Alterations of Phospholipid Metabolism in Rat Cerebral Cortex Mince Induced by Cationic Amphiphilic Drugs

Abstract Cationic amphiphilic drugs (CADs) of varied clinical use were screened to determine their capacity to alter the pattern of labeling with ³²Pj of cerebral cortex mince phospholipids. The altered phospholipid labeling patterns were qualitatively similar, the prominent features being reduced incorporation into phosphatidylcholine and increased incorporation into phosphatidic acid. Relative potencies were: (±)-propranolol > chlorpromazine = 4,4'-bis(diethylaminoethoxy) α,β -diethyldiphenylethane > desipramine > di-bucaine > pimozide > oxymetazoline = fenfluramine = haloperidol = chloroquine > amphetamine = no drug added. Propranolol was used to study the action of CADs further. Its effect was time- and dose-dependent, but in contrast with pineal gland, no label appeared in phosphatidyl-CMP (CDP-diacylglycerol), nor did dialysis of the mince to reduce diffusible substrates or exogenous addition of substrates cause appearance of liponucleotide. Thus lack of diffusible precursors is not responsible for CAD effects in vitro. Pulse-chase experiments with ³²P₁ and [2-³H]glycerol suggested that inhibition of phosphatidate phosphohydrolase may be partly responsible for the observed alterations in phospholipid labeling in the presence of CADs.     

Maintenance of the Adult Rat Superior Cervical Ganglion In Vitro: Comparison of Organ and Explant Culture Systems

Abstract: Concentrations of selected intermediates of energy metabolism whole rat superior cervical ganglia maintained in vitro by an organ culture technique were compared with values measured in small slices of this maintained under essentially the same conditions. Rates of incorporation [³H]leucine into trichloroacetic acid-precipitable material in whole ganglia mained constant for at least 48 h: however, the oxidation-reduction state tissue as indexed by (NAD):(NADH) ratios calculated from measured amounts of lactate and pyruvate decreased more than 50% within ³h in vitro . Ganglion explants prepared by cutting the tissue into ³⁰⁰-pm transverse sections played (NAD):(NADH) ratios that were about three times greater than noted in whole ganglia maintained in vitro for the same period of time. explants contained significantly higher concentrations of pyruvate and α-ketoglutarate than whole ganglia maintained in culture. Maintenance of vorable metabolic state may support the extensive growth of neurites seen explant cultures of superior cervical ganglia. Outgrowth of processes containing catecholamines could be detected readily in explant cultures of ganglia adult rats; however, this was somewhat slower and less consistent than growth observed in explants from neonatal rats. Outgrowth of neurites adult ganglia was minimal without the addition of Nerve Growth Factor.     

The effect of copper on glutathione metabolism in human leukocytes

Leukocytes incubated with Cu(II) showed a decrease in both glutathione reductase activity and reduced glutathione content. The glucose 6-phosphate dehydrogenase activity under the same conditions was not affected. Serum albumin added to mixtures prevented the loss of enzyme activity, whiled-penicillamine andl-histidine had little effect. Prior oxidation of the cell-reduced glutathione did not diminish the enzyme inhibitory action of Cu(II). The amount of regeneration of reduced glutathione in leukocytes previously treated with diamide to oxidize their reduced glutathione was a function of Cu(II) concentration in the media. No evidence was obtained that elevated serum ceruloplasmin levels in rabbits, nor incubation of leukocytes in vitro with ceruloplasmin, affect leukocyte glutathione reductase activity. It was proposed that the major mechanism by which copper affects glutathione metabolism in leukocytes is by inhibition of glutathione reductase.     

Simulation of grana stacking in a model membrane system. Mediation by a purified light-harvesting pigment-protein complex from chloroplasts

An isolated light-harvesting pigment-protein complex contains polypeptides which bind chlorophyll a and b. The individual complexes can be purified from detergent-solubilized membranes. The isolated light-harvesting complex, when dialyzed to remove detergents, was examined by freeze-fracture electron microscopy. The material consisted of planar sheets of 80-Å subunits which interacted via an edge-to-edge contact. Addition of cations caused the planar light-harvesting complex sheets to become tightly appressed in multilamellar stacks, with distinct subunits still visible within each lamellar sheet. A transition of particle organization from random to crystalline occurred in parallel with the cation-induced lamellar association. Treatment of the dialyzed light-harvesting complex subunits with low levels of the proteolytic enzyme trypsin removed a 2000 molecular weight segment of the major polypeptide of the light-harvesting complex and blocked all subsequent cation-induced changes in structural organization of the isolated light-harvesting complex lamellar sheets.To gain further evidence for mechanisms of cation effects upon the organization of the light-harvesting complex in native membranes, the light-harvesting complex was incorporated into uncharged (phosphatidylcholine) lipid vesicles. The protein complexes spanned the lipid bilayer and were arranged in either a random pattern or in hexagonal crystalline lattices. Addition of either monovalent or divalent cations to ‘low-salt’ (20 mM monovalent cation) vesicles containing light-harvesting complex caused extensive regions of membrane appression to appear. It is concluded that this cation-induced membrane appression is mediated by surface-exposed segments of the light-harvesting complex since (a) phosphatidylcholine vesicles themselves did not undergo cation-induced aggregation, and (b) mild trypsin digestion of the surface-exposed regions of the light-harvesting complex blocked cation-induced lamellar appression. The particles in the appressed vesicle membranes tended to form long, linear arrays of particles, with occasional mixed quasi-crystalline arrays with an angular displacement near 72°. Surface-mediated interactions among light-harvesting complex subunits of different membranes are, therefore, related to changes in structural organization and interaction of the particles within the lipid phase of the membrane.Numerous previous studies have implicated the involvement of the light-harvesting complex in mediating grana stocking in intact chloroplast membranes. The data presented herein provide a simulation of the membrane appression phenomena using a single class of chloroplast-derived membrane subunits. The data demonstrate that specific surface-localized regions of the light-harvesting complex are involved in membrane-membrane interactions.     

Trypanosoma theileri: In vitro cultivation in tsetse fly and vertebrate cell culture systems

Epimastigote forms of Trypanosoma theileri were grown at 25°C in insect cell culture media and in Glossina tissue cultures for more than 6 months. Doubling times of 10–14 h during exponential growth were observed. In cell cultures which had been derived from pupal tsetse flies growth rates were higher than in cell free media; in a larval cell line, however, growth of T. theileri was inhibited. Ecdysteroids and juvenile hormone I reduced multiplication of T. theileri in cell free media. When T. theileri was incubated in different sera only fetal calf serum (FCS) supported growth. Epimastigote forms transformed into trypomastigote bloodforms when cultured at 37°C in FCS, vertebrate cell cultures, and Eagle's medium, but not in insect media or Glossina cell cultures. Oxygen uptake of epimastigotes could be inhibited by rotenone antimycin A and cyanide; trypomastigotes were not affected by these inhibitors.     

Sulfite formation by wine yeasts

The enzyme catalyzing the reduction of sulfite by reduced benzyl viologen (BVH) was partially purified and characterized from two strains of wine yeasts, a sulfite-producing strain and a non-producing strain.Both enzymes showed corresponding features in pH-optima, optima of buffer and benzyl viologen concentrations.The enzymes did not catalyze the reduction of nitrite by reduced viologen dyes, but the reduction of sulfite was uncompetitively inhibited by nitrite. Compounds of sulfur metabolism such as sulfate, thiosulfate, cysteine, serine and methionine did not influence the activity of either of the enzymes. The main differences between the two enzymes exist in the specific activities in crude extracts, the K _m -values for sulfite, substrate inhibition rates, and localization in different fractions during (NH₄)₂SO₄ precipitation. The specific activity in crude extracts of the sulfite-producing strain (0.052 moles S^2- x min^-1 x mg^-1) was about three fold higher than that of the non-producing strain (0.0179 moles S^2- x min^-1 x mg^-1). On the other hand the sulfite-producing strain had a higher K _m -value for sulfite (2×10^-3 M) and was more strongly inhibited by the substrate than the non-producing strain (6×10^-3 M).     

The incorporation of nitrogen into products of recent photosynthesis in Anabaena cylindrica Lemm.

In vivo tracer studies with ¹⁴C have been performed to help determine pathways of incorporation of newly assimilated nitrogen into N₂-fixing cells of Anabaena cylindrica. After photosynthesis in Ar:O₂:¹⁴CO₂ for 30 min, the addition of N₂ or NH ₄ ⁺ resulted in increased rates of ¹⁴CO₂-incorporation both in the light and dark, and in increased incorporation of ¹⁴C into amino acids at the expense of sucrose and sugar phosphates. Evidence of enhanced sucrose catabolism and increased pyruvate kinase activity was obtained on adding nitrogen, and, of the ¹⁴C-labelling entering the tricarboxylic acid cycle, more appeared in citrate and 2-oxoglutarate than in malate and oxaloacetate. The kinetics of ¹⁴C-incorporation into various amino acids suggest that in the light and dark the most important route of primary ammonia assimilation involves glutamine synthetase and that glutamate, aspartate, glycine and probably alanine are formed secondarily from glutamine.     

The insulin receptor family in the heart: new light on old insights

Insulin was discovered over 100 years ago. Whilst the first half century defined many of the physiological effects of insulin, the second emphasised the mechanisms by which it elicits these effects, implicating a vast array of G proteins and their regulators, lipid and protein kinases and counteracting phosphatases, and more. Potential growth-promoting and protective effects of insulin on the heart emerged from studies of carbohydrate metabolism in the 1960s, but the insulin receptors (and the related receptor for insulin-like growth factors 1 and 2) were not defined until the 1980s. A related third receptor, the insulin receptor-related receptor remained an orphan receptor for many years until it was identified as an alkali-sensor. The mechanisms by which these receptors and the plethora of downstream signalling molecules confer cardioprotection remain elusive. Here, we review important aspects of the effects of the three insulin receptor family members in the heart. Metabolic studies are set in the context of what is now known of insulin receptor family signalling and the role of protein kinase B (PKB or Akt), and the relationship between this and cardiomyocyte survival versus death is discussed. PKB/Akt phosphorylates numerous substrates with potential for cardioprotection in the contractile cardiomyocytes and cardiac non-myocytes. Our overall conclusion is that the effects of insulin on glucose metabolism that were initially identified remain highly pertinent in managing cardiomyocyte energetics and preservation of function. This alone provides a high level of cardioprotection in the face of pathophysiological stressors such as ischaemia and myocardial infarction.