Inhibition of C-terminal O-methyltransferase by a rat liver cytosolic peptide

The activity of S-famesylcysteine O-methyltransferase was assayed by incubating the enzyme with a syntheticin vitro substrate, [N-acetyl-S-trans, trans-famesyl-L-cysteine (AFC)], together with S-adenosyl-L-[methyl-¹⁴C]methionine. The resulting methylesterification product, [N-acetyl-S-trans, trans-famesyl-L-cysteine (methyl-¹⁴C) ester (AFCME)], was then analyzed either directly on HPLC or by converting the AFC[methyl-¹⁴C]ME to [methyl-¹⁴C] alcohol by basehydrolysis. Employing these two analytical methods, it was established that a peptide purified from rat liver cytosol fraction [Int. J. Biochem., 25, 1157 (1993)] strongly inhibited the above enzyme activity with IC₅₀ of 7.1X10^?8 M. Also, the S-famesylcysteine O-methyltransferase from several human colon cancer cells was also equally inhibited by the peptide.     

Hydrolysis kinetics of metampicillin by high performance liquid chromatography

The hydrolysis of metampicillin to ampicillin was investigated using high performance liquid chromatography. We developed the simultaneous determination of metampicillin and ampicillin using a Zorbax CN column and 5% acetonitrile and 8% methanol in 0.02 M phosphate buffer (pH 7.0) as mobile phase. Metampicillin was hydrolyzed to ampicillin with half life of 41.5 min at physiological pH and temperature. In acidic pH, metampicillin was rapidly hydrolyzed to ampicillin within a chromatographic separation.     

Epimerization and Hydrolysis of Dalvastatin,a New Hydroxymethylglutaryl Coenzyme A (HMG-CoA) Reductase Inhibitor

In aqueous solutions, dalvastatin (1) undergoes epimerization as well as hydrolysis. The transformation of the drug was studied as a function of pH at 25°C in aqueous solutions containing 20% acetonitrile. At all pH values, first-order plots for the conversion are biphasic, indicating rapid equilibration of 1 with its epimer (2) and slower hydrolysis of 1 to the corresponding -hydroxy acid (3). Apparent first-order rate constants for the biexponential equation are given as a function of pH. The alkyl–oxygen cleavage of the lactone ring results in the epimerization of 1 to 2, whereas the acyl–oxygen cleavage results in the hydrolysis of 1 to 3. The epimerization is an S_N1 reaction reaching an equilibrium of [l] _eq/[2] _eq = 1.27. The epimerization rate is increased with an increase in the water content of the solvent. The hydrolysis of 1 to 3 is acid and base catalyzed. The hydrolysis is reversible in acidic media and irreversible in neutral and basic media. At pH values greater than 9, the hydrolysis reaction proceeds more rapidly than the epimerization.     

Studies of effects of anticancer agents in combination with/without hyperthermia on metastasized human bladder cancer cells in chick embryos using the polymerase chain reaction technique

Cultivated T24 cells derived from a human bladder cancer were inoculated into the chorioallantoic membrane vein of chick embryos. Hyperthermic treatment was performed following injection of anticancer agents 3 days after the inoculation of the T24 cells. DNA samples were obtained from the livers of the chick embryos, and the polymerase chain reaction technique was used to amplify a DNA fragment specific to the human -globin gene. The Southern hybridization method was used to evaluate the inhibitory effects of anticancer agents in combination with/without hyperthermia on T24 cells metastasized to the liver. The hyperthermia exerted an inhibitory effect on the growth of the T24 cells in the livers of the chick embryos, and this was dependent on the thermal dose. The antitumor effects of hyperthermia performed at 42.5° C for 20 min and at 43.0° C for 10 min were evidenced by 69.2% an 82.0% inhibition of the growth of the metastasized T24 cells, respectively, as compared with the growth of untreated T24 cell. Hyperthermia performed at 42.5° C for 10 min alone produced 26.7% tumor growth inhibition, and these conditions for hyperthermia were subsequently used as a criterion for evaluating the effects of its combination with various anticancer agents. Adriamycin (20 g/egg) alone, mitomycin C (10 g/egg) alone, carboplatin (10 g/egg) alone, and cisplatin (10 g/egg) alone produced 13.5%, 58.9%, 27.3%, and 29.1% tumor growth inhibition, respectively. Adriamycin and mitomycin C applied in combination with hyperthermia showed additive inhibitory effects on the growth of the metastasized T24 cells in this chick embryo model.     

Allelic loss on chromosome-13 can preceed histological-changes in head and neck-cancer

Seventy paired tumor and blood samples from patients with upper aerodigestive tract squamous cell carcinoma (UADT SCC) were tested for allelic loss on chromosome 13. Increased loss of heterozygosity (LOH) was observed at 11 of 13 different highly polymorphic microsatellite 'CA' dinucleotide repeat-containing loci. Increasing percent LOH correlated with lymph node metastasis (N Stage) (p=0.016). LOH was also detected in 10 of 16 (63%) informative samples of histologically normal mucosa adjacent to the tumors. These findings demonstrate that allelic loss on chromosome 13 is a frequent event in UADT SCC. Furthermore, these genetic alterations can be detected prior to histological changes in normal mucosa adjacent to these tumors.     

Characterization of adult human prostatic epithelial-cells immortalized by polybrene-induced DNA transfection with a plasmid containing an origin-defective sv40-genome

Normal adult human prostatic epithelial cells were infected with an adenovirus 12-SV40 virus or transfected by polybrene-induced gene transfer with a plasmid (pRSV-T) containing the SV40 early region genes or with a plasmid (pRNS-1) containing an origin-defective SV40 genome and a plasmid carrying the neomycin resistance gene. Colonies of morphologically altered cells were isolated, cultured in a serum-free medium and characterized. These cells had extended lifespan in culture compared to normal adult human prostatic epithelial cells. Both Ad12-SV40-infected and pRSV-T-transfected cultures eventually underwent senescence. pRNS-1-transfected cells (pRNS-1-1), however, have now been grown for more than 50 passages. These cells contain the SV40 genome, express SV40 T-antigen, and are not tumorigenic in nude mice. They express cytokeratins 5 and 8, like the parent cells, and are pseudodiploid. Analysis of growth regulatory processes revealed that the growth of pRNS-1-1 cells was stimulated similarly to that of normal prostatic epithelial cells by epidermal growth factor, insulin-like growth factor, and pituitary extract. The response of pRNS-1 cells to a growth-inhibitory factor, retinoic acid, was also similar to that of normal cells. However, pRNS-1-1 cells were less responsive than normal cells to growth inhibition by transforming growth factor-beta, and had lost altogether the ability of normal cells to be inhibited by tumor necrosis factor-alpha and 1,25 (OH)2 vitamin D3. Therefore transformation appeared to alter growth-inhibitory but not growth - stimulatory mechanisms. These cells should be useful in elucidating the multistep mechanism of carcinogenesis of the prostate.     

Growth suppression of human colorectal carcinoma in nude mice by monoclonal antibody C27-abrin A chain conjugate

PURPOSE: The aim of this study was to assess an immunotoxin, monoclonal antibody C27-abrin A chain conjugate (MAAC), that might be effective in the treatment of colorectal carcinoma. METHODS: The immunotoxin was prepared by a specific monoclonal antibody against carcinoembryonic antigen (CEA), monoclonal antibody C27, linked toN-succinimidyl-3-(2-pyridyldithio)propionate and then coupled covalently to the toxic abrin-A chain to synthesize MAAC. The therapeutic role of this immunotoxin in suppressing thein vitro andin vivo growth of CEA-secreting human colorectal cancer cells (LS174T) was assayed by methods of protein biosynthesis inhibition, cell colony proliferation, and treatment of tumor cells before and after inoculation in nude mice. RESULTS: We found that MAAC effectively suppressed the growth of LS174T in culture medium and completely eradicated cells in inoculated nude mice. In contrast, irrelevant immunotoxin antiferritin-abrin A chain conjugate and isotype-matched monoclonal immunoglobin (MOPC21IgG1)-abrin A chain conjugate did not cause such effects. Thein vitro toxicity was highly specific because the conjugate (MAAC) inhibitedde novo protein biosynthesis, impeded growth, and caused death of cells possessing surface CEA determinants. The 50 percent inhibition dose values of the conjugate for colonogenic survival and for protein biosynthesis in LS174T cells were 0.09 g/ml and 0.06 g/ml, respectively. Colony survival was inhibited 96.3 percent after prolonged MAAC treatment. MAAC showed selective cytotoxicity; the inhibitory effect of MAAC to the CEA-secreting LS174T cells over the CEA-nonsecreting human embryonic kidney cells was 16-fold. CONCLUSION: These results indicate that MAAC may be of benefit in therapy during or soon after resection of colorectal carcinoma or in patients who have micrometastasis.Supported by a grant from the National Science Council and the Veterans General Hospital-Taipei, Taipei, Taiwan.     

Defective RNA molecules associated with citrus tristeza virus

Preparations of single-stranded (ss) RNA extracted from particles of the Israeli VT strain of citrus tristeza virus (CTV-VT), and ss- and double-stranded (ds) RNA preparations extracted from infected Alemow (Citrus macrophylla) plants, contained a population of molecules with features that suggest that they are defective RNAs. The prototype of 2424 nt was cloned and sequenced and was found to be composed of two genomic regions corresponding to the 5' (1151 nt) and the 3' (1259 nt) termini of the genomic CTV-RNA, with two perfect direct repeats of eight nucleotides of unknown origin at the junction site. Northern hybridization analysis demonstrated that this 2.4-kb defective RNA is an abundant species among the other CTV-specific ss- and ds-RNAs in infected plants. The 2.4-kb RNA was found encapsidated by the CTV coat protein indicating that the CTV origin of assembly is located close to the 5' or 3' terminus. This is the first defective RNA to be reported for a member of the closterovirus group.     

Association of apolipoprotein ɛ4 allele and neuropathologic findings in patients with dementia

Apolipoprotein E (APOE) is a lipoprotein expressed in liver and brain as one of three isoforms (APOE 2, APOE 3 and APOE 4). Recent findings suggest that the presence of APOE 4 is associated with an increased risk for both familial Alzheimer's disease and late-onset Alzheimer's disease. We extended these observations by determining the frequency of APOE alleles in patients with pathologically confirmed Alzheimer's Disease (AD), Parkinson's disease (PD), diffuse Lewy Body disease (DLBD), AD with concomitant PD pathology, demented PD patients without or with concomitant AD pathology and in schizophrenics with a progressive dementia (SCHIZ+DEM). The APOE genotype was determined by restriction digestion of polymerase chain reaction-amplified DNA isolated from frozen brain samples. The frequency of the APOE 4 allele was highest among sporadic AD and DLBD patients (0.30 and 0.38, respectively) and lowest in the SCHIZ+DEM and non-demented PD patients (0.06 and 0.1, respectively). Thus, the APOE 4 allele is over-represented selectively in patients with dementias associated with plaques and tangles and/or cortical Lewy bodies, but not in demented schizophrenics or non-demented PD patients.