Kinetic study of azole-bridged dinuclear platinum(II) complexes reacting with a hairpin-stabilized double-stranded oligonucleotide

The cytotoxic dinuclear platinum(II) complexes [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-pz)](NO(3))(2) (pz=pyrazolate) (1) and [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-1,2,3-ta-N1,N2)](NO(3))(2) (1,2,3-ta=1,2,3-triazolate) (2), were allowed to react with the hairpin-stabilized double-stranded oligonucleotide d(TATGGCATT(4)ATGCCATA), to determine the amounts of intrastrand and interstrand DNA adducts. The reaction kinetics was investigated by reversed-phase HPLC, and the resulting products were analyzed using mass spectroscopy combined with enzymatic digestion, and Maxam-Gilbert sequencing. The reaction of 1 results in the formation of the 1,2-intrastrand d(GG) adduct as the major final product. The two most abundant products of 2 were identified as isomeric 1,2-intrastrand d(GG) adducts differing probably in platinum coordination to the triazole ring. No GG-interstrand crosslinks were detected with either compound. d(GGC)-d(GCC) sequences of DNA do thus not appear to represent significant targets for forming interstrand crosslinks with either 1 or 2.     

A sensitive method for 4-hydroxybutyric acid in urine using gas chromatography-mass spectrometry

4-Hydroxybutyric acid (4HB) was analyzed by gas chromatography-mass spectrometry. Under acidified conditions, 4HB is difficult to detect due to lactonization. Using a urine sample containing 0.01 mg creatinine, we performed trimethylsilyl derivatization without extraction, only adding dimethylsuccinic acid as an internal standard and 10 microl of 0.1 N NaOH methanol solution with adequate evaporation. Urine 4HB levels in a patient with 4-hydroxybutyric aciduria was determined to be 1258 mmol/mol Cr (control, 0.28-2.81 mmol/mol Cr) in this method. Direct derivatization of samples without extraction showed good reproducibility and linearity. Only a small sample of urine was required. Alkalinization by NaOH prevented not only lactonization of 4HB, but also loss of the compounds during evaporation.     

Evidence of isozymes for delta6 fatty acid desaturase in rat hepatocytes

The expression of delta6 fatty acid desaturase, previously identified, was suppressed almost completely by hyper expression of the corresponding antisense gene in a transformant of the rat hepatic cell line BRL-3A. Conversion rates of [1-14C] linoleic acid, alpha-linolenic acid, and tetracosapentaenoic acid into the respective delta6 fatty acids were equivalent to those in control cells. This finding suggested that all of these reactions were catalyzed by at least two delta6 desaturase isozymes in rat hepatocytes.     

Synthesis of chemotactic peptide analogs with a photoaffinity cross-linker as new probes for analysis of receptor-ligand interaction

Formylpeptide receptors are well-characterized receptors which participate in host defense responses of neutrophils. We designed and synthesized chemotactic peptide analog with p-benzoylphenylalanine (Bpa) and biotin to probe structural and mechanistic aspects of peptide-receptor interaction. These peptides possess biological activities which were dependent upon spacer residue length of and Bpa position. The covalent photoaffinity label was detected by Streptavidine-blot, which was inhibited by the parent peptide.     

Biological activity of human granulocyte colony stimulating factor with a modified C-terminus

Granulocyte colony-stimulating factor (G-CSF) undergoes receptor-mediated internalization into target cells which are normally restricted to neutrophilic granulocytes and their committed progenitor cells, suggesting that it may be applicable as a myeloid cell-targeting vehicle. To test this notion, we constructed a cDNA encoding a human G-CSF/murine stem cell factor (mSCF) chimeric molecule in a mammalian expression vector and transfected NIH3T3 cells with this plasmid. The resulting chimeric cytokine consisted of the entire G-CSF sequences fused to Lys148 of mSCF. It can be released from the surface membrane of NIH3T3 transformants through proteolytic cleavage at Ala164 of mSCF. The culture media conditioned by a number of stable transformants, which were confirmed by an enzyme-linked immunosorbent assay (ELISA) to secrete an hG-CSF derivative, were examined for their ability to stimulate CFU-G-derived colony formation as well as the proliferation of G-CSF-dependent NFS-60 cells. The results indicated that this C-terminus modified version of hG-CSF is as potent as recombinant hG-CSF in both assays.     

Sites of expression of mRNA for lysenin, a protein isolated from the coelomic fluid of the earthworm Eisenia foetida

Lysenin is a 33-kDa protein of 297 amino acids that was originally purified from the coelomic fluid of the earthworm Eisenia foetida. It binds specifically to sphingomyelin. In this study, we attempted to identify the site of synthesis of lysenin in the earthworm. We detected the expression of mRNA for lysenin and the presence of immunoreactive lysenin in the large coelomocytes and in the free large chloragocytes present in the lumen of the typhlosole, a depression in the dorsal wall of the intestine. These coelomocytes and chloragocytes seemed to be mature and separate from the chloragogen tissue that lined the typhlosole. The free large chloragocytes in the typhlosole contained numerous vacuoles. The nuclei were small and irregular in shape, and glycogen granules and mitochondria were occasionally found between vacuoles. The chloragocytes of the chloragogen tissue that surrounded the coelomic side of the intestine and the dorsal blood vessel did not react with the lysenin antiserum and no expression of lysenin mRNA was detected in these cells. Furthermore, no evidence of the protein or of the mRNA was found in the cells of the pharyngeal gland. Our findings suggest that lysenin is produced in the free large chloragocytes in the lumen of the typhlosole.     

Design and synthesis of new secretory phospholipase A2 inhibitor of a phospholipid analog

All stereoisomers of N-acyl-4,5-disubstituted oxazolidinone phospholipid analogs were synthesized by regio and stereoselective epoxide ring opening accompanied by introduction of an amino group. The (4R,5S)-derivative showed stronger inhibitory activity toward type II phospholipase A₂ than the 4-substituted oxazolidinone phospholipid analog previously reported.