In order to elucidate the physiological function of intestinal alkaline phosphatase, the characteristics of alkaline phosphatases from rat and human small intestine were compared under optimal and ...physiological pHs. The Km values of these enzymes towards p-nitrophenylphosphate at the physiological pH were lower by two orders of magnitude than those at the optimal pH. At the physiological pH, phosphate, arsenate and vanadate competitively inhibited alkaline phosphatase activity, as they did at the optimal pHs, and the Ki values of these inhibitors at the physiological pH were also lower by two orders of magnitude than those at the optimal pHs. The effects of various inhibitors and antiserum to rat intestinal alkaline phosphatase on the transport of phosphate into everted rat intestine were investigated. The results obtained from the present study indicate that a phosphate transport system operating at physiological pH exists in the upper part of the small intestine where the alkaline phosphatase is maximally concentrated. It was also found that the phosphate transport was affected by various inhibitors and antiserum to rat intestinal alkaline phosphatase, but L-homoarginine and ouabain had no effect. From the above findings, it is suggested that alkaline phosphatase may function not only as a hydrolytic enzyme of phosphomonoesters but also as a phosphate transporter in the physiological state.
Effects of sequential and combined immuno-endocrine therapies using OK-432 (Picibanil) and tamoxifen (TAM) on the growth of 7,12-dimethylbenz alpha anthracene (DMBA)-induced carcinoma were examined ...in 128 female Sprague-Dawley (SD) rats. The rats were divided into six groups: control (no treatment), tamoxifen, OK-432, simultaneous immuno-endocrine OK-432 and TAM (OK-432 + TAM) therapy, two types of sequential immuno-endocrine therapy of the OK-432 and TAM groups OK-432 (1 wk)---TAM (4 wk) and OK-432 (2 wk)---TAM (3 wk). Each group was treated consecutively for five weeks. The response rates in the TAM alone group, the OK-432 (1 wk)---TAM (4 wk) group and the OK-432 + TAM (5 wk) group were significantly higher than in the control group. When the results among the treated groups were compared, the response rate in the OK-432 (1 wk)---TAM (4 wk) group was significantly higher than in the OK-432 alone or TAM alone groups. The response rate in the OK-432 (2 wk)---TAM (3 wk) group, however, was lower than in the TAM alone group. The response rate in the OK-432 + TAM group was, moreover, not significantly superior to that in the TAM alone group. These results suggest OK-432 not to potentiate the antitumor effect of TAM since the response rate of the combined OK-432/TAM therapy was not always significantly superior to that of the TAM treatment.
Sixty-six female Sprague-Dawley (SD) rats with 7,12-dimethylbenz alpha anthracene(DMBA)-induced rat mammary cancer were divided into five groups: tamoxifen (TAM), medroxyprogesterone acetate (MPA), ...TAM + MPA, ovariectomy (Ovex), control (no treatment). An antitumor effect was shown in each treated group. Thirty-six (72%) out of 50 treated animals responded to the first endocrine therapy. Moreover, tumors disappeared completely from 21 out of the 36 animals, and no new tumors were seen until the 12th week. The facts suggest experimental hormone, when compared to clinical, therapy in DMBA-induced tumors to have a higher response rate. A total of 14 out of 50 tumors failed to respond to the first treatment (resistant tumor). There was no significant difference in the proportion of resistant tumors among the four treated groups. When other endocrine therapies were tried out of resistant tumors, seven out of the 14 responded, the resistant tumors in the TAM group responding significantly well to the other endocrine therapies compared to those in the MPA group (P less than 0.05). These results suggest the possibility of resistant tumors responding to different types of endocrine therapy.
Despite an increasing number of reports of so called 'eosinophilic otitis media', its nature and pathogenesis are not fully understood. This surveillance was performed in an attempt to investigate ...the clinical and epidemiologic characteristics of eosinophilic otitis media. Definite diagnosis was made according to the following four criteria:(a) adult patients with bronchial asthma, (b) extremely viscous middle ear effusion, (c) resistant to usual treatments such as antibiotics, myringotomy, tympanostomy tube insertion, tympanoplasty, etc., and (d) marked eosinophil infiltration in middle ear effusion and/or middle ear mucosa/granulation. Patients who met the first three criteria were judged as suggestive cases. We sent the primary questionnaires to 1409 hospitals and medical universities all over Japan in which full-time ENT doctors work, and received answers from 628 hospitals. Three hundred and forty-one definite and 446 suggestive cases were presented. The secondary questionnaires were sent to 137 hospitals to survey the details of the presented definite cases. The disease was predominant in females in their fifties and sixties. Both ears were affected in 81%, and sinusitis was associated in 74% of the patients. Forty-seven percent of the patients had sensorineural hearing loss, and 6% were deaf. The ratio of sensorineural hearing loss was not dependent on the history of sinus surgery (49% vs. 46%, p>0.1). Topical and systemic steroids were used in 78% and 66% of the patients, respectively. The ratio of sensorineural hearing loss was significantly higher in patients who were given systemic steroids than those without systemic steroids (52% vs.34%, p<0.05). Only13% of the patients underwent tympanoplasty, and showed significantly higher ratio of deafness compared to those without tympanoplasty (17%vs.4%, p<0.05). Sensorineural hearing loss was exacerbated postoperatively in most of the deaf patients who underwent tympanoplasty. These results demonstrated intractable features and poor prognosis of hearing of eosinophilic otitis media.
Among various tissue-specific alkaline phosphatases, sialic acid was detected in liver, placental and meconial alkaline phosphatases, but not in the intestinal one. The content of acidic amino acids ...was larger than that of basic amino acids in the purified enzymes. Placental, intestinal and liver alkaline phosphatases contained 4 g-atoms of zinc/mol of enzyme, but the meconial alkaline phosphatase contained 2 g-atoms of zinc / mol of enzyme. N-Terminal amino acid residues of the intestinal and meconical alkaline phosphatases were both phenylalanine, whereas that of placental enzyme was isoleucine and that of liver enzyme was leucine. The tryptic peptide patterns of human placental, intestinal, meconial and liver alkaline phosphatases were similar to one another in part. The activecenter-containing peptides labelled with 33PO4 from human placental, intestinal, meconial and liver alkaline phosphatases had the same mobility on a thin layer chromatogram.
The inactivation of human placental and intestinal alkaline phosphatases by sodium thiocyanate was investigated. Human placental alkaline phosphatase activity was completely inhibited by 2 M sodium ...thiocyanate, but human intestinal alkaline phosphatase was resistant to it. The structure of the inactivated enzyme was studied by means of the hydrophobic probe technique and circular dichroism method. Conformational changes had occurred in the secondary structure of the enzymes, and the hydrophobic regions of the inactivated enzymes had increased compared with those of the native enzymes. The addition of inorganic phosphate to the enzyme treatment system combined with sodium thiocyanate protected human placental alkaline phosphatase from inactivation, while human intestinal alkaline phosphatase was not protected. These findings suggest that the site attacked by sodium thiocyanate may be a region which induces conformational change upon binding of inorgaic phosphate at the active site in human placental alkaline phosphatase.
Human alkaline phosphatase was purified from meconium by treatment with cetylpyridinium chloride, followed by diethylaminoethyl (DEAE)-cellulose and CM-cellulose chromatography, Sephadex G-200 gel ...filtration and DEAE-Sephadex A-50 chromatography. The homogeneity of the purified enzyme was demonstrated by disc electrophoresis and immunological investigation. The molecular weight of the purified meconial alkaline phosphatase was 155000 and the enzyme was composed of two subunits of equal molecular weight. The optimum pH was found to be 10.0 and the enzyme was stable over the pH range of 4-10. The Km value was 2.2mM for p-nitrophenylphosphate as a substrate. The isoelectric point was pH 4.0, and the purified enzyme was inhibited by N-bromosuccinimide (NBS), o-phenanthroline, ethylenediaminetetraacetic acid (EDTA) and L-phenylalanine. Meconial alkaline phosphatase obtained by our method contained 2 g-atoms of zinc/mole of enzyme. The enzymic properties of the purified meconial alkaline phosphatase were compared with those of adult intestinal alkaline phosphatase.
A procedure for the determination of the heat-stable alkaline phosphatase activity in serum was developed by using antibody-conjugated paper disks and inhibition by sodium thiocyanate. The placental ...alkaline phosphatase adsorbed on a paper disk conjugated with antibody was completely inhibited by 3M sodium thiocyanate, but the activity of intestinal alkaline phosphatase adsorbed on the paper disk was unaffected by 3M sodium thiocyanate. The results obtained by the proposed method showed a good correlation with those obtained by the heat-inactivation method (γ=0.991). These results strongly suggest that the proposed method can be utilized as a routine clinical test for the determination of serum placental-type alkaline phosphatase, i. e., placental, Regan, Nagao and Kasahara isoenzymes, from patients with cancer.
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