine virus epidemiology.