Microbial asymmetric hydrolysis of N-acetyl-1-methyl-3-phenylpropylamine to optically active 1-methyl-3-phenylpropylamine

Summary The asymmetric hydrolysis of N-acetyl-1-methyl-3-phenypropylamine (MPAc) by microorganisms was investigated. Various bacteria belonging to the genera Bacillus, Agrobacterium, Corynebacterium, Arthrobacter, Brevibacterium, Cellulomonas, Acinetobacter, Nocardia and Rhodococcus showed this hydrolysing activity and yielded (S)-1-methyl-3-phenylpropylamine (MPPA). Using washed cells of N. erythropolis IAM 1440, 15.1 mg/ml of (S)-MPPA was formed, with a 38.8% conversion yield and high stereoselectivity (97.9% enantiomeric excess), in an organic solvent-water diphase system. The same (S)-amine and (S)-1-phenylethylamine were also produced in good yields from the valeryl and isovaleryl derivatives of MPPA, and N-acetyl-1-phenylethylamine, respectively.     

Isolation of a full-length cDNA encoding mouse aromatase P450

A full-length cDNA clone for aromatase P450 has been isolated from a pregnant mouse ovarian cDNA library. The insert of this clone (2394 bp) contains a 1509-bp open reading frame encoding 503 amino acid residues together with a 46-bp 5'-untranslated stretch and an 839-bp 3'-untranslated region to which a poly(A) tract is attached. Northern blot analysis of ovarian RNA from pregnant mice reveals a major mRNA band of 2.5 kb with a minor band of 2.1 kb. Comparison of mouse aromatase P450 with that of rat, human, and chicken shows 91, 81, and 69% identity in the nucleotide sequence and 92, 79, and 69% identity in the deduced amino acid sequence, respectively. The membrane-spanning domain of mouse aromatase P450 is estimated to be an extremely hydrophobic segment located within the N-terminal region of the molecule. Furthermore, a highly conserved heme-binding domain is noticed.     

Memory impairment and morphological changes in rats induced by active fragment of anti-nerve growth factor-antibody

Treatment of rats with a specific Fab' fragment of anti-nerve growth factor (NGF)-antibody (anti-NGF, 12, 120 and 400 micrograms/4 weeks, i.c.v.) impaired their learning ability. The distance of swimming of anti-NGF-treated rats in a water maze was shortened more slowly by training than that of control rats. Anti-NGF treatment altered the staining of nuclei of cells in the hippocampus, parietal cortex and dentate gyrus with hematoxylin. It is suggested that the anti-NGF-induced amnesia could be due to change in nuclear morphology.     

Synthetic sialyl glycolipids (sialo-cholesterol and sialo-diglyceride) induce granulocytic differentiation of human myelogenous leukemia cell line HL-60

When HL-60 cells were cultivated with synthetic sialyl glycolipids, sialo-cholesterol and sialo-diglyceride, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was accompanied by inhibition of cell proliferation. The differentiation-inducing activity of sialo-cholesterol was greater than that of sialo-diglyceride on a molar basis, and the alpha-anomer of each compound was more potent than the beta-anomer, suggesting that the stereospecific structure of the compounds is important for the differentiation-inducing activity.     

A novel TIMP-insensitive type IV collagen-degrading metalloproteinase from murine metastatic sarcoma cells

A novel type IV collagen-degrading metalloproteinase was purified from the conditioned media of a murine metastatic sarcoma cell line. The molecular weight of the purified enzyme was determined to be 100 kDa by SDS-PAGE, while 700 kDa by gel filtration suggesting that the enzyme has a multimer structure. This enzyme degrades type IV collagen, but neither type I collagen nor casein. The failure of trypsin treatment to enhance the enzyme activity suggested that the purified enzyme did not require activation. Although the enzyme seems to be classified as a matrix metalloproteinase, it was inhibited by neither tissue inhibitor of metalloproteinases (TIMP) nor TIMP-2 and thus represents a novel type IV collagen-degrading metalloproteinase.     

Characterization of ganglioside GM4 lactones isolated from the whale brain

Two novel ganglioside lactones were isolated from the brain of Bryde's whale (Balaenoptera edeni) and characterized. These gangliosides migrated above GM4 and were designated M4-1 and M4-2 in the order of location from the bottom of the TLC plate. These gangliosides were converted to GM4 by mild alkali treatment. Although M4-1 and M4-2 contained 1 mol each of N-acetylneuraminic acid and galactose, they behaved as neutral lipids on ion-exchange chromatography. Inner ester linkages were found in these gangliosides by secondary ion mass spectrometry and infrared spectroscopy. Two-dimensional J-correlated proton NMR spectra revealed that an inner ester linkage was formed in M4-1 between the sialic acid carboxyl group and the C-4 hydroxyl group of the galactosyl residue and another inner ester linkage was formed in M4-2 between the sialic acid carboxyl group and the C-2 hydroxyl group of galactose.     

Total synthesis of the mollu-series glycosyl ceramides alpha-D-Manp-(1----3)-beta-D-Manp-(1----4)-beta-D-Glcp-(1----1)-Cer and alpha-D-Manp-(1----3)-[beta-D-Xylp-(1----2)]-beta-D-Manp-(1----4)-beta- D-Glcp-(1----1)-Cer

The mollu-series glycosphingolipids, O-alpha-D-mannopyranosyl-(1----3)-O-beta-D-mannopyranosyl-(1----4)-O-bet a-D-glucopyranosyl-(1----1)-2-N-tetracosanoyl-(4E)-sphingeni ne and O-alpha-D-mannopyranosyl-(1----3)-O-[beta-D-xylopyranosyl-(1----2])-O- beta-D-mannopyranosyl-(1----4)-O-beta-D-glucopyranosyl-(1----1)-2-N- tetracosanoyl-(4E)-sphingenine, were synthesized for the first time by using 2,3,4-tri-O-acetyl-D-xylopyranosyl trichloroacetimidate, methyl 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-mannopyranoside, benzyl O-(4,6-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-2,3,6-tri-O-benzyl-be ta-D- glucopyranoside 9, and (2S,3R,4E)-2-azido-3-O-(tert-butyldiphenylsilyl)-4-octade cene-1,3-diol 6 as the key intermediates. The hexa-O-benzyl disaccharide 9 was prepared by coupling two monosaccharide synthons, namely, 2,3-di-O-allyl-4,6-di-O-benzyl-alpha-D-mannopyranosyl bromide and benzyl 2,3,6-tri-O-benzyl-beta-D-glucopyranoside. It was demonstrated that azide 6 was highly efficient as a synthon for the ceramide part in the coupling with both glycotriaosyl and glycotetraosyl donors, particularly in the presence of trimethylsilyl triflate.     

Super-high-affinity binding site for [3H]diazepam in the presence of Co2+, Ni2+, Cu2+, OR Zn2+

Chloride salts of Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Cr³⁺, Mn²⁺, Fe²⁺, and Fe³⁺ had no effect on [³H]diazepam binding. Chloride salts of Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ increased [³H]diazepam binding by 34 to 68% in a concentration-dependent fashion. Since these divalent cations potentiated the GABA-enhanced [³H]diazepam binding and the effect of each divalent cation was nearly additive with GABA, these cations probably act at a site different from the GABA recognition site in the benzodiazepine-receptor complex. Scatchard plots of [³H]diazepam binding without an effective divalent cation showed a single class of binding, with a Kd value of 5.3 mM. In the presence of 1 mM Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺, two distinct binding sites were evident with apparent Kd values of 1.0 nM and 5.7 nM. The higher-affinity binding was not detected in the absence of an effective divalent cation and is probably a novel, super-high-affinity binding site.     

Purification and properties of glycine N-methyltransferase from rat liver

Glycine N-methyltransferase (EC 2.1.1.20) has been purified to homogeneity from rat liver. The enzyme has a molecular weight of 132,000 by sedimentation equilibrium method. This value is in good agreement with a value of 130,000 obtained by Sephadex G-150 chromatography. The molecular weight of the denatured enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 31,500. The numbers of peptides obtained by tryptic digestion and by cyanogen bromide cleavage are one-fourth of those expected from the contents of lysine plus arginine residues and methionine residues, respectively. By Edman degradation, phenylthiohydantoin-leucine is the only amino acid derivative released from the enzyme. Neither sugar nor phospholipid is detected in the purified preparation. These data indicate that the rat liver glycine N-methyltransferase is a simple protein consisting of 4 identical subunits. The enzyme has an isoelectric pH of 6.4, and is most active at pH 9.0. From the circular dichroism spectrum, an alpha helix content of about 11% is calculated. Whereas the initial velocity as a function of glycine concentration gives a Michaelis-Menten kinetics, the enzyme shows a positive cooperativity with respect to S-adenosylmethionine. The concentrations of glycine and S-adenosylmethionine which give a half-maximum velocity are 0.13 mM and 30 microM, respectively, at pH 7.4 and 25 degrees C.