The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (), fermentation rate (v) or respiration rate () of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show K _i (ethanol inhibition constant) values for , v and , higher (K _i>1 M) than those of petite mutants or grande strains grown in anaerobiosis (K _i=0.7 M). In addition, the relationship between or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to petite mutants, the resulting strain shows K _i values similar to those of the grande strain and the inhibition of and v by increasing ethanol concentrations becomes linear.     

Endogenous phosphorylation of rat brain synaptosomal plasma membranes in vitro: Some methodological aspects

The time course of endogenous phosphorylation in vitro of total or separted synaptic plasma membrane proteins (SPM) has been correlated with that of hydrolysis of the phosphate donor (ATP) in the incubation medium. The ATP/SPM ratio in the medium was varied. In a low-ratio medium (7.5 M ATP; 2.2 g SPM/l) a complete hydrolysis of ATP occurred almost instantaneously as was measured by the release of free phosphate in and the disappearance of ATP from the medium. As a consequence, only a very short peak of phosphorylation, followed by dephosphorylation was observed. However, when higher ATP/SPM ratios were used (200 M ATP; 0.4 g SPM/l and 500 M ATP; 0.4 g SPM/l), the incorporation of phosphate into SPM proteins was linear for 20 sec, and the maximum level of phosphate incorporation was increased. Similar results were obtained after separation of³²P-labeled phosphoproteins by slab gel electrophoresis. However, analysis of the autoradiographs obtained fromone SPM preparation under different ATP/SPM ratios revealed dependence of phosphorylation of individual protein bands on the conditions used.     

Oocysts of Isospora ernsti n. sp. and Isospora blagburni n. sp. (Apicomplexa: Eimeriidae) from the black-capped bulbul,Pycnonotus xanthopygos

Oocysts of Isospora ernsti n. sp. and Isospora blagburni n. sp. are described from the black-capped bulbul Pycnonotus xanthopygos from Lincoln Park Zoo, Chicago, Illinois. The bird came from southwestern Africa seven years earlier. I. ernsti oocysts are ellipsoidal to bluntly ovoid, 28–38 × 23–31m (mean 34 × 28 m) and have a single-layered oocyst wall. Micropyle, oocyst residuum and polar granules are absent. Sporocysts are elongate ovoid, 24–30 × 11–16 m (mean 27×13 m). Stieda and substiedal bodies and sporocyst residuum are present. I. blagburni oocysts are spherical to subspherical. 21–28 × 19–26 m (mean 25 × 23 m) and have a single oocyst wall. Sporocysts are ovoid and 17–23 × 10–13 m (mean 20 × 12 m). Stieda and substiedal bodies and sporocyst residuum are present.     

Effects of cycloheximide on preprophase bands and prophase spindles in onion (Allium cepa L.) root tip cells

Summary Effects of cycloheximide (CHM) on preprophase bands (PPBs) of microtubules (MTs) and on prophase spindle MTs in root tip cells of onion (Allium cepa L.) were examined. When root tip cells were treated with 36 M CHM for 0.5–4 h, the population of cells with a PPB did not decrease markedly although the population of mitotic cells and that of prophase cells with a PPB gradually decreased to half of the control root tips. In prophase cells treated with 11 and 36 M CHM for 2 h, the width of the PPB was 1.4 times broader than that in the prophase PPB without CHM. Electron microscopic observation on the cross section of the PPB showed that the number of MTs and the distance between adjacent MTs in prophase PPBs treated with CHM were similar to those in the early developmental stage of PPBs without CHM. The bipolar spindle, that appeared in late prophase was not seen in prophase cells treated with 11 M or higher concentrations of CHM for 2 h. In order to examine differences of perinuclear MT arrangement between CHM treated and non-treated prophase cells, arrangement of perinuclear MTs was examined by confocal laser scanning microscopy. In control cells without CHM, MTs appeared on the nuclear surface with several branched or cross over type MT foci in the cytoplasm when broad PPB formation started. These MT foci were replaced by the aster type MT foci, from which several MTs radiated along the nuclear surface. The aster type MT foci gradually gathered to form a bipolar spindle. MTs connecting the spindle pole region and the PPB were seen in late prophase. In CHM-treated cells (11-360 M for 2 h), branched and cross over type MT foci were prominent, even in prophase cells with well condensed chromosomes. Neither linkages of MTs between the spindle pole region and the PPB nor aster type MT foci were seen. These observations showed that CHM prevents the bundling of MTs in the PPB and also inhibits the formation of aster type MT foci that is essential for bipolar spindle development.     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

Fine structure of early previtellogenic oocytes in Mugil (Liza) auratus Risso, 1810 (Teleostei,Mugilidae)

Summary Sequential cytological events at the onset of previtellogenesis were studied in oocytes from 12 m to 70 m in diameter of golden grey mullet. The main cytological changes observed (increase in size of cell, nucleus, nucleolus and increase in number of nucleoli, RNP particles, nuage and mitochondria) provide evidence for important synthetic processes in an early preparatory phase of oocyte development.Somatic cells (pre-follicle, follicle and thecal) are also described.     

The Opioidergic System in the Combined Regulation of Pain and Immunity

Opioidergic mechanisms are involved in responses to nociceptive and antigenic stimuli at all levels and stages (from peripheral nociceptors to the cerebral cortex and from the precursors of immunocompetent cells to mature effector cells). In most experimental and clinical studies, opioid-mediated analgesia proved to be accompanied by immunosuppression. Opioid receptors of , , and types are involved in the mechanisms of combined regulation of pain and immunity, with and receptors suppressing the immune response and receptors enhancing it. By modifying the chemical structure of opioid ligands, it is possible to preserve the analgesic effect and avoid the development of immunosuppression. The opioidergic mechanisms are coupled with nonopioid peptidergic and nonpeptide systems of pain and immunity regulation.     

Regional specialization of the sperm membrane in the tick Amblyomma hebraeum koch (Acari: Ixodidae)

Summary The spermatozoon of Amblyomma hebraeum is about 200 m long and comprises: (1) a thick, club-shaped anterior part, about 20 m long bearing at its apex a tactile hemisphere, and (2) an elongated tail-like part, about 180 m long. The surface of the tactile hemisphere is covered by numerous bulbous expansions, attached to it by short stalks. The base of the hemisphere is surrounded by a fringe of thin motile processes; the remaining surface of the spermatozoon is covered with long cellular processes which run more or less parallel to one another.The membrane-associated particles found on the membrane beneath the cellular processes are regularly arranged as groups of parallel strands. The external surface of the so-called peripheral granules, as revealed by freeze-etching, is smooth with a very small number of particles. Internally the particles exhibit a regular hexagonal pattern which has not been observed, so far, on any other membrane of these sperm cells.The regional specialization of the spermatozoon surface membrane in relation to sperm motility is discussed. The results obtained indicate that processes of three types: (1) bulbous expansions, (2) motile processes, and (3) cellular processes are regional specializations, all engaged in aspects of sperm motility.The technical assistance of Mr. R. Haemmerle of Balzers Research Laboratories, Mrs. F. Seif and Miss C. Pugin, is gratefully acknowledged     

The use of antibiotics in the culture of non-sterile plant protoplasts

Summary The use of antibiotics to control infections in cultures of protoplasts of leaf mesophyll cells has been examined. The antifungal agents nystatin and amphotericin B were non-toxic to protoplasts at concentrations that controlled fungal growth (25 units and 2.5 g/ml respectively). Of the antibacterial agents examined, only carbenicillin and, to a lesser extent, gentamicin were active against the bacteria usually encountered whilst still permitting normal protoplast metabolism and regeneration. The most satisfactory control of contaminating microorganisms was obtained with a combination of nystatin (25 units/ml) or amphotericin B (2.5 g/ml) and carbenicillin (250 g/ml).     

Inhibition of tumor growth in mice treated with synthetic muramyl dipeptide

Summary Treatment with synthetic MDP inhibited growth of transplantable, chemically induced tumors in syngeneic mice. The tumor-inhibitory effect was dependent on the schedule of MDP administration.Growth of SC transplants of a nonmetastasizing, MC-induced fibrosarcoma, MC11, was inhibited by local treatment with 200 g and 1,000 g MDP given SC 5–7 weeks before challenge. Treatment with lower (10 g and 100 g) doses of MDP and shorter (1–4 weeks) time intervals was not effective. Single doses of MDP (10–1,000 g) 1–3 weeks after challenge had no effect.Growth of IV-inoculated, metastasizing AAT-induced hepatoma A was inhibited by IV injections of 20 g MDP given 1 and 2 days prior to the challenge. Significant increases in the survival of hepatoma-bearing mice were observed only after injections of MDP incorporated in multilamellar liposomes.Abbreviations MDP n-acetylmuramyl-l-alanyl-d-isoglutamine - B10 C57BL/10ScSnPh mice - MC 3-methylcholanthrene - ATT o-amino-azotoluene - PBS phosphate-buffered saline     

Effect of aldosterone on incorporation of amino acids into renal medullary proteins

Summary Studies on the effects of pretreatment with aldosterone on the incorporation of³H leucine or³H methionine into proteins in renal slices were carried out in Joklik-modified minimal essential medium. Administration of aldosterone (2 g/100 g body wt) to adrenalectomized rats increased³H leucine incorporation into trichloroacetic acid insoluble fractions of crude homogenates of cortical slices by 15.5±0.4% and of medullary slices by 53.5±1.3%. No increase in isotope incorporation was observed in slices of renal papilla or spleen prepared from the same rats. Aldosterone had no effect on the³H-leucine content of the trichloroacetic acid-soluble fractions of all three renal zones and the spleen. The dose of aldosterone that elicited a half-maximal increase in³H-methionine incorporation into proteins of renal medullary slices (0.45 g of aldosterone/100 g body wt) was indistinguishable from that needed to elicit a halfmaximal increase in the urinary K⁺/Na⁺ ratio (0.35 g of aldosterone/100 g body wt). Dexamethasone, a potent glucocorticoid, at a dose of 0.8 g/100 g body wt did not augment³H-leucine incorporation into renal medullary proteins but was effective at 8 g/100 g body wt. Spirolactone (SC-26304), a potent anti-mineralocorticoid, abolished the effect of aldosterone on amino acid incorporation into medullary proteins when administered at a 100-fold higher dosage [i.e., 80 gvs. 0.8 g (per 100 g body wt)]. These results imply that the action of aldosterone on amino acid incorporation is mediated by the mineralocorticoid rather than the glucocorticoid pathway, presumably the mineralocorticoid receptors. Moreover, pretreatment of the rats with actinomycin D (70–80 g/100 g body wt) erased the effect of aldosterone (0.8 g/100 g body wt) on amino acid incorporation into medullary proteins.In paired experiments with³H and³⁵S methionine, aldosterone (0.8 g/100 g body wt) increased methionine incorporation into trichloroacetic acid precipitable proteins of subcellular fractions of the renal medulla. The effect of aldosterone on incorporation of methionine into medullary cytosol proteins was analyzed further by polyacrylamide gel electrophoresis at pH 8.3 in tris-glycine buffer. The gel profiles indicate that aldosterone significantly increased methionine incorporation into at least one protein (independent of the isotope) with a molecular weight of 31,000. This increase was inhibited by either pretreatment of the rat with actinomycin D (70–80 g/100 g body wt or SC-26304 (80 g/100 g body wt). Dexamethasone (0.8 g/100 g body wt) did not increase incorporation of methionine into the medullary cytosol proteins resolved by polyacrylamide gel electrophoresis.     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric plexus of the guinea-pig

Glial cells of the myenteric plexus from guinea pig small intestine were intracellulary filled with horseradish peroxidase (HRP), and histochemically stained. Camera lucida-like drawings of twenty cells were morphologically and morphometrically analyzed. The cells have very small ellipsoid, somata (85±0.7 m equivalent diameter, i.e., about 330 m³ volume), and send up to 20 thin and short processes (less than 26 to about 110 m in length). The morphology of the cells appears to depend on their location within the plexus. Glial cells located within the ganglia are similar to CNS protoplasmic astrocytes; they are star-shaped, and their very short processes are irregularly, branched. In contrast, glial cells within the interganglionic fiber tracts resemble CNS fibrous astrocytes. They extend longer processes that are parallel to the fiber tracts, and show less tendency to branch. We propose that the morphology of enteric glia is determined by the structure of the microenvironment. Both cell types form several flat endfeet at a basal lamina either surrounding blood vessels or at the ganglionic border. Furthermore, the occurrence of holes in the glial cell processes suggests that particular neuronal cell processes may be enwrapped in a specific manner. Fractal analysis of camera lucida-like drawings of the cells showed that the cells have a highly complex surface structure, comparable to that of protoplasmic astrocytes in the brain. These tiny cells may possess a membrane surface area of 2000 m², almost 90% of which are contributed by the cell processes. This geometry may enable an intense exchange of metabolites and ions between neurons, glial cells, and the capillaries and/or environment of enteric ganglia.     

α-tocopherol inhibits oxidative stress induced by cholestanetriol and 25-hydroxycholesterol in porcine ovarian granulosa cells

The cytotoxicity of oxysterols including 7-ketocholesterol, -epoxide, cholestanetriol and 25-hydroxycholesterol and the possible protecting effect of -tocopherol on cholestanetriol and 25-hydroxycholesterol-induced cytotoxicity were investigated in primary cultures of porcine ovarian granulosa cells. Cell viability as determined by % trypan blue staining and mitochondrial function as determined using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) reduction were decreased significantly after 24 h exposure to 2.5 M -epoxide, cholestanetriol and 25-hydroxycholesterol. 7-ketocholesterol (2.5 M) did not affect cell viability or mitochondrial function under the same culture conditions. The specific activities of catalase and superoxide dismutase, two antioxidant defense enzymes were increased significantly (p < 0.01) following 24 h exposure to 2.5 M concentrations of cholestanetriol while only superoxide dismutase was increased in 25-hydroxycholesterol-treated cells (p < 0.001). Specific activity of glutathione peroxidase was unchanged relative to control cells. Levels of thiobarbituric acid reactive substances remained unchanged after exposure to 7-ketocholesterol, -epoxide, cholestanetriol, 25-hydroxycholesterol and cholesterol. Administration of 1 M -tocopherol to the culture medium significantly improved cell viability and restored both superoxide dismutase and catalase activities to control levels in cholestanetriol -treated cells and only superoxide dismutase in 25-hydroxycholesterol-treated cells. These studies suggest that the cytotoxic nature of physiologically relevant concentrations of cholestanetriol and 25-hydroxycholesterol in granulosa cells is in part due to oxidative stress, but it may be reduced in the presence of a-tocopherol.     

Guanylyl Cyclase Participates in the Mechanism of Glutamatergic Modulation of Acetylcholine Non-Quantum Release in the Rat Neuromuscular Junction

It was shown that glutamate (10 M to 1 M) suppresses in a dose-dependent manner the non-quantum release of acetylcholine from rat motor nerve endings; the release intensity was estimated by the H effect. The action of glutamate was completely eliminated by the blockade of guanylyl cyclase by 1 M ODQ. An increase in the intracellular cGMP concentration by 1 M dibutyryl-cGMP reduced the H effect in a similar manner as glutamate did.     

The initiation of somatic embryos and adventitious roots from developing zygotic embryo explants of Cercis canadensis L. cultured in vitro

Zygotic embryo explants of Cercis canadensis L. cultured in vitro responded to 1 and 5 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 50 M -naphthaleneacetic acid (NAA) by initiating somatic embryos and adventitious roots. Somatic embryos and adventitous roots were formed from developing zygotic embryos, while fully developed embryos collected from mature seed initiated only adventitious roots. Following 2,4-D application, the number of somatic embryos decreased while adventitious roots increased with increasing developmental age of the explant. The greatest number of somatic embryos were initiated with a 5 to 20 day exposure to 5 M 2,4-D from zygotic embryos collected between 75 and 82 days post-anthesis in 1987. Somatic embryos formed directly from epidermal and subepidermal cells, while adventitious roots developed from interior cortex cells. Normal somatic embryos were recovered after a 20 day exposure to 5 M 2,4-D and acclimated to greenhouse conditions.     

Micropropagation and ecorestoration of Vanda spathulata,an exquisite orchid

Node explants collected from flowering plants of Vanda spathulata, an endemic and exquisite orchid of Peninsular India and Sri Lanka, were cultured in Mitra medium with combinations of 4.4–88.8 m 6-benzyl adenine (BA) and 0.0–114.2 m indole-3-acetic acid (IAA). Combinations of 44.4 m BA with 17.1 or 28.5 m IAA and 66.6 mM BA with 28.5 or 40.0 m IAA induced maximum formation of 12.6 and 12.1 shoots / node, respectively, in a 6-month period. Subcultured nodal explants produced maximum of 6.1 shoots at combinations of 22.2–44.4 m 21 BA and 5.7–28.5 m IAA. Rooting of shoots occurred in medium containing 75 g l^–1 banana pulp and 5.7 m IAA within 3–9 weeks. Plantlets of 2–5 cm length possessing two to five roots established easily in community pots at 80–90% rates without hardening. Community potted plants introduced into forest segments at Ponmudi and Palode in Southern Western Ghats of India established at a rate of 50–70%.     

Resistance to Spodoptera exigua in Apium prostratum

Two Apium accessions were compared with the commercial cultivar Tall Utah 52–70R (A. graveolens [L.]) for resistance to Spodoptera exigua (Hübner)(Lepidoptera: Noctuidae). Oviposition rate was not significantly different between the three genotypes. In all accessions, eggs were usually placed on the upper half of the plants. Implications of this oviposition pattern on S. exigua management in celery are discussed. The wild species A. prostratum ssp prostratum var filiform (A230) showed a significantly higher resistance to S. exigua than 52–70R. The levels of carcinogenic and mutagenic linear furanocoumarins in the commercial cultivar 52–70R (1.41 g/g in the petioles; 5.85 g/g in the leaves) and in the plant accession A. nodiflorum (5.40 g/g in the petioles; 2.99 g/g in the leaves) were far below the concentration reported to produce acute contact dermatitis (18.0 g/g). The levels of furanocoumarins in A. prostratum petioles (186.14 g/g) and leaves (326.45 g/g) were 10 and 18 times higher, respectively, than the concentration known to cause contact dermatitis. However, resistance in A. prostratum was primarily due to non-preference and the linear furanocoumarins did not induce non-preference. Therefore, the resistance shown by this plant accession does not appear to be furanocoumarin-based and may be suitable for transfer to commercial celery for use in S. exigua management.     

Responses of nitrogen-fixing and nitrate-supplied alfalfa (Medicago sativa L.) to iron chelates in an alkaline hydroponic medium

Ferric ethylenediamine di-(o-hydroxyphenylacetate) (FeEDDHA) and ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) were evaluated as Fe sources for hydroponic growth of alfalfa (Medicago sativa L., cv. Mesilla), either dependent on N₂ fixation or supplied with NO₃. The hydroponic medium was maintained at pH 7.5 by addition of CaCO₃. Nitrogen-fixing cultures were inoculated with Rhizobium meliloti 102 F51 and grown in medium without added nitrogen. After five to seven weeks of growth under greenhouse conditions, plants were harvested. Nitrogen fixation was measured by the acetylene reduction method.When FeEDDHA was supplied, growth of alfalfa, whether dependent on N₂ fixation or supplied with NO₃, was severely limited at concentrations typically used in hydroponic medium (10 or 20 M). Maximum yield of NO₃-supplied alfalfa was obtained at 100 M while maximum yield of N₂-fixing alfalfa was obtained in the range of 33 to 200 M FeEDDHA. Nodule fresh weights and N₂ fixation rates increased with FeEDDHA concentration up to 33 M and remained essentially constant up to 200 M. With FeHEDTA, maximum yields of both NO₃-grown and N₂-fixing alfalfa were obtained at 10 M. Growth of NO₃-supplied plants was inhibited at 200 M FeHEDTA while growth of N₂-fixing plants was inhibited at 100 M FeHEDTA. The numbers of nodules per plant increased between 3.3 and 10 M FeHEDTA; however, inhibition of nodule formation occurred at a concentration of 33 M or higher. Nodule weights per plant and N₂ fixation rates were depressed at 3.3 M as well as at 100 M FeHEDTA. The results suggest that alfalfa dependent on N₂ fixation is more sensitive to limited Fe availability than alfalfa supplied with NO₃.