MELANIN FORMATION BY HUMAN BRAIN IN VITRO

Abstract— A quantitative radiometric assay utilising incorporation of ¹⁴C from labelled precursors as a measure of melanin formation by human brain in vitro is described. The assay was validated by comparison with various criteria of melanin formation.
Catecholamines, DOPA and 5HT were precursors for brain melanin formation. Melanin formation was detected in all brain regions studied and was highest in substantia nigra and striatum.
The assay was used to evaluate various hypotheses of brain melanin formation. No evidence for enzymic activity was found and it is concluded that brain melanin formation may be a largely non-enzymic process.     

RATE OF STEROL FORMATION BY RAT BRAIN GLIA AND NEURONS IN VITRO AND IN VIVO

The ability of 11-day-old rat glial and neuronal cells to biosynthesize sterol was studied as a function of time in vivo and in vitro. The in vitro experiments utilized [2-¹⁴C]mevalonic acid as precursor. Glial-enriched cell preparations demonstrated a greater ability to incorporate [2-¹⁴C]mevalonic acid into isoprenoid material than did neuronal-enriched preparations. Approximately 4 h were required for maximal uptake of labelled mevalonate by the glial preparations. Further metabolism of the isoprenoid material, involving squalene turnover and sterol demethylation, was still evident even after 15 h of incubation. In vivo, sterol biosynthesis was studied by intraperitoneal injection of sodium [2-¹⁴C]acetate and [U-¹⁴C]glucose, sacrifice of the animals at 2 or 24 h, subsequent isolation of glial- and neuronal-cell enriched fractions and analysis of labelled isoprenoid material. Glial-enriched fractions again contained the bulk of the labelled isoprenoid material.     

KINETICS OF PLASMA CHOLINE IN RELATION TO TURNOVER OF BRAIN CHOLINE AND FORMATION OF ACETYLCHOLINE1

—[²H₄]Ch (2 μmol kg^-1 min^-1) was infused into both anaesthetized and conscious rats to study the kinetics of plasma and brain choline (Ch) and brain acetylcholine (ACh). A larger amount of endogenous Ch was found to leave the brain than enter, even in conscious animals. [²H₄]Ch was taken up into the brain where a portion was converted to [²H₄]ACh. Upon stopping the infusion, however, more [²H₄]Ch was found to leave than enter, indicating a source capable of generating Ch in brain which is labelled by infusion for 32 min. There appears, however, to be more than one source of Ch in the brain since the post mortem increase is not labelled following prolonged infusion. Thus, the brain Ch pool appears to be continually diluted by the sources within the brain to the extent of 93 per cent. During the infusion of [²H₄]Ch, the total levels of brain Ch and ACh did not increase. The brain Ch and ACh specific activities rose exponentially and appear to approach an asymptote at about 4 h. The source or sources of Ch within the brain produce Ch at a rate of 26·3 nmol g^-1 min^-1. The turnover of free Ch in the rat brain is 28·4 nmol g^-1 min^-1.     

THE UPTAKE OF PURINES BY RAT BRAIN IN VIVO AND IN VITRO

Abstract— The uptake of [¹⁴C]guanine and some of its [¹⁴C]-labelled derivatives into rat brain was studied in vivo and in vitro. In vivo guanine, guanosine, and hypoxanthine penetrated the brain of adult rats to a very small extent. Inosine was taken up somewhat better. In young animals, also, guanosine was taken up poorly, but guanine was taken up fairly well. When guanine was administered to adult animals, only guanine was found in the brain. In young animals, by contrast, radioactivity from guanine appeared in guanosine and in guanine nucleotides, but no free guanine was found. In vitro guanine was taken up much better and, in fact, remained mostly as guanine in slices from 10-day-old rats. The in vitro conversion of guanine to GMP and its incorporation into RNA was unimpaired by the addition of unlabelled guanosine, an indication that guanine was converted directly to GMP. The uptake of guanine in vitro was not subject to competitive inhibition or influenced by the presence of dinitrophenol. This finding suggested that guanine entered the slice by simple diffusion.     

THE MEASUREMENT OF TRIGLYCERIDE IN BRAIN AND THE METABOLISM OF BRAIN TRIGLYCERIDE IN VITRO

Abstract—

• 1 Triglyceride has been isolated from brain by thin-layer chromatography and determined by absorption of the carbonyl group at 1740 cm^?1. The means of yields from whole mouse brain, whole rat brain, rat brain grey matter, rat brain stem, and incubated slices of rat brain cortex were 0.15–0.17 μmole/g tissue.
• 2 The distribution of fatty esters varied from preparation to preparation. Palmitate, stearate and oleate usually occurred in greatest amounts. Hydrolysis of a preparation of triglyceride from whole rat brain with pancreatic lipase indicated that palmitate was equally distributed between the α and β esters.
• 3 [1-¹⁴C]Acetate was rapidly incorporated into triglyceride of slices of incubated rat brain cortex. When the resulting triglyceride was hydrolysed with pancreatic lipase the distribution of radioactivity amongst the hydrolysis products was consistent with both the α and β esters of the triglyceride having been radioactively labelled.

     

MULTIPLE FORMS OF CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN BRAIN OF DEVELOPING AND ADULT RATS

The multiple molecular forms of choline acetyltransferase (ChAT) were analysed during the postnatal development of rat brain. Changes in the sodium-dependent, high affinity uptake of [³H]choline (HAUC) and in the efficiency of conversion of labelled choline into ACh in vitro were also examined. Both mature and 7-day old brain contained three molecular forms of ChAT, with isoelectric points of pH 7.3, 7.9 and 8.3, but the immature brain appeared to contain smaller concentrations of the most basic form of the enzyme (pI = 8.3). Of the total choline uptake measured in slices of frontal cortex, adult samples exhibited a greater proportion of HAUC than 7-day samples and appeared to acetylate more efficiently the [³H]choline accumulated by high affinity uptake. This evidence suggests a basic molecular form of ChAT, appearing in rat brain during postnatal development, might be responsible for the efficient coupling of the high affinity uptake and subsequent acetylation of choline in cholinergic nerve terminals.     

ACETYLCHOLINE,CHOLINE AND CHOLINE ACETYLTRANSFERASE ACTIVITY IN THE DEVELOPING BRAIN OF NORMAL AND HYPOTHYROID RATS1

Abstract— Acetylcholine, choline and choline acetyltransferase activity were measured in the whole brains of normal and hypothyroid rats during development. At 1 day postpartum, brain acetylcholine was 73 per cent of adult levels. Propylthiouracil-induced hypothyroidism up to age 20 days did not alter brain acetylcholine concentrations, but at 30 days resulted in significantly decreased levels. At day 1, brain choline was 20 per cent higher than adult levels and decreased between days 8 and 10. In hypothyroid rats this phenomenon did not occur until days 15–20. At day 1 postnatally, choline acetyltransferase activity was only 7 per cent of adult levels, then between days 5 and 20 rose to 77 per cent of adult levels. Beginning at day 8, hypothyroidism resulted in significantly decreased enzyme levels. This effect could be reversed at day 17 by concurrent tri-iodothyronine substitution therapy. In hypothyroid rats, maximum brain choline acetyltransferase activity was 30 per cent less than normal adult levels.     

EFFECTS OF DI-ISOPROPYLFLUOROPHOSPHATE ON THE METABOLISM OF CHOLINE AND PHOSPHATIDYLCHOLINE IN RAT BRAIN

—The subcutaneous administration of 2·0 mg DFP per kg to rats causes a diminution in the lysophosphatidylcholine content in the brain, which is followed by a decrease of glycerylphosphorylcholine concentration and by a reduced post mortem choline increase. This supports the hypothesis that a post mortem increase in choline is due to phosphatidylcholine breakdown. Since the amount of phosphatidylcholine in brains of di-isopropylfluorophosphate-treated rats increases, it is concluded that phospholipase A is inhibited by di-isopropylfluorophosphate, which corresponds to findings of other authors in vitro. The activity of glycerylphosphorylcholine diesterase (EC 3.1.4.2) is not altered.     

FORMATION OF A SUPPLEMENTAL LONG TIME-CONSTANT RESERVOIR OF HIGH ENERGY PHOSPHATE BY BRAIN IN VIVO AND IN VITRO AND ITS REVERSIBLE DEPLETION BY POTASSIUM DEPOLARIZATION

—Brains of mice fed a diet containing 1% cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) accumulated the high energy phosphate compound cyclocreatine-P (1-carboxymethyl-2-imino-3-phosphonoimidazolidine), an analogue of creatine-P (phosphocreatine). During a 50-day feeding period mouse brain cyclocreatine-P increased linearly to 14 μmol/g fresh wt; during this time the total phosphagen level of brain, creatine-P plus cyclocreatine-P, increased from 3 to 15 μmol/g. When the blood-brain barrier was circumvented, a more rapid accumulation of synthetic phosphagen was achieved. Minced brain preparations from 11 to 15-day chick embryos incubated in vitro with 30 mm -cyclocreatine accumulated 10 μmol/g of cyclocreatine-P in 90 min, and this novel high energy phosphate pool could be depleted by incubation with 105 mm -potassium ions or 3 μm -valinomycin. Subsequent regeneration of the depleted pools could also be demonstrated. Brain tissue containing a supplemental reservoir of cyclocreatine-P, which is utilized to regenerate ATP much more slowly than creatine-P, might be better able to withstand anoxia and certain other metabolic stresses, but this has not been established. However, the marked delay of onset of rigor previously shown to occur in ischemic heart and skeletal muscle of cyclocreatine-fed animals is compatible with this suggestion.     

在胚胎发育中第三脑室室管膜细胞表达乙酰胆碱转移酶

目的在研究胚胎14天（E14）大鼠端脑放射状胶质细胞与胆碱能前体细胞的关系时发现,第三脑室室管膜细胞和侧脑室脉络丛上皮细胞均表达胆碱能神经元标志性蛋白,即乙酰胆碱转移酶（Choline acetyltransferase,ChAT）。方法E14大鼠端脑冠状连续切片,采用免疫组织化学染色法,分别对放射状胶质细胞标志性蛋白,即波形蛋白（Vi men-tin）和乙酰胆碱转移酶进行免疫组织化学染色。结果E14大鼠端脑室管膜细胞呈单层衬于第三脑室,在背中侧的室管膜细胞由单层增殖为复层。上皮基底部与邻近组织分界不清,这些细胞的游离面胞质呈乙酰胆碱转移酶免疫反应阳性,在邻近的第三脑室周围局部形成乙酰胆碱转移酶阳性细胞群。在相邻切片的同一区域内复层室管膜细胞波形蛋白免疫反应阴性。结论第三脑室背中侧室管膜细胞作为神经干细胞分裂、增殖,产生胆碱能前体细胞,并向第三脑室周围组织迁移,形成基底前脑神经核团的Ch5,Ch6胆碱能神经细胞群。     

HIGH AFFINITY CHOLINE TRANSPORT IN GUINEA PIG BRAIN AND THE EFFECT OF NOREPINEPHRINE

—The influence of 1-norepinephrine on the accumulation of [¹⁴C]choline by nuclei-free homogenates and synaptosomes of guinea-pig brain was studied. Kinetic analysis of choline accumulation by guinea-pig brain resulted in both high and low affinity Michaelis constants. Norepinephrine stimulated the high affinity choline transport process but not the low and the magnitude of its stimulation in 3 different brain regions was correlated with the choline acetyltransferase activity of those regions. Depletion of norepinephrine from the brainstem by pretreatment with the catecholamine depleter alpha-methyl-para-tyrosine significantly decreased the maximal velocity of choline transport. Both the alpha adrenergic receptor blocker phentolamine and the beta adrenergic receptor blocker propranalol inhibited norepinephrine induced stimulation of choline transport. Cocaine stimulated choline transport at low concentrations and pretreatment of animals with reserpine significantly antagonized cocaine's stimulation of choline transport. The results suggest that endogenous norepinephrine may modify the high affinity choline transport process in guinea-pig brain.     

罗汉果组织培养中愈伤组织和腋生枝的形成

用罗汉果茎段为外植体,培养在MS+BA1.0+IBA0.1毫克/升培养基上,诱导愈伤组织和芽形成。观察了罗汉果茎段愈伤组织的生长以及腋生枝的形成。罗汉果茎段培养5天后,潜伏腋芽开始萌动和生长。培养10天后罗汉果茎段的基部一端开始膨大。培养20天后产生大量白色疏松的愈伤组织,这时腋生枝已经长成3—6厘米长。培养30天后基部的愈伤组织中有少量瘤状小突起,但再分化形成芽的频率极低。结果表明,罗汉果茎段组培形成的苗均是从腋芽产生的。     

THE IN VITRO FORMATION OF ACETYL-ASPARTYL-PEPTIDO-SEROTONIN COMPLEXES BY PIG HYPOTHALAMIC TISSUE EXTRACTS

Peptido-serotonin complexes are formed on incubation of pig hypothalamic tissue extracts with acetyl-aspartate, the amino acids glu. ser, gly and ala. an ATP-regenerating system and serotonin. The purification and partial characterisation of two acetyl-aspartyl-peptido-serotonin complexes is described.     

CHOLINE ACETYLTRANSFERASE CONTENT IN DISCRETE REGIONS OF THE RAT BRAIN STEM

—Choline acetyltransferase (ChAc) content of 50 separate rat brain stem nuclei and cerebellum removed by microdissection was determined using a sensitive radiometric assay. The distribution of ChAc activity is uneven, with extremely high levels in the cranial motor nuclei and the nucleus salivatorius. Low ChAc concentrations were observed in the cranial sensory nuclei, the nuclei of the reticular formation, the raphe nuclei and the nuclei of the acoustic system. The lowest ChAc levels were measured in the cerebellum. Comparison of the distribution of ChAc with histochemical localization of acetylcholinesterase revealed generally good agreement, and notable exceptions are discussed.