The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Studies on distribution and metabolism of valproate in rat brain,liver, and kidney

Time-course studies on the distribution and metabolism of valproate (VPA) in rat brain, liver, and kidney, after intraperitoneal injection of a mixture of [¹⁴C]VPA and [³H]VPA, showed that: (1) maximal amount of radioactivity in the various tissues was observed after 30 min from the time the drug was administered; (2) at 30 min the distribution of labeled VPA in brain, liver, and kidney was 17%, 64%, and 19% of the total radioactivity, respectively; (3) at 24 hr more than 97% of the total radioactivity was lost from the tissues and the¹⁴C/³H ratios increased significantly with time. Studies on the regional distribution of the drug showed that it is relatively homogeneously distributed. Studies on the subcellular distribution of the drug showed that it is associated mostly with the soluble and mitochondrial fractions, with little radioactivity in the myelin and synaptosomal fractions. Radiochromatography of VPA metabolites in perchloric acid extracts from brain, liver, and kidney revealed the presence of four metabolites. VPA was not incorporated into phospholipids of the neuronal membranes. Furthermore, it had no significant effects on Mg²⁺-ATPase and (Na⁺+K⁺)-ATPase in synaptosomes and microsomes obtained either from control or from rats injected with VPA. It was concluded that this antiepileptic drug does not appear to act through its incorporation into neuronal membrane or through its action on the Na⁺ pump.Contribution No. 0601 from the Department of Cell and Molecular Biology, the Medical College of Georgia, Augusta, Georgia 30912.     

THE EFFECT OF MORPHINE ADMINISTRATION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEIN IN CELL-FREE SYSTEMS FROM RAT BRAIN AND LIVER

—Ribosomes isolated from the brains of rats treated with morphine in vivo were less active in promoting the incorporation of [¹⁴C]leucine into protein than ribosomes isolated from untreated rats. This inhibitory phenomenon was studied in relation to dose of morphine, time after drug administration and the pharmacological responses of hypothermia and analgesia. The inhibition of [¹⁴C]leucine incorporation into brain proteins in vitro was transient after a single injection of morphine and dose-dependent, and related to the hypothermic response, but not prevented by keeping the rats at an ambient temperature which prevented hypothermia. The incorporation of [¹⁴C]leucine into protein by liver ribosomes was also inhibited in preparations from morphine treated rats.     

Assimilation of glucose carbon in subcellular rat brain particles in vivo and the problems of axoplasmic flow

1. Rats were injected with [U-¹⁴C]glucose and the content of ¹⁴C in proteins and lipids of the cerebral P₁ (`nuclear'), P<sub>2</sub> (`mitochondrial'), P₃ (`microsomal') and high-speed supernatant fractions was measured 7, 22 and 93hr. after injection of labelled glucose. 2. The crude brain mitochondrial fractions (P<sub>2</sub>) were subfractionated on continuous sucrose gradients (0·32–1·8m-sucrose) and the <sup>14</sup>C content of the proteins and lipids of about 20 subfractions was measured. 3. About 40–50% of the <sup>14</sup>C assimilated by brain proteins was found in the P<sub>2</sub> (`mitochondrial') fraction. About 68–70% of the ¹⁴C assimilated by brain lipids was also recovered from the lipids of the P₂ fraction. 4. Between 22 and 93hr. after injection of [U-¹⁴C]glucose both the amount of ¹⁴C in the protein of the P₂ (`mitochondrial') fraction and the specific activity of this protein increased. The specific activity of the protein of all other particulate fractions (P₁, P₂ and P₃) and subfractions (obtained from sucrose-density-gradient subfractionation of fraction P₂) when related to the specific activity of the high-speed supernatant protein also increased during 93hr. after injection of [U-¹⁴C]glucose. The amount of ¹⁴C in the protein of the high-speed supernatant and the specific activity of this protein decreased during the same period. 5. The distribution of ¹⁴C in the lipids of all subcellular particulate fractions remained unchanged during the period 22–93hr. after injection of [U-¹⁴C]glucose. 6. It was concluded that a diffusion occurs of some supernatant proteins into subcellular particulate matter of the cerebrum and no significant preference for any subcellular particulate matter was observed. The lipids occur in the cerebrum mainly in a non-diffusible state, which is consistent with the view that they form almost entirely a part of the structure of the cerebrum. 7. The data obtained do not lend further support to the concept of axoplasmic flow within the cerebrum or the concept of a one-directional flow of mitochondria or other subcellular particles within the cerebrum.     

Axonal transport of RNA during regeneration of the optic nerves of goldfish

The distribution of radioactive RNA and RNA precursors in the goldfish optic tecta following intraocular injection of ³H-uridine has been studied during various stages of optic nerve regeneration. ³H-uridine was injected into the posterior chamber of the right eye 17, 30, or 60 days after both optic nerves were crushed. Fish were sacrificed at time intervals ranging from 0.5 to 21 days after injection. One day prior to sacrificing, ¹⁴C-proline was also injected into the right eye as a marker of fast axonal protein transport. Seventeen to 23 days after crushing, the approximate time of nerve reconnection, the amount of radioactive RNA appearing in the left optic tectum was increased by more than ten times control values. Approximately 30 days after crushing the nerve, when the reconnected nerve is maturing, RNA values were still elevated, but significantly decreased from the earlier stage. By 60 days after crushing the optic nerve, the amounts of RNA in the left tectum was close to normal. Evidence suggesting that, at least, some of the radioactive RNA in the tectum originated from RNA transported along optic axons rather than from RNA synthesized locally in the tectum was provided by autoradiographic experiments. Autoradiograms of paraffin sections taken from the goldfish optic tecta after the intraocular injection of ³H-uridine showed a distribution of grains in a linear pattern, suggesting a distribution over the incoming fibers during the reconnection stage of regeneration. Electron microscopic autoradiography of glutaraldehyde fixed epoxy sections confirmed that a significant number of grains (shown to be ³H-RNA) were, in fact, over regenerating optic axons. Intracranial injection of ³H-uridine, during the same stage of regeneration, on the other hand, resulted in a distribution of grains, specifically over cell perikarya. These experiments suggest that during the reconnection phase of nerve regeneration, large amounts of RNA may be carried within regenerating optic axons as they enter the optic tectum.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE: DISTRIBUTION IN SUBCELLULAR FRACTIONS AS A FUNCTION OF TIME AFTER INJECTION OF [2-14C]MEVALONIC ACID, SODIUM [2-14C]ACETATE AND [U-14C]GLUCOSE INTO 15-DAY-OLD RATS

The distribution of [¹⁴C]-labelled material into subcellular fractions of 15-day-old rat brain was studied at 2 and 24 h following intraperitoneal and intracerebral injection of [2-¹⁴C]sodium acetate, [U-¹⁴C]glucose and [2-¹⁴C]mevalonic acid respectively. The total quantity of labelled isoprenoids in the brain was, except for glucose, greater when the precursor was administered intracerebrally. The intraperitoneal route was more advantageous in the case of [U-¹⁴C]glucose. The subcellular distribution of both labelled total isoprenoid material and sterol was distinct for each labelled precursor. Intracerebrally injected [U-¹⁴C]glucose at both time periods studied suggested no dominance of labelling in any fraction. After intraperitoneal injection of [U-¹⁴C]glucose the microsomes were more prominently labelled. Both methods of administration of sodium [2-¹⁴C]acetate resulted in heavy labelling of the myelin fraction after 24 h. The total labelled isoprenoids resided mainly in the microsomes 24 h after injection of [2-¹⁴C]mevalonic acid. Labelled sterol was found to be localized more in the myelin and microsomal fractions for all three precursors than was the labelled total isoprenoids. Depending on the type of experiment to be conducted, each of these precursors can give different results, which must be interpreted accordingly.     

Localization of histamine and histamine H2-receptor antagonists in the gastric mucosa.

Synopsis Histamine stimulates acid secretion by the parietal cell and this secretion is inhibited by the histamine H₂-receptor antagonists. Whole body autoradiography showed that radioactivity from¹⁴C-histamine was localized in the artery walls of the stomach and in the muscularis mucosae, but that the level in the fundic mucosa was the same as the blood.When the H₂-receptor antagonists burimamide, metiamide and cimetidine were labelled with³⁵S,¹⁴C or³H and dosed to rats, whole body autoradiography showed that the stomach was predominantly labelled in the glandular mucosa from 5 to 120 min after administration. Microautoradiography in the rat and dog after intravenous injection of [³H] metiamide or [³H] cimetidine demonstrated an uptake of tritium in the parietal cell cytoplasm that was 3- to 4-times greater than that found in adjacent peptic cells or areas of muscularis mucosa. The preferential labelling persisted at a low level up to 6 h after injection in the rat. The localization of radioactivity from the H₂-antagonists in the parietal cell cytoplasm correlates well with their pharmacological activity in preventing acid secretion from this cell.     

Crystal methamphetamine and initiation of injection drug use among street-involved youth in a Canadian setting

Background:

Although injection drug use is known to result in a range of health-related harms, including transmission of HIV and fatal overdose, little is known about the possible role of synthetic drugs in injection initiation. We sought to determine the effect of crystal methamphetamine use on risk of injection initiation among street-involved youth in a Canadian setting.

Methods:

We used Cox regression analyses to identify predictors of injection initiation among injection-naive street-involved youth enrolled in the At-Risk Youth Study, a prospective cohort study of street-involved youth in Vancouver, British Columbia. Data on circumstances of first injection were also obtained.

Results:

Between October 2005 and November 2010, a total of 395 drug injection–naive, street-involved youth provided 1434 observations, with 64 (16.2%) participants initiating injection drug use during the follow-up period, for a cumulative incidence of 21.7 (95% confidence interval [CI] 1.7–41.7) per 100 person-years. In multivariable analysis, recent noninjection use of crystal methamphetamine was positively associated with subsequent injection initiation (adjusted hazard ratio 1.93, 95% CI 1.31–2.85). The drug of first injection was most commonly reported as crystal methamphetamine (14/31 [45%]).

Interpretation:

Noninjection use of crystal methamphetamine predicted subsequent injection initiation, and crystal methamphetamine was the most commonly used drug at the time of first injection. Evidence-based strategies to prevent transition to injection drug use among crystal methamphetamine users are urgently needed.Street-involved youth are at high risk of initiating injection drug use.¹ This situation is of concern, given that injection drug use has been associated with increased risk of transmission of HIV and hepatitis C virus^2,3 and fatal overdose,⁴ as well as a range of other serious negative health and social outcomes. Newly initiated injection drug users have also been identified as a subpopulation at particularly high risk of injection-related harm.^5–8 Unfortunately, despite recent calls to prioritize interventions to prevent the initiation of injection drug use,⁹ there are few evidence-based strategies to prevent injection initiation among street-involved youth.This situation relates, in part, to the fact that little is known about the risk factors for injection initiation within this population. Prospective research from Montréal, Quebec, has alluded to the role that crack and powder cocaine may play in promoting subsequent injection initiation,¹⁰ as has retrospective research conducted among drug users in Baltimore, Maryland.¹¹ Much less is known about the possible role that synthetic drugs, such as methamphetamine, may play in contributing to an increased risk of injection initiation.¹² Globally, amphetamine-type stimulants have emerged as one of the most commonly used groups of illicit drugs, second only to cannabis.¹³ This increase in amphetamine use is reflected in the epidemiology of drug use in some Canadian settings. For instance, in Vancouver, British Columbia, rates of injection use of crystal methamphetamine have increased significantly among adult injection drug users.¹⁴ This pattern is of substantial public health concern, given that crystal methamphetamine has been associated with a range of health and social harms, including the potential to drive high-risk drug-use patterns, including injection.¹⁵ Given these concerns and the well-established health-related harms of injection drug use, we investigated the possible role of crystal methamphetamine use in the incidence of first injection drug use within a cohort of street-involved youth in a Canadian setting.     

Microbial growth on C1 compounds: Uptake of [14C]formaldehyde and [14C]formate by methane-grown Pseudomonas methanica and determination of the hexose labelling pattern after brief incubation with [14C]methanol

1. A study has been made of the incorporation of carbon from [¹⁴C]formaldehyde and [¹⁴C]formate by cultures of Pseudomonas methanica growing on methane. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled compounds for periods of up to 1min., has been analysed by chromatography and radioautography. 3. Radioactivity was fixed from [¹⁴C]formaldehyde mainly into the phosphates of the sugars, glucose, fructose, sedoheptulose and allulose. 4. Very little radioactivity was fixed from [¹⁴C]formate; after 1min. the only products identified were serine and malate. 5. The distribution of radioactivity within the carbon skeleton of glucose, obtained from short-term incubations with [¹⁴C]methanol of Pseudomonas methanica growing on methane, has been investigated. At the earliest time of sampling over 70% of the radioactivity was located in C-1; as the time increased the radioactivity spread throughout the molecule. 6. The results have been interpreted in terms of a variant of the pentose phosphate cycle, involving the condensation of formaldehyde with C-1 of ribose 5-phosphate to give allulose phosphate.     

Metabolism of 3H- and 14C-labelled lactate in starved rats

1. [2-³H,U-¹⁴C]- or [3-³H,U-¹⁴C]-Lactate was administered by infusion or bolus injection to overnight-starved rats. Tracer lactate was injected or infused through indwelling cannulas into the aorta and blood was sampled from the vena cava (A–VC mode), or it was administered into the vena cava and sampled from the aorta (V–A mode). Sampling was continued after infusion was terminated to obtain the wash-out curves for the tracer. The activities of lactate, glucose, amino acids and water were followed. 2. The kinetics of labelled lactate in the two modes differed markedly, but the kinetics of labelled glucose were much the same irrespective of mode. 3. The kinetics of ³H-labelled lactate differed markedly from those for [U-¹⁴C]lactate. Isotopic steady state was attained in less than 1h of infusion of [³H]lactate but required over 6h for [U-¹⁴C]lactate. 4. ³H from [2-³H]lactate labels glucose more extensive than does that from [3-³H]lactate. [3-³H]Lactate also labels plasma amino acids. The distribution of ³H in glucose was determined. 5. Maximal radioactivity in ³HOH in plasma is attained in less than 1min after injection. Near-maximal radioactivity in [¹⁴C]glucose and [³H]glucose is attained within 2–3min after injection. 6. The apparent replacement rates for lactate were calculated from the areas under the specific-radioactivity curves or plateau specific radioactivities after primed infusion. Results calculated from bolus injection and infusion agreed closely. The apparent replacement rate for [³H]lactate from the A–VC mode averaged about 16mg/min per kg body wt. and that in the V–A mode about 8.5mg/min per kg body wt. The apparent rates for [¹⁴C]lactate (`rate of irreversible disposal') were 8mg/min per kg body wt. for the A–VC mode and 5.5mg/min per kg body wt. for the V–A mode. Apparent recycling of lactate carbon was 55–60% according to the A–VC mode and 35% according to the V–A mode. 7. The specific radioactivities of [U-¹⁴C]glucose at isotopic steady state were 55% and 45% that of [U-¹⁴C]lactate in the A–VC and V–A modes respectively. We calculated, correcting for the dilution of ¹⁴C in gluconeogenesis via oxaloacetate, that over 70% of newly synthesized glucose was derived from circulating lactate. 8. Recycling of ³H between lactate and glucose was evaluated. It has no significant effect on the calculation of the replacement rate, but affects considerably the areas under the wash-out curves for both [2-³H]- and [3-³H]-lactate, and calculation of mean transit time and total lactate mass in the body. Corrected for recycling, in the A–VC mode the mean transit time is about 3min, the lactate mass about 50mg/kg body wt. and the lactate space about 65% of body space. The V–A mode yields a mass and lactate space about half those with the A–VC mode. 9. The area under the wash-out curve for [¹⁴C]lactate is some 20–30 times that for [³H]lactate, and apparent carbon mass is 400–500mg/kg body wt. and presumably includes the carbon of glucose, pyruvate and amino acids, which are exchanging rapidly with that of lactate.     

EFFECT OF METHOTREXATE ON THE CELL CYCLE OF L1210 LEUKEMIA

The influence of methotrexate (MTX) on the proliferative activity of cells in different phases of cell cycle has been studied. MTX (5 mg/kg) was injected i.p. 3 days after the inoculation of 5 × 10⁶ leukemia cells into F₁ (DBA × C57 BL) mice. It was shown that MTX causes degeneration of cells, being in G₁- as well as in S-phase at the time of drug injection. Incorporation of ³H-TdR was suppressed for a period ranging from 2 to 12 hr after MTX administration, which is demonstrated by the decrease in the number of grains per cell. The number of cells labeled after ³H-TdR injection was also sharply decreased during this period. For a period of 3 until 15 hr after MTX administration the mitotic index decreased significantly as a result of inhibition of DNA synthesis. The blocking of the G₁-S transition was evident during 4 hr after MTX. Thereafter the G₁-S transition proceeds at a rate which is practically equal to that for nontreated controls. MTX did not inhibit transition to mitosis of cells being in G₂-phase and in a very late S-phase at the time of drug injection. The sensitivity of G₁-cells to the cytocidal effect of MTX shows that for L1210 leukemia cells MTX can be classified as a cycle-specific drug killing both G₁ and S-cells rather than S-phase specific agent with self-limitation.     

Determinants of the disposition of 14C-delta 9-tetrahydrocannabinol and 3H-delta 9-tetrahydrocannabinol.

Intragastric (i.g.) administration of ¹⁴C-THC plus ³H-THC to male Wistar and Fischer rats resulted in a more rapid disappearance of ¹⁴C than ³H from fresh blood or plasma. The concentrations of the two isotopes were equivalent from 1–4 hours when blood was analyzed after being dried. For each rat strain, apparent absorption of both isotopes was more rapid in fed than fasted rats and in young (130–150g) compared to older (250–260g) animals. The concentrations of ³H were significantly higher than ¹⁴C in the major organs which were analyzed fresh at 4 and 24 hours after drug administration, but the isotope levels were not different when the tissues were analyzed after lyophilization. Experiments indicate that tritiated water was produced metabolically from ³H-THC and was lost from blood and organs upon drying.The fresh blood levels of total ¹⁴C and unchanged ¹⁴C-THC were higher than total ³H and ³H-THC, respectively, from 40 min to 4 hrs following i.v. injection of ¹⁴C-THC plus ³H-THC in bile duct-cannulated rats. Similarly, the amount of ¹⁴C was higher than the amount of ³H in the urine. However, the concentration of ³H was higher than ¹⁴C in the bile after 20 min. The ³H level was higher than ¹⁴C at 4 hrs in the brain but lower than ¹⁴C in the liver, heart and spleen. Drying of body fluids and tissues before isotope analysis did not alter these results. It is concluded that the formation of tritiated water occurs in the gut after i.g. ³H-THC administration, and that the dispositions of ¹⁴C-THC and ³H-THC are not entirely equivalent following i.v. injection.     

Transfer times across the human body

The radioactivity disappearance curves of glucose-6-¹⁴C albumin-I¹³¹ after a single injection of tracer into a human subject have been determined in detail, particularly at early time intervals. The curves, expressed as sums of exponentials, have been analyzed as the infinite sum of convolutions of single passage time densities. The resultant transfer time distribution of a single circulatory pass allows examination of all delays in the system no matter how long they take. The structural detail evident by this means and the long mean time of a single pass of glucose (>5 min) supports the thesis that factors other than rapid and uniform diffusion play a role in the extravascular movements of glucose molecules. This study was supported by Public Health Service Grants CA 07123 from the National Cancer Institute and RR 00044 from the Division of Research Facilities and Resources, N.I.H.     

A comparative analysis of extracellular fluid volume of several tissues as determined by six different markers

The uptake and distribution of 6 different extracellular markers were analyzed in ten tissues of the rat. The saccharides, ³H-mannitol, ³H-raffinose, ³H-inulin, and ¹⁴C-inulin, reached a steady-state distribution in all tissues within ≈15 min after intraperitoneal injection; ²²Na and ³⁶Cl followed similar kinetics in all tissues except the choroid plexuses and the thyroid, which required > 1 hr to obtain a steady-state plateau. In most tissues, the steady-state spaces of ³H-raffinose, ³H-inulin, and ¹⁴C-inulin (60 min) were not significantly different; however, the ³H-mannitol, ²²Na and ³⁶Cl spaces were on average 45, 54, and 79%, respectively, greater than the ³H-inulin space.     

Transport of proteins and ribonacleic acid along nerve axons

The distribution of isotope in the sciatic nerve was determined at times up to 22 days after injection of (a) [¹⁴C]leucine, or (b) [³H]orotic acid, in the spinal cord of the chicken. The results indicate a distal flow of proteins, RNA precursors and perhaps RNA along the nerve axon.     

Precursors of pattern specific ommatin in red wing scales of the polyphenic butterfly Araschnia levana L.: Haemolymph tryptophan and 3-hydroxykynurenine

In the seasonally diphenic butterfly Araschnia levana¹⁴C-labelled tryptophan and 3-hydroxykynurenine, the principal precursors of ommochromes, injected into young pupae caused a pattern specific radiolabel of mature red scales. [¹⁴C]glucose and [³⁵S]methionine also labelled red scales but only when injected shortly before or during the time of pigment synthesis in the wing. In developing non-diapause pupae contents of 3-hydroxykynurenine increased until an abrupt decrease when pigments appeared in the wings. In diapausing pupae 3-hydroxykynurenine remained low but increased after injection of 20-hydroxyecdysone which induced pupal-adult development. Supply of wing scale cells with ommochrome precursors via the haemolymph was analysed after injection of [³H]tryptophan. In developing pupae haemolymph contents of [³H]tryptophan and [³H]3-hydroxykynurenine increased at the time of wing pigment formation and decreased shortly before adult emergence. In diapausing pupae haemolymph contents of [³H]tryptophan and [³H]3-hydroxykynurenine were low compared to non-diapause pupae but increased at the time of wing pigment formation after injection of 20-hydroxyecdysone. Isolated wings incubated in Grace's medium containing [¹⁴C]tryptophan and [¹⁴C]3-hydroxykynurenine incorporated radiolabel specifically into red portions of the wing colour pattern due to labelling of ommatin. Incorporation into red wing areas occurred specifically depending on different colour patterns of the spring- and the summer-morph.The results demonstrate that both tryptophan as well as 3-hydroxykynurenine are delivered via the haemolymph, and both can serve as precursors of ommatin formation in the scale cells. Therefore, the complete set of enzymes for the tryptophan-ommatin pathway is present in scale-forming cells.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.     

The cytoplasmic ribonucleoprotein particles of rat sarcoma cells

Summary The metabolism of the cytoplasmic ribosomal RNP-particles from rat sarcoma cells have been studied after intraperitoneal injection in animals of¹⁴C-leucine alone or together with H₃P³²O₄. By investigating the change of the specific activity of the ribosomes with respect to the time after injection of¹⁴C-leucine we have demonstrated that the half-life of ribosomes is 35 hours.To study the metabolic activity of ribosomes not taking part in protein synthesis, the sucrose gradient centrifugation method was used for their separation from polyribosomes. Four, seven, twelve hours after injection of the isotopes free ribosomal subunits and monoribosomes were isolated and their radioactivity was determined. As expected the label was found in the ribosomal subunits at the beginning and later on in the monoribosomal fraction. At the same time it was observed that the incorporation of H₃P³²O₄ into ribosomal subunits occurred at a much greater rate as compared with the incorporation of¹⁴C-leucine. These results indicate the existence of a pool of ribosomal proteins in sarcoma cells whose role deserves attention.an invited article     

Comparative in Vitro / in Vivo Autoradiography Using the Opiate Ligand 3H-(−)-Bremazocine

Abstract

Autoradiograms of rat brain sections were compared obtained from animals receiving a tritiated drug through intravenous injection or from precut sections incubated in vitro. The benzomorphan analogue ³H-(?)-bremazocine was used as ligand and its distribution to all different opioid binding sites was followed. Although the general distribution of opioid binding sites visualized on ³H-LKB ultrofilms was independent of the methodological approach used, the maximal number of such sites (Bmax) was greater in brain sections incubated in vitro than after in vivo drug application. Since the number of binding sites is highly dependent on the particular incubation condition used, this finding has no further relevance.     

Change in the charactertistics of 3H-spiperone binding to rat striatal membranes after acute chlorpromazine administration: effects of buffer washing of membranes.

After intraperitoneal injections of ³H-spiperone into the rat, brain membrane preparations retain the majority of the radioactivity even after several buffer washes. With ³H-spiperone as ligand, dissociation constants were significantly elevated and maximum binding unchanged in rat striatal membranes after acute intraperitoneal injection of chlorpromazine (14 mg/kg). It is suggested that in studies of post-mortem brains of schizophrenics that contain neuroleptics specific ³H-spiperone binding will be lowered by competition from residual drug in membrane preparations and valid comparisons of ³H-spiperone binding to preparations from control and schizophrenic brains can only be made if maximum binding values are determined.