LABELLING OF ACETYLCHOLINE IN THE BRAIN OF MICE FED ON A DIET CONTAINING DEUTERIUM LABELLED CHOLINE: STUDIES UTILIZING GAS CHROMATOGRAPHY—MASS SPECTROMETRY

—A method to achieve labelling of the acetylcholine stores of the brain under ideal physiological conditions is described. To this end, mice fed on a choline free diet were supplied with deuterium labelled choline in the drinking water. Labelled and unlabelled choline in plasma and in the brain as well as labelled and unlabelled acetyicholine in the brain were measured by a gas chromatographic-mass spectrometric method. It was found that after 1–25 days on the deuterium choline diet, substantial amounts of the plasma choline and brain acetylcholine were displaced by deuterium choline and deuterium acetylcholine, respectively. Already on the first day, the mole ratio of deuterium choline/total choline in plasma was 0·22, and it approached a maximum of 0·57 on the 14th day. The mole ratios of deuterium acetylcholine/total acetylcholine in the brain were slightly but significantly lower than those of deuterium choline/total choline in plasma 1–14 days, but asymptotically approached the mole ratios of deuterium Ch/total Ch in plasma by 25 days. Intact brains submitted to incubation at room temperature for 10 min increased their total choline content by about 500 per cent. Concurrently, in brains from animals kept on a deuterium choline diet for 1–2 days, the level of deuterium choline rose only by 50 per cent after incubation. Deuterium choline levels increased, however, by 200–300 per cent in the brains from animals kept on the deuterium diet for longer time periods. On the basis of these data it is suggested that: (a) choline in plasma is partly supplied from the food and partly from endogenous sources; (b) plasma choline rapidly equilibrates (less than one day) with a pool of Ch in the brain which is responsible for biosynthesis of acetylcholine; (c) the size of this choline pool is in the order of 34–40 nmol/g.     

STUDIES ON THE SITE OF MEDIATED TRANSPORT OF CHLORIDE FROM BLOOD INTO CEREBROSPINAL FLUID: EFFECTS OF ACETAZOLAMIDE

Abstract— In monkeys we measured the steady-state concentrations of Cl⁻ in endogenous CSF, in artificial CSF (which had equilibrated with the underlying exposed surface of the cerebral cortex but was not in diffusion equilibrium with endogenous CSF), and in arterial plasma. The ratio of the distribution of Cl⁻ in artificial CSF to that in plasma was consistent with a passive Donnan distribution, whereas that ratio describing Cl⁻ levels in endogenous CSF in comparison to those in plasma clearly exceeded theratio required for a passive, Donnan⁻type of distribution for Cl⁻. The kinetic analysis of the efflux of Cl⁻ from blood into endogenous CSF and into artificial CSF (perfused over the exposed surface of the cerebral cortex) indicated that the rate of efflux of Cl⁻ into endogenous CSF which was continuous with ventricular fluid was inhibited by acetazolamide [in confirmation of a similar finding described previously by M aren and B roder (1970)], whereas the rate of efflux of chloride from blood into the artificial CSF perfusate was uninfluenced by pretreatment of animals with acetazolamide. We have discussed the site of mediated (active) transport of chloride from blood into CSF in light of these findings.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VIVO STUDIES

Abstract— The total concentrations of vitamin B₆ (B₆) in plasma, choroid plexus, CSF and brain of adult New Zealand white rabbits, measured fluorometrically, were 0.30, 15.10, 0.39 and 8.90 μ mol/l or kg respectively. The mechanisms by which B₆ enters and leaves brain, choroid plexus and CSF were investigated by injecting [³H]pyridoxine (PIN) intravenously, intraventricularly and intraarterially. [³H]PIN, with or without unlabelled PIN, was infused intravenously at a constant rate into conscious rabbits. At 150 min, [³H]B₆ readily entered CSF, choroid plexus and brain. The addition of 0.5 mmol/kg carrier PIN to the infusion solution depressed the relative entry of [³H]B₆ into CSF, choroid plexus and brain by about 80%. After intraventricular injection, [³H]PIN readily entered brain from CSF. The intraventricular injection of carrier PIN with [³H]PIN decreased the amount of [³H]B₆ in brain and also decreased the percentage of [³H]B₆ in CSF and brain that was phosphorylated. During one pass through the cerebral circulation, [³H]PIN (1 μ m ) was cleared from the circulation no more rapidly than mannitol. These results were interpreted as showing that the entry of B₆ from blood into CSF and presumably the extracellular space of brain and thence into brain cells involves one or more saturable transport and/or metabolic steps.     

THE SPECIFICITY AND SULFHYDRYL SENSITIVITY OF THE INOSITOL TRANSPORT SYSTEM OF THE CENTRAL NERVOUS SYSTEM

Abstract— The transport of two cyclohexitol stereoisomers, myo-inositol (inositol) and scyllo-inositol (scyllitol), from blood into the CNS in vivo and into the choroid plexus in vitro was studied. In vitro , the uptake of [³H]scyllitol or [³H]inositol by choroid plexuses, isolated from rabbits and incubated in artificial CSF, was measured. Both scyllitol and inositol inhibited [³H]scyllitol or [³H]inositol accumulation by the choroid plexus. Inositol competitively inhibited [³H]scyllitol accumulation and both isomers had a comparable affinity (K_t= 0.1 m m ) for the single cyclohexitol accumulation system. The other 6 stereoisomers tested had an order of magnitude less affinity for the cyclohexitol accumulation system of choroid plexus. Thiol reagents that penetrate cells inhibited inositol accumulation by choroid plexus more effectively than nonpenetrating thiol reagents. In vivo , in unanesthetized rabbits. the transport of unmetabolized [³H]inositol from blood into CSF, choroid plexus and brain was readily saturated by increasing the plasma levels of myo-inositol but not by the stereoisomer d -chiroinositol. Similarly, the transport of unmetabolized [³H]scyllitol into CSF, brain and choroid plexus was readily saturated by increasing the plasma levels of myo-inositol. Beside documenting the stereospecificity and thiol reagent sensitivity of the inositol transport mechanism of the choroid plexus, these results provide further evidence that the choroid plexus is a locus for cyclohexitol transport between blood and CSF. Moreover, they show that scyllitol, which, like inositol, is present at a higher concentration in brain than plasma, can be transported from blood into CSF and brain by the same system that transports inositol.     

Influence of plasma lipid changes in response to 17β-oestradiol stimulation on plasma growth hormone, somatostatin, and thyroid hormone levels in immature rainbow trout

Plasma total lipids were significantly higher in 17β-oestradiol(E₂)-treated immature rainbow trout Oncorhynchus mykiss at week 4 after implantation, due to increases in polar and neutral lipids. The lipid classes responding were phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, sterols and sterol esters, in a proportion that approximately reflected the increase in plasma vitellogenin (VtG) levels as measured by a non-competitive enzyme-linked immunosorbent assay (ELISA). Plasma non-esterified fatty acids and triacylglycerol were not affected by E₂ treatment. Plasma growth hormone GH levels were increased, and plasma somatostatin-14 (SRIF) levels decreased in E₂-treated fish, responses which could be secondary to elevated plasma lipid (VtG) content, although a direct E₂ action on somatotroph function is possible. Plasma T₄ concentrations were not affected by E₂ treatment, but plasma T₃ concentrations were significantly lower than in controls 1 week after implantation when plasma E₂ concentrations were the highest; this is in support of the hypothesis that E₂ has a suppressive action on T₃ production.     

STIMULATED INCORPORATION OF AMINO ACIDS INTO PROTEINS OF SYNAPTOSOMAL FRACTIONS INDUCED BY DEPOLARIZING TREATMENTS

Abstract— Rat cortical synaptosome preparations incorporated l -amino acids into protein at a linear rate over 30–60 min. Synaptosomes showed large increases in incorporation after treatment with electrical pulses, veratrine or K⁺. This was inhibited (controls, 73%; electrically stimulated 58%) by cycloheximide and by chloramphenicol (28 and 44% respectively). Omission of Ca²⁺ from the medium had no effect, but the absence of Na⁺ greatly diminished the stimulus-induced increase in incorporation due to pulses. Electrical pulses, veratrine and K⁺ (in order of effectiveness), all showed their greatest proportional effects on amino acid incorporation into the Triton-X-100 insoluble portions of the synaptosomal membrane: the 'junctional complex', soluble in SDS, and the residue. Tetrodotoxin (1 μ m ), although ineffective when potassium was the stimulus, prevented or reduced the respiratory response, K⁺ loss and differential amino acid release, as well as the amino acid incorporation into protein observed with pulses and veratrine.
C-6 glioma cells showed no stimulus-dependent increase in incorporation. Preliminary data, of gel electrophoretic analysis, on the proteins labelled under our conditions is presented.     

EFFECTS OF CADMIUM TOXICITY ON GROWTH AND ELEMENTAL COMPOSITION OF MARINE PHYTOPLANKTON

Some classes of marine phytoplankton are believed to be more tolerant of high concentrations of trace metals than others, but the results of experimental tests of this hypothesis are ambiguous. Eleven species of phytoplankton representing five classes were grown in Aquil medium containing Cd concentrations between 10⁻⁸ and 10⁻⁵ M ([Cd²⁺]= 10^−9.85 to 10^−6.84 M), and growth rates and intracellular concentrations of Cd, C, N, and S were measured. The mean Cd²⁺ concentration (pCd⁵⁰) that reduced the growth rate of each species to 50% of its maximum varied by 2.5 orders of magnitude, from 10^−6.23 for Emiliania huxleyi to 10^−8.79 for Synechococcus sp. Taxonomic trends in Cd resistance were not apparent in these data. Cadmium quotas (mol Cd·L⁻¹ cell volume) were lowest in species of Bacillariophyceae (ANOVA, P < 0.001), suggesting that they might regulate Cd transport differently than other taxa. Cellular S:C molar ratios increased in four of seven phytoplankton grown at high pCd (7.37–6.84) compared to low Cd ion concentrations (no added Cd), a result of increases in S·L⁻¹ cell volume. Nitrogen:carbon molar ratios were also higher in Cd-exposed phytoplankton, as changes in N and S were highly correlated ( r = 0.98, P < 0.0001). In two species that were examined, S:C ratios increased as a linear function of increasing Cd concentration. The results demonstrate large variability in Cd resistance among phytoplankton that is primarily a function of interspecific differences in Cd detoxification.     

REGULATION OF ACETYLCHOLINE SYNTHESIS: CONTROL OF CHOLINE TRANSPORT AND ACETYLATION IN SYNAPTOSOMES

Abstract— The high affinity transport of choline (Ch) and the synthesis of acetylcholine (ACh) were measured in synaptosomes by measuring the utilization of [²H₄]Ch. The synthesis of ACh was reduced under several conditions which reduce the availability of acetyl coenzyme A (AcCoA) including no glucose added, replacement of glucose with succinate or impairment of glucose utilization by bromopyr-uvate, NaCN, or pentobarbital. These conditions did not reduce the amount of unacetylated [²H₄]Ch in the synaptosomes indicating that the high affinity transport of Ch is not directly coupled to the synthesis of ACh.     

Therapeutic effects of coenzyme Q10 (CoQ10) and reduced CoQ10 in the MPTP model of Parkinsonism

Coenzyme Q₁₀ (CoQ₁₀) is a promising agent for neuroprotection in neurodegenerative diseases. We tested the effects of various doses of two formulations of CoQ₁₀ in food and found that administration in the diet resulted in significant protection against loss of dopamine (DA), which was accompanied by a marked increase in plasma concentrations of CoQ₁₀. We further investigated the neuroprotective effects of CoQ₁₀, reduced CoQ₁₀ (ubiquinol), and CoQ₁₀ emulsions in the (MPTP) model of Parkinson's disease (PD). We found neuroprotection against MPTP induced loss of DA using both CoQ₁₀, and reduced CoQ₁₀, which produced the largest increases in plasma concentrations. Lastly, we administered CoQ₁₀ in the diet to test its effects in a chronic MPTP model induced by administration of MPTP by Alzet pump for 1 month. We found neuroprotective effects against DA depletion, loss of tyrosine hydroxylase neurons and induction of alpha-synuclein inclusions in the substantia nigra pars compacta. The finding that CoQ₁₀ is effective in a chronic dosing model of MPTP toxicity, is of particular interest, as this may be more relevant to PD. These results provide further evidence that administration of CoQ₁₀ is a promising therapeutic strategy for the treatment of PD.     

The cytotoxic and photodynamic inactivation of micro-organisms by Rose Bengal

Rose Bengal was cytotoxic to the following bacteria at the concentrations given in parentheses (highest concentrations of dye in mol/1 at which growth occurred on nutrient medium): Brochothrix thermosphacta and Deinococcus radiodurans (1 times 10^-6 or less); Streptococcus, Micrococcus, Staphylococcus, Bacillus, Arthrobacter and Kurthia spp. (1 times 10^-5–1 x 10^-4), and Pseudomonas spp. and Enterobacteriaceae (5 times 10^-3–1 x 10^-2 or greater). These organisms were killed rapidly when suspended in illuminated (170 μE/m²/s) solutions of Rose Bengal (1 times 10^-4 mol/1) providing oxygen was present. Singlet oxygen was identified as the lethal agent, because the rate of killing was increased by dissolving the dye in deuterium oxide while the organisms were protected against photoinactivation by L-histidine or crocetin. Yeasts from chilled foods were killed in illuminated solutions of Rose Bengal but a light intensity of 315 μE/m²/s was needed for a death rate comparable with that of bacteria. The yeasts present in a range of chilled meat and dairy products failed to form colonies on Rose Bengal (5 times 10^-5 mol/1) media exposed continuously to modest illumination (55–80 μE/m²/s).     

EFFECTS OF POTASSIUM AND LITHIUM ON SODIUM TRANSPORT FROM BLOOD TO CEREBROSPINAL FLUID

Abstract— The relationship between isotopic sodium entry into cerebrospinal fluid (CSF) from blood and cisternal potassium concentration was studied using ventriculo-cisternal perfusion in the rabbit. The entry of sodium into CSF was separated into 2 components. The fast component was significantly correlated with cisternal potassium concentration during perfusions with a potassium-free artificial CSF. ATPase activity in the homogenised choroid plexus was shown to be sensitive to potassium over a range of concentrations similar to that in the perfusion studies. The results are interpreted as showing a potassium-sensitive entry of ²⁴Na across the choroid plexus due to a sodium-pump situated in the apical membrane of the choroid plexus. The effects of low concentrations of lithium (0.6–1.2 mm) on ²⁴Na entry into CSF and brain and on CSF secretion were studied. When applied via the ventricles lithium caused a 30–39% stimulation of the fast component of sodium entry and a 28% stimulation of CSF secretion. When given via the blood lithium inhibited the fast component of sodium entry and CSF secretion by 43% and 40% respectively. No effects of lithium were found on the slow component of sodium entry into CSF or sodium entry into brain. The results suggest that lithium at low (0.6–1.2 mM) concentrations can stimulate the choroid plexus sodium-pump at the potassium-sensitive side and inhibit it at the sodium-sensitive side.     

Long-term ammonia exposure of turbot: effects on plasma parameters

Turbot juveniles were exposed to four ammonia concentrations [0·17 (L), 0·34 (M), 0·73 (MH) and 0·88 (H) mg l⁻¹ NH₃-N] for different exposure durations (28 days minimum to 84 days). Their physiological status and growth performances were compared to a control group [0·004 (C) mg l⁻¹ NH₃-N]. No growth was observed in the H group, and by day 57, mass increase in the MH group was only 15% of that in group C. During the first month growth in the L group was similar to that in control group while it was lower (33%) in the M group; afterwards the L and M groups had a similar growth (half that of controls). Accumulation of total ammonia nitrogen (TA-N) in plasma was dependent on ambient ammonia concentrations. Plasma urea levels in ammonia-exposed fish were lower, similar or greater than in controls (depending on ammonia concentration or exposure duration). Osmolarity, Cl^– and Na⁺ plasma concentrations were stable in the L and M groups. The increases in Na⁺, Cl^–, K⁺ and total Ca concentrations observed by the end of the experiment in the H and MH groups suggest that fish failed to adapt. There was an initial rise in plasma cortisol in all ammonia-exposed groups followed by a return to basal level (1·7–4 ng ml⁻¹) in the L and M groups. In group MH, plasma cortisol peaked at 42 ng ml⁻¹ by day 14, and after a decline at c . 1 month (14 ng ml⁻¹), it rose again.     

THE STABILITY OF VITAMIN B6 ACCUMULATION AND PYRIDOXAL KINASE ACTIVITY IN RABBIT BRAIN AND CHOROID PLEXUS

The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: (I) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B₆ accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120 mg/kg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 57% by 4-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated, intact choroid plexuses from the treated rabbits to accumulate [³H] vitamin B₆ (with [³H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B₆ accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. In vitro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a K_m , for pyridoxine of 2.0 μM Crude and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines (1 mM) or pyridoxal phosphate (5 μM). These in vitro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain in vivo.     

Effect of rearing density on thyroid and interrenal gland activity and plasma and hepatic metabolite levels in rainbow trout, Salmo gairdneri Richardson

There were significant inverse correlations between rearing density of rainbow trout, Salmo gairdneri Richardson, and final body weight, plasma L-thyroxine (T₄), trüodo-L-tryronine (T₃), cortisol and protein concentrations, plasma T₄/T₃ ratios and thyroid epithelial cell height. In addition, hepatosomatic indices and plasma free fatty acid concentrations were higher in fish reared at low (134 g 1⁻¹) density compared with groups reared at medium (210g1⁻¹) or high density (299g 1⁻¹), and the post-feeding (3.5-4h) elevation in plasma glucose and triglyceride levels evident in trout maintained at low rearing density was not found in those fish reared at higher densities. There were no significant effects of rearing density on hematocrit, carcass composition, hepatic glycogen and lipid levels and interregnal nucleus size.     

INCORPORATION OF RADIOACTIVE SULPHATE INTO SULPHATIDE DURING MYELINATION IN CULTURES OF RAT CEREBELLUM

Abstract— Explants of rat cerebellum obtained 12-24 h after birth were maintained in culture in Maximow assemblies. About 90 per cent of the cultures myelinated after 10–12 days in vitro . Cultures maintained for varying periods of time were exposed to [³⁵S]Na₂SO₄; labelled sulphatide was recovered from the total homogenate. Preparation of a subcellular fraction with density properties corresponding to those of myelin indicated that labelled sulphatide appeared in this fraction. Cultures which were poorly-myelinated always exhibited a lower rate of inccrpomtion than well-myelmated cultures from littermate animals, but the distribution of labelled sulphatide into the 'myelin' fraction was similar in the two groups. The rate of incorporation of [³⁵S]Na₂SO₄ into total sulphatide increased with the duration of the culture, with a low level of incorporation until the seventh day in vitro , followed by a sharp increase in rate up to the 21st day. This pattern resembles that observed for rat cerebellum in vivo .     

Methionine Recycling in Brain: A Role for Folates and Vitamin B-12

Abstract: The recycling of methionine via homocysteine was measured in vivo in brain. After constant intravenous infusions (5 h) of both [³H-methyl] methionine and [³⁵S]methionine into rats, the ratios of [³H-methyl]methionine to [³⁵S]methionine in liver, brain and plasma were determined, Similar experiments were performed in rabbits, except that the [³H-methyl]- and [³S]methionine were injected intraventricularly. If the methyl group of methionine was removed with the formation of homocysteine and then replaced by another (unlabeled) methyl group, the specific activity of the [³H-methyl]methionine would decrease more than that of [³⁵S]methionine; i.e., the ratio of [³H-methyl]- to [³⁵S]methionine in the tissue would decline. The results showed that the ratios of [³H-methyl]- to [³⁵S]methionine in liver and brain were less than the same ratio in plasma in the rats. The comparable ratios in the brain and CSF of rabbits were less than the ratio in the injectate. Since brain contains only one enzyme capable of remethylating homocysteine to methionine, the vitamin B-12–dependent methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13), our results for methionine recycling via homocysteine in brain strongly support the activity of this enzyme in brain in vivo.     

Kinetics of Homovanillie Acid and Determination of Its Production Rate in Humans

Abstract: Homovanillie acid (HVA) labeled with either two or five deuterium atoms (d₂-HVA or d₅-HVA) was used to label the peripheral body pool of endogenous HVA (d₀-HVA) in control humans and in neurological patients. Either d₂- or d₅-HVA was rapidly injected intravenously and concentrations of d₂- or d₅- and d₀-HVA in sequential samples of blood plasma were determined by gas chromatography-mass spectrometry (GC-MS). Parameters describing the distribution and elimination of HVA, as well as its pool size, turnover, and synthesis rate were then calculated. Results indicate that the decline of plasma d₂- or d₅-HVA with time was multiexponential in five out of six subjects. Basal levels of endogenous HVA averaged 14.4 ± 1.3 ng/ml in the control patients and 8.69 ± 2.4 ng/ml in the neurological patients. The biological half-life averaged 71.3 ± 8.0 min in the control subjects and 91.3 ± 15 min in the neurological patients. The apparent volume of distribution of HVA in the body was 31–100 liters. Plasma clearance was 243–679 ml/min. The size of the peripheral body pool, calculated from plasma kinetic parameters, was 392–673 μg. The HVA rate of production, calculated for the whole body, was 166–323 μg/h. This technique can be used to determine accurately the rate of HVA production by the whole body over short time intervals. Since sample size was very limited ( n = 3 per group) and effects of variables such as age and sex on these data have not been excluded, more thorough investigations are needed to document any differences between normal controls and neurological patients.     

APPARENT IN VIVO ACETYLCHOLINE TURNOVER RATE IN WHOLE MOUSE BRAIN: EVIDENCE FOR A TWO COMPARTMENT MODEL BY TWO INDEPENDENT KINETIC ANALYSES

Abstract— Acetylcholine turnover has been determined in whole mouse brain using a newly available high specific activity [³H]choline (70 Ci/mmol). Animals were killed at various time points (0.25–10 min) after pulse adminstration of [³H]choline (Ch) by microwave irradiation of the head. Steady-state levels of ACh were determined by radioenzymatic analysis as described by G oldberg & M c C aman (1973) as modified by M c C aman & S tetzer , 1977. Ch levels were determined by a modification of the method of M c C aman & S tetzer (1977). Radiolabelled metabolites of [³H]Ch were separated by selective extraction of [³H]Ch and [³H]ACh inio tetraphenylboron in 3-heptanone (C arroll et al. , 1977) coupled with an enzymatic separation of [³H]Ch from [³H]ACh. A precursor-product relationship was verified for Ch and ACh specific activities. Acetylcholine turnover rate was determined by the biosynthesis ratio method (S chuberth et al. , 1969, Method 1) and by the finite-differences method (N eff et al. , 1971, Method 2). Both methods of kinetic analysis revealed two distinct turnover rates for acetylcholine. In the first phase (0.25–1.5 min post-[³H]Ch), the ACh turnover rate averaged 22nmol/g/min (both methods). During the second phase, (2–10 min) acetylcholine turnover rates were significantly ( P < 0.05 and P < 0.01) lower; i.e. 7nmol/g/min (Method 1) and 5.9 nmol/g/min (Method 2). The data are consistent with a 2-compartment model for ACh turnover in whole mouse brain. Additionally, the method described for the separation of radiolabelled metabolites of [³H]Ch allows an accurate determination of ACh turnover in as little as 2 mg of tissue.     

AUTORADIOGRAPHIC CONFIRMATION OF THE MITOTIC DIVISION OF EVERY MOUSE JEJUNAL CRYPT CELL LABELLED WITH 3H-THYMIDINE

The question was investigated of whether for crypt epithelia of the jejunum of the mouse all cells labelled after a single injection of ³H-TdR subsequently divide or whether cells exist in the crypt which synthesize metabolic DNA and, therefore, do not undergo division after labelling.
A double labelling experiment was performed with a first injection of ³H-TdR followed 1 hr later by an injection of ¹⁴C-TdR. Then from double emulsion autoradiographs of isolated squashed crypts the number of ³H-only, ¹⁴C-only and double labelled cells and mitoses were counted.
The double labelling produced a narrow, 1 hr wide sub-population of ³H-only labelled cells. This subpopulation of S cells completed its division before labelled cells were lost from the crypts by migration onto the villi. The results showed that this subpopulation of ³H-only cells completely doubled within 3 hr and then remained constant through 6 hr. From this result it was concluded that every cell labelled after a single injection of ³H-TdR divides.
From the same autoradiographs the flow rate through the end of mitosis was measured. From the flow rate and the mitotic index a mitotic duration of 0·5 hr was determined. The agreement of this measured mitotic time with the value calculated from the labelling index, mitotic index and S duration is also strong evidence that every labelled cell divides.
Both experiments show that the intestinal crypt does not contain cells synthesizing metabolic DNA.     

A Circadian Clock of Drosophila: Effects of Deuterium Oxide and Mutations at the period Locus

Mutations at the period (per) locus (1:1.3; 3B1-2) in Drosophila melanogaster lengthen (per^L), shorten (per⁵), or abolish (per°) overt circadian rhythmi-city. Deuterium oxide lengthens the free-running circadian period. We tested the effects of deuterium on three mutants of the per gene (per⁵ per^L, and per°) and wild-type Drosophila melanogaster (per⁺) to assess interactions. With increasing concentrations of deuterium, the free-running circadian period of locomotor activity rhythms increased. The dose-response was linear in all genotypes tested. With increasing dosages ofdeuterium, circadian rhythms became weaker as evidenced by the signal-to-noise ratio (SNR). Genotype and deuterium changed circadian period length independently and additively, showing no interaction. SNRs for all genotypes converged on a low level as deuterium concentration increased. Deuterium increased life span, except at high concentrations (40 and 50%).