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.     

A protein-bound form of thioacetamide in liver nucleoli

The cellular distribution of ³⁵S from ³⁵S- thioacetamide was determined in rabbit liver subcellular fractions following its $\text{in vivo}$ administration. Of the various fractions isolated, only the nucleolar fraction contained ³⁵S counts that were insoluble in 10% trichloroacetic acid but soluble in trichloroacetic acid if the fraction was treated with trypsin but not RNase or DNase. These results demonstrate that a protein bound form of thioacetamide is present in the nucleolus following $\text{in vivo}$ administration of this drug.     

The phosphorylation of troponin B by phosphorylase b kinase in skeletal muscle of mice carrying the phosphorylase b kinase deficiency gene

In skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene there is < 1% of the normal activity to convert phosphorylase $\text{b}$ to $\text{a}$ in the presence of Ca⁺⁺, Mg⁺⁺, and ATP (1). Correspondingly, there is < 1% of the normal activity to phosphorylate phosphorylase $\text{b}$. Nevertheless, under the same conditions, these extracts catalyze the phosphorylation of troponin at a rate 57% of normal. Phosphorylase $\text{b}$ converting activity can be sedimented from skeletal muscle of control mice by centrifugation. This fraction isolated from I strain skeletal muscle extracts phosphorylates troponin at a rate 29–39% of the control. EGTA¹ (15 mM) inhibits troponin phosphorylation by 50–60% in this fraction from both strains. The EGTA inhibition is reversed by 15 mM Ca⁺⁺. Thus the phosphorylase $\text{b}$ kinase in skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene can phosphorylate troponin B, although it shows little or no activity with phosphorylase as a substrate. This observation is consistent with the normal muscle contractility of I strain animals.     

Enzymatic removal of O6-methylguanine from DNA by mammalian cell extracts

Extracts from various rat tissues were incubated with [³H]methylated DNA or chromatin in order to compare their abilities to catalyze the removal of labeled $\text{O}{}^6$-methylguanine from acid precipitable DNA. Liver extracts had the greatest activity. Kidney extracts had about 35% of the activity in liver and extracts from lung, colon, small intestine and brain were much less active. The enzyme responsible for this reaction does not appear to be an $\text{N}$-glycosidase because no labeled $\text{O}{}^6$-methylguanine could be detected in the supernatant fraction even though more than 50% of this base was lost from the DNA. The released radioactivity was present as methanol which is consistent with the possibility that the reaction may involve a demethylase action on either the DNA substrate or an oligonucleotide derived from it.     

Compound 4880 is a selective and powerful inhibitor of calmodulin-regulated functions

Compound $\text{48}\text{80}$, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound $\text{48}\text{80}$ was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca²⁺-transport ATPase, with IC₅₀ values of 0.3 and 0.85 μg/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 μg/ml. Inhibition of Ca²⁺ transport into inside-out red blood cell vesicles by compound $\text{48}\text{80}$ follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca²⁺-transport ATPase induced by calmodulin is inhibited by compound $\text{48}\text{80}$ according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound $\text{48}\text{80}$ bind to calmodulin in a Ca²⁺-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound $\text{48}\text{80}$ is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca²⁺-transport ATPase that has been described hitherto. In addition, compound $\text{48}\text{80}$ was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca²⁺-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound $\text{48}\text{80}$, respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca²⁺-transport ATPase or calf cardiac sarcolemma (Na⁺ + K⁺)-transport ATPase are far less influenced by compound $\text{48}\text{80}$ as compared with trifluoperazine and calmidazolium. Because of its high specificity compound $\text{48}\text{80}$ is proposed to be a promising tool for studying calmodulin-dependent processes.     

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.     

The role of the lysines in the alkaline heme-linked ionization of ferric cytochrome c.

Phage ?¹⁵ adsorbed at a low temperature (or by short-time incubation) to the outer surface of $\text{Salmonella}\text{anatum}$ gathers on further incubation at a high temperature to a certain region where the inner and outer membranes may join. This was demonstrated by separating the inner and outer membranes of the cells in sucrose gradient after addition of ³⁵S-labeled ?¹⁵ to the cells. Radioactivity adsorbed at 4° was first recovered mainly with the dense outer membrane but disappeared by further incubation at 35° within 5 min. Instead, the radioactivity was recovered with the membrane fraction which had intermediate density. Such phage translocation was not observed when phage ?¹⁵ was added to a $\text{pep}\text{mutant of}\text{S.}\text{anatum}$ to which the phage can adsorb but fail to infect. A host range mutant phage which can infect the $\text{pep}$ mutant migrated to the intermediate dense region.     

The occurrence of ethanolamine and galactofuranosyl residues attached to Penicilliumcharlesii cell wall saccharides

$\text{Penicillium}$$\text{charlesii}$ incorporates ³H or ¹⁴C from ³H- or ¹⁴C-labeled ethanolamine into an -alkali soluble, alcohol -insoluble fraction obtained from cell walls. Dansyl ethanolamine was isolated from this alcohol-insoluble fraction following dansylation and hydrolysis. The alcohol-insoluble material was non-dialyzable and contained galactofuranosyl, glucosyl, phosphoryl, amino acyl and variable quantities of uronosyl residues. The lack of detectable quantities of mannosyl residues in this material suggests that the galactofuranosyl-containing cell wall polymer is distinct from the peptidophosphogalactomannan which is obtained from culture filtrates of $\text{P}$. $\text{charlesii}$ (Gander $\text{et}$$\text{al}$., (1974) J. Biol. Chem. $\text{249}$, 2063).     

Antibacterial properties of bile acid oxazoline compounds

Chenooxazoline³ (50–100 μM) inhibited (>50%) both 7α and 7β-dehydroxylase activities in whole cells and cell extracts of $\text{Eubacterium}$ sp. V.P.I. 12708. Chenooxazoline (>50 μM) and methylchenooxazoline (>25 μM) but not lithooxazoline (≤100 μM) inhibited growing cultures of $\text{Eubacterium}$ sp. V.P.I. 12708. Chenooxazoline (100 μM) also inhibited the growth of certain members of the genera $\text{Eubacterium}$, $\text{Clostridium}$, $\text{Bacteroides}$ and $\text{Staphylococcus}$ but not $\text{Pseudomonas}$, $\text{Escherichia}$, $\text{Salmonella}$ or the eucaryotic microorganism, $\text{Saccharomyces cerevisiae}$ (_< 400 μM).     

Preliminary characterization of a small intestinal binding component for retinol and fatty acids in the rat

A component which can bind retinol and fatty acids was detected in the rat's intestinal cell cytosol following intestinal perfusion $\text{in}$$\text{vivo}$ with ³H-all-trans retinol. Following Sephadex G-100 filtration of the cytosol, the void volume concentrate was treated with 2-mercaptoethanol and SDS. Sephadex G-100 filtration of the concentrate disclosed the presence of a cytosol binder of an approximate molecular weight of 12,000–17,000. The binder contained most of the ³H-retinol eluted off the column. $\text{In}$$\text{vitro}$ incubation experiments disclosed that ³H-retinol could be displaced from its bindinf cytosol fraction by the addition of nonradioactive retinol, retinyl acetate, and the fatty acids octanoic, linoleic, and linolenic. Butyric acid addition did not displace ³H-retinol from its binding fraction. The intestinal cytosol binding fraction may be involved in the trans-cytosol transport of lipid compounds from the lipid cell membrane to the intracellular organelles.     

Isolation and partial characterization of “galactoprotein a” (LETS) and “galactoprotein b” from hamster embryo fibroblasts

The cell surface glycoproteins of hamster NIL cells, labeled with galactose oxidase and NaB³H4, were selectively solubilized by sequential extraction with Tris buffer containing 1) sucrose-ATP-EDTA, 2) zwitterionic detergent (Empigen BB), and 3) 8 M urea. The previously reported “galactoprotein b” $\text{(Gap b)}$ and “galactoprotein a” ($\text{Gap a}$ or LETS) were isolated by affinity chromatography on insoluble Ricinus communis lectin colums (RCA column) from extracts 2) and 3), respectively. The affinity-purified $\text{Gap a}$ contained an actin-like protein, whereas the other affinity-purified galactoproteins did not contain the actin-like protein. The isolated $\text{Gap b}$ was heterogeneous, and an additional glycoprotein, specific for NILpy cells was copurified on RCA-column with $\text{Gap b}$.     

Tunicamycin inhibits the differentiation of ST 13 fibroblasts to adipocytes with suppression of the insulin binding activity

ST 13 cells are a clonal line of murine fibroblasts that are capable of differentiating into adipocyte-like cells $\text{in}\text{vitro}$. When the cells were maintained as a confluent monolayer, they began to accumulate lipid droplets and to exhibit a rapid increase of insulin binding activity. Tunicamycin, a specific inhibitor of dolichol-mediated protein glycosylation, blocked this adipose conversion without affecting cell growth and total protein synthesis. The inhibitory effect of tunicamycin was dose-dependent and reversible. Enhancement of the incorporation of [¹⁴C]acetate into triglyceride fraction accompanying the adipose conversion was completely inhibited by tunicamycin, whereas the incorporation into phospholipid fraction was only partially affected. The insulin binding activity increased about 10-fold during differentiation, but was completely suppressed in tunicamycin-treated cells.     

Novel nucleotides from E.coli isolated and partially characterized

Two new nucleotides have been found in the formic acid extracts of $\text{Escherichia}\text{coli}$, $\text{Clostridium}\text{botulinum}$, $\text{Bacillus}\text{subtilis}$ and $\text{Rhodospirillum}\text{rubrum}$ isolated during log phase growth. In $\text{E.}\text{coli}$ the compounds are present at all times during cell growth but increase in amount during interruption of aeration and transition to stationary phase. They migrate close to ppGpp during one dimensional chromatography on PEI cellulose but are clearly separated from ppGpp by paper chromatography. The compounds are unstable on PEI cellulose and purification was effected by chromatography on A25 Sephadex ion exchange columns. Preliminary characterization indicates that the predominant compound is a dinucleoside polyphosphate and that both compounds contain a modified adenosine nucleoside.     

A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase beta' subunit.

$\text{Penicillium charlesii}$ extracts contain UDP-galactose:NAD⁺ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH₄ followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D-$\text{glycero}$-D-$\text{glycero}$-3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose.     

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.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.     

Selective binding of polymyxin B to negatively charged lipid monolayers

It is demonstrated by direct measurement of surface radioactivity that the cationic polypeptide antibiotic polymyxin B is specifically adsorbed to negatively charged lipid monolayers. The latter attracted the following amounts of the biologically active $\text{mono}\text{-N[}{}^{14}\text{C]}\text{acetylpolymyxin B}$ derivative (PX): lipid A from Proteus mirabilis, 0.17; phosphatidic acid, 0.12; phosphatidylglycerol and phosphatidylserine, 0.11; dicetylphosphate, 0.107; sulfoquinovosyldiglyceride, 0.104; phosphatidylinositol and cardiolipin, 0.095; and phosphatidylethanolamine, 0.017 μg/cm². Adsorption of PX to phosphatidylcholine, monogalactosyldiglyceride and stearylamine was almost or completely zero. Total lipids from Escherichia coli adsorbed 0.057 in comparison to 0.051 μg PX/cm² of an artificial mixture of phosphatidylethanolamine/phosphatidylglycerol/cardiolipin in the proportions 75 : 25 : 5. The concentration of the surface active PX at the air/water interphase was 0.091 μg/cm². These saturation surface concentrations of PXat lipid monolayers were reached at 1 μg/ml bulk concentrations in 2 mM NaCl/1 mM Tris · HCl, pH 7.2. They decreased with decreasing surface charge density of the adsorbing monolayer. In an experiment with cardiolipin/phosphatidylethanolamine mixtures it was shown that two molecules of cardiolipin induced adsorption of one molecule PX giving a 1 : 1 ratio with regard to positive and negative charges. This could be due to a similar charge density of about one charge per 40–50 Ų in PX and lipid bilayers composed of phospholipids. The electrostatic PX-lipid interaction was severely inhibited by 10^?2 and 10^?1 M Ca²⁺ and Na⁺, respectively. It is discussed that the specificity of PX against Gram-negative bacteria is caused by the occurrence of lipid A, phosphatidylglycerol and cardiolipin at the cell surface of these microorganisms.     

A rapid isotope dilution method for analysis of indole-3-acetic acid and indoleacetyl aspartic acid from small amounts of plant tissue

A method is described for analysis of indole-3-acetic acid and indoleacetyl aspartic acid in plant tissue. Methanolic extracts are purified on two small columns prior to HPLC using an electrochemical detector. $\text{In}$,$\text{vivo}$ amounts are calculated by isotope-dilution analysis. The method requires only 1 g of tissue, results in relative high recoveries, and is sensitive to the 20 pmol range or below. Typical results using light-grown $\text{Pisum}$,$\text{sativum}$ cv. Little Marvel tissue are given. This report is the first determination of the natural level of the aspartate derivative in growing plant tissues.     

Stimulation by interferon of induction of differentiation of human promyelocytic leukemia cells

F₁-ATPase was isolated from yeast $\text{S.}\text{cerevisiae}$. The constituent subunits 1 and 2 were purified by gel permeation chromatography, and their amino acid compositions determined. Both subunits have a similar composition except for $\text{1}\text{2}$ cystine, methionine, leucine, histidine, and tryptophan. When F₁ is treated for three hours with 5′-p-[³H]fluorosulfonylbenzoyl adenosine in dimethylsulfoxide, 90% of the activity is lost. Disc gel electrophoresis of the modified complex showed that over 90% of the label was associated with subunit 2. A labelled peptide from a $\text{S.}\text{aureus}$ digest of subunit 2 was isolated and sequenced. It had the following amino acid sequence: His-Try¹-Asp-Val-Ala-Ser-Lys-Val-Gln-Glu, whereby Tyr¹ is the modified amino acid residue. This sequence shows homology to other sequences obtained from maize, beef heart, and $\text{E.}\text{coli}$ F₁-ATPases.     

Specificity of the serine protease inhibitor, phenylmethylsulfonyl fluoride.

Clarified cell-free extracts were prepared from rapidly dividing $\text{Bacillus subtilis}$ cells and from rabbit liver cells. These extracts were treated with [³H]-phenylmethylsulfonyl fluoride (PMSF) and analyzed by electrophoresis in isoelectric focusing polyacrylamide gels or detergent gels. Not less than 14 proteins in the $\text{B. subtilis}$ extracts and not less than 15 proteins in rabbit liver extracts reacted covalently with PMSF. These results suggest that PMSF is not as specific for serine proteases as sometimes supposed, and its effects in physiological experiments should be interpreted with caution.