Histoenzymatic characterization of sub-types of Type I fibres in fast muscles of rats

Summary The histochemical activities of succinic dehydrogenase (SDH), myofibrillar Adenosine triphosphatase (ATPase) and alpha glycerophosphate dehydrogenase were studied in serial sections of rat vastus lateralis (red) (RVL), gastrocnemius and diaphragm muscles. Three main fibre-types were distinguished. The Type I fibres of RVL and gastrocnemius muscles fell into two distinct groups: one category-Type IA showed very low ATPase activity. The second category of Type IB fibres displayed moderate ATPase reaction. The Type IA fibres were divisible into two sub-groups when tested for SDH reaction. Type IA₁ fibres possessed a homogenous distribution of diformazan·granules throughout the fibre: Type IA₂ fibres displayed characteristic moth-eaten pattern of diformazan localization. The diaphragm muscle did not show either Type IB or Type IA₂ varieties. The great majority of Type I fibres were sub-type IA₁ in the three fast muscles studied. It is also demonstrated here that an inherent heterogeneity exists between Type I fibres of diaphragm and leg muscles in regard to -GPD localization. This histochemical data emphasizes the fact that subdivision of Type I striated muscle fibres of mammalian animals into two sub-types is only approximate and that a further subcategorization is possible.     

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     

Further histochemical properties of rabbit skeletal muscle fibres

Summary The histochemical activities of succinic dehydrogenase (SDH), creatine kinase (CK), sarcoplasmic reticular ATPase (SR-ATPase) and myosin ATPase were studied in serial sections of rabbit adductor muscle. Three fibre types were distinguished depending upon the distribution of the enzyme activities. The type II white fibres posessing minimal SDH showed high myosin ATPase, SR-ATPase and ATPase dependent CK activities. Red oxidative fibres showing high SDH fell into two distinct groups: One category had mainly a peripheral localization of SDH and showed an enzymatic profile identical to that of type II white fibres. The second category of red fibres displayed both a homogeneous distribution of small diformazan granules throughout the fibre as well as a sub-sarcolemmal collection when tested for SDH activity but possessed very low amounts of reaction product of the various enzymes of the energetic metabolism studied. Since it is well established that the myosin ATPase of a fibre correlates with its contraction time, the present histochemical investigation provides further support for this concept by demonstrating the presence of high SR-ATPase and ATPase dependent CK activities in all white and red fibres rich in myosin ATPase.     

Microphotometric determination of enzyme activities in type-grouped fibres of reinnervated rat muscle

Summary Activities of succinate dehydrogenase (SDH), glycerolphosphate oxidase (GP-OX), cytochrome oxidase (CYT-OX) and lactate dehydrogenase (LDH) were determined microphotometrically in single, actomyosin-ATPase typed (Guth and Samaha 1970) fibres within cross-sections of normal and reinnervated rat tibialis anterior muscles. SDH and GP-OX activities displayed pronounced scattering and large overlaps existed between -, -, and -fibres of normal muscle. Coefficients of variation were in the range of 16–40% for GP-OX and SDH in the different fibre populations. Enzyme activity determinations in typegrouped -, -, and -fibres of reinnervated muscle showed much less scattering than in normally distributed -, -, and -fibres of control muscles. Coefficients of variation were in the range of 10–13% for SDH, GP-OX, CYT-OX and LDH. The experimental error of the kinetic microphotometric measurement of enzyme activities in situ is in the range of 10% (Reichmann and Pette 1982). Our results therefore suggest a high degree of metabolic similarity or homogeneity of typed-grouped muscle fibres and thus support the assumption that type-groped fibres are homogeneous and correspond to regularly assembled motor units.     

Evidence of regional differences in the lectin histochemistry along the ductus epididymis of the lizard,Podarcis sicula Raf

The regional difference in the carbohydrate components of the ductus epididymis epithelium of a lizard was delineated by means of 13 lectins. Basal cells expressed only N-acetylglucosamine (GlcNAc). Throughout the ductus, the secretory cells showed oligosaccharides with terminal N-acetylneuraminic acid (Neu5Ac)(2,6)galactose (Gal)/N-acetylgalactosamine (GalNAc) and internal mannose (Man) and/or glucose (Glc) in the whole cytoplasm, oligosaccharides terminating in Neu5Ac(2,6)Gal(1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, GalNAc, GlcNAc, and fucose (Fuc) in the supra-nuclear zone, and also glycans terminating in Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal(1,3)GalNAc, Gal (1,4)GlcNAc on the luminal surface. In the caput and corpus regions, the supra-nuclear cytoplasm was characterized by terminal Gal(1,4)GlcNAc and GalNAc, the luminal surface by GalNAc and Gal. The Golgi zone, showing oligosaccharides with terminal Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal (1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, and internal GlcNAc, expressed terminal Gal (1,4)GlcNAc and GalNAc in the caput, and terminal GalNAc in the corpus. The granules showed all the investigated carbohydrates in their peripheral zone except terminal GalNAc and Fuc, whereas internal Man/Glc and terminal Gal were expressed in the central core, and Fuc throughout the ductus, terminal GlcNAc in the caput and corpus, and terminal GalNAc only in the corpus.     

Histochemical study of isolated neurons in culture from chick embryo sympathetic ganglia

Summary The dissociated sympathetic neurons maintained in culture without direct contact with glia cells keep up some enzymatic activities, like those of carboxylic esterases, of succinic-dehydrogenase (SDH), glutamic-dehydrogenase (GDH), monoamine oxydase (MAO), lactic-dehydrogenase (LDH) and alcoholic dehydrogenase (ADH) for more or less long periods.Nerve growth factor (NGF) promotes the maintenance of mitochondrial enzymes, of acetylcholinesterase (AChE) and non-specific cholinesterases (n. s. ChE), of LDH in sympathetic neurons. In absence of NGF, the more archaic enzymes, like ADH, or the less specific, like -naphtol esterases still are kept up, whereas the enzymatic mitochondrial and cholinesterasic activities are no more detected.With the technical assistance of Miss E. Darcel.This communication is part of the Doctorat de Biologie Humaine thesis.     

A successive histochemical staining for succinate dehydrogenase and “reversed”-ATPase in a single section for the skeletal muscle fibre typing

Summary A procedure is described which simplifies the classification of skeletal muscle fibres in that it allows a simultaneous evaluation of both the oxidative capacity and the intensity of reversed ATPase of the fibres, and thus enables to distinguish three fibre types — SO, FOG and FG — in one tissue section. After preincubation at pH 4.1–4.2 the cryostat section is incubated for succinate dehydrogenase (SDH) and subsequently for reversed-ATPase. This is followed by the fixation with neutral buffered formaldehyde. The results of typing of chicken, minipig and rabbit fibres in a single muscle section stained with this technique are identical to those obtained with the usual method based on a comparison of serial sections of which one is stained for SDH activity the other for reversed-ATPase activity.     

The enzyme histochemistry of developing brown fat in the fetal rat

Summary Histochemically demonstrable enzymes, glycogen, and lipids were studied during prenatal histogenesis of brown fat in fetal rats. SDH, MDH, LDH, DPN and TPN diaphorases, - and -naphthyl esterases were demonstrable in brown fat cells on day 17 of gestation. Glycogen, lipid, G-6-PDH, cytochrome oxidase, indoxyl esterase and alkaline phosphatase were detected on day 17.5. The progressive increase in MDH, LDH, SDH, DPN diaphorase and cytochrome oxidase content paralleled increases in mitochondrial population during differentiation. MAO and esterase content increased only slightly during the prenatal period studied. Alkaline phosphatase appeared in morula stage cells. These were rare at 17.5 days, but increased in number until, at day 20, they were the predominant cells in the dorsal suprascapular brown fat lobules. Further maturation resulted in the loss of alkaline phosphatase which was present in only occasional cells at birth. Reactions for acid phosphatase, ATPase, naphthol-AS esterase, leucine aminopeptidase, -glutamyl transpeptidase, cholesterol and plasmalogens were negative in prenatal and neonatal brown fat cells.Supported in part by Grant No. 375, Nutrition Foundation, Inc.Presented in part at the annual meeting of the Histochemical Society, Atlantic City, N. J., April, 1969.     

Existence and uniqueness of a sharp travelling wave in degenerate non-linear diffusion Fisher-KPP equations

In this paper we use a dynamical systems approach to prove the existence of a unique critical value c ^* of the speed c for which the degenerate density-dependent diffusion equation u _ct = [D(u)u _x ] _x + g(u) has: 1. no travelling wave solutions for 0 < c < c ^*, 2. a travelling wave solution u(x, t) = (x - c ^* t) of sharp type satisfying (– ) = 1, () = 0 ^*; '(^*–) = – c ^*/D'(0), '(^*+) = 0 and 3. a continuum of travelling wave solutions of monotone decreasing front type for each c > c ^*. These fronts satisfy the boundary conditions (– ) = 1, '(– ) = (+ ) = '(+ ) = 0. We illustrate our analytical results with some numerical solutions.     

Defective remethylation of homocysteine is related to decreased synthesis of coenzymes B2 in thyroidectomized rats

Summary. We investigated the influence of hypothyroidism on homocysteine metabolism in rats, focusing on a hypothetical deficient synthesis of FAD by riboflavin kinases. Animals were allocated in control group (n=7), thyroidectomized rats (n=6), rats with diet deficient in vitamin B2, B9, B12, choline and methionine (n=7), thyroidectomized rats with deficient diet (n=9). Homocysteine was decreased in operated rats (2.6±1.01 vs. 4.05±1.0mol/L, P=0.02) and increased in deficient diet rats (29.56±4.52 vs. 4.05±1.0mol/L, P=0.001), when compared to control group. Erythrocyte-Glutathione-Reductase-Activation-Coefficient (index of FAD deficiency) was increased in thyroidectomized or deficient diet rats (P=0.004 for both). Methylenetetrahydrofolate-reductase and methionine-synthase activities were decreased in thyroidectomized rats but not in those subjected to deficient diet. Cystathionine--synthase was increased only in operated rats. Taken together, these results showed a defective re-methylation in surgical hypothyroidism, which was due in part to a defective synthesis of vitamin B2 coenzymes. This defective pathway was overcompensated by the increased Cystathionine--synthase activity.     

Decreases in Plasma Aβ1-40 Levels with Aging in Non-Demented Elderly with ApoE-ε4 Allele

This report examines plasma amyloid proteins A40 and A42 and apolipoprotein E (apoE) levels and their relationships with age in non-demented older adults with (N = 32) or without the apoE-4 allele (N = 94). A levels did not differ between the groups whereas the 4 allele was associated with a significant reduction in plasma apoE. In subjects with the 4 allele, increasing age was associated with significant reduction in plasma A40. Subjects without the 4 allele showed a significant positive correlation between A40 and A42 levels. There was also a significant correlation between plasma A40 and apoE levels in all subjects.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

Somatic hybrid plants produced by electrofusion between dihaploid potatoes: BF15 (H1), Aminca (H6) and Cardinal (H3)

In order to regenerate somatic hybrids, mesophyll protoplasts from a dihaploid potato, BF15 (H1), were electrofused with those from two other dihaploid clones, Aminca (H6) and Cardinal (H3). Determination of the ploidy level by flow cytometry showed that 10% of plants regenerated from the fusion experiment with BF15 + Aminca were diploids, 14% triploids, 63% tetraploids and very few were mixoploids or had a higher ploidy level. Using morphological markers and vigour in plant growth, we were able to recover a total of 24 somatic hybrid plants, respectively 20 and 4 hybrids (accounting for 12% and 13% of regenerants) from the fusions BF15 + Aminca and BF15 + Cardinal. Most of the somatic hybrids were at the expected tetraploid level (2n=4x=48). The hybrid nature was confirmed by examining isoenzyme patterns for malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD).     

First controlled progenies checked by isozymic markers in cultivated yams Dioscorea cayenensis-rotundata

As tested progeny have never been obtained, breeding studies on African yams (Dioscorea cayenensisrotundata) are scarce. We report here the first progenies checked by isoenzyme markers. This was made possible by the choice of well-known genitors [one male (cv Zrezrou) and three females (cvs Sopéré, Dahomey and C 20)] and special hybridization conditions. Six enzymatic systems [esterase (EST), isocitrate dehydrogenase (ICD), malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6PGD), shikimate dehydrogenase (SDH), and phosphoglucoisomerase (PGI)] were used to check the progenies and detect outbreeding. Despite the small number of progeny, it was possible to provide information on the genetics of the isoenzymatic systems.     

Further evidence for abnormal protein kinase C regulation of macromolecule secretion in fibroblasts from cystic fibrosis patients

In comparison to skin fibroblasts from normal subjects, those from patients with cystic fibrosis (CF): (1) bound [20-³H] phorbol 12,13-dibutyrate (PDBu) with a higher affinity (K_d=25.8 vs 12.8 nM respectively) but expressed a similar number of total phorbol ester binding sites (about 2.5 pmol PDBu bound/mg of protein); (2) exhibited a faster and higher response to 4-phorbol 12-myristate 13-acetate (PMA) for the stimulation of [³⁵S]-labelled glycoconjutate release, but were equally sensitive to the synergistic effect of A23187 on this process; and (3) secreted glycoconjugates with similar [³⁵S]-sulfate and [¹⁴C]-leucine to [¹⁴C]-glucosamine labelling ratios. Taken together, these results provide further evidence for abnormal protein kinase C (PKC) regulation of macromolecule secretion in CF disease.Abbreviations BSA Bovine serum albumin - DBcAMP Dibutyryl cyclic AMP - DMEM Dulbecco's modified Eagle's medium - DMSO Dimethylsulfoxide - LDH Lactate dehydrogenase - PBS Phosphate-buffered saline - PDBu 4-phorbol 12,13-dibutyrate - 4-PDD 4-phorbol 12,13-didecanoate - PMA 4-phorbol 12-myristate 13-acetate - TCA Trichloroacetic acid     

Purification and characterization of chicken brain cytosolic aspartate aminotransferase

Aspartate aminotransferase from the cytosolic fraction of chicken brain was isolated with acceptable yield and high degree of purity. The enzyme appeared in multiple molecular forms: , , , and ( predominates), as detected by polyacrylamide gel electrophoresis with specific staining. These different forms of the enzyme were separated by DEAE-Sephacel chromatography, and showed different isoelectric points and maximal velocities values, whereas their molecular weight, optimum pH and Michaelis constants were very similar. Generation process studies suggest that minors subforms of the enzyme could be raised from form by a mechanism in which the oxidation of particular amino acid groups are involved.Abbreviations used AAT aspartate aminotransferase - c-AAT cytosolic aspartate aminotransferase - IU international units - LDH Iactate dehydrogenase - MDH malate dehydrogenase - 2-ME 2-mercaptoethanol - PAGE polyacrylamide gel electrophoresis - PLP pyridoxal-5-phosphate - S.A. specific activity     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

The cytology of phytophthora infestans

Summary Phytophthora infestans has three kinds of somatic nuclei: an oval shaped nucleus (approx. 3.1×2.7 ) which stains diffusely except for a crescent shaped Feulgen positive cap which stains intensely; a granular nucleus whose contents are organized into a fairly constant number of stained bodies, and, a deeply staining condensed nucleus. The capped nucleus is thought to be metabolic or resting and the granular nucleus is thought to be dividing as it is most commonly found in hyphal tips. Attenuated forms of all three kinds of nuclei are found.Nuclear division is mitotic and intranuclear. Eight—ten chromosomes are seen at metaphase.Sporangia have a mean of 6.3 nuclei which is constant for age and strain of culture. Sporangia become multinucleate as a result of nuclear migration and not by division in the developing sporangium. Zoospores are usually uninucleate.The nuclear cap is persistent throughout nuclear division when it also divides. It is associated with flagella production and nuclear migration and has some of the properties of a blepharoplast.     

Diel Movements of Bat Rays, Myliobatis californica, in Tomales Bay, California: Evidence for Behavioral Thermoregulation?

We used ultrasonic telemetry to examine movement patterns of 11 bat rays, Myliobatis californica, in Tomales Bay, California. Tomales Bay is long (20km) and narrow (1.4km), and is hydrographically separated into outer and inner bay regions. The outer bay (the outermost 8km) is characterized by oceanic conditions while the shallow inner bay (the innermost 12km) features wide seasonal temperature shifts. Five rays were tracked monthly from October 1990 to November 1991 and six rays (four of which carried temperature-sensing transmitters) were tracked daily from 30 June to 16 July 1992. Mean bat ray movement rate was 8.84mmin^–1 (range 4.49 to 13.40mmin^–1) and was not significantly affected by size (p=0.592), tidal stage (p=0.610), or time of day (p=0.327). Movement direction was unrelated to tidal stage (p=0.472) but showed a highly significant diel pattern (p<0.001). From 2:50–14:50h, rays moved toward the warmer and shallower inner bay, while from 14:50–2:50h they moved toward the cooler and deeper outer bay. These telemetry data, along with known bat ray foraging patterns and respiratory temperature-sensitivity, argue for behavioral thermoregulation as the primary influence on this movement pattern.     

Photoreversion und Photoprotektion bei UV-induzierten Mutationen des nichtphotoreaktivierbaren Bacteriums E.coli phr?

Summary Stationary phase cells of strain phr^–/MC 2 ofE. coli are not photoreactivable but the frequency of UV-induced mutations to low Streptomycine-resistance (S 3, 3/ml) is decreased strongly by illumination with light of fluorescence tubes (310 to 500 nm) after UV-irradiation. Also dark-reversion (DRM) of these mutations due to keeping UV-irradiated cells in saline is observed. Illumination before UV-irradiation decreases the frequency of the mutations (photoprotection against mutation=PPM) to the same extent as the combined action of photoreversion (PRM) and DRM. The lag-phase of cell division is prolonged strongly by illumination from 80 min without light to 150 min by the light-dose of highest activity. The additional lag is nearly the same if the illumination is done before, after or without UV-irradiation; this lag is about additive to the small lag caused by UV. Pre-illumination of the stationary-phase cells does not cause photoprotection against killing (PP), it even decreases the survival after high UV-doses. The observations support the hypothesis that PRM in this strain may be indirect, i.e. caused by the light-induced additional division lag which enhances the dark repair of UV-premutations. Also spontaneous premutations which are apparently present in the stationary-phase cells seem to be influenced by the light in this way.