Cycloheximide inhibition of insulin control of liver glycogen synthase b into a conversion.

Cycloheximide given to insulin-treated alloxan diabetic rats results in the inhibition of insulin-induced liver glycogen synthase $\text{b}\text{into}\text{a}$ conversion without affecting the level of synthase $\text{b}$. The effect of cycloheximide, believed to elevate cAMP in liver of normal rats, is independent of cAMP levels of the insulin-treated diabetic rat. The inhibition of insulin-mediated synthase $\text{b}$ to $\text{a}$ conversion by cycloheximide does not appear to be the result of a cycloheximide-induced cAMP-dependent phosphorylation of synthase $\text{a}$ to $\text{b}$ and suggests that insulin control of synthase $\text{b}$ and $\text{a}$ interconversions is dependent upon cycloheximide-sensitive protein synthesis.     

Cooperativity in ligand binding: a new graphic analysis.

When analyzing binding of ligands to macromolecules, the existence of site-site interactions complicates a straightforward interpretation of the binding parameters obtained through classical analytical methods, such as the Scatchard plot. For describing site-site interactions, we propose a new parameter, the $\text{average affinity}$ of the receptor sites, $\text{K}$, calculated as $\text{(}\text{B}\text{F}\text{)/(R}{}_{\mathrm{o}}\text{?B)}$. Plotting $\text{K}$ as a function of fractional occupancy ($\text{B}\text{R}{}_{\mathrm{o}}$), reveals that: (1) at very low occupancy a limiting high $\text{K}$ is obtained ($\text{K}$_e) (“empty sites” conformation); (2) when the fraction of sites filled increases above a certain threshold, $\text{K}$ begins to fall due to increasing site-site interactions until (3) a limiting low $\text{K}$ ($\text{K}$_f) is obtained (“filled sites” conformation). This method has been successfully applied to the negative cooperativity of insulin receptors.     

Molecular characterization of a stable Flac plasmid

F$\text{lac}$S is a thermostable extrachromosomal element isolated in $\text{Salmonella typhimurium}$ which is altered in its replication as compared to its precursor F_ts114$\text{lac}$. Sedimentation of both these plasmids in alkaline sucrose gradients has indicated a difference in their sizes. Contour length measurements of open circular plasmid DNA molecules photographed in the electron microscope have revealed the estimated molecular weight of F_ts114$\text{lac}$ to be 81 × 10⁶ daltons while that of F$\text{lac}$S is 109 × 10⁶ daltons. F$\text{lac}$S may carry a segment of $\text{S. typhimurium}$ chromosomal or cryptic plasmid DNA.     

1-Aminoglycosides, a new class of specific inhibitors of glycosidases

1-Aminoglycosides represent a new class of specific and relatively potent inhibitors of glycosidases. These compounds are specific against those enzymes which act upon glycosides that correspond to glycone of the inhibitor. Thus α- and β-$\text{D}\text{=}$- are inhibited by $\text{D}\text{=}$-glucosidases but not by $\text{D}\text{=}$-galactosylamine and $\text{D}\text{=}$-mannosylamine. α- and $\text{D}\text{=}$-galactosidases are inhibited by $\text{D}\text{=}$-galactosylamine but not by the other two glycosylamines. $\text{D}\text{=}$-Mannosylamine inhibits mannosidase.     

Isolation of a complementing activity for a dna-B mutant

A cell free extract which displays temperature sensitive DNA synthesis in the presence of single strand DNA and ATP was prepared from a $\text{dna-B}$ mutant. Following an activity which would reverse this temperature sensitivity, a protein fraction was isolated. The absence of this fraction in a $\text{dna-B}$ mutant indicates that this protein corresponds to the $\text{Dna-B}$ product.     

The detection of a bound ferredoxin in the photosynthetic lamellae of blue-green algae and other oxygen evolving photosynthetic organisms

The presence of an electron transport component with an EPR spectrum similar to that of a ferredoxin has been demonstrated in the blue-green alga $\text{Anabaena}$$\text{cylindrica}$, the green alga $\text{Euglena}$$\text{gracilis}$, and in chloroplasts from sorghum ($\text{Sorghum}$$\text{bicolour}$) and beans ($\text{Phaseolus}$$\text{vulgaris}$). The component is photoreduced at 77°K and is very similar to that previously reported in spinach. It seems likely that this component is a primary electron acceptor in photosynthesis in all of these organisms.     

Resistance of Tetrahymena to ricin, a toxic enzyme from Ricinus communis.

The protozoan $\text{Tetrahymena}\text{pyriformis}$ was found to be resistant to the toxic action of ricin $\text{in}\text{vivo}$. Isolated $\text{Tetrahymena}$ ribosomes were strongly resistant to the A subunit of ricin when tested in a cell free protein synthesis system under different conditions and also lacked the ability to bind A chain stoichometrically. This suggests that $\text{Tetrahymena}$ is resistant $\text{in}\text{vivo}$ because it contains a ribosome which is not susceptible to the toxic action of ricin.     

Antimicrobial activity of juncusol, a novel 9-10-dihydrophenanthrene from the marsh plant Juncus roemerianus

The 9,10-dihydrophenanthrene phenolic compound juncusol, from the marsh plant $\text{Juncus}$$\text{roemerianus}$, has been shown to be inhibitory to four species of naturally occurring $\text{Bacillus}$ and to two ATCC species $\text{Bacillus}$$\text{subtilis}$ and $\text{Staphylococcus}$$\text{aureus}$. Juncusol may regulate populations of bacillus bacteria in the marsh and has potential as an antimicrobial agent particularly to gram positive microorganisms.     

The biosynthesis of phosphorylated tyrosine hydroxylase by organ cultures of rat adrenal medulla and superior cervical ganglia: a correction.

Determination of the complete amino acid sequence of the rubredoxin isolated from the sulfate reducing bacterium $\text{Desulfovibrio}$$\text{gigas}$ showed that the molecule consists of a single polypeptide chain of 52 residues. The sequence of the first 42 residues was determined using an automatic Protein Sequencer. Peptides derived from tryptic hydrolysis and from specific cleavage at tryptophan residue were used to construct the total sequence. Compared with the sequence of $\text{Desulfovibrio}$$\text{vulgaris}$ rubredoxin, 37 positions are identical, and with the sequences of $\text{Clostridium}$$\text{pasteurianum}$, $\text{Peptostreptococcus}$$\text{elsdenii}$, $\text{Micrococcus}$$\text{aerogenes}$ and $\text{D.}$$\text{vulgaris}$ rubredoxins, 20 matching residues occur. A crystallographic study of the $\text{D.}$$\text{gigas}$ rubredoxin is in progress.     

Fluorescence properties of a benzo(a)pyrene 7,8 dihydrodiol 9,10-oxide-DNA adduct. Conformation and effects of intermolecular DNA interactions

The pyrene-like fluorescence of the covalent benzo(a)pyrene diol-epoxide-DNA complex prepared by reacting 7,8,-dihydrodiol 9,10-epoxy benzo(a)pyrene (BPDE) with DNA in aqueous solution $\text{in vitro}$, has been investigated. It is shown that this fluorescence is $\text{sensitive}$ to molecular oxygen, to the concentration of $\text{native}$ DNA and to the ionic strength (KCl concentration), but is $\text{insensitive}$ to the concentration of $\text{denatured}$ DNA. These effects are related to the conformation of the pyrene-like chromophore of BPDE. Most of the fluorescence of a $\text{dilute}$ solution of the DNA-bound benzo(a)pyrene derivative originates from binding sites in which the pyrene moiety is not intercalated between the DNA base pairs, but is located on the outside of the DNA double helix.     

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.     

Solid phase peptide synthesis of alpha-factor, a yeast mating pheromone.

Based on analysis of highly purified preparations of natural $\text{α}$-factor and on the sequence recently reported by others, oligopeptides of the following structures were chemically synthesized by the solid phase method of Merrifield: N-Trp-His-Trp-Leu-Lys-Pro-Gly-G1N-Pro-Met-Tyr-C N-His-Trp-Leu-Lys-Pro-Gly-G1N-Pro-Met-Tyr-C Both synthetic species arrested $\text{a}$ cells in G1, inhibited their DNA synthesis, caused them to elongate markedly, and induced an increase in their adhesivity toward $\text{α}$ cells. Neither synthetic material caused any of these effects in $\text{α}$ cells or in $\text{a}\text{α}$ diploids.     

Myelin basic protein: a substance that releases immunoreactive growth hormone in vitro.

A protein has been isolated from ovine hypothalamus on the basis of its ability to stimulate release of growth hormone by $\text{in}$$\text{vitro}$ cultures of dispersed pituitary cells. This protein has been identified as being myelin basic protein. With no similar biological activity $\text{in}$$\text{vivo}$, myelin basic protein is thus to be recognized as a potentially interfering substance in any search for the physiological growth hormone releasing factor using $\text{in}$$\text{vitro}$ assay systems.     

Infertility in a bull with a nuclear sperm defect: A case report

A Simmental bull with a history of low fertility, both by natural service and artificial insemination, was presented for examination. Two previous semen evaluations had revealed no specific semen abnormalities that would support the breeding history. A comprehensive cytochemical analysis of the bull's ejaculate revealed a complex nuclear lesion affecting over 80% of sperm. This condition was expressed in abnormal shaping of the nuclei, with deficient distribution, condensation and stabilization of the nucleoplasm. These abnormalities were associated with various-sized intranuclear pouches or depressions. The acrosome was moderately involved and the tail was relatively free of abnormalities resulting in normal sperm motility.Two controlled breeding trials utilizing a total of 15 super-ovulated females were conducted to evaluate the bull's fertilization rate. Combined data demonstrated an 18% ($\text{23}\text{128}$) fertilization rate of recovered ova. At the same time, the fertilization rate of seven bulls classified as satisfactory potential breeders was 72% ($\text{353}\text{490}$).Data from two embryo transplant units regarding ova collected from eight donor females inseminated with semen from this bull revealed a fertilization rate of 41% ($\text{30}\text{73}$). Of the fertilized ova, 37% ($\text{11}\text{30}$) were degenerate and were not transferred. A pregnancy rate of 57% ($\text{11}\text{19}$) resulted from the transfer of 19 fertilized ova.A natural breeding pregnancy rate of 5% ($\text{2}\text{42}$) and artificial breeding pregnancy rate of 8% ($\text{15}\text{180}$) support the breeding trial results.     

Cysteine as a system-specific substrate for transport system ASC in rat hepatocytes.

The rapid transport of $\text{L}$-cysteine into isolated rat hepatocytes escapes detectable inhibition by 2-(methylamino)-isobutyric acid at levels up to 50 mM. The system transporting cysteine instead is convincingly similar to the $\text{ASC}$ system described for the Ehrlich cell in structural and steric specificity and in pH sensitivity. The Na⁺-dependent uptake of 2-aminoisobutyric acid is almost evenly divided between Systems $\text{A}$ and $\text{ASC}$, showing better accommodation of its two α-methyl groups by $\text{ASC}$ than in the Ehrlich cell. The hepatocyte $\text{ASC}$ system tolerates Li⁺-for-Na⁺ substitution better than does System $\text{A}$, although the tolerance depends on amino acid structure. Adaptive regulation and insulin and glucagon stimulation were not seen under conditions producing these effects for System $\text{A}$.     

Stereoselectivity in the metabolism of drobuline (d1-1-(isopropylamino) -4,4-diphenyl-2-butanol) a new anti-arrhythmic agent

The metabolism of drobuline has been examined in the dog, rabbit, rat, guinea pig and hamster. In the dog, unlike the other species, glucuronide conjugation is the major route of metabolism. The structure of the conjugate has been established as an O-glucuronide by isolation using HPLC following by field desorption mass spectral analysis. When the separate $\text{d}$- and $\text{l}$-isomers of drobuline were administered to a series of dogs the $\text{l}$-isomer reached plasma levels approximately three time higher than those of the $\text{d}$-isomer. Deuterium labeled drobuline was synthesized and resolved by multiple crystallizations of the malate salts. Racemic mixtures containing $\text{d}$₆-$\text{d}$ and $\text{h}$₆-$\text{l}$ drobuline and $\text{d}$₆-$\text{l}$ and $\text{h}$₆-$\text{d}$ drobuline were prepared and analyzed by GC-MS as the pentafluoropropionate derivatives. When either racemic mixture was administered to dogs (10 mg/kg, p.o.) the plasma levels of the $\text{l}$-isomer were found to be approximately three times those of the $\text{d}$-isomer. Using these deuterium labeled mixtures the disposition of the two isomers has been examined in the isolated perfused dog liver, in hepatocytes and isolated microsomes. The results indicate that the difference in plasma levels of the $\text{d}$- and $\text{l}$-isomers is not dependent upon stereospecific absorption or excretion but rather it is caused by metabolism of the $\text{d}$-isomer at a faster rate than that of the $\text{l}$-isomer.     

Ribosomal proteins L7/L12 localized at a single region of the large subunit by immune electron microscopy.

Ribosomal proteins $\text{L}\text{7}\text{L}\text{12}$ have been mapped by immune electron microscopy. These multiple copy proteins are located at a single region extending from the large subunit, known as the $\text{L}\text{7}\text{L}\text{12}$ stalk. The $\text{L}\text{7}\text{L}\text{12}$ stalk is approximately 100 Å long, about 40 Å wide and extends at an angle of approximately 50 ° from one side of the central protuberance of the large subunit. In the monomeric 70 S ribosome, the portion of the $\text{L}\text{7}\text{L}\text{12}$ stalk proximal to the 50 S subunit is located in the vicinity of the 30 S-50 S interface.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibody binding to the stalk was shown to be solely dependent upon the presence of $\text{L}\text{7}\text{L}\text{12}$ by the following experiments. Sucrose gradient analysis was used to demonstrate that large subunits depleted of $\text{L}\text{7}\text{L}\text{12}$ were unable to bind anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ restored the ability of $\text{L}\text{7}\text{L}\text{12}$-depleted cores to react with anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies. Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies pre-absorbed with $\text{L}\text{7}\text{L}\text{12}$ did not react with 50 S subunits.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies used in these experiments reacted only with the $\text{L}\text{7}\text{L}\text{12}$ stalk and with no other region of the subunit. This was shown by electron microscopy and by immune electron microscopy in the following ways. Electron microscopy of 50 S subunits, $\text{L}\text{7}\text{L}\text{12}$-depleted 50 S cores, and reconstituted 50 S subunits was used to demonstrate that stripping removes the $\text{L}\text{7}\text{L}\text{12}$ stalk from more than 95% of the subunits, and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ into depleted cores restores the $\text{L}\text{7}\text{L}\text{12}$ stalk. Double-labelling experiments, using monomeric subunits with two or more attached anti-$\text{L}\text{7}\text{L}\text{12}$ immunoglobulins, were used to demonstrate, independently of 50 S subunit morphology, that $\text{L}\text{7}\text{L}\text{12}$ are located only on the $\text{L}\text{7}\text{L}\text{12}$ stalk.     

Biosynthesis of 5-methylaminomethyl-2-thiouridylate. I. Isolation of a new tRNA-methylase specific for 5-methylaminomethyl-2-thiouridylate

A new enzyme, which catalyzes the transfer of a methyl group to tRNA to form 5-methylaminomethyl-2-thiouridylate, was isolated from $\text{E.}$$\text{coli}$ by a procedure including affinity chromatography. The purified enzyme was nearly homogeneous upon disc electrophoresis. Using methyl-deficient tRNA^Glu of $\text{E.}$$\text{coli}$ as substrate, the 5-methylaminomethyl-2-thiouridylate residue synthesized was mostly found in the anticodon loop, showing a coincidence of the modification site $\text{in}$$\text{vitro}$ with that $\text{in}$$\text{vivo}$.     

On the defect of a dCMP hydroxymethylase mutant of bacteriophage T4 showing enzyme activity in extracts

Infection by $\text{L}\text{13}$, a temperature-sensitive mutant of gene 42 of phage T4, the structural gene for dCMP hydroxymethylase, previously was shown not to form T4 DNA at nonpermissive temperatures. Yet the enzyme activity was found in extracts. Since inactivation of the enzyme was not reversible, we have examined acid-soluble extracts of cells infected at nonpermissive temperature by $\text{tsL}\text{13}$ for 5-hydroxymethyldCMP in order to determine whether the enzyme functioned $\text{in vivo}$. A double mutant of $\text{tsL13}$ and $\text{amB}\text{24}$ (5-hydroxymethyldCMP kinase) did not form the nucleotide at nonpermissive temperature, but the control, $\text{amB}\text{24}$, formed large quantities. From these results and previous temperature-shift studies it is suggested that the enzyme is normally activated to function $\text{in vivo}$ between 5 and 8 minutes after infection.