Biosynthesis of a P1-2-acetamido-2-deoxy-D-glucosyl P2-polyisoprenyl pyrophosphate by calf pancreas microsomes

Incubation of UDP-[¹⁴C]-$\text{N}$-acetylglucosamine with calf pancreas microsomes in the presence of Mn⁺⁺ and potassium thiocyanate gave a labeled glycolipid, tentatively identified as $\text{P}$¹-2-acetamido-2-deoxy-$\text{D}$-glucosyl $\text{P}$²-dolichyl pyrophosphate on the basis of cochromatography with synthetic $\text{P}$¹-2-acetamido-2-deoxy-α-$\text{D}$-glucopyranosyl $\text{P}$²-dolichyl pyrophosphate, similar chemical and enzymic hydrolyses of the biosynthetic and synthetic compounds, and stimulation of the biosynthesis by addition to the incubation mixture o dolichyl phosphate or a crude lipid fraction extracted from microsomes.     

The role of the sites for ATP of the Ca2+-ATPase from human red cell membranes during Ca2+-phosphatase activity

1. In the presence of ATP, the Ca²⁺ pump of human red cell membranes catalyzes the hydrolysis of $\text{p-}\text{nitrophenyl}$ phosphate. The requirement for ATP of the Ca²⁺-$\text{p-}\text{nitrophenylphosphatase}$ activity was studied in relation to the two classes of site for ATP that are apparent during Ca²⁺ -ATPase activity. 2. (a) The $\text{K}{}_{0.5}$ for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ extrapolated at 0 mM PNPP is equal to the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the Ca²⁺ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca²⁺ -ATPase or as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 4. At ATP concentrations that almost saturate the high-affinity site, Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity increases as a function of PNPP along an S-shaped curve, while Ca²⁺ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca²⁺ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca²⁺ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca²⁺ -ATPase activity at the high-affinity size persists during Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity.     

Demethylation of O6-methylguanine in a synthetic DNA polymer by an inducible activity in Escherichiacoli

$\text{O}$⁶-Methyl[8-³H]deoxyguanosine in a synthetic DNA polymer, poly(dC, dG, m⁶dG), is demethylated by cell-free extracts of $\text{Escherichia}\text{coli}\text{B}\text{r}$ adapted by exposure to $\text{N}$-methyl-$\text{N}$′-nitro-$\text{N}$-nitrosoguanidine, as shown by the appearance of ³H-labeled deoxyguanosine in hydrolysates of the recovered DNA. The demethylating activity could not be detected in extracts of nonadapted $\text{E}\text{.}\text{coli}$. These results provide direct evidence that a previously described inducible repair activity in $\text{E}\text{.}\text{coli}$ acts by demethylating $\text{O}$⁶-methylguanine at the DNA level.     

Somatostatin analogs which inhibit glucagon and growth hormone more than insulin release

Three analogs of somatostatin, [$\text{D}$-Cys¹⁴] -, [Ala², $\text{D}$-Cys¹⁴] - and [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, were synthesized by the solid phase method, characterized by several means, and tested for their effects on the release of insulin, glucagon, and growth hormone. The peptides sharply suppressed the release of growth hormone $\text{in vitro}$ and glucagon $\text{in vivo}$, but had less effect on insulin secretion $\text{in vivo}$. These analogs, particularly [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, could possibly be useful for the treatment of diabetes mellitus.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

Peptide chain initiation with chemically formylated Met-tRNAs from E. coli and yeast

Chemically formylated Met-tRNA_m^Met and Met-tRNA_f^Met species from $\text{E.}$$\text{coli}$ and yeast were tested for their capacity to serve as chain-initiators in a cell-free system from $\text{E.}$$\text{coli}$. In the presence of R 17 mRNA, initiation factors and $\text{E.}$$\text{coli}$ ribosomes, all four Met-tRNAs could form functional initiation complexes as measured by ribosomal binding kinetics, fMet-puromycin formation and synthesis of a dipeptide fMet-Ala. Unformylated Met-tRNA_f^Met from $\text{E.}$$\text{coli}$ displayed significantly less activity as a peptide chain-initiator than the formylated Met-tRNA_m^Met species from $\text{E.}$$\text{coli}$ and yeast. Although the latter tRNAs were less effective initiators than the “physiological” initiator tRNAs, the data seem to indicate that a blocked α-amino group represents the major token of identification by which Met-tRNA is admitted to function in $\text{E.}$$\text{coli}$ peptide chain initiation.     

Behavioral comparisons of the stereoisomers of tetrahydrocannabinols

The potencies of (?)-$\text{trans}$-Δ⁹-THC, (+)-$\text{trans}$-Δ⁹-THC, (+)-$\text{cis}$-Δ⁹-THC, (?)-$\text{trans}$-Δ⁸-THC and (+)-$\text{trans}$-Δ⁸-THC were compared in several different species. (?)-$\text{trans}$-Δ⁹-THC was 100 times more potent than (+)-$\text{trans}$-Δ⁹-THC in depressing schedule-controlled responding in monkeys. The (+)-$\text{trans}$ isomers were less effective than their corresponding (?)-$\text{trans}$ isomers in the dog static-ataxia test, but potency ratios could not be determined due to a lack of dose-responsiveness of the (+)-$\text{trans}$ isomers. However, it appeared that their potency differed by at least ten fold. The potency of (+)-$\text{cis}$-Δ⁹-THC in the dog static-ataxia test was comparable to that of (+)-$\text{trans}$-Δ⁹-THC. The hypothermia in mice produced by the (?) isomers of $\text{trans}$-Δ⁹-THC and $\text{trans}$-Δ⁸-THC were 9.1 and 30.4 times greater than that produced by their respective (+)-isomers. Also, the potency ratio of the (+)- and (?)-$\text{trans}$-Δ⁹-THC was 5.6 as measured by depression of spontaneous activity in mice. The magnitude of the potency ratios of the THC stereo-isomers is dependent upon the species and the pharmacological test used.     

Complexation of Pr3+ by two different ionophores enhances markedly its transport rate

Transport of Pr³⁺ across phosphatidylcholine vesicles, monitored through ³¹P nmr, is first-order in monensin ($\text{1}$), second-order in etheromycin ($\text{2}$) or in lasalocid A ($\text{3}$). When $\text{1}$ and $\text{2}$ (or $\text{2}$ and $\text{3}$) are incorporated $\text{together}$ in 1:1 ratio into the lipidic phase, transport is faster than with each ionophore alone. For instance, assuming that the complexes $\text{2}$.Pr³⁺.$\text{2}$, $\text{3}$.Pr³⁺.$\text{3}$, and $\text{2}$.Pr³⁺$\text{3}$ are equiprobable, they effect transport at intrinsic relative rates of 1, 2, and 13.5, $\text{i.e.}$ a remarkable synergism is set up.     

Phosphorylation of high mobility group proteins 14 and 17 by nuclear protein kinase NII in rat O6 glioma cells

High mobility group (HMG) proteins 14 and 17 of rat C₆ glioma cells are phosphorylated $\text{in}\text{vivo}$ on both serine and threonine. In HMG 14 about 60% of the total [³²P]phosphate was identified as phosphoserine and 40% as phosphothreonine. In HMG 17, there was 88% phosphoserine and 12% phosphothreonine. Glioma cell nuclear protein kinase NII phosphorylates HMG 14 and 17 $\text{in}\text{vitro}$ on serine as well as threonine and the relative percentages of [³²P]phosphoamino acid are similar to those seen $\text{in}\text{vivo}$. Nuclear protein kinase NI and the type I and II cAMP-dependent protein kinases exhibit only minor phosphorylating activity towards HMG 14 and 17. We conclude that nuclear protein kinase NII is responsible for the phosphorylation of HMG 14 and 17 $\text{in}\text{vivo}$.     

Enkephalin analogues and naloxone modulate the release of growth hormone and prolactin - evidence for regulation by an endogenous opioid peptide in brain

Met⁵-enkephalin amide, D-Ala²-Met⁵-enkephalin amide, D-Ala²-Leu⁵-enkephalin amide, morphine sulfate and naloxone hydrochloride were examined for their effects on growth hormone and prolactin release $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. Subcutaneous injection of D-Ala²-Met⁵ enkephalin amide^a, D-Ala²-Leu⁵ enkephalin amide^b and morphine sulfate, but not Met⁵-enkephalin and amide^c, resulted in significant elevations in the serum growth hormone and prolactin of immature female rats. Naloxone blocked the hormone-stimulatory effect of the opioid receptor agonists and when administered alone significantly reduced serum growth hormone and prolactin concentrations. None of the drugs demonstrated a direct action on anterior pituitary tissue growth hormone or prolactin release $\text{in}$$\text{vitro}$.     

The incision and strand rejoining step in the excision repair of 5,6-dihydroxy-dihydrothymine by crude E. coli extracts

Strand resealing in the $\text{in}$$\text{vitro}$ excision repair of 5,6-dihydroxy-dihydrothymine in osmium tetroxide oxidized polyd(A-T) by crude $\text{E.}$$\text{coli}$ extracts is accomplished by polynucleotide ligase. Osmium tetroxide oxidized polyd(A-T)_serves as a chemically well defined model substrate containing damage of the kind introduced into DNA by ionizing radiation. In the first incision step of excision repair approximately one endonucleolytic nick is introduced into the polymer by extracts of $\text{E.}$$\text{coli}$ endoI^? and $\text{E.}$$\text{coli}$ endoI^?$\text{uvr}$A6^? per ring damaged thymine residue removed.     

Respiratory control and mitochondrial monovalent cation permeability of isolated liver cells

Uncoupling agent releases the respiratory control of rat hepatocytes to approximately the same degree as in isolated mitochondria indicating that mitochondria $\text{in situ}$ possess a low H⁺ conductance as $\text{in vitro}$. Mitochondria also have no detectable natural K⁺ conductance since the ionophore, valinomycin, is required for K⁺ ions to uncouple. Na⁺ but not K⁺ or choline inhibits the uncoupled respiration of liver cells. This is consistent with operation of neutral mitochondrial Na⁺ for H⁺ exchange $\text{in vivo}$. These results indicate a considerable similarity between certain functional and permeability properties of mitochondria $\text{in vitro}$ and $\text{in situ}$. These similarities form the basis for discussion of the role of mitochondrial ion transport in metabolic regulation.     

Amino acid uptake by yeasts: IV. Effect of thiol reagents on l-leucine transport in Saccharomyces cerevisiae

(1) $\text{N-}\text{Ethylmaleimide}$ (a penetrating SH- reagent) inactivated l-[¹⁴C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with $\text{N-}\text{ethylmaleimide}$, $\text{N-}\text{ethylmaleimide}$ concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With d-glucose-energized yeast, $\text{N-}\text{ethylmaleimide}$ inhibited l-[¹⁴C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit l-[¹⁴C]leucine binding, except when the cells were preincubated with l-leucine. With the rho^? respiratory-deficient mutant (energized cells), $\text{N-}\text{ethylmaleimide}$ inhibited l[¹⁴C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the $\text{N-}\text{ethylmaleimide}$ effect as a function of l-[¹⁴C]leucine concentration showed a significant decrease of $\text{J}{}_{\mathrm{<mtext>max</mtext>}}$ values of the high- (S₁) and low- (S₂) affinity amino acid transport systems, but $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ values were not significantly modified. (3) When assayed in the presence of d-glucose, $\text{N-}\text{ethylmaleimide}$ inhibition of d-glucose uptake and respiration contributed significantly to inactivation of l-[¹⁴C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of l-[¹⁴C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate l-[¹⁴C]leucine entrance, while $\text{p-}\text{chloromercuribenzoate}$, a slowly penetrating SH- reagent, inactivated it to a limited extent. When compared with the effect of $\text{N-}\text{ethylmaleimide}$, these negative results indicate that thiol groups of the l-[¹⁴C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.     

The relationship between the transport of glucose and cations across cell membranes in isolated tissues XI. The effect of vanadate on 45Ca-efflux and sugar transport in adipose tissue and skeletal muscle

(1) The effects of vanadate of hexose transport, ⁴⁵Ca-exchange and (Na⁺, K⁺)-contents have been characterized in isolated adipose tissue and skeletal muscles of the rat. (2) In whole epididymal fat pads, vanadate (0.5–5.0 mM) markedly stimulated the uptake of 2-deoxyl[¹⁴C]glucose as well as the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$. (3) Within the same concentration range, vanadate induced an early increase in ⁴⁵Ca-washout from preloaded fat pads. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.001}$, $\text{r = 0.98}$). (4) In extensor digitorum longus and soleus muscles, vanadate (0.5–5.0 mM) stimulated the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and this effect was preceded by a rise in the washout of ⁴⁵Ca. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.005}$, $\text{r = 0.98}$). (5) In extensor digitorum longus and soleus muscles, vanadate increased K⁺-contents and decreased Na⁺ contents. (6) The stimulation of ⁴⁵Ca-washout presumably reflects an increase in the cytoplasmic Ca²⁺ level, brought about by an inhibitory effect of vanadate on the Ca²⁺-sensitive ATPase of the sarcoplasmic or the endoplasmic reticulum. As demonstrated for most other insulin-like agents (Sørensen, S.S., Christensen, F. and Clausen, T. (1980) Biochim. Biophys. Acta 602, 433–445), the stimulating effect of vanadate on glucose transport appears to be associated with or mediated by a rise in the cytoplasmic Ca²⁺ level.     

Conversion of N6-isopentenyladenine to zeatin by Actinidia tissues

Tissue cultures grown from stem explants of three $\text{Actinidia}$ species and a hybrid species rapidly converted N6-isopentenyladenine (i⁶Ade) to zeatin (io⁶Ade), a potent hydroxylated cytokinin. Within 24 h on 50 uM i⁶Ade, callus tissues of $\text{A.}\text{chinensis}\text{×}\text{arguta}$ accumulated 83 ± 6 nmol/g io⁶Ade which was purified using HPLC and identified by its characteristic UV and mass spectra. Activity converting i⁶Ade to io⁶Ade was also demonstrated in stem segments from intact plants where it was low in the tip (3 cm), highest in the region corresponding to rapid leaf growth and very low in the mature stem. Root segments converted i⁶Ade to io⁶Ade almost as rapidly as the most active region of the stem while leaf petioles produced little io⁶Ade. Fruits of $\text{A.}\text{arguta}$ and $\text{A.}\text{chinensis}$ produced little or no io⁶Ade, respectively.     

A new category of ovulation inhibitors: linear LH-RH analogues having more than ten residues.

A linear analogue of the luteinizing hormone-releasing hormone, longer than a decapeptide, is described for the first time, which is equivalent in potency to the best known inhibitors of ovulation, and which constitutes an important new lead to the design of inhibitors of even greater potency. At a dosage of 200 μg/rat, the undecapeptide [(1, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH caused 100% inhibition of ovulation. The related analogues, [(1, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH and [(Gly-Pro)¹, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH, were less active, $\text{in}\text{vivo}$. All of these undecapeptides inhibited the action of 0.6 ng/ml of LH-RH by greater than 50% at the very low level of 10 ng/ml.     

Roles of sodium and potassium ions on p-aminohippurate transport in rabbit kidney slices

This investigation was principally undertaken to test the ionic gradient hypothesis as applied to active $\text{p-}\text{aminohippurate}$ uptake in the rabbit kidney cortical slice preparation. Efflux of $\text{p-}\text{aminohippurate}$ from the slice was shown to be independent of external Na⁺ concentration. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing $\text{p-}\text{aminohippurate}$ increased intracellular concentrations of both Na⁺ and K⁺, and $\text{p-}\text{aminohippurate}$ accumulation occurred. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing ouabain and $\text{p-}\text{aminohippurate}$ resulted in a net increase in intracellular Na⁺ concentration but no $\text{p-}\text{aminohippurate}$ accumulation occurred. Different combinations of preincubation and incubation media gave a high to low array of intracellular Na⁺ concentrations and these directly reflected their respective $\text{p-}\text{aminohippurate}$ uptake. These results suggest that the Na⁺-gradient hypothesis does not adequately explain the transport of organic acids in rabbit kidney. These results also suggest that Na⁺ possibly has an intracellular role through its stimulation of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ channeled to energizing the $\text{p-}\text{aminohippurate}$ accumulative mechanism.     

Acylated des-(Ala1-Gly2)-somatostatin analogs: prolonged inhibition of growth hormone secretion

The following eight analogs of somatostatin were synthesized by solid phase: des-[Ala¹-Gly²]-somatostatin (I); des-[Ala¹-Gly²]-H₂somatostatin (II); $\text{N}$-acetyl-Cys³-somatostatin (III); $\text{N}$-acetyl-Cys³-H₂somatostatin (IV); $\text{N}$-pyvalyl-Cys³-H₂somatostatin (V); $\text{N}$-acrylyl-Cys³-H₂somatostatin (VI); $\text{N}$-benzoyl-Cys³-H₂somatostatin (VII); $\text{N}$-hexanoyl-Cys³-H₂somatostatin (VIII). Deletion of the N-terminal dipeptide Ala¹-Gly² is compatible with high biological activity. A single s.c. injection of these analogs as a microsuspension in saline inhibits for 24–72 hours (depending on the compound) the secretion of growth hormone normally stimulated in rats by pentobarbital.     

Synthesis and relative stereochemistry of the four mercapturic acids derived from styrene oxide and N-acetylcysteine

The chemical reaction between (±)-styrene oxide and N-acetylcysteine produces both positional isomers ($\text{1}$ and $\text{2}$) as a mixture of diastereoisomers with a preference for the benzylic thioether isomer $\text{1}$ (2 : 1). Synthesis of the mercapturic acid conjugates from either (+)- or (?)-styrene oxide produces only two of the four possible stereoisomers. The single diastereoisomers of $\text{1}$ and $\text{2}$ were separated by high pressure liquid chromatography (HPLC) and identified by ¹H- and ¹³C-nuclear magnetic resonance (NMR). The relative stereochemistry at the benzylic carbon center of the mercapturic acid conjugates was assigned on the basis of the established chemical correlation between optically pure styrene oxide and its precursor mandelic acid, and considerations on the mechanism of ring opening of epoxides by sulfur nucleophiles. The stereochemical definition of the isomers $\text{3}$–$\text{6}$ should prove useful in investigations of the biotransformation of the glutathione (GSH) conjugates of styrene oxide.