Investigation of phycocyanin-membrane interaction: Promotion of membrane interactions

• 1.1. In a continuing investigation of phycocyanin-membrane surface interaction, fluorescence quenching experiments were performed with a mixture of two populations of fluorescence probe-encapsulated phospholipid bilayer vesicles in the presence and absence of phycocyanin.
• 2.2. These membrane vesicles were prepared with 1,2-dimyristoyl phosphatidylcholine (DMPC), cholesterol and a probe molecule.
• 3.3. A fluorophore was encapsulated in one population of membrane vesicles, while a quencher was encapsulated in another population of membrane vesicles.
• 4.4. The result was compared with those of experiments in the presence of other biomolecules, including albumin, cytochrome c, hemoglobin, myoglobin or RNA.
• 5.5. Interestingly, a one-third reduction of the fluorescence intensity was observed in the mixture of these two populations of membrane vesicles in phycocyanin's presence.
• 6.6. In contrast, the other biomolecules caused no significant reduction in the fluorescence intensity.
• 7.7. These findings were evidence of a phycocyanin-induced membrane perturbation.
• 8.8. This was further demonstrated by a phycocyanin-induced change in the thermotropic behavior of DMPC vesicles, as measured by differential scanning microcalorimetry.
• 9.9. Such a unique property of phycocyanin is believed to be associated with its known membrane surface-interacting character.
• 10.10. A possible phycocyanin-modulated membrane-membrane interaction was discussed.

     

The qualitative and quantitative analysis of insect hemolymph sugars by high performance thin-layer chromatography

• 1.1. A procedure is described for the analysis of sugars in insect hemolymph by high performance thin-layer chromatography.
• 2.2. Densitometric scanning of spots after plate developments shows linear responses with most sugars in an analytical range of 125ng-2.0 /gmg; detection limits range from 30/2–60 ng.
• 3.3. Quantitative measurements of trehalose, glucose and fructose can be made on hemolymph sample volumes of less than one microliter, allowing blood sugar analysis of individual insects.
• 4.4. Hemolymph sugar determinations on six species of insects agree well with values reported in the literature, but mean values for a species often showed higher variability because samples were taken from individuals.

     

Driving forces for the uphill transport of amino acids into epidermal brush border membrane vesicles of the sea anemone,Anemonia sulcata (Cnidaria,anthozoa)

• 1.1. The transport of amino acids into membrane vesicles prepared from epidermal tentacle tissue of the sea anemone, Anemonia sulcata, depends on an electrochemical potential difference caused, e.g. by sodium chloride gradients.
• 2.2. Potassium or choline chloride gradients energized the transport less effectively than sodium chloride gradients. Both Na⁺-ions and Cl⁻-ions were required for the amino acid transport.
• 3.3. The uphill transport of amino acids along the downhill movement of driver ions (sodium chloride gradient conditions) was characterized by an overshoot; under sodium chloride equilibrium conditions, however, an accumulation of amino acids within the vesicles could not be measured.
• 4.4. Potassium diffusion potentials in combination with valinomycin indicated that hyperpolarization (vesicle inside negative) and hypopolarization (vesicle inside positive) enhanced or depressed the accumulation of amino acids within the vesicles.
• 5.5. Being at the phylogenetic base of the Eumetazoa, cnidarians show characteristics for the transmembrane transport of amino acids comparable to those established for vertebrates.

     

Lipolysis post mortem in North Atlantic krill

• 1.1. Three common species of North Atlantic krill, Meganyctiphanes norvegica (M. Sars), Thysanoessa inermis (Krøyer) and T. raschii (M. Sars), have been stored at 0°C post mortem, and the lipolytic activity followed by measuring changes in the lipid composition during storage.
• 2.2. Both phosphoglycerides and triacylglycerols were subjected to extensive hydrolysis with the formation of free fatty acids in all krill species examined, whereas wax esters, constituting a considerable proportion of the lipids in the Thysanoessa species, were not hydrolysed at all.
• 3.3. In M. norvegica the triacylglycerols and phosphoglycerides were hydrolysed at similar rates, whereas in T. inermis and T. raschii the phosphoglycerides were hydrolysed most rapidly.
• 4.4. For all krill species examined, the rate of production of free fatty acids was nearly constant during the initial phase of storage, and subsequently declined on prolonged storage.
• 5.5. At the end of the storage period of 16–24 days, the free fatty acids constituted about 35% of the total lipid in M. norvegica, and about 50% in the Thysanoessa species.
• 6.6. The rate of production of free fatty acids was about the same in all the three species of krill and seemed to be independent of the total lipid content.

     

Properties and topology of enzymes methylating phosphatidylethanolamine to phosphatidylcholine in sarcoplasmic reticulum

• 1.1. The synthesis of phosphatidylcholine (PC) by stepwise methylation of phosphatidylethanolamine (PE) is carried out by two enzymes in sarcoplasmic reticulum (SR) membrane of rabbit fast-twitch skeletal muscles.
• 2.2. Two methyltransferases (Met I and Met II) have a different pH optimum and affinity for methyl donor—5-adenosyl-L-methionine (SAM).
• 3.3. Met I is an integral SR membrane protein which active site faces the cytoplasmic surface of the membrane.
• 4.4. Met II is a peripheral, loosely bound protein, localized mainly on the extracytoplasmic (luminal) part of the SR membrane.

     

BioID Performed on Golgi Enriched Fractions Identify C10orf76 as a GBF1 Binding Protein Essential for Golgi Maintenance and Secretion

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Highlights

• •BioID with Golgi fractions identified C10orf76 as proximal to GBF1.
• •Tagged C10orf76 overlaps with Golgi markers.
• •C10orf76 binds GBF1 and exchanges rapidly between free and bound forms.
• •C10orf76 is essential for maintenance of the Golgi and for secretion.

     

Biphasic action of growth hormone on intestinal amino acid absorption in striped bass hybrids

• 1.1. Weekly injections of bovine growth hormone (bGH) increased the maximal transport rate of both Na⁺-dependent and Na⁺ -independent l-leucine transport with little effect on the affinity constants in the intestine of striped bass hybrids.
• 2.2. The Na⁺-dependent and the Na⁺-independent transport of the non-metabolizable analog cycloleucine was also stimulated by bGH.
• 3.3. The Na⁺ -dependent active transport was stimulated 2 days after the hormone treatment, while the stimulation of the Na⁺-independent diffusional transport was not observed until after 2 weeks of treatment.
• 4.4. Studies of intestinal morphometry and l-leucine transport using brush border membrane vesicles suggested that bGH affects intestinal amino acid absorption initially by increasing the number of transporters per cell.
• 5.5. This phase is followed by a general increase of the intestinal mass after long-term treatment with the hormone.

     

Distribution of beta-adrenergic binding in fractionated porcine adipocytes

• 1.1. The subcellular distribution of the porcine adipocyte beta-adrenergic receptor was studied in fractionated adipocytes.
• 2.2. The 30,000 g pellet obtained from hypotonically lysed cells contained membrane vesicles and mitochondria; it yielded approx 200–300 fmol dihydroalprenolol-bound receptors/mg protein.
• 3.3. Activity was increased to about 1000 fmol/mg protein after isolation of a plasma membrane fraction on a Percoll gradient.
• 4.4. The 5'-nucleotidase, succinate dehydrogenase and lactate dehydrogenase activities were usually enriched in compartments different from the ligand-binding activity.
• 5.5. Activity of porcine adipocyte 5'-nucleotidase, a purported plasma membrane marker enzyme, was not distributed in the same manner as the beta-adrenergic receptor.

     

2-Deoxyglucose transport by the frog sartorius: Effects of electrical stimulation and N-carbobenzoxy-glycyl-l-phenylalaninamide

• 1.1. Studies characterizing glucose transport in the frog sartorius were performed.
• 2.2. For nonstimulated and stimulated muscles, intracellular 2-deoxyglucose exceeded 2-deoxyglucose-6-phosphate at 15 min, showed little further increase, and was maintained below the extracellular concentration for 2 hr.
• 3.3. Accumulated 2-deoxyglucose-6-phosphate did not inhibit glucose transport.
• 4.4. Unlike in adipocytes, basal and stimulated 2-deoxyglucose transport showed no difference in sensitivity to N-carbobenzoxy-glycyl-l-phenylalaninamide.
• 5.5. Phenylarsine oxide blocked contraction-enhanced 2-deoxyglucose uptake.
• 6.6. These results suggest that the glucose transporter of the sartorius exhibits auto-regulation, and that basal transport is not regulated by the same process as in adipocytes.

     

Phosphatidylethanolamine: Ceramide-ethanolaminephosphotransferase activity in synaptic plasma membrane vesicles. Influence of some cations and phospholipid environment on transferase activity. Further proof of its location

• 1.1. Synaptic plasma membrane vesicles (SPMV) from rat brain synthesized ceramide-phosphoethanolamine (SpE), an analogue of sphingomyelin (SpC) from phosphatidylethanolamine (PE) and ceramide.
• 2.2. This reaction was catalyzed by PE: ceramide-phosphotransferase.
• 3.3. The presence of PC did not modify the SpE synthesis and PI and PS at twice PE concentration seemed to be activators; only PG was an inhibitor at all concentrations.
• 4.4. Some cations (Mg²⁺, Mn²⁺) were without effect, while Ca²⁺ increased transferase activity, so was interesting to study.
• 5.5. Transferase was compared with sialidase (external enzyme).
• 6.6. Kinetics other than those already performed by us were undertaken in order to confirm its location.

     

Activation of gastric mucosal calcium channels by epidermal growth factor

• 1.1. Gastric mucosal calcium channel complex was isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin.
• 2.2. The complex following labeling with [³H]PN200-110 was reconstituted into phosphatidylcholine vesicles which exhibited active ⁴⁵Ca²⁺ uptake into intravesicular space as evidenced by La³⁺ displacement and osmolarity measurements. The ⁴⁵Ca²⁺ uptake was independent of sodium and potassium gradients indicating the electroneutral nature of the process.
• 3.3. The gastric mucosal channels on epidermal growth factor binding in the presence of ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170 kDa subunits of calcium channel.
• 4.4. The phosphorylated channels following reconstitution into vesicles displayed at 48% greater ⁴⁵Ca²⁺ uptake, thus indicating the tyrosine kinase involvement in EGF dependent activation of calcium channel.
• 5.5. The results point towards the importance of epidermal growth factor in the maintenance of gastric mucosal calcium homeostasis.

     

Decreased Antibiotic Susceptibility Driven by Global Remodeling of the Klebsiella pneumoniae Proteome

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Highlights

• •Global proteomic remodeling alters antibiotic susceptibility in K. pneumoniae.
• •Drug specific transport, sugar utilization, central metabolism, capsule synthesis.
• •Common pathways of reactive oxygen scavenging, turnover of misfolded proteins.
• •Integrated adjustments and unique drug-specific features for drug combinations.

     

The influence of calcium and magnesium on sea bass (Dicentrarchus labrax) intestinal brush border membrane purification and activity

• 1.1. The activity of brush border enzymes (alkaline phosphatase, maltase, sucrase, trehalase, leucine amino peptidase) was higher in purified membranes prepared with calcium. The contamination of these membranes with basolateral membranes was also lower (1.27 for Na-K-ATPase activity ratio).
• 2.2. The extraction of brush border lipids was carried out according to Folch adapted method. Two dimensional thin layer chromatography was used to separate the phospholipidic fractions. Fatty acids of phospholipids were analysed using gas chromatography after acid transmethylation (column SP 2330).
• 3.3. Phospholipids are composed of phosphatidylcholine (PC: 33%), phosphatidylethanolamine (PE: 30%), sphingomyeline (SM: 21%), phosphatidylserine (PS: 14%) and phosphatidylinositol (PI: 2%). 4. PC, PE and PS are characterized by high levels of unsaturated fatty acids (monounsaturated MUFA: 21.5% and polyunsaturated PUFA: 34.9%). The most abundant PUFA belong to the (n-3) family [18:3 (n-3), 20:5 (n-3) and 22:6 (n-3)].
• 4.5. Fatty acids from sphingomyelin of purified membranes have low proportions of PUFA (13.5%) but higher proportions of MUFA (39.5%).
• 5.6. No specific differences were found between calcium and magnesium prepared membranes.
• 6.7. The low content in LPC and the absence of LPE confirmed the absence of major structural lipids transformation during the membrane purification with calcium or magnesium.
• 7.8. Glycine transport was measured during 10 sec at different temperatures using the rapid filtration technique. Glycine transport was higher with Na⁺ than with K⁺. In the presence of Na⁺, this transport increases with temperature.
• 8.9. Arrhenius curves were mono phasic without obvious breakpoint and indicated no phase transition in the lipid bilayer.
• 9.10. A significant Na⁺ dependent glycine transport has been characterized at low temperatures (0°C) which suggests a possible role of membrane polyunsaturated fatty acids in the control of glycine transport.

     

The role of lipoproteins in the delivery of tumour-targeting photosensitizers

• 1.I. Serum lipoproteins play an important role in the in vivo transport of several porphyrinoid derivatives having a moderate or high degree of hydrophobicity.
• 2.2. There appears to exist a correlation between the extent of photosensitizer association with low-density lipoproteins (LDL) and the efficiency of tumour targeting by some classes of photosensitizers, such as differently sulphonated porphyrins and phthalocyanines, haematoporphyrin dialkylethers and unsubstituted phthalocyanines and naphthalocyanines.
• 3.3. In all cases, LDL-carried photosensitizers are preferentially released to malignant cells; hence, direct cell damage appears to be the major determinant of tumour damage consequent to photodynamic therapy.
• 4.4. Present evidence suggests that the LDL-associated photosensitizer is accumulated by tumour cells largely via a receptor-mediated endocytotic process.
• 5.5. Thus, the use of delivery systems for orientating a systemically injected photosensitizer towards lipoproteins has been explored; promising results have been obtained by incorporation of the dye into liposomal vesicles, oil emulsions or inclusion complexes, as well as by precomplexation of the dye with LDL.
• 6.6. Moreover, a suitable choice of the chemical constituents of the delivery system and the experimental conditions allows one to modulate the photosensitizer distribution among the different lipoproteins.
• 7.7. The occurrence of tumour-targeting strategies other than the LDL pathway is briefly discussed.

     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

l-Leucine intestinal transport at different ages in chicken: Effect of anticoccidial drugs

• 1.1. l-Leucine transport by everted slices of duodenum, jejunum ileum and cecum of 1-, 2- and 3-week-old chickens has been determined.
• 2.2. The effects of the administration with the diet of three anticoccidial drugs: monensin, maduromicin ammonium and nicarbazine have been studied.
• 3.3. In the control animals, there is a reduction on intestinal transport with age, except in the duodenum which is not age-dependent.
• 4.4. Momensine induces an increase on leucine transport in the duodenum and cecum at all ages studied and only in the 3-week-old animals in the jejunum and ileum.
• 5.5. Nicarbazine reduces jejunal absorption of leucine and does not affect the function of the other segments.
• 6.6. Maduromicin ammonium has almost no effect on the absorptive process.

     

Effects of long- and medium-chain triglycerides on amino acid uptake in rat intestinal brush border membrane vesicles

• 1.1. Uptake of l-leucine, l-phenylalanine, l-proline and l-lysine into brush border membrane vesicles from rats fed either a medium-chain triglyceride (MCT) or a long-chain triglyceride (LCT) diet was studied under conditions of the presence or absence of a Na⁺ gradient.
• 2.2. From the results of initial rate, Na⁺-dependent transport in LCT feeding were lower than in feeding MCT. The Na⁺-independent transport did not vary in either group except for l-lysine uptake.
• 3.3. For l-leucine, l-phenylalanine and l-proline in Na⁺ dependence, kinetic analysis revealed 4–6-fold smaller V_max values in LCT group than in MCT group. l-Lysine in Na⁺-independent transport was 10-fold lower in LCT group than in MCT group. The K_m values were not affected by feeding the LCT or MCT diet.
• 4.4. It is clear that amino acid transport is regulated by different types of dietary fat. We consider that the alteration of transport activity is attributable to the changes in number of membrane-bound transport carriers but not to their affinity.

     

Hepatic Mitochondrial Defects in a Nonalcoholic Fatty Liver Disease Mouse Model Are Associated with Increased Degradation of Oxidative Phosphorylation Subunits

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Highlights

• •Stability of oxidative phosphorylation subunits are reduced in a diet-induced mouse model of NAFLD.
• •These changes are associated with impaired activities of electron transport chain complexes and ATP synthesis.
• •Increased mitophagy contributed to enhanced degradation of mitochondrial proteins.

     

Amino acid sequences around exofacial proteolytic cleavage sites of band 3 from bovine and porcine erythrocytes

• 1.1. Amino acid sequences of bovine and porcine band 3, an erythrocyte anion transporter, were determined.
• 2.2. The sequence of bovine band 3 was positioned to residues 519–599 (the numbering is based on human band 3), in which probably 6 residues were unidentified.
• 3.3. Binding site of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate), a potent anion transport inhibitor, was identified as Lys-539 in the bovine case.
• 4.4. A loop (residues 551–567), which provides exofacial proteolytic cleavage sites, contains only 53% homology between human and bovine, whereas the residues flanking it on either side are >84% homologous.
• 5.5. Furthermore, the loop of porcine band 3 was indicated to consist of a 6 or 7-residues short peptide as compared with those of other species.

     

Regulation of spermidine transport in l1210 cells

• 1.1. 1 mM 2-amino isobutyric add (AIB), glutamine or asparagine when preincubated for 3 hr with L1210 cells promoted a marked increase in the rate of spermidine uptake.
• 2.2. Cycloheximide also increased the transport rate and completely prevented the increase due to AIB.
• 3.3. Trifluoperazine and iso-H7 inhibited the uptake of spermidine, much less the uptake of AIB.
• 4.4. Adenosine promoted an increase in the uptake of AIB, a decrease in that of spermidine.
• 5.5. Hypotonic stress also increased the rate of spermidine transport. This modification was only partially prevented by cycloheximide.
• 6.6. Okadaic arid had no effect on this increase, whereas it prevented the increase of ODC activity.