The regulated catabolism of endogenous and exogenous phosphatidylinositol by Saccharomyces cerevisiae leading to extracellular glycerophosphorylinositol and inositol.

It was previously shown that phosphatidylinositol catabolism leads to the accumulation of glycerophosphorylinositol in the culture medium of Saccharomyces cerevisiae. We now find that lack of an energy source (glucose) reduces the formation of glycerophosphorylinositol and increases extra-cellular inositol. This situation is reversed by refeeding glucose. [3H]Phosphatidylinositol is the precursor of extra-cellular [3H]inositol with energy-starved cells. Extracellular glycerophosphorylcholine and glycerophosphorylethanolamine accumulate more slowly than glycerophosphorylinositol in the growth medium and do not appear to be a strongly affected by energy starvation. Phosphatidylinositol deacylation appears to occur at the cell surface in a regulated manner. Exogenously added phosphatidylinositol apparently does not mix randomly with the endogenous pool since it is not converted to either inositol-containing sphingolipid or to diphosphoinositide, both previously shown to be derived in part from cellular phosphatidylinositol. Labeled exogenous phosphatidylinositol is, however, quantitatively converted to glycerophosphorylinositol with the probable intermediat formation of monoacyl-glycerophosphorylinositol. Breakdown of exogenous phosphatidylinositol requires an energy source and does not lead to free inositol. Deacylation of exogenously added 1-acyl-glycerophosphorylinositol occurs much faster than deacylation of phosphatidylinositol and does not require an energy source. Glycerophosphorylethanolamine formation from exogenous phosphatidylethanolamine occurs about as fast as the breakdown of phosphatidylinositol and is also inhibited in the absence of energy source. The much slower deacylation of exogenous phosphatidylcholine was also affected by an energy source. Glycerophosphorylinosiyolaccumulates in the culture medium of Kloeckera apiculata, Saccharomyces carlsbergenis, and Neurospora crassa.


Equilibrium and water proton relaxation rate enhancement properties of formyltetrahydrofolate synthetase-manganous ion-substrate complexes.

Binding of Mn(pi)-nucleotide complexes to the enzyme formyltertrahydrofolate synthetase (EC 6.3.4.3) from Clostridium cylindrosporum has been examined in the presence and absence of other substrates by solvent proton relaxation mearurements. MnADP and MnATP form ternary complexes with the enzyme with highly enhanced proton relaxation rates for water. The enhancement parameters, epsilont, for the MnADP and MnATP ternary complexes are 19.8 and 12.5, respectively at 24.3 MHZ and 25 degrees. Titration curves with constant total concentrations of enzyme and Mn(pi) with variable nucleotide concentration are similar to those observed in similar titrations with the endp and MnATP are 175 muM and 64 muM, respectively at 25 degrees. Addition of tetrahydrofolate to solutions of the MnADP OR MnATP ternary complexes lowers the observed relaxation enhancement markedly. An analysis of titration curves with constant total concentrations of enzyme, Mn(pi), and nucleotide with variable tetrahydrofolate concentration gives the dissociation constant for tetrahydrofolate from the respective quaternary complexes. The affinity of the enzyme for tetrahydrofolate is increased 6-fold when MnADP is present at the active site whereas a 3-fold increase is observed with MnATP present. Furthermore, there is a 20-fold increase in the enzyme's affinity for tetrahydrofolate when both MnADP and the third substrate, formate, are present. The observed relaxation rate of water for solutions of the complex, enzyme-MnADP-tetrahydrofolate-formate, is deenhanced with respect to the rate observed for the simple aquo-Mn(pi) solution. Addition of nitrate to solutions of the above complex increases the affinity of the enzyme for tetrahydrofolate and MnADP by an additional factor of 5 and lowers the relaxation rate further to a value which approaches that for solutions of the enzyme and substrates which lack the paramagnetic cation.


Electron paramagnetic resonance and water proton relaxation rate studies of formyltetrahydrofolate synthetase-manganous ion complexes. Evidence for involvement of substrates in the promotion of a catalytically competent active site.

Conformational properties of the active site of formyltetrahydrofolate synthetase from Clostridium cylindrosorum have been examined by EPR spectroscopy and by solvent proton relaxation rate (PPR) studies of manganous complexes with the enzyme. Ternary enzyme-Mn-nucleotide complexes give EPR spectra which are very similar to those for the binary Mn-nucleotide complexes. However, upon addition of tetrahydrofolate to form the quaternary complexes, enzyme-MnADP-tetrahydrofolate and enzyme MnATP-tetrahydrofolate the EPR line shapes are changed substantially. Spectra for the quaternary complexes exhibit narrow line widths, and the splitting patterns are characteristic of a slightly asymmetric electronic environment for the bound Mn(II). Addition of formate to the ADP quatenary complex induces a further significant narrowing of the EPR line widths, although in the absence of tetrahydrofolate, formate does not influence the EPR spectrum for the enzyme-MnADP species. Both Pi and nitrate cause changes in the EPR patterns for the higher complexes of the enzyme which involve both ADP and tetrahydololate. However, the Pi effect is not influenced by the presence of formate whereas the characteristic effect of nitrate is potentiated only when formate is present. EPR sectra for the thernary complex with the beta, gamma-methylene analog of ATP App(CH2)p differ significantly from spectra for the binary App(CH)p complex is not influenced by further additions of tetrahydrofolate and of tetrahydorfolate and formate. The failure of spectra for the App(CH)p complex to respond to additions of the other substrates for the reaction is in marked contrast to the behavior found for the natural nucleotide substrates and is tentatively attributed to the lack of a protein-mediated interaction between the nucleotide and tetrahydrofolate binding sites in the analog complex. The frequency dependence of solvent PRR in the presence of the various complexes allows an estimate of the correlation times for electron-nuclear dipolar interaction and thereby the extent of hydration of the bound Mn(II) among the various complexes..


Reduced diphosphopyridine nucleotide peroxidase. Intermediates formed on reduction of the enzyme with dithionite or reduced diphosphopyridine nucleotide.

DPNH peroxidase is a flavin adenine dinucleotide-containing flavoprotein. Anaerobic titration of enzyme with dithionite has shown that the active site of the enzyme contains 2 mol of flavin and in addition 1 mol of a non-flavin electron acceptor that is tentatively identified as a disulfide group. Thus complete reduction of the enzyme requires 3 mol of dithionite per mole of active site. The first mole of dithionite reduces the non-flavin acceptor; complex formation between the reduced acceptor and one of the bound flavin molecules causes the formation of a long wavelength absorption band between 500 and 670 nm. The second mole of dithionite reduces the flavin that interacts with the reduced non-flavin group, and the long wavelength band disappears. The third mole of dithionite reduces the second mole of flavin. All groups are reoxidized in the presence of air. DPNH reacts with only two of the enzyme-bound electron acceptors. The first mole of DPNH reduces the non-flavin group to form an intermediate (I) that is almost identical with that formed by dithionite. The second mole of DPNH complexes with the second flavin of Intermediate I to form Intermediate II. This reaction causes a further absorbance increase in the long wavelength region; the tail of the absorption band now extends to 960 nm. The titration data (potassium phosphate, 0.05 M, pH 7.0) can be fitted with dissociation constants of 1 times 10-7 M for the formation of I, and 3 times 10-6 M for the conversion of I to II. In air, species II is oxidized to I; I is stable in air, but is oxidized stoichiometrically to oxidized enzyme by H2O2. Present evidence suggests that bound DPN-plus is responsible for the air stability of species I. Intermediate I, but not oxidized enzyme, reacts slowly with phenylmercuric acetate. This reaction causes loss of the air-stable intermediate and parallel loss in enzyme activity. The inactive enzyme cannot be reduced by DPNH to Species I; DPNH can, however, still react with the second flavin to form the autoxidizable complex. With other methods of enzyme inactivation there is also a direct correlation between residual enzyme activity and the ability of enzyme to form the air-stable intermediate. It is concluded that the air-stable intermediate is an important catalytic species.


Hydrophobic binding sites on human interferon.

Human interferon binds to a omega-carboxpentyl-agarose column at low ionic strength (0.15 M NaCl) and is still retained when the ionic strength is raised (to 1.0 M NaCl). The binding can be reversed, however, by ethylene glycol, indicating a hydrophobic interaction. The binding of human interferon to omega-aminohexyl-agarose is weak, even at a low ionic strength, and is probably exclusively electrostatic. This disparate binding behavior may be caused by the presence of a positive charge, adjacent to the hydrophobic binding site, on human interferon. The interaction of human interferon with omega-carboxypentyl-agarose is quite selective, inasmuch as the majority of proteins present in interferon preparations pass through the column unretained. Hydrophobic chromatography of human interferon may thus be useful in its purification.


On the fidelity of DNA replication. Characterization of polynucleotides with errors in base-pairing synthesized by avian myeloblastosis virus deoxyribonucleic acid polymerase.

Polynucleotide templates were copied by avian myeloblastosis virus DNA polymerase ("reverse transcriptase") and the frequency and distribution of errors were determined. The error rate with [r(pA)2500-d(pT)12-18] template-initiator under a variety of conditions was approximately 1/600, i.e. one incorrect dCMP incorporated for 600 correct dTMP polymerized. Addition of the metal chelator o-phenanthroline to the reaction inhibited the incorporation of correct and incorrect nucleotides proportionately. The enzyme inhibited a pH optimum of 8.5 and the error rate remained constant over a range of pH (6.0 to 10.0). The rate of polymerization was greater at higher temperatures and approximately doubled for every 10 degrees increase. The error rate was constant at all temperatures. These results indicate that the purified avian myeloblastosis virus DNA polymerase synthesizes polydeoxynucleotides with an unusually large number of errors in base-pairing. Velocity sedimentation of the products of the reaction obtained at various times during the course of synthesis indicate that: (a) the entire length of the 14 S template was copied, and (b) the incorporation of the incorrect nucleotide did not terminate chain propagation. Isopyknic banding in neutral and alkaline cesium sulfate gradients showed that the noncomplementary nucleotides are incorporated into the polydeoxynucleotide product. Stepwise degradation and nearest neighbor analysis of the reaction product indicated that (a) the correct and incorrect nucleotides are present in phosphodiester linkages, (b) the errors are not concentrated at either termini; and (c) the errors are uniformly distributed throughout the newly synthesized polydeoxynucleotide.


Carbamylphosphate synthetase from Salmonella typhimurium. Regulations, subunit composition, and function of the subunits.

Carbamylphosphate synthetase was purified to homogeneity from a derepressed strain of Salmonella typhimurium by a procedure based on affinity chromatography employing immobilized glutamine. The enzyme catalyzes the synthesis of carbamylphosphate from either ammonia or glutamine together with ATP and bicarbonate. The ATP saturation curve of either nitrogen donor is sigmoidal (n equals 1.5) but the affinity for ATP is higher with ammonia. In addition to the feedback inhibition by UMP and activation by ornithine which we previously reported (1), the activity was found to be stimulated by IMP and phosphoribosyl-1-pyrophosphate. Evidence from pool measurements in enteric bacteria by others suggests that of the latter two compounds only phosphoribosyl-1-pyrophosphate is physiologically significant. All effectors regulate enzyme activity by altering its affinity for ATP. Glutamine also modulates the affinity for ATP; it is increased as glutamine concentratiions decrease, an effect that could serve to insulate the cell against major changes in carbamylphosphate synthesis in response to fluctuations in concentration of glutamine. The molecular weight of the holoenzyme was estimated to be 150,000 by sucrose density gradient centrifugation in triethanolamine and Tris-acetate buffers in which the enzyme is a monomer. In the presence of ornithine in potassium phosphate buffer, the enzyme is an oligomer with a molecular weight of 580,000. This transition has been exploited as an alternate route of purifying the enzyme to homogeneity using successive sucrose density centrifugation. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate shows that the enzyme consists of two unequal subunits with molecular weights of 110,000 and 45,000. The two subunits were separated by gel filtration in the presence of 1 M potassium thiocyanate, ATP, MgCl2, glutamine, NH4Cl, ornithine, and UMP. The heavy subunit catalyzes the synthesis of carbamylphosphate from ammonia but not glutamine. The ATP saturation curve for the separated heavy subunit is still sigmoidal (n equals 1.4 and So.5 equals 0.3 mM). The ammonia dependent activity of the heavy subunit is stimulated by the activators ornithine, IMP, and phosphoribosyl-1-pyrophosphate but is only marginally inhibited by high concentrations of UMP. The addition of the light subunit restored full ability to utilize glutamine as well as normal sensitivity to UMP. Purified subunits were used for in vitro complementation studies with strains carrying mutations in pyrA, the structural gene encoding carbamylphosphate synthetase. The results indicate that the pyrA region encodes both subunits and that the structural genes for the two polypeptides are linked. A deletion mutant lacking both subunits of carbamylphosphate synthetase also lacked any ability to synthetize carbamylphosphate from ammonia. Hence, unlike certain other bacteria, S. typhimurium does not possess a carbamate kinase.


Characteristics of 5'-guanylyl imidodiphosphate-activated adenylate cyclase.

Characteristics of adenylate cyclase stimulation by the GTP analog 5'-guanyl imidodiphosphate Gpp(NH)p have been examined in intact frog erythrocytes, frog erythrocyte membranes, and solubilized canine myocardial preparations. Gpp(NH)p caused marked enzyme activation in the erythrocyte membranes and in solubilized myocardial preparations, but had much lesser effects in intact cells. Enzyme activation by Gpp(NH)p exhibited a definite lag period, requiring 10 to 15 min for complete activation at 37 degrees. Activation was essentially irreversible after a 5-hour dialysis sufficient to reduce the Gpp(NH)p levels below threshold for stimulation. Gpp(NH)p-"activated" enzyme differed from native enzyme in several respects, such as its greater temperature stability, and its insensitivity to further stimulation by other activators, such as catecholamine or fluoride. These differences suggest that the enzyme, once fully activated by Gpp(NH)p, may have undergone some modification that is not subject ot facile reversal.


Purification and regulatory properties of chicken heart prostaglandin E 9-ketoreductase.

Prostaglandin E 9-ketoreductase was purified from chicken heart by ammonium sulfate fractionation, and DEAE-Sephadex, hydroxylapatite and phosphocellulose chromatography. Two peaks of activity were resolved during the phosphocellulose chromatographic step. Both peaks were stimulated by a substance that was not bound to the phosphocellulose column. This stimulatory substance was destroyed by treatment with phosphodiesterase and 0.1 M NaOH. It was heat-stable (100 degrees, 2 min), nondialyzable, and resistant to treatment with pronase, ribonuclease, and deoxyribonuclease; but it was dialyzable after heating or digestion with pronase. Sodium pyrophosphate also enhanced the activities of the prostaglandin E 9-ketoreductases as did angiotensin I; but not angiotensin II. In the presence of 3':5'-cyclic AMP, AMP, or several other ribonucleotides, the enhancing effects of the natural stimulatory substance, sodium pyrophosphate or angiotensin I were blocked, but these ribonucleotides themselves had little effect on the enzymes activity. The substrate specificities of the two prostaglandin E 9-ketoreductases were also studied. Both the 9-keto group and the 15-keto group of 15-ketoprostaglandin F2 alpha could be converted to the corresponding hydroxyl group; the 15-keto group was reduced faster than the 9-keto group. Prostaglandin D2, a prostaglandin with a 9-hydroxyl and an 11-keto group, could not be converted to prostaglandin F2 alpha nor could cyclohexanone be converted to cyclohexanol by the prostaglandin E 9-ketoreductase.


The role of Ca-2+ and cyclic adenosine 3':5'-monophosphate in insulin release induced in vitro by the divalent cation ionophore A23187.

Insulin release from isolated perifused pancreatic islets was stimulated by the divalent ionophore A23187 in the absence of exogenous glucose. In addition, A23187 produced a 2-fold elevation of cyclic adenosine 3':5'-monophosphate (cAMP) levels in isolated perifused islets. The elevation of cAMP levels coincided with peak insulin release. Ionophore-induced insulin release was unaffected by pretreatment of the islets with theophylline (5 mM). Stimulation of insulin release produced by the ionophore occurred either in the presence or absence of extracellular Ca-2+; however, cAMP accumulation required the presence of extracellular Ca-2+. The ionophore (10 muM) had no effect on adenylate cyclase activity of homogenates of isolated islets. The results of this study are interpreted as indicating that intracellular Ca-2+ has an essential role in the insulin releasing mechanism, whereas the cAMP system has a modulatory effect on this process.


The kinetics of glucocorticoid binding to the soluble specific binding protein of mouse fibroblasts.

The kinetics of binding of glucocorticoids to the soluble, specific binding protein of mouse fibroblasts has been examined. The rate at which both potent and weak glucocorticoids achieve binding equilibrium is very slow. Second order rate constants of association range from 3 times 10-5 M- minus 1 min- minus 1 for cortisol to 6.7 times 10-5 M- minus 1 min- minus 1 for triamcinolone acetonide. Studies of the rates of binding at high steroid concentrations suggest that the slow rate of binding may be explained by a two-step mechanism. Active glucocorticoids, regardless of their potency, bind initially in a rapid manner to form a weak complex with the binding protein. The dissociation constant for the weak binding reaction is 0.87 times 10- minus 7 M for triamcinolone acetonide and 2.4 times 10- minus 7 M for cortisol. The weak binding complex becomes converted slowly to a tight complex. The first order rate constants for this conversion and the rate constants of dissociation from the tight complex have been determined for cortisol, dexamethasone and triamcinolone acetonide. The binding affinity of steroids of different biological potency is correlated with their rate of dissociation from this second tight binding state.


Chemical synthesis and sequence studies of deoxyribooligonucleotides which constitute the duplex sequence of the lactose operator of Escherichia coli.

We have synthesized the deoxyribooligonucleotide fragments, constituting the sequence of the lac operator of Escherichia coli. Two of these fragments, d(pApApTpTpGpTpTpApT) (nonamer) and d(pApApTpTpGpTpGpApG) (nonamer), corresponding to the 5' termini of lac operator have been synthesized by the phosphodiester method. The remaining four fragments, d(ApCpApApTpT) (hexamer), d(ApTpApApCpApApTpT) (nonamer), d(ApApTpTpGpTpGpApGpCpGpG) (dodecamer), and d(ApApTpTpGpTpTpApTpCpCpGpCpTpC) (pentadecamer), have been synthesized by an improved phosphotriester method. All of the compounds were first characterized by venom and spleen phosphodiesterase digestion to obtain their base composition. The sequence of these oligonucleotides was fully confirmed by the characteristic mobility shifts of their partial venom phosphodiesterase digestion products on two-dimensional homochromatography. A comparative study of the two methods for the synthesis of oligonucleotides has revealed that the phosphotriester method is more convenient than the phosphodiester method because of higher yields and ease of handling large scale preparations.


DNA sequence analysis. Terminal sequences of bacteriophage phi80.

Sequences of the cohesive ends and the 3'-terminal regions of phi80 DNA have been determined. Sequences of the cohesive ends were obtained through the use of two standard methods. The first method involved the incorporation of all four labeled deoxyribonucleotides into the phi80 cohesive ends using DNA polymerase I. The DNA was then partially digested with micrococcal nuclease or pancreatic DNase. The products were separated by two-dimensional electrophoresis and characterized by composition, 3'-terminal, and nearest neighbor analyses. The second method involved partial incorporation using one, two, or three labeled deoxyribonucleotides followed by similar analyses. Sequences of the double-stranded regions adjacent to the cohesive ends were determined by three new methods. These methods were: (a) the DNA was specifically labeled at the 3' terminus and then partially degraded. Labeled oligonucleotide products were sequenced by their mobilities on various separation systems. (b) The cohesive ends were enlarged by limited degradation with exonuclease III. After this treatment, the DNA was partially repaired with labeled nucleotides, digested, and the products were analyzed. (c) A synthetic ologonucleotide primer was bound to phi80 DNA which had been repaired with DNA polymerase I, and then partially digested with lambda-exonuclease. The primer was extended into the region of interest by partial repair with labeled nucleotides. The extended primer was isolated and analyzed.


Cytochrome P-450 of adrenal mitochondria. Spin states as detected by difference spectroscopy.

Adrenal mitochondrial cytochrome P-450 which functions in cholesterol side chain cleavage (P-450scc) exhibited type I (lambdamax 385, lambdamin 420 nm) and inverse type I (lambdamin 385, lambdamax 420 nm) difference spectra with several steroids. The magnitude and type of response were dependent on the particular steroid and on the extent to which cholesterol was bound to the cytochrome in the intact mitochondrion. the inverse type I difference spectrum induced by 3beta-hydroxy-pregn-5-ene-20-one (pregnenolone) was dependent on the proportion of high spin cholesterol-cytochrome P-450scc complexes. With rat adrenal mitochondria cholest-5-ene-3beta, 20alpha-diol (20alpha-hydroxycholesterol) invariably induced a smaller inverse type I response and, under conditions where cytochrome P-450scc was nearly free of cholesterol, even produced a small type I response. Two distinct steroid binding sites on cytochrome P-450scc were detected by, respectively, the slow type I response to cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and the rapid type I response to a subsequent addition of cholest-5-ene-3beta, 20alpha, 22 R-triol (20alpha, 22R-dihydroxycholesterol). The relative proportions of the spectral responses to these steroids were dependent on the previous extent of adrenal activation by adrenocorticotropic hormone (ACTH), because this stimulatory process altered the combination of mitochondrial cholesterol with cytochrome P-450scc. It is proposed that the two steroid binding sites on cytochrome P-450scc interact with steroids in the following way: site I binds cholesterol, 25-hydroxycholesterol, and 20alpha, 22R-dihydroxycholesterol with formation of a partially high spin cytochrome; site II binds both pregnenolone and 20alpha-OH cholesterol resulting in a low spin cytochrome. Interactions between sites I and II are not competitive, and occupancy of site II ensures a low spin state irrespective of the occupancy of site I. A second mode of interaction by 20alpha, 22R-dihydroxycholesterol stabilizes a high spin cytochrome and is competitive with site II binding by 20alpha-hydroxycholesterol or pregnenolone. Formation of a maximally high spin cytochrome follows occupancy by 20alpha, 22R-dihydroxycholesterol at both sites.


Cytochrome P-450 of adrenal mitochondria. In vitro and in vivo changes in spin states.

Steroid-induced difference spectra have been used to examine the combination of cholesterol with adrenal mitochondrial cytochrome P-450 which participates in cholesterol side chain cleavage (P-450scc) and the depletion of cholesterol from the cytochrome which results from turnover of the enzyme system. Type I difference spectra-induced by cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and cholest-5-ene-3beta, 20 alpha, 22R-triol (20alpha, 22R dihydroxycholesterol) have been used to quantitate binding of cholesterol to two sites (I and II) on cytochrome P-450scc. The action of adrenocorticotropic hormone (ACTH) in vivo and the action of calcium or phosphate ions on isolated mitochondria stimulate the combination of cholesterol with site I but not site II. Cholesterol derived from lecithin-cholesterol micelles, however, binds to both sites. Malate-induced cholesterol depletion occurred at a comparable rate to the transfer of cholesterol from lecithin-cholesterol micelles. However, a residual proportion of cholesterol-cytochrome P-450scc complexes remained, even after 10 min of exposure to malate, and was of similar magnitude in mitochondria from both cycloheximide-treated and stressed rats. It is suggested that this reflects a less reactive form of cholesterol-cytochrome complex. Steroid-induced difference spectra indicate that sites I and II on cytochrome P-450scc are similarly depleted after metabolism of mitochondrial cholesterol in vitro and after inhibition of the action of ACTH in vivo. Anaerobiosis of adrenal cells after excision of the accumulation of cholesterol at cytochrome P-450cc. When anaerobiosis was prevented, cytochrome P-450scc in the freshly isolated mitochondria was apparently essentially free of complexed cholesterol, irrespective of the extent of ACTH action. For 30 min after suspension of the mitochondria in 0.25 M sucrose at 4 degrees, cholesterol combines with cytochrome P-450scc. The extent of this process was not affected by the presence of cycloheximide during ether stress treatment of the rats. It is concluded that there are at least two pools of mitochondrial cholesterol with access to cytochrome P-450scc but that ACTH stimulates only the pool which most readily interacts with the cytochrome.


Insulin binding to liver plasm membranes in the obese hyperglycemic (ob/ob) mouse. Demonstration of a decreased number of functionally normal receptors.

In previous studies, the insulin resistance of the obese hyperglycemic mouse (ob/ob) was found to be associated with decreased insulin binding to liver, fat, and lymphocytes. The present study demonstrates that the insulin receptors in the liver membranes of the ob/ob mouse are decreased in number, but are indistinguishable from normal by other criteria including affinity, kinetics of association and dissociation, temperature dependence of binding, and biological specificity of the binding reaction. The receptor in liver membranes of the ob/ob mouse is also indistinguishable with respect to insulin receptor site-site interactions. Degradation of both insulin and of receptor sites was studied and did not account for differences observed in binding. We conclude that the insulin receptor from the ob/ob mouse is a functionally normal receptor and that its presence in diminished number accounts for the observed decrease in insulin binding to liver plasma membranes.


The role of calcium in regulation of cyclic nucleotide content in human umbilical artery.

In term gestational human umbilical artery segments incubated in room air at 37 degrees, histamine, acetylcholine, bradykinin, K+, and serotonin (agonists that cause contraction) cause accumulation of guanosine 3':5'-monophosphate (cGMP) without altering the content of adenosine 3':5'-monosphophate (cAMP); prostaglandin E1 (PGE1), which relaxes the artery, causes cAMP accumulation without affecting the cGMP content (Clyman, R. I., Sandler, J.A., Manganiello, V.C., and Vaughan, M. (1975) J. Clin. Invest., in press). It has been postulated that Ca-2+ is important in the regulation of cyclic nucleotides in other tissues. In the umbilical artery the control of cAMP content by PGE1 was independent of Ca-2+. After incubation in Ca-2+-free medium, the c GMP contentof the artery segments was decreased by 50% and was unaffected by histamine, acetylcholine, bradykinin, and K+. Readdition of Ca-2+ (2.7 mM) or Sr-2+ (3.6 mM) to the medium partially restored the basal cGMP content and the agonist effects on the cGMP content. However, Sr-2+ was not as effective as Ca-2+ in this regard. Ionophores A23187 and X537A (agents that facilitate Ca-2+ movement through membranes) mimicked the effects of these Ca-2+-dependent agonists on cGMP content. Incubation with the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (0.1 mM) increased both the basal content of cGMP and the histamine-induced accumulation 3-fold. This effect was dependent on the presence of Ca-2+ also. Accumulation of cGMP induced by serotonin, on the other hand, was not diminished in Ca-2+-depleted arteries and, in fact, seemed to be inhibited by 2.7 mM Ca-2+. These observations are consistent with the existence in the umbilical artery of two separate mechanisms for control of cGMP synthesis that are influenced differently by Ca-2+.


The cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum. Binding characteristics of aggregation-competent cells and variation of binding levels during the life cycle.

Both cyclic guanosine 3':5'-monophosphate and dithiothreitol stimulate binding of cyclic adenosine 3':5'-monophosphate (cAMP) to aggregation-competent amoebae. Both compounds appear to function solely by preventing the hydrolysis of cAMP by the cell-bound phosphodiesterase. The dissociation constant for binding of cAMP is 36 nM. Both cAMP binding and membrane-bound phosphodiesterase activities increase dramatically as cells develop aggregation competence, reach a maximum at about 11 hours, and remain at high levels for up to 48 hours if cells are maintained in shaken suspension. When amoebae are allowed to aggregate and develop naturally, binding of cAMP increases during aggregation, decreases during tip formation, and disappears during culmination. Phosphodiesterase activity parallels binding activity except that the decreased level after tip formation is retained throughout culmination. Two N-6-modified cAMP derivatives compete with cAMP for binding sites. One derivative is fluorescent (1,N-6-etheno-cAMP); the other is photolyzable [N-6(ethyl-2-diazomalonyl)cAMP]. This result opens the possibilities of using fluorescence quenching for assay of in vitro binding and of affinity labeling of binding sites. Competition by the derivatives is only partial, indicating possible heterogeneity of binding sites. Both compounds inhibit hydrolysis of cAMP by the membrane-bound phosphodiesterase.


Preparation and characterization of liver cells made permeable to macromolecules by treatment with toluene.

Isolated individual liver cells were made permeable to charged molecules and macromolecules by treatment with toluene, and the properties of such cells were examined in detail. The optimal conditions of toluene treatment, as determined by assay of aminoacyl-tRNA synthetase activities, was 7 to 9% toluene for 2 min at 0 degrees. Conditions are also described for maintaining the treated cells for periods up to 1 hour. Toluene treatment was found to be as efficient as various disruptive methods for making internal enzymes accessible to exogenous substrates, and this was true for enzymes in the cytosol, membrane, or organelle fractions. Electron microscopic and biochemical examination of toluen-treated cells indicated that they were relatively intact and lost only small amounts of cellular constituents to the suspension medium. The data in this paper suggest that toluene treatment of individual cells might prove useful for studies of macromolecular synthesis in liver;


Characterization of gastric mucosal membranes. VIII. The localization of peptides by iodination and phosphorylation.

Two fractions of gastric mucosal membranes obtained by Ficoll-sucrose density gradient centrifugation were studied by a variety of techniques to localize the polypeptides. Gel electrophoresis showed the presence of five major polypeptides and several minor ones. Only one of these, 82,000 daltons, was available for iodination in the intact tissue. The two membrane fractions differed in their accessibility to peroxidase. The denser fraction showed two major defined iodination peaks at 82,000 and 102,000 daltons. Freeze-thawing and iodinating with 131-I produced additional labeling of peaks as well as relabeling the 82,000-dalton component, showing it was accessible from both sides of the membrane. The two major components were also sensitive to cross-linking, the 102,000 polypeptide being especially sensitive to --SH oxidation. Proteolysis with trypsin removed both components in the denser membrane fraction, in addition to inhibiting the K+-ATPase and K+-p-nitrophenylphosphatase of that fraction. Phosphorylation with [gamma-32-P]ATP labeled the 102,000-dalton component and K+, HCO3- minus and p-nitrophenylphosphate reduced the level of labeling. Hence the 102,000 region contains a subunit of the ATPase, is readily iodinated in inside-out vesicles, and is the most available for interpeptide S--S cross-linking.


Oligo(A) not coded by DNA generating 3'-terminal heterogeneity in a lambda phage RNA.

An RNA species from Escherichia coli infected with phage lambda was purified by hybridization to lambda 1-strand DNA and shown to have variable length. This variability was due to a variable number of adenylate residues attached to the 3' end of the molecule. Pancreatic RNase treatment of the RNA-DNA hybrid removed the 3'-terminal adenylate residues, generating a homogeneous RNA molecule terminating with -Up. The results indicate the presence of adenylate residues not coded by the DNA template at the terminus of this transcript from intact cells.


Gonadotropin receptors. Solubilization and purification by affinity chromatography.

Specific gonadotropin receptors of the rat testis were purified 15,000-fold after detergent extraction by a single affinity chromatography step on agarose coupled to human chorionic gonadotropin. Receptors were eluted in the free form at pH 3.2 and did not aggregate in the absence of detergent. The purified receptors displayed about 50% of the maximum theoretical binding activity, and retained the high binding affinity and hormonal specificity of the original gonadotropin receptors of the cell membrane.


Glycogenolytic response to glucagon of cultured fetal hepatocytes. Refractoriness following prior exposure to glucagon.

The glycogenolytic effect of glucagon has been studied in fetal hepatocytes cultured for 3 to 4 days in the presence of cortisol (10 muM). The hepatocytes, when transplanted from young fetuses (15-day-old), contain only minute amounts of glycogen, whereas when cultured 3 to 4 days in the presence of cortisol, they contain high levels of stored glycogen. Glucagon induced a rapid but partial mobilization of glycogen, which was maximal after 2 hours. The half-maximal response was observed with  about 0.1 nM glucagon. The glycogenolytic effect of glucagon in fetal hepatocytes is probably mediated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) as in adult liver. This effect was mimicked by cyclic AMP and N-6, O-2-dibutyryl cyclic AMP, (dibutyryl cyclic AMP), and potentiated by theophylline. Glucagon addition was followed by accumulation of cyclic AMP in the cells within 2 min. Glucagon produces a marked stimulation of the rate of glycogen breakdown and an inhibition of the rate of incorporation of [14-C] glucose into glycogen. The glycogeneolytic effect of a single addition of glucagon was reversed within 4 hours. A second addition of glucagon at this time was unable to induce a new glycogenolytic response. A resistance to glucagon stimulation appeared in the cells after a first exposure to the hormone. This refractoriness was also shown by the loss of glucagon-dependent cyclic AMP accumulation and was not linked to the release by the cells of a "hormone antagonist" into the medium. The hepatocytes resistant to the action of glucagon retained their response to cyclic AMP, dibutyryl cyclic AMP, and norepinephrine. Finally, glycogenolytic concentrations of cyclic AMP and of its dibutyryl derivative failed to induce a refractoriness to glucagon.


Kinetics of adenosine triphosphate synthesis in bovine heart submitochondrial particles.

The kinetics of ATP synthesis by submitochondrial particles were studied with a continuous flow mixing technique allowing measurements of ATP synthesis between 15 and 120 ms after mixing for estimation of the initial rate. During the initial 100 ms, ATP synthesis proceeded at a faster rate when driven by an artificially imposed electrochemical proton gradient than when driven by the respiratory substrate NADH. The rate of ATP synthesis driven by the artificial electrochemical proton gradient was dependent upon the magnitude of the gradient, and was affected similarly by variations of either the pH gradient or membrane potential. Initiation of ATP synthesis driven by NADH oxidation with ADP plus Pi resulted in an initial rate of phosphorylation equivalent to that during steady state respiration. However, a lag in phosphorylation was observed when ATP synthesis was initiated by oxygen or NADH. Valinomycin caused transient uncoupling of ATP synthesis driven by respiration since it strongly inhibited ATP synthesis during the initial 100 ms but it had no effect during steady state phosphorylation. The kinetic results strongly suggest that an electrochemical proton gradient is an obligate intermediate between electron transport and ATP synthesis on the main pathway of energy transduction.


Inactivation of Salmonella phosphoribosylpyrophosphate synthetase by oxidation of a specific sulfhydryl group with potassium permanganate.

Phosphoribosylpyrophosphate synthetase from Salmonella typhimurium contains four cysteine residues per subunit. Three of these react readily with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB), forming an active derivative with kinetic and physical properties similar to the native enzyme, but one reacts only under denaturing conditions. Stoichiometric amounts of KMnO4 inactivate the DTNB-treated enzyme. The loss of activity is correlated with the oxidation of the remaining cysteinyl group to cysteic acid by KMnO4. Amino acid analysis indicates that no other residues are altered. The rate of inactivation of the enzyme is decreased 30-fold by saturatin g concentrations of the substrate ATP. Inorganic phosphate also protects substantially against KMnO4. Titration of the native enzyme with limiting amounts of KMnO4 shows that the sulfhydryl group essential for activity competes effectively with the other sulfhydryl groups for KMnO4. These results suggest that the essential sulfhydryl group is near the active site, and that KMnO4, a phosphate analogue, can act as an active site-directed reagent at the ATP binding site of the enzyme. The KMnO4-oxidized enzyme is more highly aggregated than untreated enzyme and fails to bind ATP appreciably.


The interaction of human plasma glycoaminoglycans with plasma lipoproteins.

Modifications of existing methods have allowed for the isolation and purification of various species of plasma glycosaminoglycans on the basis of their sulfate content and molecular size. All of the preparations precipitated human plasma low density lipoproteins (LDL); maximal precipitation occurred with amounts of glycans corresponding to 50 mug of hexuronate and 12 mg of LDL. The interaction of glycans with pyrene-labeled lipoproteins was also studied, measuring variations of the fluorescence emitted by the monomer (M) and excimer (E) species of the bound pyrene. The ratio IE/IM is proportional to c/eta, where c is the microscopic concentration of the pyrene confined to the hydrocarbon region of the lipoprotein and eta is the microviscosity of that region. To 0.12 mg of pyrene-labeled LDL, very low density lipoproteins (VLDL) or high density lipoproteins (HDL) were added increasing amounts of the various glycan preparations. The sulfate-rich species decreased the IE/IM ratio of LDL and HDL but not that of VLDL. This finding suggests that the glycan caused a change in lipoprotein conformation associated with either an increased volume or increased microscopic viscosity of the hydrocarbon region. The modification of LDL conformation could be prevented by proteolytic treatment of the sulfate-rich species or by addition to the system of suitable amounts of sulfate-poor species or of chrondroitin-4-sulfate, but could not be prevented by increased ionic concentration. These results suggest that the two main species of plasma glycans are important in maintaining adequate rheological properties of plasma lipoproteins.


Stimulation of synthesis of free chondroitin sulfate chains by beta-D-xylosides in cultured cells.

Beta-Xylosides stimulate  2- to 6-fold the synthesis of glycosaminoglycans by three types of nonconnective tissue cells (RG-C6, NB41A, and rat hepatoma cells, and normal and simian virus 40 (SV40)-transformed normal human skin fibroblasts. The effect, which is specific for the anomeric linkage and the glycone, is observed in the presence and absence of puromycin. Beta-Xylosides may substitute for xylosylated core protein as initiators of synthesis of chondroitin sulfate chains. No stimulation of synthesis of heparan sulfate was observed. With the use of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside, it was demonstrated that the free chondroitin sulfate chains secreted into the medium bear the xyloside at the reducing end, and have an average molecular weight of 16,500.


Regulation of glycogen synthetase.  Specificity and stoichiometry of phosphorylation of the skeletal muscle enzyme by cyclic 3':5'-AMP-dependent protein kinase.

Complete conversion of skeletal muscle glycogen synthetase from the I form to the D form requires incorporation of 2 mol of phosphate per enzyme subunit (90,000 g). Incubation of sythetase I with low concentrations of adenosine 3':5'-monophosphate(cAMP)-dependent protein kinase (10 units/ml) and ATP (0.1 to 0.3 mM) plus magnesium acetate (10 mM) results in incorporation within 1/2 hour of 1 mol of phosphate persubunit concomitant with a decrease in the synthetase activity ratio (minus glucose-6-P/plus glucose-6-P) from 0.85 to 0.25.  Further incubation for 6 hours does not greatly increase the phosphate content of the synthetase or promote conversion to the D form.  This level of phosphorylation is not increased by raising the concentration of protein kinase to 150 units/ml and is not influenced by the presence of glucose-6-P, UDP-glucose, or glycogen.  However, at protein kinase concentrations of 10,000 to 30,000 units/ml a second mol of phosphate is incorporated per subunit, and the sythetase activity ratio decreases to 0.05 or less.  In addition to the 2 mol of phosphate persubunit which are required for formation of sythetase D, further phosphorylation can be observed which is not associated with changes in synthetase activity.  This phosphorylation occurs at a slow rate, is increased by raising the ATP concentration to 2 to 4mM, and is not blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase.  These data indicate that skeletal muscle glycogen synthetase contains multiple phosphorylation sites only two of which are involved in the synthetase I to D conversion.


Studies and sequences of Escherichia coli 4.5 S RNA.

4.5 S RNA, a biologically stable species with electrophoretic properties intermediate between 5 S and transfer RNAs, has been isolated from Escherichia coli and characterized.  No function has yet been found for this molecule.  Its primary structure and behavior suggests an unusually stable and possibly unique secondary structure.  Even from single species of E. coli, there is some sequence heterogeneity within the molecule.  The sequence of a major species from MRE 600 is: (see article).  Methods for getting sequence overlaps on this highly structured RNA are described, and a possible functional role for 4.5 S RNA is discussed.


Nucleotide sequence studies of normal and genetically altered glycine transfer ribonucleic acids from Escherichia coli.

The total nucleotide sequence of tRNAGGA/G -Gly2 from Escherichia coli is pG-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-C-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-AOH, where T- at position 53 is ribothymidylic acid, and psi- at position 54 is pseudouridylic acid; N- at position 36 is an unidentified derivative of uridylic acid, and is present in modified form in a portion of tRNAGGA/G -Gly 2 molecules isolated from E. coli cells. The missense suppressor mutation, glyTsuA36(HA), results in a C yields U base substitution at the 3' end of the anticodon of tRNAGGA/G -Gly 2 (nucleotide position 38). A secondary effect of this base substitution is the modification of the A residue directly adjacent to the 3' end of the anticodon of tRNAsuA36(HA), -Gly 2 suggesting that the enzymes responsible for this modification recognize the anticodon sequences of prospective tRNA substrates. The creation of a missense-suppressing tRNA, tRNAsuA36(HA), -Gly 2 by an alteration of the anticodon sequence of tRNAGGA/G -Gly 2 is analogous to mechanisms whereby other suppressor tRNAs have arisen. The high degree of nucleotide sequence homology between the amino acid acceptor stems and anticodon regions of four glycine isoaccepting tRNAs specified by E. coli and bacteriophage T4 suggests that these regions may be recognized by the glycyl-tRNA synthetase; the involvement of the anticodon region in the synthetase recognition process is supported by the greatly decreased rate of aminoacylation of tRNAsuA36(HA) -Gly 2.


The nucleotide sequence of a precursor to the glycine- and threonine-specific transfer ribonucleic acids of Escherichia coli.

The primary nucleotide sequence of an Escherichia coli tRNA precursor molecule has been determined. This precursor RNA, specified by the transducing phage lambdah80dglyTsuA36 thrT tyrT, accumulates in a mutant strain temperature-sensitive for RNase P activity. The 170-nucleotide precursor RNA is processed by E. coli extracts to form mature tRNA Gly 2 suA36 and tRNA Thr ACU/C. The sequence of the precursor is pG-U-U-C-C-A-G-G-A-U-G-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-U-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-A-A-G-A-U-G-U-G-C-U-G-A-U-A-U-A-G-C-U-C-A-G-D-D-G-G-D-A-G-A-G-C-G-C-A-C-C-C-U-U-G-G-U-mt6A-A-G-G-G-U-G-A-G-m7G-U-C-G-G-C-A-G-T-psi-C-G-A-A-U-C-U-G-C-C-U-A-U-C-A-G-C-A-C-C-A-C-U-UOH(tRNA sequences are italicized). It contains the entire primary nucleotide sequences of tRNA Gly2 suA36 and tRNA Thr ACU/C, including the common 3'-terminal sequence, CCA. Nineteen additional nucleotides are present, with 10 at the 5' end, 3 at the 3' end, and the remaining 6 in the inter-tRNA spacer region. RNase P cleaves the precursor specifically at the 5' ends of the mature tRNA sequences.


The nucleotide sequence in the promoter region of the gene N in bacteriophage lambda.

The sequence of 18 nucleotides in the region preceding the initiation of transcription of the gene N of bacteriophage lambda has been determined to be as follows (see article). The basic approach used for the sequence determination involved Escherichia coli DNA polymerase I-catalyzed elongation of the octadecanucleotide primer, dT-C-A-G-T-G-C-G-T-C-C-T-G-C-T-G-A-rU, possessing the appropriate polarity and nucleotide sequence corresponding to the 5' end of the gene N transcript. Following hybridization of the primer to the r-stand of bacteriophage lambda CI85657, sequences of the newly grown ollgonucleotide chains were determined by a) partial exonuclease digestion followed by two-dimensional fingerprinting; b) determination of pyrimidine tracts; and c) nearest neighbor analyses. Primer elongation was carried out in a controlled manner, the size of the newly grown chains being kept short by the following techniques: a) insertion of a ribonucleotide unit as the 3' terminus of the primer; b) use of a limited number of deoxynucleoside 5'-triphosphates in the elongation reaction; and c) enlongation of the primer using all the four nucleoside triphosphates with one of the triphosphates being supplied in a limiting concentration.


The regulation of phosphoenolpyruvate carboxykinase (GTP) synthesis in rat kidney cortex. The role of acid-base balance and glucocorticoids.

The effects of metabolic acidosis and of hormones on the activity, synthesis, and degradation of renal cytosolic P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) were studied in the rat using isotopic -immunochemical procedures. At normal acid-base balance, the synthesis of the enzyme accounted for between 2 and 3.5% of the synthesis of all soluble protein in the kidney cortex. P-enolpyruvate carboxykinase synthesis was selectively stimulated in acute metabolic acidosis, so that the relative rate of synthesis of the enzyme was increased to 7% 13 hours after oral administration of ammonium chloride. The stimulation of P-enolpyruvate carboxykinase synthesis preceded any increase in the assayable activity of the enzyme. The administration of sodium bicarbonate to acutely acidotic rats returned the rate of enzyme synthesis to normal in 8 hours. The effect of acidosis on both the synthesis and the activity of P-enolpyruvate carboxykinase was prevented by actinomycin D, cordycepin, and cycloheximide. The degradation in vivo of pulse-labeled P-enolpyruvate carboxykinase was not affected by acidosis. Thus, the stimulation of P-enolpyruvate carboxykinase synthesis is the major mechanism for the increase in the level of the enzyme observed in metabolic acidosis. The administration of glucocorticoid triamcinolone resulted in an increase in the relative rate of P-enolpyruvate carboxykinase synthesis and a commensurate increase in the activity of the enzyme in the renal cortex. Both changes were abolished by actinomycin D. Fasting was characterized by a high enzyme activity and a rapid rate of enzyme synthesis in the kidney cortex. This high rate of synthesis was reduced after the administration of sodium bicarbonate, but not after glucose feeding. Moreover, the injection of insulin to diabetic rats did not repress P-enolpyruvate carboxykinase synthesis in the renal cortex. Theophylline plus N-6, 0-2'-dibutyryl adenosine 3':5'-monophosphate stimulated P-enolpyruvate carboxykinase synthesis in the kidney of intact rats. However, the latter effect was probably due to glucocorticoid secretion, since it did not occur in adrenalectomized animals. The administration of parathyroid extracts did not result in the induction of the enzyme. Thus, the hormonal regulation of cytosolic P-enolpyruvate carboxykinase synthesis in the kidney differs markedly from that in the liver.


A spin label study of egg white avidin.

Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized, noninteracting spin label. Spin labels III and IV interacted with avidin in a similar fashion to that described for II with the exception that their dipolar splittings were 11.9 G and 14.2 G, respectively. From these splittings it was estimated that the distance between adjacent avidin-bound nitroxides was 16.7 A for labeled III and 15.7 A for label IV. The electron spin resonance spectrum of label V bound to avidin was characteristic of a noninteracting highly immobilized nitroxide with a maximum splitting of 62 G. The spectrum of V bound to avidin was independent of both time and the amount of bound label. The rate of dissociation of V from a 4:1 complex with avidin was monophasic. A model is proposed in which the recognition site for the heterocyclic ring system of biotin is represented as a cleft located within a hydrophobic depression in the surface of avidin.


Enhanced differential synthesis of proteins in a mammalian cell-free system by addition of polyamines.

Addition of the polyamines spermidine, spermine, or putrescine to a fractionated mammalian cell-free protein-synthesis system programmed by a variety of mRNAs results in a 3- to 5-fold stimulation of amino acid incorporation over that found in the absence of added polyamine. The mRNAs used as template were adenovirus mRNA, globin 9s mRNA, and RNA from the bacteriophages R17, Qbeta, and MS2. The relative amounts of 10 adenovirus polypeptides synthesized in vitro are altered by the addition of polyamines to the translation system to reflect more closely the relative amounts of these polypeptides synthesized in vivo. This qualititive improvement in translation products on addition of polyamines allow the analysis of a number of products which are at best only marginally synthesized in the absence of added polyamines. The low level of synthesis due to endogenous mRNA is stimulated by spermidine and spermine but a lesser extent by putrescine.


The interaction of NADH-cytochrome b5 reductase and cytochrome b5 bound to egg lecithin liposomes.

Incubation of liposomes prepared by sonication of egg lecithin with the amphipathic form of cytochrome b5 results in the binding of a maximum of 244 molecules of cytochrome b5 per liposomal vesicle. Interactions of the phospholipid with the hydrophobic segment of cytochrome b5 are involved in this binding which does not disrupt the liposome. When a small amount of NADH-cytochrome b5 reductase is bound liposomes simultaneously with cytochrome b5, the two proteins catalyze the reduction of cytochrome c by NADH. A qualitative kinetic analysis reveals that all of the cytochrome b5 interacts with reductase, a result consistent with these protein undergoing translational diffusion in the plane of the membrane. This system and the purified stearyl coenzyme A desaturase provide a model to study the dynamics of protein andlipid interactions in this membrane-bound oxidative sequence.


Subsensitivity of adenylate cyclase and decreased beta-adrenergic receptor binding after chronic exposure to (minus)-isoproterenol in vitro.

In vitro incubation of frog erythrocytes with (minus)-isoproterenol, 0.1 mM, at 23 degrees for 10 to 24 hours caused a 63% decline (rho less than 0.001) in the maximum (minus)-isoproterenol-stimulated adenylate cyclase activity in the erythrocyte membranes. Affinity for (minus)-isoproterenol as judged by the concentration which half-maximally stimulated the enzyme was not markedly altered. Basal enzyme activity and stimulation by fluoride or prostaglandin E1 remained unaltered. The number of beta-adrenergic receptor binding sites, assessed by binding studies with the beta-adrenergic antagonist (minus)-[3-H] alprenolol, declined by 50% (rho less than 0.005) in the (minus)-isoproterenol-treated cells. The binding affinity of the sites was not changed. Regulation of the concentration of functionally active beta-adrenergic receptors in membranes may be one of the mechanisms by which chronic exposure to catecholamines desensitizes tissues to beta-adrenergic stimulation.


Isolation of a unique peptide inhibitor of hormone-responsive adenylate cyclase.

We have perfused isolated rat livers with hypocalcemic (4.4 mg 100 ml) Krebs-Ringer bicarbonate albumin buffer. After 15 min of perfusion, a substance appeared in the perfusate which decreased rat renal adenylate cyclase activation by parathyroid hormone (PTH). The material in the perfusate was purified greater than 50,000-fold by Bio-Gel P-10 chromatography. The purified antagonist decreased the activation of rat renal cortical adenylate cyclase by PTH, glucagon, and epinephrine 75 to 100%. Concentration response curves for each of the hormones indicated a noncompetitive interaction of the inhibitor with the hormone. The inhibition was not species-specific, as the activation of the parathyroid hormone-responsive adenylate cyclase in cat renal cortex was also abolished by the inhibitor from the perfused rat liver. The inhibitor is a peptide, Mr equal to similar to 1000, which is heat-stable, acid-stable, alkai-labile, and is destroyed by trypsin, leucine aminopeptidase, and elastase. It is not destroyed by phosphodiesterase, 5'-nucleotidase, alkaline phosphatase, neuraminidase, RNase, or phospholipase A. The inhibitor is not produced by isolated rat livers perfused with normocalcemic perfusion media. It is unclear whether the peptide is synthesized by the liver or whether it is a breakdown product of a larger peptide or protein in the liver. This is the first reported peptide inhibitor of adenylate cyclase.


The effectiveness of various antibiotics in methyl methacrylate.

The effectiveness of different antibiotics mixed with Simplex P has been tested in vitro. A laboratory model was designed to simulate the constant immersion of bone cement in tissue fluid. Clindamycin, and to a lesser extent Cephalothin, were shown to be effective when used in this manner against Staphylococcus aureus and epidermidis. Effective inhibition of Gram-negative organisms could not be demonstrated with any of the antibiotics tested. The addition of up to 3 grams of antibiotic powder per unit of 40 grams of Simplex P did not appear to alter the expansive properties of the cement. Such release of antibiotics as did occur was thought to be related to the slow absorption of water by the slightly porous methyl methacrylate.


Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions.

The recirculating perfusion of adult rat liver with a Ca-++-free Hanks' solution produces a release of the adhesiveness of cells and a cleaving of the desmosomes. The addition of collagenase and hyaluronidase to the perfusion medium leads to complete dissociation of the liver tissue into a mixture of isolated cells and cell cords in which the hepatocytes remain connected with specific junctional differentiations, namely the gap and tight junctions. Individual cells are released by submitting the suspension of cell trabeculae to a gentle rolling. The gap junctions are ruptured at least in one of the two adjacent cells and remain generally attached to the other cell taking with them a small portion of cytoplasm. This technique of isolation of hepatocytes yields about 60-65% of the parenchymal cells contained in a liver; endothelial cells and other cells of the connective tissue are not recovered. The ultrastructural preservation of the isolated hepatocytes is excellent and the glucose-6-phosphatase activity, confined to the endoplasmic reticulum, appears unaltered in most cells. Protein, DNA and RNA recovery in the preparations of isolated hepatocytes is satisfactory, amounting to 70% of that found in liver homogenate; glycogen, the most labile component examined, is partly lost or degraded during the manipulations. Cell diameters measured by different methods confirm the preservation of the original volume of the in situ hepatocytes and the presence of more than one type of parenchymal cell. By submitting this heterogeneous cell population to an isopycnic density gradient centrifugation, two types of hepatocytes can be distinguished: the light hepatocytes, with a mean diameter of 20.5 mum and a mean density of 1.10, are characterized by an extended smooth-walled endoplasmic reticulum entrapping dispersed alpha-glycogen particles; the heavy hepatocytes, with a mean diameter of 19.0 mum and a mean density of 1.14, present a relatively reduced compartment of smooth endoplasmic reticulum, but large accumulations of glycogen. It is suggested that the cell fraction of low density is enriched in centrolobular cells and the high density fraction in perilobular hepatocytes.


Isolation of centrolobular and perilobular hepatocytes after phenobarbital treatment.

Daily phenobarbital (PB) injections, on 3-7 consecutive days, induce an intense proliferation of smooth endoplasmic reticulum (ER) associated with a decrease of the glucose-6-phosphatase activity. This situation first affects the centrolobular hepatocytes, enhancing the degree of liver lobule heterogeneity. This experimental model was used for isolation and further subfractionation of hepatocytes on Ficoll density gradients, as described in the preceding paper. Profiles of protein, DNA, RNA, glycogen, phosphorylase, and glucose-6-phosphatase were determined all along the gradient. Two liver cell populations were distinguished: (a) light hepatocytes (mean density 1.10) present the same morphological characteristics as centrolobular cells, i.e., an abundant smooth ER composed of tubular elements, numerous small mitochondria, and few glycogen particles; (b) heavy hepatocytes (mean density 1.14) are characterized by large and compact glycogen areas and prominent rough endoplasmic cisternae, as are the perilobular cells. After incubation in the Wachstein-Meisel medium, Centrolobular hepatocytes exhibit dispersed reaction sites of glucose-6-phosphatase activity, whereas perilobular cells present  a continuous and intense reaction. Morphometric determinations were carried out for both cell populations. Centrolobular PB hepatocytes are considerably enlarged (mean diameter: 23.7 mum); perilobular hepatocytes have a significantly smaller mean diameter of 19.2 mum, which is close to the values of control liver cells.


Receptor mobility and the binding of cells to lectin-coated fibers.

The ability of cells to bind to nylon fibers coated with lectin molecules interspaced with varying numbers of albumin molecules has been analyzed. The cells used were lymphoma cells, normal lymphocytes, myeloid leukemia cells, and normal and transformed fibroblasts, and the fibers were coated with different densities of concanavalin A or the lectins from soybean or wheat germ. Cells fixed with glutaraldehyde did not bind to lectin-coated fibers. The number of cells bound to fibers could be increased by increasing the density of lectin molecules on the fiber, the density of specific receptors on the cell, or the mobility of the receptors. It is suggested that binding of cells to fibers involves alignment and binding of specific cell surface receptors with lectin molecules immobilized on the fibers, and that this alignment requires short-range rapid lateral mobility (RLM) of the receptors. The titration of cell binding to fibers coated with different densities of lectin and albumin has been used to measure the relative RLM of unoccupied cell surface receptors for the lectin. The results indicate a relationship of RLM to lectin-induced cell-to-cell binding. The RLM or receptors for concanavalin A (Con A) was generally found to be higher than that of receptors for the lectins from wheat germ or soybean. Receptor RLM could be decreased by use of metabolic inhibitors or by lowering the temperature. Receptors for Con A had a lower RLM on normal fibroblasts than on SV40-transformed fibroblasts, and trypsinization of normal fibroblasts increased Con A receptor RLM. Normal lymphocytes, lymphoma cells, and lines of myeloid leukemia cells that can be induced to differentiate had a high receptor RLM, whereas lines of myeloid leukemia cells that could not be induced to differentiate had a low receptor RLM. These results suggest that the RLM of Con A receptors is related to the transformation of fibroblasts and the ability of myeloid leukemia cells to undergo differentiation


The relation between sporulation and the induction of antibiotic synthesis and of amino acid uptake in Bacillus brevis.

The induction and localization of tyrocidine-synthesizing enzymes is shown to be parallel, during growth of Bacillus brevis (ATCC 8185, American Type Culture Collection, Rockville, Md.), with the induction of uptake of constitutive amino acids and of components of pantetheine, a coenzyme of tyrocidine synthesis. Antibiotic synthesis appears at the end of logarithmic growth when the first soluble enzymes may be obtained from homogenates. During this period, binding proteins for metabolite uptake were isolated by intensive sonication which, when studied by chromatography, were identified by the appearance of low molecular weight fractions binding the radioactively marked metabolites; their induction was prevented by addition of rifampicin. The major purpose of this study was a comparison of antibiotic production and sporulation, the progress of which was followed by electron microscopy. The onset of tyrocidine synthesis and metabolite uptake coincided with the appearance of septum formation indicating that sporulation had progressed to stage II. With the progress of spore encapsulation, the tyrocidine production migrated from the soluble fraction into the forespore, terminating with the separation of forespores from the sporangium membrane. The resulting concentration of antibiotic in the forespore may indicate its function in sporulation, the nature of which, however, was not explored.


"Ultramicroinjection" of macromolecules or small particles into animal cells. A new technique based on virus-induced cell fusion.

A new method is described for the introduction of macromolecules and small particles into animal cells. The first step in this procedure is the trapping of particles in ghosts of human erythrocytes. This is achieved by the gradual hemolysis of erythrocytes in the presence of the particles to be trapped. The second step is the Sendai virus-induced fusion of the ghosts containing the particles with cells. By this method, ferritin and latex spheres (diameter 0.1 mum) have been "injected" into cells.


An ultracytochemical study of the respiratory potency, integrity, and fate of the sea urchin sperm mitochondria during early embryogenesis.

Cytochrome oxidase activity via cytochrome c, as demonstrated by the diaminobenzidine procedure, has been employed in this electron microscope cytochemical study to determine the respiratory potency, integrity and fate of the Arbacia sperm mitochondrion at fertilization and during early embryogenesis. The sperm mitochondrion remained intact and was intensely positive for cytochrome oxidase activity both during and after penetration into the egg. The mitochondrion remained highly reactive throughout zygote formation, up to the eight-cell stage. The sperm mitochondrion formed many projections and buds in the cytoplasm of immature oocytes, monospermic and polyspermic eggs, and in blastomeres. At all stages of early embryogenesis, close juxtaposition and structural contact were observed between the highly reactive sperm mitochondrion and the less reactive egg mitochondria. The results suggest that following fertilization the mitochondrion of the sea urchin spermatozoon retains some degree of metabolic autonomy within the ooplasm. The structural integrity of the paternal mitochondrion is maintained along with a functional respiratory enzyme system (cytochrome c-a3). The hypothesis that the fertilizing sperm mitochondrion may have some relevance to sea urchin development is discussed.


Antagonism by dibutyryl adenosine cyclic 3',5'-monophosphate and testololactone of concanavalin A capping.

Exposure of CHO-K1 cells in vitro to dibutyryl adenosine cyclic 3',5'-monophosphate (DBcAMP) plus testololactone produces a rapid, reversible antagonism of ligand-induced collection of initially dispersed concanavalin A (Con A) binding sites into a caplike mass. Morphologically, as Con A capping occurs, the cells become less spread and then round completely. With prolonged Con A exposure, cells cultured in either the absence or the presence of DBcAMP plus testololactone cap and round. Capping is blocked by cold treatment and respiratory inhibitors. Colcemid at concentrations greater than 1 muM promotes both Con A capping and cell rounding. Cytochalasin B at similar concentrations inhibits both capping and cell rounding. Treatment of cells with Con A has little effect on intracellular cAMP concentration. Possible mechanisms by which cAMP may modulate the movement of Con A binding sites are discussed.


Galactose and glucose metabolism in galactokinase deficient, galactose-1-P-uridyl transferase deficient and normal human fibroblasts.

Despite the genetic interruption of the Leloir pathway both galactosemic patients and galactosemic fibroblasts can convert galactose to CO2 and TCA precipitable products, although at less than the normal rate. These observations stimulated investigations into the identity of the alternative metabolic routes which allows for galactose metabolism in the absence of in vitro galactose-1-P-uridyl transferase. Four lines of galactosemic cells, each without detectable gal-transferase, produced 14CO2 from [1-14C]-galactose (0.094 mumoles in 20 cc of medium) at approximately 39% +/- 16% the rate of transferase positive cells over a 48-hour period. However, galactokinase deficient fibroblasts produced 14CO2 and TCA precipitable products from [1-14C]-galactose or [U-14C]-galactose at only 3% to 9% the rate of normal fibroblasts. Therefore it seems likely that gal-transferase deficient fibroblasts must first synthesize galactose-1-P for further metabolism of galactose.


Somatic genetic analysis of cyclic AMP action: selection of unresponsive mutants.

Dibutyryl cyclic AMP and theophylline kill S49.1 mouse lymphoma tissue culture cells. When cells are grown in soft agar with these drugs, the few clones that survive are resistant to cytolysis. The rate of mutation to resistance is 1-3 times 10-7/cell/generation in both diploid and tetraploid cells. The incidence of mutants is increased by treatment with a chemical mutagen, ICR 191. The mutation is consistently associated with greatly reduced or absent cytoplasmic cyclic AMP binding protein. These results suggest that a somatic mutation leads to a defect of the protein kinase regulatory subunit and that activity of this kinase is required for induction of cell death by cyclic AMP.


Somatic genetic analysis of cyclic AMP action: characterization of unresponsive mutants.

N-6,O-2'-dibutyryl adenosine 3',5'-monophosphate kills cultured mouse lymphosarcoma cells, but not resistant mutants derived by a single-step clonal selection. Resistant clones lack the cyclic AMP binding proteins present in wild type, cyclic AMP sensitive clones. Both endogenous cyclic AMP, accumulated in response to isoproterenol or cholera toxin, and exogenous dibutyryl cyclic AMP induce cyclic AMP phosphodiesterase, slow growth, and eventually kill wild type cells. In the resistant mutants, however, the endogenous and exogenous cyclic nucleotides appear to be completely inactive. These results indicate that an intracellular receptor for cyclic AMP, previously shown to be associated with a cyclic AMP-dependent protein kinase, mediates cyclic AMP's regulation of growth and phosphodiesterase synthesis.


The relation of endogenous adenosine cyclic 3':5'-monophosphate to the antagonistic effects of adenosine and colchicine on cell shape.

Adenosine and colchicine have antagonistic effects on cell shape. When Chinese Hamster lung fibroblasts (CHE36-6) or SV40 transformed 3T3 (SV3T3) cells are incubated with colchicine (1 muM) for one hour at 37 degrees C, they round up into spheres with short spikes. Cells treated with adenosine (1 muM-minus 4 mM) for one hour become refractile and develop spindly processes. However, when the two compounds are added simultaneously, the characteristic responses to either drug are abolished and the cells appear normal. The counteraction is specific for adenine and its derivatives, adenosine being the most effective of the compounds we tested. Accumulation of colchicine or adenosine is not altered significantly by the presence of the other drug, ruling out decreases in uptake as the basis of the mutual antagonism. The morphological changes can be observed under conditions where there are no changes in intracellular cAMP levels (such as incubation with low concentrations of adenosine or cordycepin, an adenosine analog that cannot be directly converted to cAMP). Colchicine does not alter cAMP content of control or adenosine-treated cells. These data show that adenine compounds have potent effects on cell shape, and the antagonistic effects of adenosine and colchicine on cell shape are not mediated through changes in intracellular cAMP levels.


Difference in transport of leucine in attached and suspended 3T3 cells.

3T3 cells in subconfluent culture take up leucine through a transport system which has a relatively high affinity for leucine (M system). When the culture becomes confluent, the M system is turned off and leucine is transported by another system which has a low affinity for leucine (S system). The M system is reactivated by transferring the cells into subconfluent cultures. In suspension cultures 3T3 cells, initiated from confluent cultures, the M system is not activated and leucine is transported by the S system. In cells suspended from subconfluent culture, the M system continues to operate at a high level for four hours and then is gradually turned off. Tumor virus transformed 3T3 cells (SV3T3 and Py3T3) grow quite well in suspension culture and transport leucine both in monolayer and suspension through a high affinity system, with a high Vmax value. A derivative of 3T3, 3T3/41, which grows in suspension much more slowly than tumor virus transformed 3T3 cells, also takes up leucine through a high affinity transport system both in monolayer and suspension but its Vmax value is lower than that of the transformed cells.


Effects of discrete nuclear u.v-microbeam irradiation on herpes virus and SV40 infection.

The requirement for a nucleolus in the expression of structural genes of nuclear viruses was examined by means of experiments in which the nucleolus or other parts of the nucleus was inactivated by a microbean of unltraviolet light. These experiments showed that the expression of such genes is not dependent on cucleolar function. This conclusion is discussed in the light of previous experiments in which similar inactiviation of nucleolus was shown to prevent the expression of cellular structural genes.


The binding of the mucoprotein from gastric mucus to cells in tissue culture and the inhibition of cell adhesion.

The mucoprotein, which is responsible for the formation of gastric mucous gel in pig, has been shown to bind equally well to suspensions of baby hamster kidney cells, polyoma-virus-transformed baby hamster kidney cells and HeLa cells. The binding of the mucoprotein to the cells is dependent on Ca 2


Phase-contrast and electron-microscopic observations on a membranous complex in primary spermatocytes of the mouse.

A prominent cytoplasmic inclusion present in living mouse primary spermatocytes has been observed by both light and electron microscopy. It began to form at prometaphase and continued to increase in thickness and length as the cells developed. By metaphase it was a distinct sausage-shaped boundary that enclosed a portion of the cytoplasm between the spindle and the cell membrane. At the end of metaphase, the inclusion reached its maximum length. At telophase, it was divided between the daughter secondaries. The inclusion persisted as a circular contour in the interphase secondary spermatocyte. Electron microscopy of the same cultured cells that were previously observed with light microscopy revealed that the inclusion was a distinctive formation of membranes. It consisted of agranular cisternae and vesicles, and was therefore a membranous complex. Many of the smaller vesicles in the membranous complex resembled those found in the spindle. The cisternae in the membranous complex were identical to the cisternal endoplasmic reticulum of interphase primary spermatocytes. Nevertheless, the organization of vesicles and cisternae into the membranous complex was unique for the primaries in division stages, since such an organization was not present in their interphase stages.


Trimethylsilylation of penicillic acid and patulin, and the stability of the products.

Penicillic acid and patulin have been effectively silylated by N,O-bis(trimethylsilyl)acetamide, alone or with trimethylchlorosilane in benzene. The silylation products are stable, if stored under refrigeration, for at least 15 days.


Determination of penicillic acid and patulin by gas-liquid chromatography with an electron-capture detector.

Quantitative and concurrent determinations of penicillic acid and patulin have been achieved after separation of these compounds, as their trimethylsilyl derivatives, by gas-liquid chromatography with electron-capture detection and a column of 10% of DC-200 plus 15% of QF-1 on Gas-Chrom Q; the minimum determinable amounts of penicillic acid and patulin were 0.05 and 0.1 ng, respectively. The procedure devised for determining these compounds in grain involves extraction with solvents, followed by further clean-up by thin-layer chromatography; the detection limit of this procedure is about 0.02 ppm, and recovery at the level of 0.05 ppm is about 90%. The significant separation of penicillic acid and patulin produced in moldy rice is demonstrated on the gas chromatograms, and this makes possible a micro-assay for these toxic compounds in contaminated grain.


Inorganic saltsin the fluorometric detection of presticides.

A method is described for the detection and quantification of pesticides on silica gel ghromatograms by in situ fluorometry. Simple inorganic salts are used to induce fluorescence in a number of pesticidal compounds. Intensified fluorescence is shown for one naturally fluorescent pesticide. Spectral data and instrumental limits of detection are given.


Chromatographic-fluorometric analysis of antileprotic sulfones.

Modifications to the power supply system of a spectrophotofluorometer are described. These modifications stabilize the output of the xenon are lamp and permit the determination of nanogram quantities of antileprotic sulfones. The sulfones are removed from plasma by a single extraction with ethyl acetate, then separated by high-pressure liquid chromatography on silica, and detected in the effluent by their fluorescence. The method is specific, rapid, and reproducible.


Rapid identification of amino acid derivatives from the sequenator.

A simple procedure is described for converting thiazolinones, from the protein sequenator, to the more stable phenylthiohydantoins without using the conventional HCI conversion procedure. The thiazolinones are applied to a silica gel plate and converted to phenylthiohydantoins by heating at 140 for 5-10 min, in the presence of heptafluorobutyric acid, prior to chromatography. After chromatography, measurements of the yields of the derivatives on the plate can be made with a variable-wave-length thin-layer chromatography scanner. This is shown to be a useful adjunct for identifications.


Analysis for organophosphorus insecticides and metabolites.

Extraction and cleanup methods used for the analysis of organophosphate pesticides are similar to those used for chlorinated hydrocarbons but differ in some important details. Many organophosphates are lost during extraction of acetonitrile solutions containing organophosphates with petroleum ether; dichloromethane is substituted. Also, cleanup by column chromatography on mixed adsorbents containing charcoal results in better recoveries than can be obtained on Florisil alone. The detectors used for measurement of organophosphates by GLC are almost unique for this group of compounds. TLC is very useful for confirming the identities of the compounds, and several promising optical methods are available for their measurement. Methods for the analysis of metabolites of this group are basically similar, since all organophosphates undergo dealkylation reactions and cleavage of the acid anhydride bond. However, measurement of the polar group which is removed during phosphorylation or other reactions involving cleavage of the acid anhydride bond is a problem unique for each compound.


Analysis of fumigants and fumigant residues.

The terms fumigant and fumigant residue are defined. Interrelationships between physically and chemically bound residues, storage environments, nature of the substrate and other influencing factors are outlined. Analytical methods include polarography by DME and RPE, titrimetry, spectrophotometry, and GC with microthermal conductivity, hydrogen flame ionization, electron capture, microcoulometric, thermionic, and flame photometric detector systems, with backup by enzymatic, radiometric, NAA and X-ray flourescence methods. Various aspects are illustrated with different fumigants used commercially. Supplementary methods to extend the power and usefulness of analytical methods in fumigant research are indicated.


Control and localization of rat adrenal cyclic guanosine 3', 5'-monophosphate.  Comparison with adrenal cyclic adenosine 3', 5'-monophosphate.

Cyclic AMP and cyclic GMP were measured in rat adrenal glands after either hypophysectomy alone or after hypophysectomy and treatment with ACTH. Adrenal cyclic GMP levels rise in acutely hypophysectomized rats to a maximum at 1 h of approximately 200% of control levels; there is a return to base line at 4-12 h after hypophysectomy. In contrast, adrenal cyclic AMP falls immediately to about 50% of control levels after hypophysectomy and remains at approximately 1 pmol per mg tissue. Doses of ACTH beyond the physiological range markedly suppress adrenal cyclic GMP while producing a 50-fold or greater rise in cyclic AMP in hypophysectomized rats. This pattern of adrenal cyclic GMP rise was unchanged in acutely hypophysectomized animals treated with desamethasone. N-6-2'-0 dibutyryl cyclic AMP acted similarly to the effect of ACTH in bringing about a suppression of adrenal cyclic GMP levels. Physiological i.v. pulse doses of ACTH produced a rapid dose related increase in adrenal cyclic GMP. In vitro incubation of quartered adrenal pairs with 500 mU ACTH produced elevated cyclic AMP levels and suppression of cyclic GMP. Whereas adrenal cyclic AMP fell rapidly to 50% of control levels after hypophysectomy and remained at about 1 pmol per mg tissue for 7 days, adrenal cyclic GMP showed a biphasic rhythm in long-term hypophysectomized animals. After an initial peak at 1 h after hypophysectomy, adrenal cyclic GMP declined to baseline at 4-12 h but thereafter progressively rose with time, eventually reaching levels over 1 pmol per mg tissue. Fluorescent immunocytochemical staining of rat adrenal zona fasciculata showed cyclic AMP largely confined to cytoplasmic elements with little fluorescence contained in nuclei. In constant, cyclic GMP was found discretely positioned in nuclei with prominent fluorescence in nucleoli in addition to cytoplasmic localization. It is concluded that in hypophysectomized rats ACTH, either directly or in conjunction with altertion of adrenal cyclic AMP, appears to be one factor which regulates adrenal cyclic GMP. The direction of cyclic GMP change and the different subcellular localization of the nucleotides suggest divergent roles for cyclic AMP and cyclic GMP in adrenocortical function. Furthermore, our observations suggest a role for adrenal cyclic GMP in nuclear directed events.


Changes in the cyclic 3', 5'-adenosine monophosphate system of rat mammary gland during lactation cycle.

Several aspects of the cyclic 3', 5'-adenosine monophosphate system of rat mammary glands were investigated including effects of stage of pregnancy and lactation upon tissue cyclic 3', 5'-adenosine monophosphate amounts and adenyl cyclase, cyclic 3', 5'-adenosine monophosphate phosphodiesterase, and protein kinase activities. Cyclic 3', 5'-adenosine monophosphate decreased at early lactation, and this decrease coincided with an increase in phosphodiesterase activity. Adenyl cyclase activity remained unchanged from late pregnancy to end of lactation. At late pregnancy, activity of protein kinase was about the same as during lactation indicating that increase in protein kinase activities in the glands precedes increases in activities of other major enzymes and the increase in ribonucleic acids in late pregnancy or early lactation. Epinephrine, prolactin, growth hormone, thyroxine, and prostaglandine caused 60, 80, 140, 200, and 270% increases in adenyl cyclase activity in vitro.


Adrenal responsiveness in lactating holstein cows.

Ten Holstein cows, in second to fifth lactation, received 200 IU adrenocorticotrophin at approximately 4, 8, 16, 24, 32, and 40 wk of lactation. Blood samples were collected from indwelling jugular catheters at -60 and 0 min pre-injection and at 2, 6, 10, 15, 20, 30, 45, 60, 120, 180, 240, 360, 540, and 720 min postinjection. Post-injection concentrations of glucocorticoids were influenced by milk yield, stage of lactation, age, and mean environmental temperature but not by pre-injection concentration of glucocorticoids or days pregnant. As milk yield, cow age, and daily temperature (range 6.4 to 26.9 C) increased, glucocorticoid response decreased whereas it increased with advancing stage of lactation. Curves of adrenal response for the six injection times during lactation were not parallel, and average response adjusted for significant independent variables increased roughly in linear fashion from 15 to 53 ng/ml between 4 and 40 wk of lactation. Adrenal responsiveness was less in cows injected on days when temperatures were above 21.1 C than in those injected below this temperature. There was no evidence that adrenal function or milk production was altered in high producing cows at higher environmental temperatures differently from in low producing cows. The change in adrenal function during advancing lactation was not associated with pre-treatment peripheral plasma glucocorticoid concentrations which did not change. There appears to be a well mainained adrenal potential and sustained adrenocortical activity throughout lactation.


Transport of dietary cholesterol into blood and milk of the goat.

Position 4 labeled carbon-14 cholesterol was placed in abomasums (stomachs) of two lactating goats. Blood and milk samples were collected from the animals for 5 to 13 days. Specific activities of cholesterol and cholesteryl esters in various fractions of blood serum and milk were determined to reveal pathways by which dietary cholesterol enters milk. Results with the two animals showed similar trends. Within 24 h both cholesterol and cholesteryl esters of the three principal serum lipoproteins of the goat were labeled, and this labeling persisted in substantial degree for the 13-day experiment. Specific acities for cholesteryl esters in  milk fat globules exhibited several remarkable attributes: they fluctuated in intensity with a 3-to 4-day cycle reaching a maximum at 7 to 8 days after tracer injection; they exceeded cholesteryl ester specific activity in the skim milk by an order of magnitude; and at their maximum they exceeded all specific activities for serum components. The results of this investigation exemplify the ease with which dietary cholesterol enters and crosses membranes in the animal body.


Origin of cyclic adenosine monophosphate in saliva.

The level of cyclic adenosine monophosphate (AMP) in duct saliva from the dog submandibular gland was increased after cyclic AMP was administered intravenously in vivo. Isoproterenol increased the level of cyclic AMP in plasma and saliva in vivo and in salivary gland slices in vitro, but increased the level only slightly in saliva in a prefused dog submaxillary gland.


A procedure for removing red cells and dead cells from lymphoid cell suspensions.

A procedure is described for simultaneously removing red cells and dead cells from lymphoid cell suspensions, based on the observation that when populations of lymphoid cells are centrifuged on a mixture of Isopaque/Ficoll, dead cells and red cells sediment whereas viable cells float. The technique very efficiently removed red cells from a wide range of lymphoid cell suspensions and eliminated lymphocytes killed by mechanical stress, by antibody and complement and by prolonged tissue culture. The depletion of red cells was greater than 99% and the recovery of viable lymphocytes usually greater than 90%, the resulting cell suspensions being around 95-100% viable. The immunological activity of B cells, helper T cells and cytotoxic T cells virtually unimpaired by the separation procedure.


Epidermal synthesis of prostaglandins and their effect on levels of cyclic adenosine 3', 5'monophosphate.

Extracts of guinea-pig and human skin epidermis were analyzed for prostaglandins PGE1, E2, and F2alpha by radioimmunoassy, and found to contain a total of 62.0 (guinea pig) and 144.7 (human) ng/gm wet weight. the three prostaglandins occurred in approximately equal amounts. Guinea=pig epidermal homogenates converted labeled arachidonic acid to PGE2 and PGF2alpha, the rate of formaiton being 10 and 2.5 pmoles per mg protein in O.K hr, respectively. Conversion in the dermis occurred to a much smaller extent. Homogenates of univolved and involved epidermis from 10 subjects with psoriasis produced PGE2 from arachidonic acid at rates of 6.48 and 2...


An in vitro and in vivo investigation of the phototoxic effect and its amelioration with radioprotective compounds.

Electron spin resonance spectroscopy has been used to demonstrate that the phototoxic antimalarial drug, 6,8-dichloro-2-phenyl-a-2-piperidnylquinolinemethanol (WR 7930), when irradiated with long-wave ultraviolet (UV) light (lambda greater than 320 nm) while held in a glassy matrix at 73 degrees K, enters a triplet state and releases hydrogen atoms in its environment. The steady-state concentration of triplet WR 7930 molecules and of hydrogen atoms is reduced 2 to 3 times when mercaptoethylamine (MEA) is also present in the UV-irradiated glass. Organosulfur radicals form on MEA while hydrogen atoms and triplet-state molecules are reduced in number. Hydrogen atoms and triplet WR 7930 molecules are considered as mediators of the phototoxicity of the antimalarial drug. Thus, hydrogen atom scavanging and chemical quenching of the triplet state are possible mechanisms by which protection against phototoxic effects could be gained. Protection is demonstrated in mice receiving 20 mg per kg WR 7930 intraperitoneally and exposed to long-wave UV for 20 hr when the radioprotective aminothiol-forming compound, 2-(3-aminopropylamino) ethyl dihydrogen phosphorothioate (WR 2721), is administered at 400 mg per kg immediately before irradiation. When no protective drug is administered concurrently, WR 7930 administration results in intense erythema, edema, and eventual necrosis of ear tissues.


Non-chromaffin paraganglioma of the nasopharynx.

A case of non-chromaffin paraganglioma of the nasopharynx is presented. Previously reported cases are discussed and classified, and theories of origin are reviewed in the light of new evidence suggesting a common origin for this rare tumour and juvenile angiofibroma of the nasopharynx.


Effects of glucagon and dibutyryl adenosine 3', 5'-cyclic monophosphate on oxidative desaturation of fatty acids in the rat.

The present work was undertaken to study the effect of anti-insulinic and glycogenolytic factors on the oxidative desaturation of fatty acids. The effects of glucagon and dibutyryl cyclic AMP on the desaturation of linoleic acid to gamma-linolenic acid, alpha-linolenic acid to octadeca-6,9,12,15-tetraenoic acid, stearic acid to oleic acid, and eicosa-8,11,14-trienoic acid to eicosa-5,8,11,14-tetraenoic acid by rat liver microsomal preparations were investigated. Fasted rats had low desaturating activity, but refeeding a fat-free diet enhanced the activity. Administration of glucagon or dibutyryl cyclic AMP abolished the increase of the 6-desaturase activity elicited by refeeding. However, a similar effect on the 9-desaturase and 5-desaturase activity was not observed. The relationship between these effects and glucose metabolism is discussed.


Turnover and tissue distribution of 125-I-labeled low density lipoprotein in swine and dogs.

Swine plasma low density lipoprotein (LDL) isolated ultracentrifugally (d 1.019-1.063) was labeled with 125-I, dialyzed, and reisolated by centrifugation at d 1.063. Over 96% of the radioactivity was shown to be associated with the apoprotein. After reinjection into the donor animal, disapperance of 125-I was followed for up to 122 hr. At all time intervals examined, over 95% of the total plasma 125-I was recovered in LDL (D 1.006-1.063), i.e., there was apparently no transfer of radioactivity to high density or very low density lipoproteins. The disappearance curve was biexponential, with half-lives of 0.83 plus or minus 0.06 and 22.5 plus or minus 1.7 hr for the first and second phases, respectively (13 studies). The mean calculated fractional catabolic rate was 0.041 plus or minus 0.003 hr-minus 1. Similar results were obtained in three dogs using autologous LDL of density 1.020-1.050; fractional catabolic rates were 0.031, 0.031, and 0.029 hr-minus 1. Tissue distribution of 125-I was determined in swine killed at various time intervals after [125-I]LDL injection with corrections for radioactivity in trapped plasma. Of the tissues examined, the liver showed by far the highest concentration. Total hepatic radioactivity, expressed as a percentage of total plasma radioactivity, was rather constant and independent of the time of killing from 3 to 122 hr (15.8 plus or minus 1.9%). The total extravascular LDL pool calculated from analysis of the plasma disappearance curves was about 20-30% of the size of the plasma LDL pool. These data are consistent with the conclusion that the liver accounts for a very large fraction of the total extravascular LDL pool. These data are consistent with the conclusion that the liver accounts for a very large fraction of the total extravascular LDL pool and that it is infairly rapid equilibrium with the plasma pool. To what extent the liver is involved in irreversible degradation cannot be inferred from these findings.


Influence of the pituitary gland on insulin secretion in the genetically obese (ob/ob) mouse.

The influence of the pituitary gland of lean and genetically obese (ob/ob) mice on insulin secretion from microdissected pancreatic islets of lean and ob/ob mice has been studied by perifusing the pituitaries of these animals in series with the isolated islets and measuring insulin secretion at 5-min intervals over a period of 60 min. It has been shown that the pituitary perifusate of both lean and obese mice stimulate insulin secretion from lean mouse islets but not from obese mouse islets. The maximum stimulation occurs in the first 10 min and with the lean mouse pituitaries returns to the basal level in about 20 min, whereas with the obese mouse pituitaries insulin secretion is about double that from the control islets even after 40 min. A concentration of pure porcine ACTH equivalent to about three times the amount released from the pituitary gland under the experimental conditions used, caused only a small stimulation of insulin release. Possible interpretations of these findings and further lines of investigation are discussed.