Kinetic behaviour of succinate dehydrogenase of three fibre types in skeletal muscle. I. Effects of temperature and a competitive inhibitor

Summary The kinetic behaviour of succinate dehydrogenase [EC 1.3.99.1] in three fibre types of rat gastrocnemius was examined by a quantitative histochemical method without disruption of the cellular structure. 2-(2-Benzothiazolyl)-3-(4-phthalhydrazidyl)-5-styryl-tetrazolium chloride (BPST) and phenazine methosulphate were used as electron acceptors. On measurement of the absorbance value at 530 nm of BPST formazan, produced by the succinate dehydrogenase reaction in sections, it was found that the staining intensity of succinate dehydrogenase was linearly proportional to both the incubation time and the thickness of the slice therefore, the initial velocity of the staining could be calculated. By Michaelis-Menten (1913) treatment of the dependence of the initial velocity on the substrate concentration in the absence and the presence of a competitive inhibitor, malonate, the Km andVmax values for succinate and the Ki value for malonate were obtained. The Km and Ki values of the three fibre types were similar. The ratio of theVmax values of type A, B and C fibres was 1.02.03.3. The temperature dependence of the kinetic parameters was very similar in the three fibre types. These findings confirm that the differences in the staining intensity of the three fibre types reflect differences in the amounts, but not the properties, of succinate dehydrogenase.     

Molecular species of membrane phospholipids containing arachidonic acid and linoleic acid contribute to the interindividual variability of red blood cell Na+-Li+ countertransport: In vivo and in vitro evidence

Summary Previous studies indicate a particular sensitivity of red blood cell Na⁺-Li⁺ countertransport activity to small variations in the fatty acid composition of membrane phospholipids. To assess whether the interindividual variability of Na⁺-Li⁺ countertransport is related to differences in the species pattern of erythrocyte phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vivo, the molecular species composition of PC and PE as well as the kinetics of Na⁺-Li⁺ countertransport were analyzed in parallel in normo- and hyperlipidemic donors. Both in diacyl PC and in diacyl-PE the species 160/204 and 160/182 were, respectively, positively and negatively related to the apparent maximal velocity of Na⁺-Li⁺ countertransport. The sum of all species with 204 at sn₂ of diacyl-PE exhibited a strong positive (r = 0.82, 2p < 0.001), and those containing 182 a negative correlation (r = –0.63, 2p < 0.01) to the transport activity. Essentially similar connections were observed between these species and the apparent affinity of the transport system for intracellular Na⁺. To evaluate whether the associations between molecular species of membrane phospholipids and Na⁺-Li⁺ countertransport activity were indicative of a causal relationship, the species 160/204-PC and 160/182-PC were selectively introduced into the erythrocyte membrane by means of the PC-specific transfer protein. Replacement of 11% of native PC by 160/182-PC inhibited the transport rate by about 25%. Exchange of 6 and 9% of PC with 160/204-PC, in contrast, accelerated the transport rate by 30 and 60%, respectively. The accordance between the in vivo relations and the results of the in vitro modification strongly suggests that elevations and reductions in the arachidonic acid and linoleic acid content of membrane PC and PE contribute to the interindividual variability of red blood cell Na⁺-Li⁺ counter-transport activity and its acceleration in hyperlipidemias.The authors wish to thank Dr. W.O. Richter (II. Medizinische Klinik, Klinikum Großhadern, Universität München) for selection of the patients and Dr. T. Brosche (Universität ErlangenNürnberg) for gaschromatographic analyses. This study was supported in part by a grant of the Wilhelm-Sander-Stiftung to B.E.     

Genetics of fertility restoration in hybrid rice

Summary The cross combination involving 14 male-sterile lines in rice, when crossed with different maintainers, showed fertility restoration in certain combinations. When F₂ segregating populations were classified based on spikelet fertility, fertility restoration was shown to be governed by 31, 9331, and 1231, due to allelic differences. This indicated that the cytosterility of the same group showed monogenic fertility restoration, whereas crossing plants belonging to different cystosterile groups showed a digenic pattern of segregation.     

Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres

Summary A histochemical technique was developed for the quantitative determination of succinic dehydrogenase (SDH) activity in muscle cross-sections using 1-methoxyphenazine methosulphate (mPMS) as the exogenous electron carrier, and azide as an inhibitor of cytochrome oxidase. The optimal composition of the incubation medium for the SDH reaction was determined. This histochemical procedure was compared to one using phenazine methosulphate (PMS) instead of mPMS and cyanide instead of azide. The substitution of mPMS and azide resulted in a substantial decrease in the non-specific reduction of nitroblue tetrazolium (NBT; the reaction indicator), i.e., nothing dehydrogenase activity. With mPMS and azide in the reaction medium, the production of NBT formazan was linear for at least 9 min during the enzymic reaction. This compared to a non-linear reduction of NBT during the initial stages of the reactions (SDH and nothing dehydrogenase) when using PMS and cyanide. The use of both mPMS and azide also eliminated the production of NBT monoformazan which occurred with PMS and cyanide. This procedure was shown to meet various criteria established for the quantification of histochemical reactions.     

Isozymic patterns and functional states of cell lines ofDrosophila melanogaster cultured in vitro. II

Summary Our previous isoenzyme investigation ofDrosophila melanogaster cell lines in vitro has been completed with twelve further enzyme systems. The enzyme profiles seem to be in good agreement with a previous hypothesis concerning the precise origin of these cell lines (probably from imaginal discs or nervous tissues). Our results have been summarized with reference to the biochemical genetic map ofDrosophila melanogaster in order to consider a possible functional organization of the genome.Abbreviations NAD nicotine adenine nucleotide - NADP nicotine adenine nucleotide phosphate - NBT nitroblue tetrazolium - PMS phenazine methosulfate - EDTA ethylene diamine tetraacetic acid - GOT Glutamate-oxaloacetate transaminase - PGK Phosphoglycerate kinase - GPDH -glycerophosphate dehydrogenase - MDH Malate dehydrogenase - PGM Phosphoglucomutase - Aph Alkaline phosphatase - MDH-NADP Malic enzyme - Lap Leucine Amino-Peptidase - LDH Lactate dehydrogenase - -1-OHDH L-3-hydroxyacid dehydrogenase - ADH Alcohol dehydrogenase - Aldox Aldehyde oxydase - 6PGD 6 Phosphogluconate dehydrogenase - G6PD Glucose-6-Phosphate dehydrogenase - Hex3 Fructokinase - IDH Isocitrate dehydrogenase - Est 6 Esterase 6 - Est C Esterase C - ODH Octanol dehydrogenase - XDH Xanthine dehydrogenase - AcPh Acid Phosphatase 1     

Succinic dehydrogenase activity in liver,kidney and brain of rat

Summary A method is described for histochemical quantification of the activity of succinic dehydrogenase in various tissues of rat by means of Nitroblue tetrazolium. This method can be used for comparison of enzyme activities; the activities calculated correspond to values obtained by biochemical methods. The necessity to quantify the nothing dehydrogenase is established as well as the amount of half-formazan.Accepted as doctoral dissertation by the Faculty of Medicine, Westfälische Wilhelms-University Münster     

Malarial dihydroorotate dehydrogenase mediates superoxide radical production.

Dihydroorotate dehydrogenase purified from mitochondria of Plasmodium berghei, a rodent malaria parasite, mediates production of superoxide radical during oxidation of dihydroorotate to orotate. Reduction of dichlorophenolindophenol or cytochrome c or nitroblue tetrazolium was significantly inhibited by superoxide dismutase or theonyltrifluoroacetone, a specific iron chelator of the enzyme. These results, together with the recent evidence of manganese-superoxide dismutase activity in malarial mitochondria [Ranz, A., and Meshnick, S.R. (1989) Exp. Parasitol. 69, 125-128], suggest that the production of superoxide radical may occur in vivo.     

Isolation of mutants ofLactobacillus bacterias,grown in skim milk,that resist fatty acid inhibition

The effect of saturated fatty acids from 60 to 160 and oleic acid was determined onLactobacillus leichmannii growing in skim milk. The growth of this strain was markedly inhibited by fatty acids from 80 to 120 but not by straight chain fatty acids greater than 130 or less than 70 and oleate. Laurate was the fatty acid with the highest bactericidal effect. Similar results, with little changes depending on strains, were obtained withL. casei, L. plantarum, L. bulgaricus, L. lactis, L. helveticus. Mutants from theseLactobacillus organisms, resistant to fatty acid inhibition, were isolated by a recycling selection procedure. These mutants exhibited high levels of oxidation for laurate. The presence of 2 mM of this compound in the skim milk culture increased the fatty acid oxidation activity four to ten times higher than was exhibited by the parent strains. The practical implications of these observations are discussed.     

Brain-gonad axis and photoperiodically-stimulated sexual maturation in the slug,Limax maximus

Summary Physiological and endocrine mechanisms mediating long-day (LD 168) triggered sexual maturation were studied in the terrestrial slug,Limax maximus. Our findings were: (1) Maturation was induced in immature slugs seeing only short days (LD 816) after implantation of whole brains from maturing donors, but no development was found in sham-operated control slugs or in animals receiving implants of muscle from mature donors (Table 1). (2) Removal of the optic tentacles did not block maturation in LD 168 or promote maturation in LD 816 (Fig. 1). (3) Gonadectomy (castration) abolished penis development in 9 of 11 slugs exposed to LD 168 for periods of up to 31 weeks (Table 2). The results are consistent with a model forLimax reproductive tract development in which the perception of long days by extraocular receptors results in the secretion of a maturation hormone by the brain followed by the production of a separate male-phase sex hormone by the developing gonad.Abbreviations ASO accessory sex organs - DB dorsal bodies - FPSH female-phase sex hormone - MH maturation hormone - MPSH male-phase sex hormone This work was supported in part by grants from NSF (BNS 78-01408) and NIH (MH 27948) to P.G.S. The assistance of Mr. T.M. Gordon, Mr. J.L. Broyles, and Mr. M. Bullock is gratefully acknowledged.     

Interaction of free fatty acids with the erythrocyte membrane as affected by hyperthermia and ionizing radiation

The interference of hyperthermia and ionizing radiation, respectively, with the effects of capric (100), lauric (120), myristic (140), oleic (cis-181) and elaidic (trans-181) acids on the osmotic resistance of human erythrocytes was investigated. The results are summarized as follows: (A) not only at 37°, but also at 42° and 47°C lauric acid (120) represents the minimum chain length for the biphasic behaviour of protecting against hypotonic hemolysis at a certain lower concentration range and hemolysis promotion at subsequent higher concentrations; (B) with increasing temperatures the protecting as well as the hemolytic effects occur at lower concentrations of the fatty acids; (C) the increase of temperature promotes the extent of hemolysis and reduces the extent of protection against hypotonic hemolysis; (D) Gamma-irradiation of erythrocytes selectively affects the concentration of oleic acid at which maximum protection against hypotonic hemolysis occurs, without altering the minimum concentration for 100% hemolysis.     

Bacterial regeneration of ammonium and phosphate as affected by the carbon:nitrogen:phosphorus ratio of organic substrates

The effect of carbonnitrogenphosphorus (CNP) ratio of organic substrates on the regeneration of ammonium and phosphate was investigated by growing natural assemblages of freshwater bacteria in mineral media supplemented with the simple organic C, N, and P sources (glucose, asparagine, and sodium glycerophosphate, respectively) to give 25 different substrate CNP ratios. Both ammonium and phosphate were regenerated when CN and NP atomic ratios of organic substrates were 101 and 161, respectively. Only ammonium was regenerated when CN and NP ratios were 101 and 10–201, respectively. On the other hand, neither ammonium nor phosphate was regenerated when CN and NP ratios were 151 and 51, respectively. In no case was phosphate alone regenerated. As bacteria were able to alter widely the CNP ratio of their biomass, the growth yield of bacteria appeared primarily dependent on the substrate carbon concentration, irrespective of a wide variation in the substrate CNP ratio.     

The circadian rhythm of intra-acinar profiles of alcohol dehydrogenase activity in rat liver: a microquantitative study

Summary Using microquantitative measurements of alcohol dehydrogenase activity in microdissected samples of liver tissue along the sinusoidal length, the intra-acinar distribution profiles were studied in seven groups of female rats at different times during 24h with a light phase from 630h to 1830h. The mean values of alcohol dehydrogenase activity showed a circadian rhythm with a minimum at 13.30h and a maximum at 17.30h (p<0.0001). However, the intra-acinar gradients remained almost unchanged, indicating that increase and decrease in enzyme activity takes place simultaneously in all parts of the liver acinus. This observation, together with data from the literature, suggests that the circadian rhythm of alcohol dehydrogenase activity reflects variations in different liver cell consituents, rather than enzyme protein synthesis or proteolysis.     

A tetrazolium technique for the histochemical localization of ATP: Creatine phosphotransferase

Summary A tetrazolium technique is presented that permits the study of ATP: Creatine phosphotransferase, or creatine kinase, in fixed skeletal muscle tissue sections, within the limits imposed by the properties of the chosen ditetrazole, nitro blue tatrazolium. There is a variation in creatine kinase activity between the muscle fibres. Those with high creatine kinase activity also have high succinate dehydrogenase activity.List of Abbreviations ADP Adenosine-5-diphosphate - ATP adenosine-5-triphosphate - CK creatine kinase - G-6-P glucose-6-phosphate - G-6-P-DH glucose-6-phosphate dehydrogenase - HK hexokinase - NADP nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PMS phenazine methosulphate - SDH succinate dehydrogenase     

Comparative analysis of glycoprotein and glycolipid composition of virulent and avirulent strain membranes ofMycoplasma hyopneumoniae

The glycoproteins and glycolipids from membranes of virulent strain Z and avirulent strain M ofMycoplasma hyopneumoniae have been compared. The proteins and the glycoproteins were identified by SDS-polyacrylamide gel electrophoresis and concanavalin A-biotin labeling, respectively. The membrane preparation contained approximately 34 protein bands with molecular weights between 20 KD and 100 KD. The concanavalin A-biotin system reacted with a glycoprotein of a molecular weight of approximately 28,000 from avirulent strain M and did not react with the correspondent band from virulent strain Z. The membrane glycolipids of both strains consisted of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and the percentages of 160, 180, and 181 fatty acids comprised more than 80% of the total fatty acids of membrane glycolipids. The 180 fatty acid of MGDG in avirulent strain M was twofold higher than that of virulent strain Z.     

Disjunction types and their frequencies in two heterozygous reciprocal translocations of Blattella germanica (L.)

Adjacent-1, alternate-1, adjacent-2, and alternate-2 disjunction configurations were observed cytologically at metaphase I in heterozygotes from two reciprocal chromosome translocations in the German cockroach, Blattella germanica (L.). T(7; 12) shows random disjunction and the frequencies of the above four types were in a ratio of 2112 (p>0.90). T(3; 12) has directed disjunction (about 70% alternate) which is attributable to a heavy preponderance of alternate-2. No interpretable ratio occurs here except for the equality of alternate-1 and adjacent-2 types. These observations confirm the existence of two types of alternate disjunction, and provide insight into the basis for random vs directed disjunction.     

Crcadian pacemaker in theBursatella eye: Properties of the rhythm and its effect on locomotor behavior

Summary The eye of the frilled sea hare,Bursatella leachi plei, expresses a circadian rhythm in the frequency of spontaneously occurring optic nerve impulses. The rhythm will free-run for at least 3 cycles in vitro (Fig. 2) and can be entrained by light cycles provided in vivo (Fig. 4 A). While bothBursatella andAplysia eyes contain circadian pacemakers the two rhythms differ in several respects: (1) the peak impulse frequency forBursatella eyes is only 96/h (±36 SD) compared with 247/h (±61 SD) forAplysia. (2) The ocular waveform of theBursatella rhythm exhibits a steep rise and fall from peak frequencies and lacks the delayed falling phase which creates a shoulder on the ocular waveform inAplysia (Fig. 2). (3) The in vitro free-running period of theBursatella ocular rhythm is 21.2 h (±0.6 SD) compared with 24.3 h (±0.9 SD) for theAplysia rhythm (Fig. 2). (4) The steady state phase angle for entrainment differs withBursatella eyes showing a median activity peak at +3 Z.T. compared with a medianAplysia peak at –1 Z.T. (Fig. 4).We also investigated the locomotor rhythm.Bursatella were found to be predominantly diurnal when exposed to LD, 1212 (Fig. 5A) and to exhibit anticipatory locomotor activity when maintained on LD), 915 (Fig. 6). The eyes appear to play a minor role, if any, in timing the locomotor rhythm. EyelessBursatella remained diurnal on LD, 915 and most animals continued to exhibit anticipatory behavior (Fig. 6). These results suggest that theBursatella eye plays a less prominent role than theAplysia eye in controlling locomotor behavior.Abbreviations DD constant darkness - LD 1212 24 h light cycles 12 h light, 12 h dark - EST Eastern Standard Time - Z.T. Zeitgeber Time We would like to thank L. Baird, W. Kilmartin and S. Wallace for help with animal maintenance, data presentation and photography. We also thank T. Breeden for our computer programs. This work was supported by NIH grant NS-15264 to G. Block.     

Edge preference of retinal and tectal neurons in common toads (Bufo bufo) in response to worm-like moving stripes: the question of behaviorally relevant ‘position indicators’

Summary Previous experiments have shown that during prey-catching behavior (orienting, snapping) in response to a worm-like moving stripe common toads.Bufo bufo (L.) exhibit a contrast-and direction-dependent edge preference. To a black (b) stripe moving against a white (w) background (b/w), they respond (R^*) preferably toward the leading (l) rather the trailing (t) edge (R _l ^* > R _t ^* ), thus displaying head preference. If the contrastdirection is reversed (w/b), the stripe's trailing edge is preferred (R _l ^* < R _t ^* ), hence showing tail preference. In the present study, neuronal activities of retinal classes R2 and R3 and tectal classes T5(2) and T7 have been extracellularly recorded in response to leading and trailing edges of a 3 ° × 30 ° stripe simulating a worm and traversing the centers of their excitatory receptive fields (ERF) horizontally at a constant angular velocity in variable movement direction (temporo-nasal or naso-temporal).The behavioral contrast-direction dependent edge preferences are best resembled by the responses (R) of prey-selective class T5(2) neurons (R_l R_t=101 for b/w, 0.31 for w/b) and T7 neurons (R_lR_t=61 for b/w, 0.41 for w/b); the T7 responses may be dendritic spikes. This property can be traced back to off-responses dominated retinal class R3 neurons (R_lR_t=61 for b/w, 0.51 for w/b), but not to class R2 (R_lR_t =1.21 for b/w and 0.91 for w/b). The respective edge preference phenomena are independent of the direction of movement.When stimuli were moved against a stationary black-white structured background, the head preference to the black stripe and the tail preference to the white stripe were maintained in class R3, T5(2), and T7 neurons. If the stripe traversed the ERF together with the structured background in the same direction at the same velocity, the responses of tectal class T5(2) and T7 neurons were strongly inhibited, particularly in the former. Responses of retinal R2 neurons in comparable situations could be reduced by about 50%, while class R3 neurons responded to both the stimulus and the moving background structure.The results support the concept that the prey feature analyzing system in toads applies principles of (i) parallel and (ii) hierarchial information processing. These are (i) divergence of retinal R3 neuronal output contributes to stimulus edge positioning and (in combination with R2 output) area evaluation intectal neurons and to stimulus area evaluation and (in combination with R4 output) sensitivity for moving background structures inpre tectal neurons; (ii) convergence of tectal excitatory and pretectal inhibitory inputs specify the property of prey-selective tectal T5(2) neurons which are known to project to bulbar/spinal motor systems.Abbreviations ERF excitatory receptive field - IRF inhibitory receptive field - N nasal - T temporal - R _w response to a worm-like stripe moving in the direction of its longer axis - R _A response to an antiworm-like stripe whose longer axis is oriented perpendicular to the direction of movement - R _l response to the leading edge of a worm-like moving stripe - R _t response to the trailing edge of a worm-like moving stripe - b/w black stimulus against a white background - w/b white stimulus against a black background - sm structured moving background - ss structured stationary background - u minimal structure width of a structured background consisting of rectangular black and white patches in random distribution - HRP horseradish peroxidase     

Cellular fatty acid composition of six fastidious,gramnegative, xylem-limited bacteria from plants

Composition of cellular fatty acids was determined for strains of fastidious, Gramnegative, xylem-limited bacteria causing or associated with Pierce's disease of grapevine, phony disease of peach, plum leaf scorch, stunt of ragweed, elm leaf scorch, and periwinkle wilt. The most abundant fatty acids were straight-chain 150, 160, 170, and 180, unsaturated 161, 181, and unsaturated 17-and 19=carbon homologs. Minor fatty acids included straightchain 120, 140, 190, and 200; an unsaturated 15-carbon homolog; hydroxy-substituted 2-OH 120, 3-OH 120, and 3-OH 140; and branched chain iso-140 and iso-200. Cyclopropane acids were not detected. Physiological age had no effect on fatty acid composition. Class analysis of data indicated relative uniformity within the group. Saturated even-carbon chains comprised 31%–42%, unsaturated acids 41%–52%, saturated odd-carbon chains 10%–18%, hydroxysubstituted chains 2%–7%, and branched-chains 1%–4% of total fatty acids. The ratio of saturated-unsaturated acids ranged from 0.8 to 1.2.     

Observations on the use of Nitro Blue tetrazolium in the detection of early myocardial changes

Synopsis Reserpine-induced cardiomyopathy in guineapigs was studied by means of histochemical dehydrogenase reactions using succinate as substrate and tetrazolium salts as electron acceptors. Succinate: Nitro Blue tetrazolium oxidoreductase activity in the ventricular muscle of reserpine-treated animals was localized cytochemically in a blue granular pattern instead of the purple myofibrillar pattern typical of the normal myocardium. A series of experiments designed to investigate their mechanism showed that the blue granular pattern is a physicochemical sequel secondary to the fatty deposition in the disordered myocardial cells and, moreover, the preferential deposition of the blue dinitroformazan is not indicative of augmented succinate: Nitro Blue tetrazolium oxidoreductase activity. It is concluded that the histochemical reaction for this enzyme is a very sensitive index of myocardial alterations associated with an early deposition of fat, not easily detectable by organotropic dye techniques.     

Generation of succinyl-coenzyme A in photosynthetic bacteria

Pathways of succinyl-Coenzyme A (succinyl-CoA) formation in various photosynthetic bacteria were investigated through several approaches, including determination of activity levels of relevant enzymes. Extracts of photosynthetically grown cells of representative Rhodospirillaceae and Chromatium vinosum showed -ketoglutarate dehydrogenase (KGD) activities sufficient to account for generation of the succinyl-CoA required for biosynthetic metabolism. Except as noted below, the observed ratios of fumarate reductase/succinate dehydrogenase activities were low, consistent with the conclusion that these organisms produce succinyl-CoA oxidatively from -ketoglutarate (KG), rather than by reductive metabolism of fumarate. On the other hand, the green bacterium Chlorobium limicola appears to produce succinyl-CoA by the reductive pathway; in this organism, KGD activity could not be detected, and a high fumarate reductase/succinate dehydrogenase ratio was observed. Results obtained with Rhodopseudomonas gelatinosa suggest that this otherwise typical member of the Rhodospirillaceae may be able to generate succinyl-CoA via both arms of the citric acid cycle, that is, oxidatively from KG, and reductively from fumarate. To further explore the several physiological roles of the conversion: KGsuccinyl-CoA in Rhodopseudomonas capsulata, a mutant (strain KGD 11) almost completely blocked in KGD activity was isolated and studied in detail. Under anaerobic photosynthetic conditions, KGD 11 grows readily on succinate as the sole carbon source; in contrast to the wild type parent, however, it cannot grow with l-glutamate as the source of carbon. The R. capsulata parental strain can grow in darkness as an aerobic heterotroph on various carbon/energy sources including pyruvate, D,L-malate, or succinate. Mutant KGD 11, however, is unable to grow aerobically on the substrates noted. These results indicate that the energy for aerobic dark growth of R. capsulata is provided by respiratory phosphorylation fueled by citric acid cycle function, and that this requires a substantial level of KGD activity. The present findings also indicate that citric acid cycle sequences in most of the Rhodospirillaceae prominently used in current research are geared to operate in the oxidative direction, as in nonphotosynthetic aerobic heterotrophs.Abbreviations CoA coenzyme A - FR fumarate reductase - KG -ketoglutarate - KGD -ketoglutarate dehydrogenase - SD succinate dehydrogenase