A hollow-fibre column system to effectively prepare washed platelets.

BACKGROUND AND OBJECTIVES: We developed a hollow-fibre column system specifically adapted to prepare washed platelet concentrates (WPCs). This study was performed to evaluate the efficacy of the hollow-fibre column system for preparing WPCs. MATERIALS AND METHODS: First, the percentages of platelet (PLT) recovery and remaining plasma proteins were calculated by determining the PLT count, volume and plasma protein levels in both the prewash and postwash. Secondly, washed PLTs and unwashed control PLTs were stored for 5 days, and the changes during this 5-day storage of in vitro PLT characteristics were determined. RESULTS: The hollow-fibre column system effectively removed >98% of plasma in platelet concentrates (PCs), and the PLT recovery was 97% on an average. The CD62P-expression level on washed PLTs immediately after washing was approximately twofold higher than that on prewashed PLTs as well as on PLTs washed via manual methods or cell washing devices. Until day 5 during storage, PLT aggregability, hypotonic shock response and swirling scores of washed PLTs were not significantly different from those of the control PCs. CONCLUSION: Our novel hollow-fibre column system proved valuable in preparing washed PLTs with <2% of residual plasma proteins and high recovery of PLTs.

Primary biliary cirrhosis associated with idiopathic retroperitoneal fibrosis.

Idiopathic retroperitoneal fibrosis (IRF) and primary biliary cirrhosis (PBC) are distinct clinical disorders which rarely occur in the same patient. We report a 79-year-old man with the coexistence of both conditions. The patient had antibodies to both centromere and mitochondria, as indicated by indirect immunofluorescence. Diagnoses of IRF and PBC were confirmed histologically. Although the association between IRF and PBC is obscure, IRF may be involved in many autoimmune diseases associated with PBC.

Contribution of alpha 1-acid glycoprotein to species difference in lincosamides-plasma protein binding kinetics.

Protein binding kinetics of lincomycin (LM) and clindamycin (CM) were studied using plasma, albumin and alpha 1-acid glycoprotein (AGP) derived from humans, dogs, cattle and sheep. Based on Rosenthal plots of LM and CM, drug-binding property in plasma presented specific and non-specific binding, except for LM in cattle and sheep and for CM in sheep, where only non-specific binding was demonstrated. Dissociation constant (Kd) and binding capacity (Bmax) for specific binding and proportionality constant (PC) for non-specific binding were as follows: Kd = 3.14 mumol/L, Bmax = 15.28 mumol/L, PC = 0.19 for humans; Kd = 3.84 mumol/L, Bmax = 6.55 mumol/L, PC = 0.14 for dogs; PC = 0.12 for cattle; PC = 0.16 for sheep in LM and Kd = 0.94 mumol/L, Bmax = 12.24 mumol/L, PC = 4.98 for humans; Kd = 1.48 mumol/L, Bmax = 9.52 mumol/L, PC = 2.91 for dogs; Kd = 1.22 mumol/L, Bmax = 4.45 mumol/L, PC = 2.40 for cattle; PC = 1.48 for sheep in CM. The specific binding for each species was different, showing more difference in Bmax compared with Kd. The non-specific binding of LM was similar among species whereas that of CM was different, implying species difference. The drug-binding property of AGP for each species was all specific binding and the Kd was comparable to that obtained from plasma, indicating that AGP is a major specific binder in plasma. The lack of detection of specific binding for LM in cattle and sheep and for CM in sheep plasma could be attributable to a higher Kd and lower plasma AGP concentration compared with other species. The drug-binding property of albumin was characterized as all non-specific, without a great difference among species. Except for CM in sheep, the lower PC in albumin solution compared with that in plasma suggested the presence of another non-specific binder in plasma, i.e. lipoprotein. From the simulation of drug-binding percentage to AGP concentrations, AGP could be a major contributor to drug-plasma protein binding in pathological states. The degree of AGP-drug binding for each species could vary according to the degree of increase of AGP concentrations from a healthy to a pathological state, inducing a decrease in the unbound fraction (fp): 6.1 fold for dogs, 4.6 fold for humans, 1.8 fold for sheep and 1.4 fold for cattle in LM; 5.8 fold for dogs, 5.7 fold for cattle, 4.0 fold for humans and 1.5 fold for sheep in CM. Therefore, the disposition and efficacy of lincosamides affected by fp can be modified differently by the change of fp attributable to the alteration of plasma AGP concentration in each species.

[Successful eradication of H. pylori in an elderly patient with gastric cancer after effective UFT-E therapy].

The patient was a 83-year-old female with 2' T2-type gastric cancer associated with positive H. pylori in the lesser curvature of the stomach. The patient was treated with oral UFT-E alone in a daily dose of 400 mg. The tumor exhibited an O' IIa + IIc-like appearance 4 weeks after the start of administration and became scarred 8 weeks later, revealing marked tumor reduction in a short period of time. At 8 weeks, biopsy showed marked polymorphonuclear cells infiltration of gastric mucosa with no evidence of malignancy. In an attempt to eradicate H. pylori, 30 mg of lansoprazole, 400 mg of clarithromycin, and 2.0 g of ecabet-Na (3.0 g of Gastrom) were administered for 2 weeks. H. pylori was found to have been successfully eradicated, and the inflammatory lesions were no longer visible histologically. UFT-E was highly effective in this patient, and the eradication of H. pylori may contribute to the prevention of cancer recurrence.

Thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus: crystallization and site-directed mutagenesis.

The gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli. The synthase was purified to homogeneity and crystallized. The enzyme carried only two cysteine residues in contrast to its counterparts from other sources, which have four to six cysteine residues. Either or both of the cysteine residues can be replaced with serine without causing a loss of the catalytic activity. The conserved arginine residue that occupies the third position from the C-terminus was also replaced with valine without significant loss of activity, but the valine mutant showed a weakened affinity for isopentenyl diphosphate.

Crystallization and preliminary X-ray diffraction studies of Bacillus stearothermophilus farnesyl diphosphate synthase expressed in Escherichia coli.

Thermostable farnesyl diphosphate synthase (EC 2.5.1.10) from Bacillus stearothermophilus, which was overexpressed in Escherichia coli, has been crystallized by the vapor-diffusion procedure. Tetragonal crystals were obtained using ammonium sulfate as a precipitant. The crystals diffracted X-rays to about 3 A resolution. The diffraction pattern indicated that the space group is I4(1)22 with unit-cell dimensions of a = b = 114 A and c = 247 A. It is thought that the asymmetric unit comprises two or three molecules of farnesyl diphosphate synthase.

Thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus: molecular cloning, sequence determination, overproduction, and purification.

The structural gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli cells. A 1,260-nucleotide sequence of the cloned fragment was determined. This sequence specifies an open reading frame of 891 nucleotides for farnesyl diphosphate synthase. The deduced amino acid sequence shows a 42% similarity with that of E. coli FPP synthase [Fujisaki et al. (1990) J. Biochem. 108, 995-1000]. Comparison with prenyltransferases from a wide range of organisms, from bacteria to human, revealed the presence of seven highly conserved regions. In contrast to thermolabile prenyltransferases, which have four to six cysteine residues, the thermostable farnesyl diphosphate synthase carries only two cysteine residues. This enzyme is also unique in that some of the amino acids that are fully conserved in equivalents from other sources are replaced by functionally different amino acids. Construction of an overproducing strain provided a sufficient supply of this enzyme and it was purified to homogeneity. The purified recombinant enzyme is immunochemically identical with the native B. stearothermophilus enzyme, and it is not inactivated even after treatment at 65 degrees C for 70 min.

[Effect of tranilast (N-5) on cellular immunity--delayed type hypersensitivity].

Tranilast (N-5) as a mast-cell stabilizer is widely used in cases of bronchial asthma and nasal allergy. Some reports showed that N-5 is effective against keloids and ulcerative colitis. So, it is considered that N-5 inhibits cell-mediated immunity. N-5 inhibited BCG-CWS-stimulated lymphocyte proliferation which was proved to be mainly operated by OKT4 positive T cells. Production of the lymphokine from BCG-CWS-stimulated lymphocytes was suppressed by the addition of N-5. Thus, it is demonstrated that N-5 inhibits delayed type hypersensitivity in vitro.

Urinary metabolites of N-nitrosodibutylamine and N-nitrodibutylamine in the rat: identification of N-acetyl-S-alkyl-L-cysteines.

N-Acetyl-S-butyl-L-cysteine, N-acetyl-S-3-oxobutyl-L-cysteine and N-acetyl-S-3-hydroxybutyl-L-cysteine have been isolated and identified (as their methyl esters) from the urine of rats given N-nitrosodibutylamine (NDBA), N-nitrodibutylamine (NO2DBA) and their 1-acetoxy derivatives. Greater amounts of these N-acetyl-S-alkyl-L-cysteines were detected in the urine after administration of NDBA than of NO2DBA, and greater urinary excretion of the three N-acetyl-S-alkyl-L-cysteines was observed with 1-acetoxy NDBA than with 1-acetoxy NO2DBA. This suggests that the markedly different biological activities of NDBA and NO2DBA might be due, in part, to a difference in their alkylating abilities in vivo.

Urinary metabolites of N-nitrosodibutylamine in the rat: identification of N-acetyl-S-butyl-L-cysteine derivatives.

N-Acetyl-S-(butyl, 3-oxobutyl and 3-hydroxybutyl)-L-cysteines have been isolated and identified (as their methyl esters) from the urine of rats given N-nitrosodibutylamine (NDBA), N-nitrodibutylamine (NTDBA) and their corresponding alpha-acetoxy derivatives, N-nitroso-N-butyl(1-acetoxybutyl)amine and N-nitro-N-butyl(1-acetoxybutyl)amine, respectively. Greater amounts of these L-cysteine derivatives were detected in urine after administration of NDBA than of NTDBA. This suggests that the markedly different biological activities of NDBA and NTDBA might be due, in part, to a difference in their alkylating abilities in vivo.

Directly-acting mutagens formed from N-nitroso-N-(formylmethyl)alkylamines.

Directly-acting mutagens formed from N-nitroso-N-(formylmethyl)alkylamines (I) were isolated and identified as N-nitroso-N-alkyl-1-hydroxyimino-2-oxoethylamines (II). Their structures were elucidated on the basis of nuclear magnetic resonance spectra and confirmed by leading to their crystalline 2,4-dinitrophenylhydrazone. II (alkyl = ethyl and n-butyl) were strongly mutagenic to Salmonella typhimurium TA1535 and Escherichia coli WP2 hcr- without metabolic activation, while II with a tert-butyl group was not mutagenic. The formation of II from I is considered to proceed by the nitrosation of I, indicating a possible involvement of a formylmethyl metabolite in the carcinogenesis of nitrosamines with a 2-hydroxyethyl group.

In vitro metabolism of N-nitrodialkylamines.

The in vitro metabolism of N-nitramines was investigated in order to compare it with that of N-nitrosamines and to elucidate the mode of mutagenic action. N-Nitrodibutylamine (NO2DBA) and N-nitrodiethylamine (NO2DEA) were incubated with liver microsomes and hepatocytes prepared from rats treated with phenobarbital, and the products were analyzed by high-performance liquid chromatography and gas-liquid chromatography. The in vitro metabolic pattern of these nitramines was similar to that of the corresponding nitrosamines, except that N-nitro-N-alkylamines (produced via alpha-hydroxylation) were identified after incubation of the nitrodialkylamines. In the case of NO2DBA, besides N-nitro-N-butylamine, several nitramines produced by omega, omega-1, and omega-2 oxidations were identified as metabolites. NO2DBA and NO2DEA were mutagenic to Escherichia coli WP2 hcr- but not to Salmonella typhimurium TA1535. They were mutagenic only in the presence of hepatic microsomes, whereas their metabolites, N-nitro-N-butylamine and N-nitro-N-ethylamine, were direct mutagens. Thus, N-nitrodialkylamines are also metabolically activated to mutagens through alpha-hydroxylation.

Mutagenicity of alpha-hydroxy N-nitrosamines in V79 Chinese hamster cells.

N-Nitrosodialkylamines are activated metabolically by alpha-hydroxylation. Chemical properties and bacterial mutagenicity of alpha-hydroxy N-nitrosamines have been reported previously. This paper describes potent and direct mutagenicity of four N-nitroso-N-(hydroxymethyl)alkylamines in V79 Chinese hamster cells, using ouabain resistance as an indicator. The mutagenic potency depended on the alkyl group, decreasing in the following order: methyl greater than ethyl greater than propyl, butyl. A similar order was observed for cytotoxicity. Mutagenic and cytotoxic potencies of these alpha-hydroxy N-nitrosamines in V79 cells were well correlated not only with those of model compounds (alpha-acetoxy and alpha-hydroperoxy N-nitrosamines), but also with their alkylating ability, measured by alkylation of thiophenol. The mutagenic activity of the alpha-hydroxy N-nitrosamines in V79 cells was shown to be parallel to that in Salmonella typhimurium TA1535 and to that of N-nitrosodialkylamines in V79 cells, after metabolic activation by rat hepatocytes. The results obtained here further support the conclusion that the alpha-hydroxy N-nitrosamine is the active species in the metabolic activation of carcinogenic and mutagenic N-nitrosodialkylamines.

Mutagenicity of alpha-hydroxy N-nitrosamines in V79 Chinese hamster cells.

Carcinogenic and mutagenic N-nitrosodialkylamines are metabolically activated through alpha-hydroxylation. The synthesis, chemical properties, and microbial mutagenicity of alpha-hydroxy N-nitrosamines have been reported previously. Potent mutagenicity of four N-nitroso-N-(hydroxymethyl)-alkylamines (alkyl = methyl, ethyl, propyl, and butyl) was demonstrated in the present study in V79 Chinese hamster cells, ouabain resistance being used as an indicator. All the compounds were strong mutagens in the absence of metabolic activation systems. The mutagenic and cytotoxic potencies correlated well with each other, and depended on the alkyl group, decreasing in potency in the following order: methyl greater than ethyl greater than propyl = butyl. Their alkylating reactivity was measured by alkylation of thiophenol, and a good linear relationship was observed between the mutagenic and cytotoxic potencies and their alkylating reactivity. The mutagenic and cytotoxic potencies of the alpha-hydroxy N-nitrosamines in V79 cells were well correlated with those of alpha-acetoxy and alpha-hydroperoxy N-nitrosamines with respect to the effect of alkyl group. The results obtained here supported further that alpha-hydroxy N-nitrosamine is the active species in the metabolic activation of N-nitrosodialkylamine.