Incorporation of 3H-adenine into free cytokinins by cytokinin-autonomous tobacco callus tissue

Cytokinin-autonomous tobacco callus was incubated in defined mineral medium containing ³H-adenine for 60 minutes. Radioactivity was incorporated into the four predominant free cytokinins, ribosyl-$\text{trans}$-zeatin, $\text{trans}$-zeatin, N⁶-(Δ²-isopentenyl) adenosine and N⁶-(Δ²-isopentenyl) adenine. The bases were more abundant than their respective ribosides, N⁶-(Δ²-isopentenyl) adenine being the most abundant cytokinin. No discrete peaks of radioactivity could be detected on the HPLC column eluate corresponding to the elution volumes of $\text{cis}$-zeatin and ribosyl-$\text{cis}$-zeatin.     

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.     

The stereochemistry of hydrogen addition by a delta1-steroid reductase in baker's yeast

The reduction of the Δ¹ double bond in 5α-androstan-1-en-3-one was investigated utilizing a reductase present in fermenting baker's yeast. The stereochemistry of the hydrogen addition was found to be $\text{trans}$ diaxial, as determined by suitable deuterium labeling and lanthanide induced shift nmr spectroscopy.     

Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli

The cytoplasmic and outer membranes containing either trans-Δ⁹-octadecenoate, trans-Δ⁹-hexadecenoate or cis-Δ⁹-octadecenoate as predominant unsaturated fatty acid residues in the phospholipids were prepared from a fatty acid auxotroph, Escherichia coli strain K1062. Order-disorder transitions of the phospholipids were revealed in both fractions of the cell envelope by fluorescent probing or wide angle X-ray diffraction. The mid-transition temperatures, $\text{T}\text{t}$, and the range of the transition, $\text{ΔT}$, are similar in the outer and cytoplasmic membrane. Relative to the corresponding extracted lipids, 60–80% of the hydrocarbon chains take part in the transition in the cytoplasmic membrane whereas in the outer membrane only 25–40% of the chains become ordered. The results suggest that in the outer membrane part of the lipids form fluid domains in the form of mono- and/or bilayers.     

Griseofulvin: Association with tubulin and inhibition of in vitro microtubule assembly

Griseofulvin—shown previously to disrupt the mitotic apparatus $\text{in vivo}$—inhibited the $\text{in vitro}$ microtubule assembly reaction completely at 8 × 10^?4M griseofulvin. In a gel filtration assay, randomly tritiated griseofulvin associated stoichiometrically with purified tubulin, as determined by chromatography on Sephadex G-25. No detectable drug binding was observed when bovine serum albumin was used as a control in an identical column assay. Both gel filtration chromatography and a kinetic analysis of the inhibition of assembly by griseofulvin suggest that the drug interacts directly and stoichimetrically with the tubulin dimer, and that the interaction is both rapid and independent of temperature.     

13C magnetic resonance study of the ionization of N-acetyl-dl-proline in aqueous solution

NMR titration curves have been recorded for all the ¹³C resonances of cis and trans$\text{N-}\text{acetyl}\text{-}\text{dl}\text{-}\text{proline}$ in ²H₂O. the measured pK_2H values are 3.4 ± 0.8 and 4.13 ± 0.08 respectively; the free energy of ionization for the trans isomer being (3.8 kJ/mole) greater than for the cis. The ionization shifts of the two isomers differ significantly only at the acetyl carbonyl and C_γ positions. It is suggested that these are related to conformational changes which stabilize the trans form at low p²H.     

Chymotrypsin stimulated development of delayed implantation mouse blastocysts

Chymotrypsin-like enzyme activity increases transiently in the uterine lumen of ovariectomized mice upon administration of progesterone and estrogen (1). This is one of the few known macromolecular changes associated with conditions which result in activation of delayed implantation blastocysts $\text{in}$$\text{utero}$. $\text{In}$$\text{vitro}$, α-chymotrypsin (100 μg/ml) was found to shorten the time required for these embryos to attach to the glass culture dish and then form outgrowths in fetal calf serum-supplemented medium. Higher concentrations of the enzyme (250 μg/ml) prevented embryo attachment probably by digesting the fetuin present in fetal calf serum. Nevertheless, 250 μg/ml α-chymotrypsin could apparently replace fetal calf serum as a stimulator of development during the first 24 hours of culture. In contrast, bovine serum albumin (3.0 mg/ml) seemed to slow development of blastocysts $\text{in}$$\text{vitro}$. It is suggested that chymotrypsin-like enzyme activity may stimulate development of delayed implantation blastocysts $\text{in}$$\text{utero}$ (a) indirectly by removing inhibitory proteins such as albumin and (b) by directly affecting these embryos in a manner yet to be determined.     

Hydrolysis of inulin from Jerusalem artichoke by inulinase immobilized on aminoethylcellulose

Purified inulinase (inulase, 2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) of Kluyveromyces fragilis has been immobilized on 2-aminoethyl-cellulose by treatment with 2% glutaraldehyde in 0.05 m phosphate buffer, pH 7.0, for 2 h at room temperature. The immobilized enzyme preparation had 39.3 units inulinase activity per gram dried matrix, with 53.4% recovery yield of activity, and showed good operational stability in the presence of substrate, inulin or the tuber extract of Jerusalem artichoke. Optimum pH and temperature were 5.5 and 45°C, respectively. In a batch reactor, the conversion was 90% ($\text{d}\text{-fructose/}\text{d}\text{-glucose}\text{= 76/24}$) and 34 mg d-fructose per ml was produced from the artichoke tuber extract by the immobilized inulinase in 20 h. In column reactor packed with 28 ml immobilized enzyme, the following conditions were found to be optimal: height/diameter ratio of column, 10.3; space time, 3.8 h; temperature, 40°C. Operation under these conditions gave 90% conversion of a 7% inulin solution and the productivity was 102 mmol l^?1 h^?1.     

Secondary structure of peptides: 15. 13C n.m.r. CP/MAS study of solid elastin and proline-containing copolyesters

In order to study the chemical shifts and the cis—trans isomerism of prolyl units neighbouring glycine or other amino acids, 75.4 MH_z¹³C nuclear magnetic resonance (n.m.r.) cross-polarization/magic angel spinning (CP/MAS) spectra of the following solid oligopeptides and sequence polypeptides were measured: Z-Gly-Pro-OH,Z-Gly-Pro-Gly-Gly-OEt,Z-Gly-Pro-Ala-Ala-OMe,(Gly-Pro-Gly)_n,(Gly-Pro-Ala)_n,(β-Ala-Pro)_n and $\text{(δ-Ava-Pro)}{}_{\mathrm{<mtext>n</mtext>}}\text{(δ-Ava=δ-}\text{aminovaleric acid}$). Whereas all these oligo- and polypeptided contain exclusively trans X-Pro bonds, both cis and trans peptide bonds were found in a polypeptide prepared by copolymerization of glycine- and $\text{proline-}\text{N}\text{-carboxyanhydrides}$ in pyridine. On the basis of these model compounds, the ¹³C n.m.r. CP/MAS spectra of solid elastin allows the following conclusions. Almost all X-Pro bonds assume the trans conformation, most alanine and leucine units form α-helical chain segments, whereas only a small fraction of β-sheet structure is present. A 30.3 MHz ¹⁵N n.m.r. CP/MAS spectrum of solid elastin confirms that ～25% of all amino acids assume the α-helical structure. A model of elastin is discussed consisting of an amorphous phase, α-helical chain segments and helical segments of still unknown pitch.     

Juvenile hormones radiobiosynthesised by corpora allata of adult female locusts in vitro

When corpora allata from adult female $\text{Schistocerca}$$\text{gregaria}$ are incubated $\text{in}$$\text{vitro}$ with either ³H-$\text{trans}$, $\text{trans}$ farnesenic acid or ³H-$\text{trans}$, $\text{trans}$, $\text{cis}$ bishomo-farnesenic acid and [$\text{methyl}$-¹⁴C] methionine, they fabricate large quantities of the corresponding double labelled methyl 10, 11-epoxy esters. Radio GLC of these products indicates retention of geometric configuration at the C-2 and C-6 double bonds. Separate analyses of the contents of the glands and medium after incubation show that the epoxy esters are rapidly released from the glands into the medium and that only the glands contain the corresponding unepoxidized esters. We suggest that unepoxidized esters are the intracellular intermediates in the formation of juvenile hormones from the unsaturated acids. Gel filtration shows that the epoxy esters are not released as stable protein complexes but as simple solutes into the medium. Using this method of promoting the synthesis of juvenile-hormone-active compounds, rates of biosynthesis of epoxy esters of up to 33 ng. per pair of glands per hour have been achieved.     

A radioimmunoassay for 11-oxotestosterone: Its application in the measurement of levels in blood sebum of rainbow trout (S. Gairdneri).

17β-Hyd.roxyandrost-4-ene-3,11-dione was linked via its 3-(O-carboxymethyl) oxime to bovine serum albumin to give a conjugate which was used to generate antiserum in rabbits. The antiserum, at an overall dilution of 1 in 16,000, together with [1,2-³H] 17β-hydroxyandrost-4-ene-3,11-dione synthesized from [1,2-³H] cortisone have been used to develop a radioimmunoassay for the parent steroid. The assay incorporates a purification step in which serum or plasma extracts are chromatographed on silica gel layers bound to plastic or aluminium sheets and the steroid, containing zones cut out and eluted directly with assay buffer. The cross-reactivities of several steroids with the antiserum and the specificity, sensitivity, accuracy and precision of the assay are described. Blood sera from Immature male rainbow trout contain $\text{ca}$ 0.2–0.4 μg/100 ml of 17β-hydroxyandrost-4-ene-3,11-dione. As male fish mature, serum levels rise sharply to reach values of 2 to >9 μg/100 ml. Levels in immature females rarely exceeded the assay sensitivity but serum from three ripe females showed low but detectable levels ($\text{ca}$ 0.2 μg/100 ml) of steroid. The assay has found application in sexing live fish for experimental purposes.     

Compensation for measuring error produced by finite response time in determination of rates of oxygen consumption with a Clark-type polarographic electrode

In the determination of the rates of oxygen consumption with a Clark-type oxygen electrode, and experimental error is caused by finite response time of the oxygen electrode for a rapid oxidation reaction. A theoretical equation between the observed pseudo first-order rate constant (k_obs) and the true rate constant (k)

$\text{1}\text{k}{}_{\mathrm{obs}}\text{=}\text{1}\text{k}\text{+T}$

where T is a time constant for a first-order response of the oxygen electrode, was derived and found to hold up to k = 23 min^?1 in oxidation of hydroquinone at pH 7.60–8.63.     

Aureobasidium pullulans metabolism of prostaglandins of the A-type

$\text{Aureobasidium pullulans}$, originally introduced as an inadvertent contaminant in solutions used for evaluating the stability of prostaglandins, proved to lead to the rapid disappearance of the cyclopentenone unit of PGA₂ (as monitored by circular dichroic spectroscopy). The cyclopentenone unit is converted, in various metabolites, to a 9-keto, 9α or 9β-hydroxy group lacking the ring unsaturation. The major EtoAc-soluble 9-hydroxy metabolite (Compound-I) was shown to be 9α, 15α-dihydroxy-2,3,4,5-tetranor-13-$\text{trans}$-prostenoic acid. Similar tetranor 9-hydroxy metabolites with one additional degree of unsaturation, and with a 9β-hydroxy group, also occur but these have not been fully characterized. Only two of the wide range of 9-keto metabolites are fully characterized by mass spectral (MS) data: 9,15-oxo-2,3,4,5-tetranorprostanoic acid and 9,15-oxo-2,3,4,5-tetranor-13-$\text{trans}$-prostenoic acid. The water soluble metabolites have not been characterized further.The fully characterized metabolites together with MS data from mixtures of minor metabolites indicate that $\text{A. pullulans}$ can perform the following transformations: β-oxidation, dehydrogenation at C-15, reduction of the enone carbon-carbon double bonds (both Δ^10,11 and Δ^13,14), reduction of the 9-ketone, and possibly migration of the cyclopentyl double bond (Δ^10,11 → Δ^11,12). $\text{A. pullulans}$ metabolizes 15-epimeric PGA₂ equally readily with the production of similar products. PGA₁ affords less 9-keto metabolites with compound I constituting 33% of the product by HPLC analysis. $\text{A. pullulans}$ displays some enantioselectivity, PGA₂ and 15-epi-PGA₂ are each metabolized more rapidly than their enantiomers. Other prostaglandins appear to be less readily metabolized.     

Leukotriene A: stereochemistry and enzymatic conversion to leukotriene B

Leukotriene A was assigned the structure 5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}\text{-11,14-}\text{cis}$-eicosatetraenoic acid by the enzymatic conversion of a synthetic product of known stereochemistry into the naturally occurring isomer of 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid in human polymorphonuclear leukocytes.     

Solid-phase extraction on Styrosorb cartridges as a sample pretreatment method in the stereoselective analysis of propranolol in human serum

Sample pretreatment using solid-phase extraction (SPE) on cartridges filled with small-particle Styrosorb porous polystyrene-based sorbent has been used in the analysis of propranolol enantiomers in human serum by high-performance liquid chromatography (HPLC) with fluorescent detection. SPE on Sep-Pak C₁₈ cartridges was used as a reference pretreatment method. The propranolol content of the samples was determined by achiral normal-phase HPLC and the enantiomeric ratio of propranolol (S/R) was then determined by chiral HPLC on a column with silica-bonded cellulose-tris(3,5-dimethylphenyl carbamate). Recoveries of propranolol from serum using SPE on Styrosorb and C₁₈ phases were 97±5% and 96±5%, respectively. Detection and quantification limits for propranolol enantiomers were 4 and 7 ng/ml, respectively.     

Comparative studies between microbiological culture and uptake of uridine/uracil to detect mycoplasmal infection of cell cultures.

Controlled, prospective studies were performed to compare detection of cell culture mycoplasmas by ratio of uptake of tritiated uridine (UdR) to tritiated uracil (U) and by microbiological culture. Culture was by standard agar and broth inoculation with aerobic and anaerobic incubation; immunofluorescent staining of indicator cell cultures was used to detect M. hyorhinis. The ratio of uptake of UdR to U ($\text{UdR}\text{U}$) and interpretation of test results were by standard published methods and performed in triplicate. 115 cell cultures were simultaneously assayed by the two techniques. 84 cultures (73.1%) yielded agreement between the 2 methods; 2 cultures (1.7%) yielded conflicting results, and 29 cultures (25.2%) yielded $\text{UdR}\text{U}$ results in the questionable range. Conflicting results consisted of two negative $\text{UdR}\text{U}$ tests in mouse cell cultures infected with M. orale. In separate studies, 3T-6 cultures freshly infected with M. orale yielded negative $\text{UdR}\text{U}$ results 3 days after infection, questionable results after 10 days and a positive $\text{UdR}\text{U}$ 17 days after infection. $\text{UdR}\text{U}$ detected infection in fibroblast, epithelial, and lymphocyte cell cultures. Highest $\text{UdR}\text{U}$ ratios were detected in human skin fibroblasts at early population doubling levels (PDLs), 4064 in one culture at PDL4. $\text{UdR}\text{U}$ was determined for IMR-90, a human diploid fibroblast at 12 different PDLs using the same lot of media. $\text{UdR}\text{U}$ gradually decreased throughout the life of the culture, from 2 125 at PDL6 to 340 at PDL36. Cultures in phase III and others exhibiting poor growth frequently yielded questionable or false-positive $\text{UdR}\text{U}$ results and were not included in tabulations of these results. $\text{UdR}\text{U}$ determined in endothelial cell cultures decreased as population density increased. In a representative experiment performed over a 4-day period, the $\text{UdR}\text{U}$ values were 1 808, 955 and 356 when the number of endothelial cells in culture were 5.3 × 10⁵, 6.6 × 10⁵ and 1.1 × 10⁶, respectively.     

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.     

The metabolic fate of 14C-labeled immunoadjuvant peptidoglycan monomer: II. In vitro studies

Peptidoglycan monomer (GlcNAc-MurNac-L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala), labeled with ¹⁴C both in the disaccharide and pentapeptide portions, was incubated with slices of mouse liver, kidney or spleen as well as with mouse and human blood cells, plasma and serum. Peptidoglycan monomer was isolated unchanged after incubations with mouse organs and blood cells. However, upon incubation with mouse or human blood, 10–50% of the peptidoglycan monomer underwent hydrolysis to the corresponding disaccharide and pentapeptide. After incubations with plasma and serum more than 90% of the [^14C]peptidoglycan monomer was metabolized: about 50% of the administered radioactive dose was recovered in the disaccharide unit and about 35% in the pentapeptide part. These results suggest that in blood, plasma and serum of mouse and man, an $\text{N-}\text{acetylmuramoyl-}\text{L}\text{-alanine}$ amidase (mucopeptide amidohydrolase, EC 3.5.1.28) exists which splits the amide bond between the lactyl carboxyl group of the muramyl residue and the amino group of the peptide moiety in the peptidoglycan molecule.     

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.     

Preparation and redox properties of the complexes trans-[ReL2(dppe)2]BF4 (L = CO or isocyanide). Estimate of the oxidation potential of octahedral 18-electron complexes with 14-electron square planar metal centres and of related electrochemical parameters for derived 16-electron sites

The complexes trans-[ReL₂(dppe)₂]BF₄ (I, L = CO, CNR with R = Me,Bu^t, C₆H₄OMe-4, C₆H₄Me-4, C₆H₄Cl-4 and C₆H₃Cl₂-2,6) were prepared from the reaction of the parent dinitrogen complex trans-[ReCl(N₂)(dppe)₂] with the appropriate ligand, in thf and in the presence of TlBF₄.Their redox properties were studied by cyclic voltammetry at a Pt electrode in [Bu₄N][BF₄]/thf or acetonitrile; they undergo a one-electron reversible oxidation and the observed E^OX_{$\text{1}\text{2}$} values were applied to test the validity of a proposed general expression —derived from the electrochemical ligand (P_L) and 16-electron metal site (E_s, β) parameters [6]— to estimate E^OX_{$\text{1}\text{2}$} for 18-electron octahedral complexes of the type [M′_sL₂] with a square planar 14-electron metal centre {M′_s}. E_s and β parameters were also estimated for the auxilliary {ReL(dppe)₂}⁺ (L = CO or CNR) centres.