SUBCELLULAR LOCALIZATION OF TRYPTOPHAN-5-MONO-OXYGENASE IN BOVINE PINEAL GLANDS AND RAPHE NUCLEI

Abstract— Tryptophan-5-mono-oxygenase from both bovine raphe nuclei and pineal glands was activated by preincubation with dithiothreitol and ferrous ion at pH 8.5. The optimum pH for the enzyme activity lies between pH 6.4 and 7.3. Preincubation increased the activity of the enzyme from raphe nuclei by about 20 times in both the homogenate and 105,000 g precipitate prepared from it. Activity in the 105,000 g supernatant fraction was about trebled. Corresponding increases in pineal gland enzyme activity were noted: 100 times in homogenate and 105,000 g precipitate and 15 times in 105,000 g supernatant fluid. Total recoveries of activated enzyme from the homogenate prepared in hypo-osmotic medium, in the 105,000 g supernatant and precipitate, were 87.1% and 79.0% for raphe nuclei and pineal glands respectively. Of this, 89.5-91.3% in the case of the raphe nuclei and 76.0-82.0% in the case of the pineal glands, was found in the precipitate. In contrast, 85-90% of the lactate dehydrogenase activity was found in the supernatant fraction. The results of subcellular fractionation revealed that the raphe nuclear enzyme was located in both 'mitochondrial' and 'microsomal' fraction while the pineal gland enzyme was effectively restricted to the 'mitochondrial' fraction. The structural characteristics of the fraction were confirmed by electron microscopy.     

LIPID COMPOSITION OF GLIAL CELLS ISOLATED FROM BOVINE WHITE MATTER

Abstract— —Glial cells were isolated from bovine white matter by differential centrifugation with'Ficoll'and their lipid composition was analysed. The preparations contained 20.8 per cent lipid and 792 per cent protein. The major lipid components were cholesterol, ethanolamine glycerophosphatides (EGP), cerebroside and serine glycerophosphatides (SGP). Sphingomyelin, cerebroside sulphate and inositol glycerophosphatide were present in lower proportions. EGP contained the largest proportion of aldehydes (17 per cent) and SGP contained 12 per cent. Choline glycerophosphatides contained only a trace of aldehyde. No gangliosides were present in the filial cell preparations.     

THE PURIFICATION AND PROPERTIES OF BOVINE PINEAL S-ADENOSYLMETHIONINE: N-ACETYLSEROTONIN O-METHYL TRANSFERASE

Abstract— Bovine pineal gland S-adenosylmethionine: N-acetylserotonin O-methyltransferase has been purified about 2800-fold using cell fractionation, ammonium sulphate treatment, Sephadex G-200 gel filtration and anion exchange chromatography. The enzyme has been found to be a polymer; the smallest unit observed had a mol. wt. of 21,800 and the other polymers' molecular weights were multiples of this figure. In the gland extract polymers of 83,000, 100,000, 125,000 and 150,000 mol. wt. were more abundant than the others; they showed also higher specific activity. One of the products of the reaction, S-adenosylhomocysteine was found to be a potent inhibitor, whereas the other product, melatonin, did not inhibit the bovine pineal gland enzyme, even at much higher concentrations. Homocysteic acid, cysteic acid, GSG and GSSG inhibited the enzyme. The required concentrations for this effect was 100 times higher than that of S-adenosylhomocysteine. The addition of GSH to the medium during purification led to complete loss of activity. Adenosine, homocysteine and other thio compounds had little or no effect. The enzyme was found to be activated by its substrates and also by certain anions. Among various organic acid salts, citric acid cycle intermediates were found to be good activators; their nonsubstituted analogues were not as effective. The activator effect of oxaloacetate and bicarbonate was the highest, and was brought about by relatively low concentrations of these anions (1–5 × 10^?3 M), hence their effect was considered specific. The degree of activation caused by oxaloacetate was decreased by increasing substrate concentrations and vice versa. The S-adenosylhomocysteine inhibition could not be reduced by increasing the substrate concentration; S-adenosylhomocysteine also inhibited the oxaloacetate-activated enzyme. These observations have been explained by the allosteric behaviour of the enzyme. The kinetic behaviour of various polymers was also investigated. The highest substrate and oxaloacetate activation and the highest S-adenosylhomocysteine inhibition was observed for polymers of 83,000, 100,000, 125,000 and 150,000 mol. wt. The K_m values for S-adenosylmethionine and ^N-acetylserotonin calculated for the oxaloacetate activated enzyme were also lower for these polymers than others.     

THE ULTRASTRUCTURE AND HISTOPHYSIOLOGY OF HUMAN ECCRINE SWEAT GLANDS

The electron microscopy of human eccrine sweat glands has been studied before and after stimulation by pilocarpine iontophoresis. The identity of the dark and clear cells in the secretory segment as defined by Montagna et al. (23) was determined by studying serial sections, thin for electron microscopy and thick for light microscopy. Cells with numerous apical secretory vacuoles are termed mucoid (dark) cells, since these vacuoles stain positively for acid mucopolysaccharide. Clear cells are intimately associated with intercellular canaliculi. The "cuticular border" of surface cells of the duct is a condensation of tonofilaments and granules. Numerous mitochondria are concentrated in basal cells of the duct. The presence of mucoid cells in the secretory segment may bear on the interpretation of the pathologic findings in the disease cystic fibrosis of the pancreas, and suggests that this disease may be due to a basic disorder of mucopolysaccharide production. The possible roles of the various cellular components in the elaboration of sweat are discussed.     

LIPID COMPOSITION AND METABOLISM OF CULTURED HAMSTER BRAIN ASTROCYTES

Abstract— The lipid composition and metabolism of confluent cultures of cells derived from newborn hamster brain and having morphology characteristic of immature astrocytes or spongioblasts was investigated and compared to that of newborn hamster brain dispersions and cloned glioma cells (C6). The cells displayed stable morphology for at least 30 subcultures; thereafter spontaneous transformation occurred. No appreciable changes were observed in either composition or metabolic characteristics of any major neutral lipid or phospholipid class in successive subcultures or following transformation. The overall lipid composition of the hamster astrocyte cultures closely resembled that of newborn hamster brain, but the phospholipid composition showed substantial differences. The cells contained as a percent of lipid P relatively more ethanolamine plasmalogen, choline plasmalogen and sphingomyelin and somewhat less phosphatidylcholine and phosphatidylethanolamine. The phospholipids of the hamster astrocyte and C6 cells were similar. Of the lipid precursors examined, [U-¹⁴C]glucose was incorporated best into all preparations. C6 glioma cells incorporated both [U-¹⁴C]glucose and [1-¹⁴C]acetate most actively. From 69–88% of ³²P incorporated into hamster astrocyte phospholipids was present in choline phosphoglycerides, whereas the corresonding figure for hamster brain dispersions was 53%. The ratio of specific activities of phosphatidylcholine to phosphatidylinositol was substantially higher in the cultured cells than in the brain preparations. The small pool of choline plasmalogen in the hamster astrocytes usually achieved the highest specific activity of any phospholipid. When [U-¹⁴C]glucose and [1-¹⁴C]acetate were precursors, the bulk of label in the astrocytes appeared in choline phosphoglycerides and triacyglycerol. Our results indicate that the hamster astrocyte cell line as grown expresses distinctive features of lipid composition and metabolism which are nearly constant through many generations.     

PURIFICATION OF BOVINE PINEAL HYDROXYINDOLE O-methylTRANSFERASE BY IMMUNOADSORPTION CHROMATOGRAPHY

A procedure is described for the use of immunoadsorption chromatography of hydroxyindole O-methyltransferase (HIOMT). HIOMT was purified from bovine pineal extract by affinity chromatography on immunoglobulins (Ig)-Sepharose. The overall purification was about 45-fold; the yield was 84%. This enzyme constitutes about 2.0% of the soluble proteins in the pineal gland. The enzyme represented a single precipitin line on Ouchterlony double diffusion plate and immunoelectrophoresis. Ultracentrifugation analysis indicated the existence of molecular aggregates of enzyme and disc gel electrophoresis showed one main protein band and several minor bands. However sodium dodecyl sulphate (SDS) gel electrophoresis showed a single protein band with subunit molecular weight 38,000 demonstrating bovine pineal HIOMT to be polymer enzyme of a single subunit. The properties of the purified enzyme including disc gel electrophoretic pattern, the effect of pH, chemicals and substrates and immunological properties were identical with those of the crude enzyme.     

14种微藻总脂含量和脂肪酸组成研究

比较分析了14种微藻的总脂含量和脂肪酸组成,结果表明：除小球藻、亚心形扁藻、极微小环藻、微绿球藻外,其他微藻的总脂含量均超过其干重的10％。每一纲的微藻脂肪酸组成都有各自特点,绿藻纲中16：0、16：1(n-7)、18：1(n-9)含量较高,但微绿球藻中16：1(n-7)、20：5(n-3)(EPA)含量远高于其他绿藻;金藻纲中含大量14：0、16：0、18：3(n-3)、22：6(n-3)(DHA);硅藻纲中14：0、16：0、16：1(n-7)、EPA含量较高;黄藻纲的异胶藻富含16：0、16：1(n-7)和EPA。     

LIPID,FATTY ACID,AND STEROL COMPOSITION OF EIGHT SPECIES OF KARENIACEAE (DINOPHYTA): CHEMOTAXONOMY AND PUTATIVE LIPID PHYCOTOXINS1

The lipid class, fatty acid, and sterol composition of eight species of ichthyotoxic marine gymnodinioid dinoflagellate (Karenia, Karlodinium, and Takayama) species was examined. The major lipid class in all species was phospholipid (78%–95%), with low levels of triacylglycerol (TAG; 0%–16%) and free fatty acid (FFA; 1%–11%). The common dinoflagellate polyunsaturated fatty acids (PUFA), octadecapentaenoic acid (OPA 18:5ω3), and docosahexaenoic acid (DHA 22:6ω3), were present in all species in varying amounts (14%–35% and 8%–23%, respectively). The very‐long‐chain PUFA (VLC‐PUFA) 28:7ω6 and 28:8ω3 were present at low levels (<1%), and the ratio of these fatty acids may be a useful chemotaxonomic marker at the species level. The typical dinoflagellate sterol dinosterol was absent from all species tested. A predominance of the 4‐methyl and 4‐desmethyl Δ⁸⁽¹⁴⁾ sterols in all dinoflagellate species included 23‐methyl‐27‐norergosta‐8(14),22‐dien‐3β‐ol (Karenia papilionacea A. J. Haywood et Steid, 59%–66%); 27‐nor‐(24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol, brevesterol, (Takayama tasmanica de Salas, Bolch et Hallegraeff 84%, Takayama helix de Salas, Bolch, Botes et Hallegraeff 71%, Karenia brevis (C. C. Davis) G. Hansen et Moestrup 45%, Karlodinium KDSB01 40%, Karenia mikimotoi (Miyake et Kominami ex Oda) G. Hansen et Moestrup 38%); and (24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol, gymnodinosterol, (K. mikimotoi 48%, Karenia umbella de Salas, Bolch et Hallegraeff 59%, Karlodinium veneficum (D. L. Ballant.) J. Larsen 71%–83%). In Takayama species, five steroid ketones were identified, including for the first time the 3‐keto form of brevesterol and gymnodinosterol. These results indicate a biochemical link between sterol and steroid ketone biosynthesis, suggesting that selected dinoflagellates can make a significant contribution to ketones in marine sediments. The presence of steroid ketones, specific sterols, and fatty acids, and the ratio of VLC‐PUFA may prove to be a useful chemotaxonomic tool for distinguishing between morphologically similar species. The relative levels of the PUFA, OPA, and DHA, coupled with the potential inhibitory action of Δ⁸⁽¹⁴⁾ sterols, may provide an insight into the ichthyotoxicity of these bloom‐forming dinoflagellates.     

REGULATION OF GUANOSINE 3'',5'' CYCLIC MONOPHOSPHATE IN THE RAT PINEAL AND POSTERIOR PITUITARY GLANDS

Potassium and norepinephrine stimulate the accumulation of cyclic AMP and cyclic GMP in rat pineal glands and their efflux into the medium. The efflux of both cyclic nucleotides was blocked by probenecid. The accumulation and efflux of cyclic GMP, but not of cyclic AMP, depends upon the presence of intact nerve endings and extracellular calcium. The calcium-dependent release of norepinephrine caused by veratridine was accompanied by the efflux of both cyclic AMP and cyclic GMP. In contrast, the calcium-independent release of norepinephrine caused by tyramine was accompanied by the efflux of cyclic AMP but not cyclic GMP. Changes in cyclic GMP therefore, may be related to exocytosis from the sympathetic nerve endings in the gland. High concentrations of potassium also increased tissue levels of cyclic GMP in the posterior pituitary gland. Veratridine and potassium, but not norepinephrine, stimulated the efflux of cyclic GMP from this neurosecretory gland. Thus, the relationship between cyclic GMP and exocytosis may extend beyond sympathetic nerve endings. The enhanced accumulation of cyclic GMP in the pineal gland after potassium does not appear to be mediated by extracellular (released) norepinephrine. Desmethylimipramine blocked the norepinephrine-stimulated changes in cyclic GMP, but not those caused by potassium. Investigation of the possible relationship between cyclic GMP and release of neurotransmitters is complicated by the apparent seasonal variation in the response of pineal cyclic GMP to potassium or norepinephrine.     

BRAIN LIPID COMPOSITION OF IMMATURE THIAMINE-DEFICIENT AND UNDERNOURISHED RATS1

The brain lipid composition of 25-day-old offspring of rats exposed to dietary thiamine (vitamin B₁) deficiency from the 14th day of gestation was examined and compared to normal and pair-fed (undernourished) controls. Thiamine-deprived rats displayed neurological signs and a marked diminution of growth at 25 days of age. No changes in brain lipids of either whole brain or selected brain areas (brain stem, cerebellum, diencephalon) which were distinct from the effects of undernutrition (pair-fed controls) were observed in the thiamine-deficient group. Undernutrition, as exemplified by the pair-fed control group produced a highly significant depression of all lipids expressed per total brain and a significant deficit of whole brain and regional lipid, cerebroside and cholesterol concentrations indicating a deficiency in myelinogenesis. Ganglioside NeuNAc concentration was shown to be significantly greater in whole brain and certain brain areas of the same group while no changes were evident in total phospholipid concentration and the distribution of individual phospholipids. The implications of these findings in terms of the pathophysiology of thiamine-deficiency encephalopathy and undernutrition in early life are discussed.     

RADIOMETRIC ASSAY OF TOTAL TRYPTOPHAN HYDROXYLATION BY INTACT CULTURED PINEAL GLANDS

Abstract— A new method is described for the analysis of total tryptophan hydroxylation by intact tissue in culture. This method differs from classical measurements of tryptophan hydroxylase because it allows the detection of changes in tryptophan hydroxylation due to alterations in tryptophan transport, cofactor regeneration and protein synthesis. The rat pineal gland was studied. It was found to hydroxylate tryptophan linearly for a 48-h period. A direct proportional relationship between the concentration of tryptophan in the culture medium and the amount of tryptophan hydroxylated persists between 0.05 and 0.5 m m . As the medium concn of tryptophan is increased above 0.5 m m the amount of tryptophan hydroxylated decreases. Tryptophan hydroxylation is substantially inhibited by 1 m m p-chlorophenylalanine and 0.03 m m cycloheximide. No difference in the total amount of tryptophan hydroxylated was detected between male and female rat pineal glands, between chronically denervated and normal rat pineal glands, or between control glands and glands treated with 0.1-0.001 m m norepinephrine, 0.01 m m isoproterenol, or 0.01 m m mescaline.     

LIPID COMPOSITION OF OPTIC NERVE MYELIN

Abstract— Myelin was isolated from bovine optic nerves by differential ultracentrifugation and its lipid composition was analysed. Optic nerve myelin contained 76·3 per cent lipid. The major lipids were cholesterol, ethanolamine glycerophosphatides (EGP) and cerebroside. Serine glycerophosphatides (SGP), sphingomyelin and cerebroside sulphate were present in smaller proportions. EGP and SGP contained 34·6 and 0·5 per cent aldehydes. The major fatty aldehydes were palmitaldehyde, stearaldehyde and octadecenaldehyde. The fatty acids of EGP, SGP and choline glycerophosphatides (CGP) were chiefly 16:0, 18:0 and 18:1, with small proportions of 20 and 22 carbon polyunsaturates. The sphingolipids contained predominantly saturated and monounsaturated fatty acids of chain lengths of 20–26 carbon atoms. Optic nerve myelin and white matter myelin resembled one another closely in overall lipid composition and in the fatty acid compositions of their constituent lipids. Optic nerve myelin and white matter myelin are chemically similar membranes, but both of these differ in their lipid composition from spinal root myelin.