Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/10⁵ cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.     

A new immobilization technique of whole cells and enzymes with colloidal silica and alginate

A mixed gel composed of colloidal silica and alginate (As gel) was prepared for the immobilization of enzymes or microorganisms. The physical strength of AS gel increased with the amount of colloidal silica. The ethanol production rate of Saccharomyces cerevisiae (IFO 0224) immobilized in AS gel was higher than in alginate gel (Al gel) in the early phase of growth. At a concentration of glucose of more than 10%, the ethanol production of immobilized yeast in AS gel was higher than in Al gel. Any difference was not recognized in the diffusion coefficient of glucose between AS and Al gels. The AS gel had an ability to retain proteins such as bovine serum albumin and gamma-globulin. The alkaline protease and beta-galactosidase in AS gel continued their function for a long time, but those immobilized in Al gel did not. Immobilized beta-galactosidase in AS gel had a higher thermal stability than in Al gel or free enzymes.     

Pharmacokinetics of methimazole in normal subjects and hyperthyroid patients

Serum and urinary concentrations of methimazole (MMI) were measured by high-performance liquid chromatography (HPLC) with an electrochemical detector (ECD) in 10 normal subjects and 43 hyperthyroid patients after intravenous and oral administration of the drug. The pharmacokinetic parameters of MMI were estimated in 5 normal subjects and 15 hyperthyroid patients according to a two-compartment model after intravenous injection of a 10 mg dose. The mean half-life of the distribution phase (T1/2 alpha) was 2.7 +/- 1.0 h (mean +/- SD) and 3.1 +/- 1.4 h and that of the slower-phase (T1/2 beta) was 20.7 +/- 9.6 h and 18.5 +/- 12.9 h in normal subjects and hyperthyroid patients, respectively. There were no significant differences between pharmacokinetic parameters of normal subjects and those of hyperthyroid patients. No correlations between free T4 index (FT4I) and pharmacokinetic parameters were observed. Maximum serum MMI concentrations (Cmax) (213 +/- 84 and 299 +/- 92 ng/ml) were attained 1.8 +/- 1.4 h and 2.3 +/- 0.8 h after a single dose of 10 mg in 5 normal subjects and in 15 hyperthyroid patients, respectively. In hyperthyroid patients the time taken to reach the peak concentration (Tmax) after a single dose of 10 mg was similar to that after a single 15 mg and 30 mg dose. The pharmacokinetic parameters, except Cmax and the area under the curve (AUC), were not affected by the administered dose and those, except Cmax, were not affected by the thyroid function. All urine was collected at intervals of 3 h for the first 12 h and then at 24 h and 48 h after intravenous and oral administration of MMI. In all subjects, MMI rapidly appeared in the urine and the rate of excretion was highest in the first 3 h. The cumulative urinary excretion of MMI was 5.5-8.5% of administered doses in normal subjects and hyperthyroid patients. These findings in the present study are compatible with the assumption that the extent of absorption of MMI is high, if not complete, and hyperthyroidism does not affect the kinetics of MMI, and that interindividual variation is observed in the time taken to reach the peak concentration after oral administration.     

Hydroxyl-radical production and ethanol oxidation by liver microsomes isolated from ethanol-treated rats.

In order to distinguish between the mechanism of microsomal ethanol oxidation and hydroxyl-radical formation, the rate of cytochrome P-450 (P-450)-dependent oxidation of dimethyl sulphoxide (Me2SO) was determined in the presence and in the absence of iron-chelating compounds, in liver microsomes from control, ethanol- and phenobarbital-treated rats. Ethanol treatment resulted in a specific increase (3-fold) of the microsomal ethanol oxidation and NADPH consumption per nmol of P-450. A form of P-450 was purified to apparent homogeneity from the ethanol-treated rats and characterized with respect of amino acid composition and N-terminal amino acid sequence. Specific ethanol induction of a cytochrome P-450 species having a catalytic-centre activity of 20/min for ethanol and consuming 30 nmol of NADPH/min could account for the results observed with microsomes. Phenobarbital treatment caused 50% decrease in the rate of ethanol oxidation and NADPH oxidation per nmol of P-450. The rate of oxidation of the hydroxyl-radical scavenger Me2SO was increased 3-fold by ethanol or phenobarbital treatment when expressed on a per-mg-of-microsomal-protein basis, but the rate of Me2SO oxidation expressed on a per-nmol-of-P-450 basis was unchanged. Addition of iron-chelating agents to the three different types of microsomal preparations caused an 'uncoupling' of the electron-transport chain accompanied by a 4-fold increase of the rate of Me2SO oxidation. It is concluded that ethanol treatment results in the induction of P-450 forms specifically effective in ethanol oxidation and NADPH oxidation, but not in hydroxyl-radical production, as detected by the oxidation of Me2SO.     

Measurement of receptor-independent metabolism of low-density lipoprotein. An application of glycosylated low-density lipoprotein

Incubation of human low-density lipoprotein (LDL) with glucose results in a nonenzymatic formation of a Schiff base between the monosaccharide and lysyl residues of apolipoprotein B. Increasing the percentage of lysyl residues of apolipoprotein B modified by glycosylation decreases the fractional catabolic rate of the glycosylated LDL, and decreases the metabolism of the glycosylated LDL by human skin fibroblasts. The glycosylated LDL, containing 20-40% of total lysyl residues of apoprotein B modified, was metabolized at a slow rate by both human skin fibroblasts and mouse peritoneal macrophages. These results led to the suggestion that glycosylated LDL is primarily catabolized via a receptor-independent process. Assuming LDL catabolism occurs via receptor-dependent and receptor-independent processes, the ratio of (fractional catabolic rate of glycosylated LDL)/(fractional catabolic rate of native LDL) should be an estimate of the percentage of LDL catabolism via the receptor-independent process. From the fractional catabolic rates of glucose-LDL (20-40% of lysyl residues modified) and galactose-LDL (30-60% of lysyl residues modified) 41% and 30% respectively, of LDL catabolism occurred by a receptor-independent process.     

Histochemical demonstration of O-glycosidically linked,type 3 based ABH antigens in human pancreas using lectin staining and glycosidase digestion procedures

Summary Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo-and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with -N-acetylgalactosaminidase and -L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with -galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B₄ reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo--N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from nonsecretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(1-3)-N-acetyl-D-galactosamine1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.     

Comparison of enzyme phenotypes in human bladder tumours and experimentally induced hyperplastic and neoplastic lesions of the rat urinary bladder. A combined histochemical and immunohistochemical approach

The expression of a number of enzymes involved in drug metabolism, membrane function etc. was compared in hyperplastic and neoplastic lesions of the rat bladder and in human bladder tumours. Transitional cell carcinomas (TCC) in both rat and Man were characterized by decreased alkaline phosphatase (ALP) and increased gamma-glutamyl transpeptidase (GGT), beta-glucuronidase (beta-G1), succinate dehydrogenase (SD) and glucose-6-phosphate dehydrogenase (G6PD) activities. In addition, binding for antibodies specific for different cytochrome P-450 species (UT50, PB3a, MC1, MC2) and microsomal epoxide hydrolase (mEHb) was elevated in both murine and human tumours. Comparison of the enzyme phenotype in hyperplastic lesions induced by freeze ulceration or uracil administration with that in preneoplastic papillary or nodular hyperplasia (PNH) and TCC suggested, however, that most of the alteration in enzyme content or activity was non-specific and related to requirements for epithelial cell proliferation. On the other hand, the decreased ALP, and increased GGT and beta-G1 activity appeared more directly related to neoplastic transformation. The results suggested that qualitative differences exist between reactive hyperplasia and preneoplastic or neoplastic lesions in the urinary bladder. The finding of increased cytochrome P-450, in clear contrast to the reduction characteristic of preneoplastic hepatic lesions, may be important with regard to the observed difference in neoplastic transformation between the bladder and liver in response to drug metabolising enzyme inducers.     

Molecular cloning and nucleotide sequence of the gramicidin S synthetase 1 gene

The entire gene for gramicidin S synthetase 1 (GS 1) was cloned into the plasmid vector pUC18, and the nucleotide sequences of the GS 1 gene and its flanking region were determined. The full-length clone was 4,539 base pairs long and had an open reading frame of 3,294 nucleotides coding for 1,098 amino acids. The calculated molecular weight of 123,474 agreed with the apparent molecular weight of 120,000 found in SDS-PAGE of GS 1 from B. brevis. The nucleotide sequence of GS 1 gene was highly homologous to that of tyrocidine synthetase 1. The overall similarity between the deduced amino acid sequences of the two genes was 57.5%. The gene product of clone GS309 was easily purified to an essentially homogeneous state by ammonium sulfate fractionation followed by DEAE-Sepharose CL-6B, Ultrogel AcA-34, and second DEAE-Sepharose CL-6B column chromatography. The purified protein catalyzed the D-phenylalanine-dependent ATP-32PPi exchange reaction which is specific for GS 1 activity, and the specific activity of the purified product was nearly the same as the purified GS 1 from B. brevis. The product also showed a weak phenylalanine racemase activity.     

Induction of human microvascular endothelial tubular morphogenesis by human keratinocytes: involvement of transforming growth factor-alpha.

Transforming growth factor-alpha(TGF-alpha), homologous to epidermal growth factor(EGF), is closely involved in hyperproliferation of human keratinocytes. Psoriasis is a common hyperproliferative skin disease characterized by hyperproliferation of keratinocytes and abnormal development of dermal capillary networks. In this study, we have examined whether keratinocytes could enhance angiogenesis. TGF-alpha or EGF efficiently stimulated formation of tubular-like structures of human omental microvascular endothelial(HOME) cells in type I collagen gels. Human keratinocytes produced TGF-alpha. To examine whether co-cultured keratinocytes could induce tubulogenesis of HOME cells in collagen gel, we have developed a co-culture system with human keratinocytes. Surprisingly, there appeared new development of many tubular-like structures of HOME cells in collagen gels when co-cultured with keratinocytes. This keratinocytes-dependent tubulogenesis was almost completely blocked when anti-TGF-alpha-antibody was present. The TGF-alpha molecules derived from keratinocytes appeared to enhance tubulogenesis of human microvascular endothelial cells. We propose the hypothesis that secretory TGF-alpha from human keratinocytes may promote an autocrine loop to proliferate the skin keratinocytes and also a paracrine loop to induce the skin angiogenesis.