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The effect of shifting zygotes from a rich medium into a poor medium (shift-down) on the frequency of recombination has been examined; our results suggest that protein syntheses other than those required for growth may be involved. Experiments with chloramphenicol support this notion, and further suggest that there is a competition between genetic integration and degradation in the recipient cells. By inhibiting protein synthesis one can shift the competition in favor of either integration or segregation, depending on the physiological state of the zygotes. By calculating the time required for segregation as one generation, we conclude that the recombination chromosome is homozygous at the time of integration.

Cell extracts of a thermophilic strain of Bacillus grown on phenol metabolise catechol by the meta -cleavage route to yield 2-hydroxymuconic semialdehyde. The product of ring-fission may be further converted by a NAD+-dependent semialdehyde dehydrogenase or by a non-NAD+-dependent enzyme reaction. Both enzymes are induced in phenol-grown cells and high levels of activity are detectable at 50°.

In the presence of glucose, respiration a d l v ls cit i i cycle intermediates n washed hu n sp atozoa ar low Cit t ev ls o m rkedly re se bpy uva , actt t e tr dth c mpound c s r spir tio . S cina h ch oxidiz g , it b ut f fo ; w v r iration t ibu ucc ate-f marate n r lo g r l i ul fc i lc Td ti u i l a p ns t in ng 50100-l \g=a\-k glu or ly y i l bl s u zh c o p i d re rfu i t i hw hyd o a ps u l d c equiv l t r ig gm z i r y cycle.ummary. In the presence ofglucose, respiration and the levels ofcitric acid cycle intermediates in washed human spermatozoa are low. Citrate levels are not markedly increased by pyruvate, lactate or acetate, nor do these compounds increase respiration. Succinate, which is oxidized at high rates, increases citrate levels about fourfold; however, the increased respiration is attributed to the succinate\p=m-\fumarateconversion alone and not to a general stimulation of the citric acid cycle. The addition of succinate, malate, or fumarate to sperm suspensions containing pyruvate increases citrate levels 50to 100-fold, markedly stimulates \g=a\-ketoglutarateformation, decreases glycolysis and almost doubles the rate ofpyruvate utilization. The increased rates of the synthesis of citrate and other intermediates of the citric acid cycle are not accompanied by increased rates of respiration. These data argue for the presence of a powerful pyruvate dismutation pathway in human spermatozoa in which lactic dehydrogenase competes successfully for the reducing equivalents generated by pyruvate oxidation and argues against the idea that oxidation in spermatozoa is limited by substrate entry into the citric acid cycle.

During growth of Hydrogenomonas eutropha H 16 on 2-ketogluconate, 2-ketogluconate kinase and 2-keto-6-phosphogluconate reductase were formed. These enzymes were absent from cells grown on fructose, gluconate, acetate, succinate or autotrophically. There was no evidence for extracellular oxidation of glucose, fructose or gluconate with the formation of ketogluconic acids. The utilization of 2-ketogluconate is neither subject to catabolite inhibition by hydrogen nor is 2-keto-6-phosphogluconate reductase inhibited by ATP, ADP or phosphoenolpyruvate. The enzyme is characterized by a high affinity for its substrates.

Glycyrrhetinic acid (carbenoxolone) inhibited cyclic nucleotide phosphodiesterase (PDE) activity in a number of tissues from rat, guinea pig and dog. In the fundic mucosa, it was more active when cyclic AMP rather than cyclic GMP was used as substrate in the PDE assays. Glycyrrhetinic acid also raised the intracellular levels of cyclic AMP in rat pyloric and fundic mucosae in vivo with little effect on cyclic GMP levels. This effect, at least in the rat, was apparently mediated via PDE-inhibition, since the drug did not affect adenylyl cyclase activity from either mucosa. Studies with theophylline and papaverine, in addition to glycyrrhetinic acid, suggested that the effects of PDE inhibitors on gastric acid secretion could be related to their relative activities on cyclic AMP-PDE's or cyclic GMP-PDE's, owing to the possibly opposite effects mediated by the two cyclic nucleotides on acid secretion by the stomach.

The steady state concentrations of arginine and related intermediary metabolites of the arginine biosynthetic pathway in the eukaryote Neurospora crassa were varied and the concurrent de-repression of the enzymes ornithine transcarbamylase, argininosuccinate synthetase and argininosuccinase was measured. Pool variation was achieved endogenously by the introduction and combination of mutant enzymes with reduced specific activities. Measurements of activities of the mutationally unaltered enzymes showed various degrees of de-repression. The highest activity level for each of the three enzymes was about five times that found in the fully repressed wild-type strain. The variations observed in the pools were as follows: ornithine, 7-fold; citrulline, 700-fold; argininosuccinic acid, 400-fold; arginine, 300-fold. By this means a quantitative analysis of the process of repression is made possible. A strong correlation was found between the degree of de-repression of the three enzymes and the concentration of arginine. The de-repression follows a sigmoid curve with respect to arginine concentration. This is consistent with the interpretation that the pathway enzymes are subject to a repression system with arginine, or a simple derivative of it, acting as a co-repressor.

A procedure has been devised that allows selection of mutants defective in the β-methylgalactoside transport system ( mgl ) of Escherichia coli . This procedure utilizes the compound 2R-glyceryl-β- d -galactopyranoside (glycerylgalactoside), which is known to be transported by only two transport system in E. coli , namely, the lactose and the β-methylgalactoside transport systems. Mutants lacking glycerol-3-phosphate dehydrogenase ( glpD ) are sensitive to glycerol. Similarly, mutants lacking uridine diphosphate-galactose-4-epimerase ( galE ) are sensitive to galactose. Glycerylgalactoside is an inducer of the lactose operon and also a substrate for β-galactosidase. Thus, a mgl + glpD galE lacY strain will not grow in the presence of glycerylgalactoside owing to accumulated glycerol-3-phosphate, galactose-1-phosphate, and uridine diphosphate-galactose. We have constructed such a strain and shown that mgl mutants can be obtained by selecting for those that grow in the presence of glycerylgalactoside.

A radioimmunoassay for human plasma corticosterone has been developed. Antisera were obtained by immunizing rabbits with corticosterone-21-hemisuccinate-BSA. An antiserum titer of 1:4000 was used for standard curves ranging from 0–1000 pg. Interfering steroids were removed from plasma extracts by paper chromatography. Plasma blanks obtained from adrenalectomized or Addisonian patients ranged from 29 to 42 ng/dl. Recovery of radioactive corticosterone through the entire method was 67.6 ± 5.2%. The coefficient of variation within assays was 19% and between assays 13%. The average 8 a.m. value in males was 396 ± 228 ng/dl and in females it was 655 ± 271 ng/dl. Corticosterone was found to be secreted episodically, in parallel with cortisol. Secretion of this steroid was suppressed by dexamethasone and stimulated by ACTH infusion.

This paper continues the description of transketolase mutants of Escherichia coli ; they are absolutely unable to grow on pentoses, but slightly “leaky” with respect to their aromatic requirement (B. L. Josephson and D. G. Fraenkel, 1969). Several experiments have explored the degree of leakiness and shown it to be low. There is little conversion of radioactive xylose to carbon dioxide. The labeling of ribose in cells grown on [1- 14 C]glucose and [2- 14 C]glucose accords with its origin being chiefly by the oxidative pathway. A mutant lacking both transketolase and gluconate-6-phosphate dehydrogenase has been constructed; it requires supplementation with pentose. Pentoses are inhibitory to growth of transketolase mutants, but high levels of pentose phosphates do not accumulate in this situation. Several experimental results are suggestive of regulation of metabolic flow in the oxidative branch of the hexose monophosphate shunt.

— The metabolism of a tricarboxylic acid cycle (cycle) intermediate, [1.4-'14C]succinate, was studied in the brain at 2 20 min after intracerebral injection. The oxidation of [14C]succinate was rapid, as shown by the incorporation of 14C into cycle amino acids which accounted for about 30 per cent and 70 per cent of the tissue -“Cat 2 and 10 min respectively. During the whole experimental period the specific radioactivity of glutamine was about three times higher than that of glutamate. Thus exogenous [14C]succinate elicited signs of metabolic compartmentation similar to those seen after the administration of short chain fatty acids or amino acids. A computer programme, based on data obtained previously on the metabolic compartmentation of acetate and of glucose in the brain, was used to simulate the kinetics of labelling of cycle amino acids after an input of [1.4-14C]succinate. The correspondence of the simulated data with the experimental results was good in the first 10 min after injection, although the deviations were significant at later time points. Incorporation of 14C into GABA was very low (< 1 per cent of the amino acid -14C) after the injection of [1.4-14C]succinate. Further, labelled GABA formation was not detected in the decapitated rat brain labelled in vivo with [1.4-14C]succinate 2 min beforehand. Since the oxidation of [l,4-14C]succinate via the cycle yields unlabellcd GABA. whereas the reversal of the reactions in the GABA bypath may introduce 14C from succinate into the GABA pool, the results indicate that this reversal is negligible even under the most favourable conditions, i.e. post mortem when both the NADH/NAD+ ratios and [14C]succinate concentrations arc high. The observations are therefore consistent with the view that glutamate is the predominant and probably the only source of GABA carbon in the brain both in vivo and post mortem.

1. The interrelationship of acidosis and Ca(2+) on the stimulation of gluconeogenesis by rat kidney-cortex slices was studied. 2. Ca(2+) stimulated gluconeogenesis from glutamine, glutamate, 2-oxoglutarate, succinate, malate, pyruvate, lactate and fructose, but not from galactose. 3. The [Ca(2+)] needed for optimum gluconeogenesis was about 2mm, but at this concentration, acidosis, produced in vitro by a decrease of [HCO(3) (-)] in the medium at constant pCO(2) or by an increase in pCO(2) at constant [HCO(3) (-)], did not stimulate gluconeogenesis. 4. In the absence of Ca(2+), acidosis (low [HCO(3) (-)]) stimulated gluconeogenesis from glutamine, glutamate, 2-oxoglutarate, succinate, malate, pyruvate and lactate but not from fructose or galactose. With succinate as substrate, the stimulatory effect of acidosis (low [HCO(3) (-)]) disappeared at Ca(2+) concentrations above 1.0mm. 5. The [HCO(3) (-)] was the most important determinant of the acidosis effect since a decrease of pH caused by an increase in pCO(2) did not uniformly stimulate gluconeogenesis, whereas a decrease in [HCO(3) (-)] without a change in pH consistently stimulated glucose formation in a way similar to the stimulation produced by acidosis (low [HCO(3) (-)]) in the absence of Ca(2+). 6. Acidosis in vitro inhibited the rate of decrease of activity of phosphoenolpyruvate carboxylase in slices, and Ca(2+) caused an increase in the activity of fructose 1-phosphate aldolase. 7. Respiratory acidosis in vitro caused an increase in the activity of phosphoenolpyruvate carboxylase in kidney cortex and an increase in gluconeogenesis from glutamine. 8. Possible points of interaction between Ca(2+), H(+) and HCO(3) (-) with the gluconeogenic sequence are discussed.

Various mitochondrial functions have been studied at different stages of preservation of dog livers incubated in Krebs-plasma solution at 4°C. under normal or hyperbaric oxygen pressure. Hyperbaric preservation protects the deterioration of the liver mitochondrial functions for a period of no longer than 12 hr. The correspondence between the mitochondrial impairment times found by us and the limit for good hepatic viability found by other authors using hyperbaric hepatic preservation suggest that one of the roles of hyperbaric oxygen could be the protection of mitochondrial functions. Final mitochondrial deterioration occurs during hepatic preservation possibly due to other factors which are beyond the protective influence of oxygen.

Cell-free extracts of an organism capable of degrading the synthetic ether carboxymethyloxysuccinic acid were used to study the mechanism of degradation of this compound. The initial degradation products were acidic and could be separated by high-voltage electrophoresis. Extended incubation of [14C]carboxymethyloxysuccinate with the cell-free extracts gave rise to radioactive carbon dioxide and to glycolic, malic, fumaric, and pyruvic acids which were identified by electrophoresis. Identification of the malic and glycolic acids was confirmed by the addition of non-radioactive carrier malic and glycolic acids and recrystallization to constant specific radioactivity. The identification of pyruvate was substantiated by thin-layer chromatography of the 2,4-dinitrophenylhydrazine derivative. The presence of fumaric acid was substantiated by its ultraviolet absorbance. Shorter incubations give rise to only malic, glycolic, and fumaric acids. When the time course of the reaction was followed carefully over the first 15 min of the reaction, it was found that initially nearly equimolar quantities of glycolic and fumaric acids were produced. The early production of fumaric acid was also demonstrated by following the increase in 245-nm absorbance of the degradation mixture. The fumaric acid was subsequently hydrated to malic acid, and the quantity of fumaric acid present decreased slightly until it reached a steady-state level. The presence of fumarase in the extract was demonstrated. It was concluded that the initial attack on the carboxymethyloxysuccinic acid was a cleavage of the ether linkage by an elimination mechanism catalyzed by a carbon-oxygen lyase giving rise to glycolic and fumaric acids.

Pseudomonas Pxy was isolated on p -xylene as sole source of carbon and energy. Substrates that supported growth were toluene, p -methylbenzyl alcohol, p -tolualdehyde, p -toluic acid, and the analogous m -methyl derivatives, including m -xylene. Cell extracts prepared from Pseudomonas Pxy after growth with either p -xylene or m -xylene oxidized the p - and m -isomers of tolualdehyde as well as p -methylbenzyl alcohol. The same cell extracts also catalyzed a “meta” fission of both 3- and 4-methylcatechol. Treatment of Pseudomonas Pxy with N -methyl- N ′-nitro- N -nitrosoguanidine led to the isolation of two mutant strains. Pseudomonas Pxy-40, when grown on succinate in the presence of p -xylene, accumulated p -toluic acid in the culture medium. Under the same conditions Pseudomonas Pxy-82 accumulated p -toluic acid and also 4-methylcatechol. When Pseudomonas Pxy-82 was grown on succinate in the presence of m -xylene, 3-methylcatechol and 3-methylsalicylic acid were excreted into the culture medium. A pathway is proposed for the initial reactions utilized by Pseudomonas Pxy to oxidize p - and m -xylene.

1. The NADP-dependent glutamate dehydrogenase of Neurospora crassa undergoes slow reversible structural transitions, with half-times in the order of a few minutes, between active and inactive states. The inactive state of the enzyme, which predominates at pH values below 7.0, has an intrinsic tryptophan fluorescence 25% lower than that of the active state, which predominates at pH values above 7.6. The inactive state can be activated either by an increase in pH or by addition of activators such as succinate. 2. The kinetics of the slow transitions that follow activating and inactivating rapid changes in conditions have been monitored by measurements of protein fluorescence. The results show that the slow reversible conformational change detected by the change in fluorescence is the rate-limiting process for enzyme activation and inactivation. 3. In both directions this conformational change follows apparent first-order kinetics and the rate constant is independent of protein concentration. These kinetics and published measurements of molecular weight are indicative of an isomerization process. 4. In both directions the changes show a large energy of activation and a large positive entropy of activation, consistent with a considerable disturbance of conformation in the transition state. 5. Comparisons of the fluorescence emission spectra of the active and inactive states indicate that the difference in fluorescence is produced by quenching, possibly intramolecular, in the inactive conformation. Iodide ions cause similar quenching. 6. In some mutationally altered forms of the enzyme comparable but modified conformational changes can be followed by protein fluorescence.

Rat-liver mitochondria, devoid of outer membrane, were subjected to the action of pure phospholipase A2 from porcine pancreas. Degradation products were removed by treatment with bovine serum albumin. Arrhenius plots of succinate-cytochrome c reductase and cytochrome c oxidase activities revealed two breaks, in the temperature region 10–30 °C. The exact values of the break temperatures were dependent on the extent of phospholipid hydrolysis, particularly in the case of succinate-cytochrome c reductase. The higher temperature break probably reflects a phase change of the lipids in close proximity to the enzyme molecules, while the lower temperature break can possibly be explained by a reorganization of the lipids in the gel phase. The values of the apparent activation energies varied with the extent of phospholipid hydrolysis and, consequently, with the relative phospholipid composition, indicating that the magnitude of the activation energy of a lipid-dependent enzyme is not merely governed by the physical state, but also by the composition of the lipid environment. Differences were observed in the effect of phospholipase treatment between the two enzyme activities studied. This suggests a certain degree of heterogeneity in the distribution of the lipids which are directly involved in the functioning of the enzyme molecules.

Orange, chlorophyll-deficient cells of Chlorella fusca were obtained by prolonged exposure (6 wk) to light and CO 2 (1.5% in air) in a nitrogen-sparse medium: growth ceased after 6 days, chlorophyll formation after 3 days, and then chlorophyll degradation followed with a drop in chlorophyll a :b ratio. When the 6-wk-old cells were exposed to light in a nitrogen-rich medium and sparged with CO 2 (1.5% in air) rapid chlorophyll synthesis ensued with preferential synthesis of chlorophyll a . Regreening under these conditions was complete in approximately 24–30 hr and during this period no cell division occurred. We were unable to demonstrate 5-aminolaevulinate synthase (EC 2.3.1.37) in cell-free extracts of regreening Chlorella but demonstrated aminolaevulinate formation by whole regreening cells incubated in the presence of laevulinate, an inhibitor of aminolaevulinate dehydratase (EC 4.2.1.24). Chlorophyll synthesis was almost completely inhibited by 100 m m laevulinate, and a stoichiometric relationship exists between aminolaevulinate formation and the chlorophyll deficit caused by the presence of laevulinate: thus, the use of the inhibitor provides a true indication of the ability of the cells to form aminolaevulinate. Using this technique we found that chlorophyll synthesis during regreening appears to be regulated by the availability of aminolaevulinate since there was a correlation between the rate of aminolaevulinate and chlorophyll synthesis: both reached a maximum about halfway through the regreening period. It was not possible to decide whether the availability of aminolaevulinate was limited by the level or activity of aminolaevulinate synthase or by the supply of succinyl CoA. Regreening of orange Chlorella was inhibited by cycloheximide. Regreening of Chlorella can occur in the dark if vigorously sparged with oxygen so differing from greening of higher plants which is light dependent. Both [1,4- 14 C]succinate and [2- 14 C]glycine were incorporated into aminolaevulinate by partly regreened Chlorella fusca cells incubated in the presence of laevulinate.

The interaction of chemically modified rabbit anti-trinitrophenyl (TNP) macrophage cytophilic antibodies with guinea pig macrophages was studied. The antibodies were modified with dodecylsuccinic anhydride which had no effect on antibody activity, but rendered the modified antibodies inactive both in the passive direct and passive indirect tests for cytophilic activity. However, the modified proteins had the ability to block macrophage receptors and inhibit rosette formation. It was found that the modified antibodies were bound to macrophages, but were released after combining with TNP-coated erythrocytes. No such release was observed when soluble antigen trinitrophenylated (bovine serum albumin) or TNP-hapten, TNP-ϵ-aminocaproic acid were used. The implication of this effect is that combination of the Fab portion of antibody molecules with an insoluble antigen might produce a conformational change in the Fc region. However, other possibilities, e.g. the effect of modification on the cytophilic activity of the Fc region and the disruption of rosettes by thermal motion, are also discussed.

Using a polarographic method (oxygen electrode), the effects of 1−Δ 9 -tetrahydrocannabinol (an active constituent of marihuana), DL-amphetamine and pentobarbital were compared in regard to their action on oxygen consumption by mouse brain and heart homogenates. This study was unique in that the drugs were injected in vivo , while measurement of oxygen consumption was conducted in vitro for up to 8 hr. This allowed for the true active forms of these drugs—after normal biotransformation, if necessary—to exert the effects later measured. Pentobarbital was found to have no significant effect on cerebral or cardiac oxygen consumption. Both DL-amphetamine and 1−Δ 9 -tetrahydrocannabinol caused significant stimulation of oxygen consumption in the brain and heart for up to at least 2·5 hr after administration. This could indicate that both drugs cause increased synthesis and utilization of high energy compounds (i.e. ATP) via oxidative phosphorylation in the two organs. However, DL-amphetamine and 1−Δ 9 -tetrahydrocannabinol also induced limited depression of oxygen consumption in the brain and heart, respectively, at 8 hr after administration.

Affinity chromatography of 3-oxosteroid Δ4-Δ5-isomerase (EC 5.3.3.1) of Pseudomonas testosteroni on 19-nortestosterone-17-O-succinyl diaminodipropylamino-agarose constitues a powerful and highly effective step in the purification of the enzyme in preparation for crystallization.

The distributions of phosphate and of a dicarboxylate were compared in rat liver mitochondria. The phosphate can be divided into an ionised part, assuming a Donnan relation holds between it and the other anion, and a non-ionised part which is found to be stoichiometrically related in the ratio 1:1.8 to the mitochondrial Ca content. When arsenate and phosphate distributions are compared, the distribution calculated for ionised phosphate corresponds to that measured for the arsenate. The ionised phosphate was deduced from the total by deduction of an amount calculated to be equivalent to the Ca content. Both sets of results support the concept of mitochondria normally carrying non-ionised phosphate as the Ca salt. A competition between arsenate and ionised phosphate for accumulation is described.

Incubation of washed human spermatozoa in the presence of 6 mM concentrations of EDTA, histidine and cysteine induces a release of about 75% of the zinc bound to the cells. No zinc is released by human spermatozoa when incubation is done in the absence of the mentioned reagents. No detectable amounts of calcium or magnesium were found to be released by the sperm cells under any of the experimental conditions tested. Zinc release induced by the presence of EDTA, histidine and cysteine is accompanied: 1) by a significant increase in oxygen uptake, both under basal conditions and in the presence of some substrates (glucose, pyrubate and succinate) and 2) by a significant increase in motility. This increase was greater with cysteine than with histidine, and greater with the latter than with EDTA. This behavior of human spermatozoa resembles that previously described for invertebrate spermatozoa and is related to the regulation of energy metabolism and probably to sperm capacitation.

The diglyceride kinase activity of isolated E. coli membrane vesicles appears to be mediated in part by succinyl CoA synthetase. Upon incubation of vesicles with [γ- 32 P]ATP, or with 32 P i and succinyl CoA, radioactivity appears in membrane-bound phosphatidic acid. CoA inhibits incorporation of radioactivity from [γ- 32 P]ATP. Succinate lowers the concentration of CoA required to inhibit this incorporation, although succinate alone stimulates incorporation. At high [γ- 32 P]ATP concentrations, inhibition by succinate and CoA is overcome. The results suggest that diglyceride kinase can use either ATP or the phosphorylated form of succinyl CoA synthetase as phosphate donor for the phosphorylation of diglyceride.

The activities of three enzymes specifically involved in naphthalene metabolism have been measured in Pseudomonas NCIB 9816 after induction with salicylate or 2-hydroxybenzyl alcohol. The results indicate that naphthalene oxygenase, 1,2-dihydroxynaphthalene oxygenase and salicylaldehyde dehydrogenase are induced coordinately and it is suggested that all of the enzymes converting naphthalene to salicylate are regulated coordinately.

Pseudomonas putida NCIB 10015 metabolizes phenol and the cresols (methylphenols) by the meta pathway and metabolizes benzoate by the ortho pathway. Growth on catechol, an intermediate in the metabolism of both phenol and benzoate, induces both ortho and meta pathways; growth on 3- or 4-methylcatechols, intermediates in the metabolism of the cresols, induces only the meta pathway to a very limited degree. Addition of catechol at a growth-limiting rate induces virtually no meta pathway enzymes, but high levels of ortho pathway enzymes. The role of catechol and the methylcatechols as inducers is discussed. A method is described for assaying low levels of catechol 1,2-oxygenase in the presence of high levels of catechol 2,3-oxygenase and vice versa.

SYNOPSIS Drug sensitivities of a lower trypanosomatid, Leptomonas sp. from the hemipteran Dysdercus, to antitrypanosomatid agents were estimated by growth inhibition and inhibition of polarographically measured O2 uptake of cells and particulate fractions. Several standard trypanocides, antimalarials, and electron-transport inhibitors were used. Acriflavine, quinacrine, primaquine, pentamidine, and ethidium, each at ∼1 mM, inhibited cellular O2 uptake by 50% after 3-hr incubation. Suramin and melarsen were inactive. Crude particulate fractions oxidized L-α-glycerophosphate at 80% the rate of succinate oxidation and L-proline at 55% the succinate rate, leading to choice of succinate as standard respiratory substrate. Concentrations for 50% inhibition of growth were (μg/ml): ethidium bromide 0.009, acriflavine HCl 0.12, Antrycide 0.83, pentamidine diisethionate 1.5, and stilbamidine isethionate 1.1. Possible use of this trypanosomatid as model organism for screening antitrypanosomatid agents, and use of ethidium and acriflavine as initial model compounds are discussed.

1. 1. Wing epidermis from diapausing and non-diapausing silkmoths (Antheraea pernyi) was assayed for NADH [nicotinamide adenine dinucleotide (reduced form)] oxidase, NADH- and succinate-cyctochrome c reductases, and cytochrome c oxidase. 2. 2. Diapausing pupae showed a marked decline in activities after pupation and the same subsequent profile of very low activities reported for diapausing Cecropia silkmoths. 3. 3. Non-diapausing pupae showed only a slight and transient decline after pupation and otherwise retained relatively high activities throughout metamorphosis.

Reconstitution of tobacco mosaic virus from its constituents, the coat protein and RNA, was investigated by means of ultracentrifugation and circular dichroism measurement. Tobacco mosaic virus protein forms a 20S double-layer disc under conditions favorable for tobacco mosaic virus reconstitution. Dibromination of the tyrosine 139 residue of tobacco mosaic virus protein prevents formation of the 20S disc. Acidification of the tobacco mosaic virus protein solution causes 20S discs to polymerize into long helical rods. Changes in the CD spectra of tobacco mosaic virus protein in the near-ultraviolet region suggest that stacking of the aromatic sidechains of amino acid residues stabilizes the helical rod. The dibrominated tobacco mosaic virus protein also has the ability of rod elongation under acidic condition. CD studies reveal that assembly of tobacco mosaic virus particles from its constituents is stabilized by the stacking effect between the base residues of RNA and the aromatic residues of tobacco mosaic virus protein. Cucumber green mottle mosaic virus protein, which acts as a substituent for tobacco mosaic virus protein in tobacco mosaic virus reconstitution, was also investigated.

The effects of 2-phenylisatogen and menadione on some mitochondrial reactions have been studied. The respiration with NAD+-linked substrates was stimulated, but respiration with succinate was not affected. The stimulation of respiration was inhibited by p-hydroxymercuribenzoate, but not by other respiratory chain inhibitors. Potassium cyanide and sodium sulphide stimulated NADH oxidation in the presence of 2-phenylisatogen or menadione. 2-Phenylisatogen was found to be converted to 2-phenylindolone. It is concluded that both 2-phenylisatogen and manadione are acting as electron acceptors in the NADH dehydrogenase region of the respiratory chain. Possible mechanisms for the reaction and for the stimulation observed in the presence of cyanide are discussed.

Using a new dialysis membrane highly permeable to middle molecules and a closed loop dialysate delivery system, the author has tested the middle molecule hypothesis in a tentative way for understanding the uremic syndrome through hemodialysis.

1. The slow enhancement of the fluorescence of the complex between aurovertin and Mg-ATP particles, induced by succinate oxidation in the absence of adenine nucleotides and phosphate, is completely accounted for by dissociation of the natural ATPase inhibitor from the particles. 2. The large increase in the aurovertin fluorescence induced by an anaerobicaerobic transition requires the presence of all State-3 components (substrate, ADP, phosphate and Mg 2+ ). This change is very rapid with a half-time of 20 ms. 3. There are two aurovertin-binding sites on Mg-ATP particles, one with high affinity and one with low affinity. Binding of aurovertin to the strong sites is positively cooperative only under State-3 conditions. 4. The quenching of the fluorescence of the particle-aurovertin complex induced by an uncoupler is rapid and extensive in the presence of ATP. The rapid quenching induced by an uncoupler is followed by a slow increase in the aurovertin fluorescence, that may be attributed to a redistribution of bound adenine nucleotides. All uncoupler-induced changes are prevented by oligomycin. 5. Excess oligomycin added to Mg-ATP particles oxidizing succinate under State-3 conditions initially induces a rapid enhancement of the aurovertin fluorescence. The pH dependency of this change suggests that this rise may be attributed to the induction of a maximally ‘coupled’ membrane.

A clonal strain of epithelial cells has been established from the transplantable Morris hepatoma 7800 and is designated 7800C1. The cells grow with a population doubling time of about three days in serum-supplemented synthetic medium. Cells of the 7800C1 strain have maintained measurable activities of all the enzymes of the urea cycle during 17 months in continuous culture. The activity of argininosuccinate lyase is approximately that found in normal rat liver, while argininosuccinate synthetase, carbamoyl phosphate synthetase, arginase and ornithine carbamoyl transferase activities are, respectively, 40%, 28%, 6%. and 1% of normal values. Treatment of 7800C1 cells with glucagon, dibutyryl 3′,5′-cyclic adenosine monophosphate or hydrocortisone did not increase the activity of any of the five enzymes.

The effects of N,N ′-dicyclohexylcarbodiimide (DCCD) on the growth of Streptococcus faecalis , and on the growth, β-galactosidase synthesis, and various membrane-mediated processes, were studied in wild-type Escherichia coli JE1011 and its lipopolysaccharide-defective mutant NS1. DCCD (0.1 mM) completely inhibited the growth of S. faecalis and E. coli NS1 but had little effect on strain JE1011. The same amount of DCCD with E. coli NS1, but not with E. coli JE1011, inhibited the induction of β-galactosidase, increased the permeability of the cells to o -nitrophenyl-β- d -galactoside without causing extensive cell lysis or release of ultraviolet-absorbing materials, and inhibited the oxidation of certain intermediates of the tricarboxylic acid cycle. Inhibition of the oxidation of malate, fumarate, and α-ketoglutarate by DCCD appeared to be at the level of the transport system for these compounds. Inhibition of the membrane-bound adenosine triphosphatase by DCCD was not entirely responsible for these effects, since oxidation of these substances, and transport of [ 14 C]succinate and [ 14 C]fumarate, was inhibited by DCCD in a mutant, N 144 , which lacked adenosine triphosphatase activity. It is concluded that lipopolysaccharide forms a barrier to DCCD in wild-type E. coli , and that DCCD can inhibit several processes in the cell.

Antisera were raised in rabbits using conjugates of albumin with 11-hemisuccinates of 11α-OH E1 and 11α-OH E2. These antisera were compared with antisera to 6-oxo E2 6-CMO-, E2 17-succinyl- and E3 16, 17-disuccinyl-BSA by radioimmunoassay using a statistically designed three-point assay. Sheep anti-(rabbit γ-globulin) coupled to cellulose was used for the separation of antibody-bound and free labeled hapten. Antisera obtained with haptens linked to the carrier at the 17(16) position poorly discriminated between the various estrogens. Antisera obtained with 6- and 11-conjugates showed a much better specificity. In addition the specificity was influenced by using either estrone, estradiol or estriol as tritiated labels. This gives the possibility to determine different parameters by employing different labeled hormones.