Synthesis and enantiomeric analysis of cyclotriphosphazene derivatives with one centre of chirality

A series of chiral cyclotriphosphazene compounds 2-9 in which the spiro 3-amino-1-propanoxy moiety provides the one centre of chirality have been synthesised and characterised by elemental analysis, MS, ¹H and ³¹P NMR spectroscopies. The enantiomers of newly synthesised compounds have been analysed by the changes in the ³¹P NMR spectra on addition of a Chiral Solvating Agent (CSA), (S)-(+)-2,2,2-trifluoro-1-(9′-anthryl)ethanol. HPLC methods have been developed for the enantiomeric separations of chiral cyclotriphosphazenes containing one centre of chirality. It is found that chiral HPLC gave a good resolution of enantiomers of the racemic compounds 2-9 with resolution factors between 2.49 and 7.50 making them good candidates for enantiomeric separations and determination of absolute configuration.     

Sapogenins of Yucca glauca seed pods

From the fruiting pods of Yucca glauca Nutt. devoid of seeds the following sapogenins have been isolated and identified: neo-tigogenin, hecogenin, gitogenin, manogenin, Δ⁹-manogenin and sarsasapogenin. A small amount of a material believed to be Δ²-desoxysarsasapogenin was also isolated and is apparently an artifact arising from the hydrolysis and extraction procedure. Manogenin and Δ⁹-manogenin have not been previously detected in Y. glauca, and Δ⁹-manogenin has not been reported in any Yucca species.     

Molecular characterization of a human cation‐Cl− cotransporter (SLC12A8A,CCC9A) that promotes polyamine and amino acid transport

Cation‐Cl^? cotransporters (CCCs) belong to a large family of proteins that includes 9 isoforms, two of which have still not been ascribed a transport function (CCC8 and CCC9) while the others are all known to promote Cl^?‐coupled Na⁺ and/or K⁺ movement at the cell surface. The CCCs are also included in a larger family termed amino acid‐polyamine‐organocation carriers (APCs). In contrast to the CCCs, however, polyamine (PA) transporters have thus far been isolated from unicellular species exclusively and do not all belong to the APC family. In this work, we have found that a splice variant of CCC9 (CCC9a) promotes PA‐amino acid transport at the surface of HEK‐293 cells. We have also found that the influx of PAs in CCC9a‐expressing cells is inhibited by pentamidine as well as furosemide, and that it increases further in the presence of specific amino acids but not of Na⁺, K⁺, or Cl^?. Hence, a group of substrates that are directly transported by CCC9 and the molecular identity of a PA transport system in animal cells may have been uncovered for the first time. These findings are of special interest given that intracellular PAs play a key role in cell proliferation. J. Cell. Physiol. 220: 680–689, 2009. © 2009 Wiley‐Liss, Inc.     

<Emphasis Type="Italic">In vitro</Emphasis> Detection of Enhancing Antibody by the Macrophage Migration Test

THE immunological basis of enhanced allograft survival in inbred strains of mice is well established: humoral antibody, by some mechanism which is not yet clear, interferes with the cell-mediated host response to alloantigens¹. Various in vitro correlates of transplantation immunity in the mouse have been developed^2–9 and we have adapted one, the technique of macrophage migration from capillary tubes⁹, as an in vitro method to detect enhancing antibody.     

The effect of gramicidin on ATP synthesis in pea chloroplasts: two modes of phosphorylation

The effect of gramicidin on the phosphorylation rate, electron flow and light-induced H²⁺ uptake (ΔH⁺) in chloroplasts with methyl viologen (MV) or phenazine methosulfate (PMS) added has been studied. In the presence of MV low concentrations of gramicidin (~10⁻⁹ M) were shown to inhibit the phosphorylation rate up to 80–90% of the initial value, without changing either the electron transport rate or ΔH⁺ value. Two components of phosphorylation have been identified in the presence of PMS - the first is DCMU-sensitive, which was suppressed by low gramicidin concentrations (< 10⁻⁹ M) and the second - DCMU-insensitive component, which was suppressed by high gramicidin concentrations (~10⁻⁷ M) exclusively, the high gramicidin concentration inducing the ΔH⁺ value fo fall.     

Some characteristics of new tissue-binding proteins for metabolites of vitamin D other than 1,25-dihydroxyvitamin D

Protein(s) have been found in a wide range of tissues which have a high affinity for 25-hydroxycholecalciferol. Of the tissues examined only erythrocytes do not have this protein. The properties of the protein have been examined and it has been found that the association constants range from 2 · 10⁹ to 5 · 10⁹ M⁻¹ and the sedimentation constants between 5.0 and 6.0 S. It was not possible to distinguish the proteins from the different tissues by their S values, mobility on gel electrophoresis or behaviour on ion-exchange chromatography. These techniques were all used, however, to show that the tissue 25-hydroxycholecalciferol binding protein is distinct from the main plasma binding protein for this steroid and from the intestinal 1,25-dihydroxycholecalciferol-binding protein. A protein has been found in the plasma of rachitic animals but not of normals, which is apparently indistinguishable from this new tissue 25-hydroxycholecalciferol-binding protein. The steroid specificity of this new binding protein has been shown to be dependent upon a C-25 hydroxyl group, and an intact conjugated double bond system. Possible functions for this protein have been briefly discussed.     

Pyrrolizidine alkaloids from Senecio cacaliaster

Four pyrrolizidine alkaloids have been isolated from Senecio cacaliaster and their structures analysed by spectroscopic methods (IR, mass, ¹H, ¹³CNMR). One of them is new and the name sencalenine (3) is proposed. Alkaloids O⁷-senecioylretronecine (1) and 7-senecioyl-9-sarracinylretronecine (2) have recently been identified elsewhere. The fourth is bulgarsenine (4) which was isolated from a Senecio species before.     

Activity of substance P analogs substituted at the Gly9 and Gln6 positions

We have prepared peptide derivatives of Substance P in which Gly⁹ or Gln⁶ in the C-terminal part of the molecule have been substituted and we have examined the activity of these peptides using the guinea pig ileum bioassay. Gly⁹ can be substituted without a significant effect on the activity by Ala or Sar but not with DAla or βAla. Derivatives of the C-terminal pentapeptide were prepared and were almost as potent as the undecapeptide Substance P. The results presented are of particular relevance for the future design of radioactive receptor ligands of high affinity, of Substance P antagonists and of affinity labels.     

A fluorescence study of Tn10-encoded Tet repressor

Steady-state fluorescence quenching and time-resolved measurements have been performed to resolve the fluorescence contributions of the two tryptophan residues, W43 and W75, in the subunit of the homodimer of the Tet repressor fromEscherichia coli. The W43 residue is localized within the helix-turn-helix structural domain, which is responsible for sequence-specific binding of the Tet repressor to thetet operator. The W75 residue is in the protein matrix near the tetracycline-binding site. The assignment of the two residues has been confirmed by use of single-tryptophan mutants carrying either W43 or W75. The FQRS (fluorescence-quenching-resolved-spectra) method has been used to decompose the total emission spectrum of the wild-type protein into spectral components. The resolved spectra have maxima of fluorescence at 349 and 324 nm for the W43 and W75 residues, respectively. The maxima of the resolved spectra are in excellent agreement with those found using single-tryptophan-containing mutants. The fluorescence decay properties of the wild type as well as of both mutants of Tet repressor have been characterized by carrying out a multitemperature study. The decays of the wild-type Tet repressor and W43-containing mutant can be described as being of double-exponential type. The W75 mutant decay can be described by a Gaussian continuous distribution centered at 5.0 nsec with a bandwidth equal to 1.34 nsec. The quenching experiments have shown the presence of two classes of W43 emission. One of the components, exposed to solvent, has a maximum of fluorescence emission at 355 nm, with the second one at about 334 nm. The red-emitting component can be characterized by bimolecular-quenching rate constant,k _q equal to 2.6×10⁹, 2.8×10⁹, and 2.0×10⁹ M^?1 sec^?1 for acrylamide, iodide, and succinimide, respectively. The bluer component is unquenchable by any of the quenchers used. The W75 residue of the Tet repressor has quenching rate constant equal to 0.85×10⁹ and 0.28 × 10⁹ M^?1 sec^?1 for acrylamide and succinimide, respectively. These values indicate that the W75 is not deeply buried within the protein matrix. Our results indicate that the Tet repressor can exist in its ground state in two distinct conformational states which differ in the microenvironment of the W43 residue.     

Nature of the Spermagglutinin in the Normal Serum of Rabbits

THE agglutination of homologous spermatozoa^1,2 by heat-inactivated rabbit serum (56° C for 30 min) seems to be an immunological reaction. Although complement fixing activity of normal rabbit serum by rabbit spermatozoa was reported^3,4, antibody-like activity was not proved. Edwards³ obtained neither immobilization of spermatozoa nor a positive reaction with passive cutaneous anaphylaxis and precipitin tests. Immunofluorescence⁶ and mixed conglutination⁷ gave similar results for spermagglutinin activity in normal rabbit serum. Germinal cells of guinea-pig testis have been lysed by the animal's own serum⁸, but contrary findings have been reported^9–11 in which reaction between serum and homologous spermatozoa by immunofluorescence was negative. I have now investigated whether the spermagglutinin activity of normal rabbit serum is associated with antibody protein.     

Initiation of Cellulose Biosynthesis

IN spite of much investigation the problem of the molecular mechanism of cellulose synthesis remains unsolved¹. Hexose phosphates², sugar nucleotides^3–6 and a glycolipid^7–9 have been suggested as the precursor of cellulose. Implicit in all these investigations is the supposition that a single substrate suffices for the synthesis. We describe here some preliminary observations which seem to throw new light on the possible mechanism.     

Synthesis of thiazol-2-imines from the reduction of single enantiomer 2-imino-thiazolidin-4-ones followed by a spontaneous water elimination

Chiral hemiaminals ( 5-8RR and 5-8SS ) have been synthesized from the corresponding 2-iminothiazolidine-4-ones ( 1-4RR and 1-4SS ) by LiAlH₄ reductions stereoselectively and were then converted to single enantiomer thiazol-2-imines ( 9-12RR and 9-12SS ) by a water elimination reaction. The kinetics of the dehydration reactions which occurred spontaneously both in the solid state and in the solution have been followed by time dependent ¹H nuclear magnetic resonance spectroscopy. The corresponding first order rate constants and free energies of activation values for the conversions have been reported.     

Possible Cytoplasmic Precursor of Haemoglobin Messenger RNA

THE “rapidly labelled” RNA of immature erythroblasts includes a rather homogeneous high specific activity RNA in the 9S region of linear sucrose gradients, but when the same RNA is assayed for ability to stimulate protein synthesis in a cell-free system, the peak of activity is found just trailing the 18S ribosomal RNA^1,2. Evidence has been assembled to support the contention that the 9S species of RNA is the haemoglobin messenger RNA³. While investigating the 9S RNA of chicken erythroblasts, we have found conditions in which a well defined rapidly labelled RNA peak could be observed in the 9S and/or the 17S region of the gradient. The concentration of pulse labelled RNA in the 17S region has been reported in diverse systems⁴ and may be a general phenomenon. It is particularly striking in the erythroblast system in which background ribosomal RNA synthesis is at a minimum.     

The physiologic disposition of marihuana in man

Marihuana and hashish are the most widely used illicit drugs. They are derived from the hemp plant, $\text{Cannabis sativa}$. Among the diverse chemicals in the plant, more than 20 so-called cannabinoids have been isolated and their chemical structures elucidated (1). In 1965, Mechoulam and coworkers (2,3) isolated △⁹- tetrahydrocannabinol (△⁹-THC) from cannabis extract and demonstrated that it was responsible for the psychopharmacologic effects of cannabis in animals. Later, Isbell (4) and Hollister $\text{et al.}$ (5) confirmed these findings in man. This paper reviews the physiologic disposition of △⁹-THC in man; the disposition of △⁹-THC in animals has been reviewed elsewhere (6, 7, 8).     

Independence of Protein Synthesis and Drug Uptake in Nerve Cell Bodies and Glial Cells isolated by a New Technique

A severe handicap in any study of the cellular biochemistry of the brain is the unavailability of sufficient amounts of pure neurones and glial cells. Many efforts to separate one from the other have been made¹, but only four basic types of isolation procedures have been described for routine use: (1) brain is treated with a mixture of acetone-glycerol and water before mincing and centrifugation^3–5; (2) brain mince is sieved and subjected to gradient centrifugation¹,^6–8—this separates neuronal cell bodies from neuropil⁶ or from intact but rather contaminated glial cells⁸; (3) whole brain is disrupted in a tissue press and zonal centrifugation is used to separate cellular and subcellular components as they leave the zonal centrifuge rotor⁹; and (4) chopped brain is incubated in oxygen at 37° C in the presence of 1 % (w/v) trypsin¹⁰, a treatment which, in our estimation, severely prejudices this technique's general usefulness, because it precludes meaningful comparative investigations of enzymes and non-enzymatic structural and/or soluble cell proteins. Since investigations of the latter type are the aim in our laboratory, we needed a technique in which brain is not subjected to deleterious treatments such as immersion in acetone-glycerol-water mixtures^3–5 or trypsinization¹⁰ and in which no special instrumentation⁹, tissue presses⁹ or homogenizers⁸ are required. We now report a technique which successfully accomplishes this objective, for it makes possible the isolation of highly purified neuronal perikarya and of intact glial cells from a few grams of brain, in yields which permit the subsequent subcellular fractionation of the isolated cells. We also report two applications of the new technique which have enabled us to determine the in vivo time course of protein synthesis in and the uptake of the radioactive convulsant agent methionine sulphoximine^11,12 by, the neurones and the glial cells of the immature rat brain cortex.     

Curtius Rearrangement Using Ultrasonication: Isolation of Isocyanates of Fmoc-Amino Acids and Their Utility for the Synthesis of Dipeptidyl Ureas

An efficient conversion of N^α-[(9–fluorenylmethyl)oxy]carbonyl (Fmoc) amino acid azides to the corresponding isocyanates using ultrasound is described. The Curtius rearrangement was accomplished using acid azides in toluene solution as well as solid powder at room temperature. All isocyanates synthesized have been obtained as crystalline solids and were characterized. Coupling of isocyanates with amino acid methyl ester hydrochloride salts in presence of N-methylmorpholine (NMM) resulted in Fmoc-protected dipeptidyl urea esters, which have been well characterized by ¹H NMR, ¹³C NMR and mass spectrometry.     

Synthetic,chemical spectroscopic (infrared and electronic) and magnetic studies on dizirconiumenneaisopropoxide complexes of iron(II)

Bimetallic alkoxide derivatives of iron(II) with zirconium of the types [Fe{Zr₂(OPrⁱ)₉}₂], [ClFe- {Zr₂(OPrⁱ)₉}], [(RO)Fe{Zr₂(OPrⁱ)₉}], and [(acac)- Fe{Zr₂(OPrⁱ)₉}] have been synthesized and characterized by elemental analyses, infrared and electronic spectral as well as magnetic susceptibility studies.     

Digenic Inheritance in Cystinuria Mouse Model

Cystinuria is an aminoaciduria caused by mutations in the genes that encode the two subunits of the amino acid transport system b^0,+, responsible for the renal reabsorption of cystine and dibasic amino acids. The clinical symptoms of cystinuria relate to nephrolithiasis, due to the precipitation of cystine in urine. Mutations in SLC3A1, which codes for the heavy subunit rBAT, cause cystinuria type A, whereas mutations in SLC7A9, which encodes the light subunit b^0,+AT, cause cystinuria type B. By crossing Slc3a1 ^-/- with Slc7a9 ^-/- mice we generated a type AB cystinuria mouse model to test digenic inheritance of cystinuria. The 9 genotypes obtained have been analyzed at early (2- and 5-months) and late stage (8-months) of the disease. Monitoring the lithiasic phenotype by X-ray, urine amino acid content analysis and protein expression studies have shown that double heterozygous mice (Slc7a9 ^+/- Slc3a1 ^+/-) present lower expression of system b^0,+ and higher hyperexcretion of cystine than single heterozygotes (Slc7a9 ^+/- Slc3a1 ^+/+ and Slc7a9 ^+/+ Slc3a1 ^+/-) and give rise to lithiasis in 4% of the mice, demonstrating that cystinuria has a digenic inheritance in this mouse model. Moreover in this study it has been demonstrated a genotype/phenotype correlation in type AB cystinuria mouse model providing new insights for further molecular and genetic studies of cystinuria patients.     

6-Oxodendrolasin, 6-hydroxydendrolasin, 9-oxofarnesol and 9-hydroxyfarnesol,stress metabolites of the sweet potato

Four sesquiterpene stress metabolites, 6-oxodendrolasin, 6-hydroxydendrolasin, 9-oxofarnesol, and 9-hydroxyfarnesol have been isolated from mercuric chloride-treated sweet potatoes. The metabolites have been synthesized and feeding studies have been carried out to determine the extent of incorporation of ¹⁴C-labelled 6-oxodendrolasin and 9-hydroxyfarnesol into ipomeamarone.