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Modified constructions of a microsomal cytochrome P450, of NADPH-cytochrome P450 reductase, and of a P450/reductase fused enzyme were prepared to analyze the function of the amino-terminal hydrophobic regions of these enzymes and the hinge region of the fused enzyme. Expression plasmids for delta P450c, delta reductase, and the delta P450/reductase fused enzyme, all of which lacked their amino-terminal hydrophobic regions, were constructed by inserting each of the corresponding cDNAs between the yeast alcohol dehydrogenase I promoter and the terminator of the expression vector pAAH5. Yeast transformed with plasmids encoding delta P450 and the delta P450/reductase fused enzyme produced smaller amounts of the respective enzymes and showed lower monooxygenase activity toward 7-ethoxycoumarin than did yeast transformed with plasmids encoding the complete enzymes. Both delta P450 and delta P450/reductase were found in the microsomal fraction of the yeast cells. Yeast transformed with the expression plasmid for delta reductase produced 20 times more enzyme than did yeast transformed with the plasmid for the complete enzyme. delta Reductase was present in the soluble fraction and was 33 times more active in reducing cytochrome c than was the complete enzyme. The results suggest that the amino-terminal hydrophobic regions of P450c and the P450/reductase fused enzyme play an important role in their stability and function in the yeast microsomes. By contrast, the amino-terminal-containing P450 reductase appears to be unstable in yeast cells. Altering the size of the hinge regions does not affect the activity of the P450/reductase fused enzyme significantly, but some amino acid changes in this region increase the stability of the fused enzyme slightly.

For the purpose of resolving the mechanism of the ill-effect of refluxed pancreatic juice, due to the anomalous pancreaticobiliary ductal union, canine models were produced. Mongrel adult dogs and puppies were used. Pancreatico-cholecystostomy was performed on 5 adult dogs and 5 puppies. Pancreatico-choledochostomy was performed on 10 adult dogs and 5 puppies. In both procedures, the cylindrical dilatation of the choledochus resulted. The pathological changes in the choledochus were very similar to the cylindrical choledochal dilatation in the human. Elastic fibers diminished universally and in some cases they vanished completely. The changes in the papilla of the bile duct were very slight. There were no cases with hardened papilla. The pathological changes were greater in the pancreatico-cholecystostomy. Enzymatic analysis of pancreatic juice in bile was very important in explaining the etiology. Amylase was proven to be very high throughout the cases, over 10,000 IU/1 by enzymatic assay. Trypsin and elastase were proven to be activated in pancreatico-cholecystostomy, naturally assuming the existence of enterokinase. They were aso proved to be activated in the pancreatico-choledochostomy, without the existence of enterokinase. Though the mechanism was not clear, the activated proteases were the actual cause of the ill-effect in this anomaly. Thus we have succeeded in producing the ideal animal models of the reflux of pancreatic juice into the bile duct in adult dogs and puppies.

Three forms of cytochrome P-450, designated as P-450MC-I, P-450MC-II, and P-450MC-III, were isolated from liver microsomes of rats treated with 3-methylcholanthrene (MC) by using a high performance liquid chromatography (HPLC) technique. The major MC-inducible forms, P-450MC-I and P-450MC-II showed a single protein band on SDS-polyacrylamide gel electrophoresis giving a minimum molecular weight of 56,000 daltons. The oxidized absolute spectra of both cytochromes P-450 were of low spin type, having a Soret absorption peak at 417 nm. The CO-reduced difference spectra of these two cytochromes P-450 showed a peak at 447 nm. In a reconstituted system, both cytochromes P-450 exhibited similar high levels of catalytic activity for benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation. Anti-P-450MC-I IG and anti-P-450MC-II IG, which were produced against the corresponding cytochromes P-450, each formed a single continuous precipitin line with both P-450MC-I and P-450MC-II in Ouchterlony double diffusion tests. Amino acid sequence analysis revealed that the sequence of the NH2-terminal 18 amino acids of both enzymes was the same. Therefore, the major MC-inducible forms, P-450MC-I and P-450MC-II, were highly homologous, being indistinguishable from each other in terms of apparent molecular weight, spectral properties, substrate specificity and the NH2-terminal 18 amino acid residues, but clearly separable by HPLC. The characteristics of both P-450 forms appear to correspond to those of the previously reported P-450c (1). On the other hand, a minor form, P-450MC-III was different from P-450MC-I and P-450MC-II in chromatographic properties, apparent molecular weight, substrate specificity and immunochemical properties, and did not correspond to any P-450 species previously purified from MC-treated rat liver microsomes.

Cholinergic modulation and effects of dynorphin on the non-adrenergic non-cholinergic inhibitory potentials (NANC i.p.s) in the longitudinal smooth muscle cells of the guinea-pig duodenum were studied intracellularly. Atropine (1.4 X 10(-7)-1.4 X 10(-5) M) and scopolamine (3.3 X 10(-8)-3.3 X 10(-7) M) increased the amplitude of the evoked i.p.s while physostigmine (3.7 X 10(-7)-3.7 X 10(-6) M) and neostigmine (4.8 X 10(-8) M) decreased it. The frequency of the spontaneous action potentials in the longitudinal smooth muscle cells was increased by dynorphin (6 X 10(-8)-3 X 10(-7) M) without continuous membrane depolarization. The excitatory effect of dynorphin on the spontaneous electrical activity was not blocked by atropine (1.4 X 10(-6) M). Dynorphin (6 X 10(-8)-2.4 X 10(-7) M) increased the amplitude of the i.p.s evoked in the presence of atropine (1.4 X 10(-7)-1.4 X 10(-6) M) and in the propranolol (3.9 X 10(-6) M) solution containing atropine while dynorphin decreased the amplitude of the i.p.s evoked in the absence of atropine. In the high calcium solution containing atropine, the amplitude of the i.p.s increased. Further increase in the amplitude of the i.p.s was observed by additively applied dynorphin. However, the amplitude of the i.p.s evoked in the high calcium solution without atropine was decreased by dynorphin. These results suggest the cholinergic inhibitory modulation on the NANC inhibitory nerves in the guinea-pig duodenum, the non-cholinergic excitatory action of dynorphin on the spontaneous electrical activity of the longitudinal smooth muscle and the excitatory action of dynorphin on the NANC inhibitory nerves in the presence of muscarinic blocking agents.

Rat liver cytochrome P-450MC cDNA was inserted between the ADH1 promoter and terminator regions of the yeast expression vector pAAH5. On introduction of the resulting recombinant plasmid pAMC1, Saccharomyces cerevisiae cells synthesized up to 8 X 10(5) molecules per cell of the cytochrome P-450MC protein, most of which was localized in yeast microsomes. Approximately half of the synthesized cytochrome contained heme in the enzyme molecule. These formed a functional electron-transport chain in the microsomes which exhibited aryl hydrocarbon hydroxylase activity toward benzo[a]pyrene.

Two human malignant tumors, which we previously reported to produce colony-stimulating factors (CSFs), were found to be accompanied by remarkable hypercalcemia. A patient with a CSF-producing lower jaw cancer (squamous cell carcinoma) developed a marked granulocytosis (150,000/microliters) and hypercalcemia (more than 215 mg/dl). The tumor was successfully transplanted into nude mice, which developed marked granulocytosis (300,000/microliters) and hypercalcemia (20 mg/dl). White blood cell and serum calcium concentrations of these mice decreased promptly to normal levels when the tumor was excised. Treatment with prednisolone (1.5 mg/kg) or indomethacin (5 mg/kg) had no effect on the serum calcium level of these mice. Parathyroid hormone or prostaglandin E was not increased in the serum of the mice or in the tumor tissue. However, the mice bearing the tumor excreted extremely large amounts of calcium in their urine, and their bony tissues contained less calcium and phosphorus than controls. Moreover, histology of bony tissues of these nude mice clearly demonstrated the decrease in trabecular tissues and cortical thickness as well as remarkable activation of osteoclasts. Another patient with a CSF-producing bronchogenic squamous cell carcinoma showed mild granulocytosis and hypercalcemia. The biopsied tumor tissue was transplanted into nude mice, which developed marked granulocytosis (300,000/microliters) and also severe hypercalcemia (18 mg/dl). These results suggest the presence of a new syndrome of granulocytosis and hypercalcemia associated with CSF-producing tumors. The causal mechanism of the hypercalcemia was shown to be some humoral factor which activates osteoclasts other than parathyroid hormone. Neither prostaglandins nor osteoclast-activating factor seemed to be the cause of the hypercalcemia.

For assessing the participation of mucus glycoproteins in the cytoprotective process, mucus glycoprotein content in the rat gastric mucosa was measured after treatment with 70% ethanol with or without prostaglandin (PG) E derivatives or 20% ethanol pretreatment. Oral administration of two synthetic PGE derivatives did not cause any significant changes in mucus glycoprotein content. Seventy percent ethanol administration caused marked reduction in mucus glycoprotein content (about 50% of control) as well as severe gastric mucosal damage. Treatment with two PGE derivatives (10-100 micrograms/kg) prior to 70% ethanol administration markedly inhibited the gross mucosal lesion, whereas the glycoprotein content under these conditions was significantly less than that in the untreated control group (ranging from 67-88% of control). Pretreatment with 20% ethanol markedly inhibited the gross mucosal damage caused by 70% ethanol dosing but the inhibition in the reduction of mucus glycoprotein content was restricted to about 80% of the untreated controls. In summary, cytoprotection induced by PG was not accompanied by entire conservation of intramucosal mucus glycoprotein in the gastric mucosa.

Spontaneous electrical activity of the intestine smooth muscle of the cat consisted of slow waves and spikes. The spike activity and the slow wave generation were inhibited by pentagastrin and tetragastrin. Phasic contraction corresponding with the electrical activity was also abolished by pentagastrin and tetragastrin. Adrenaline showed the inhibitory action on the spike activity and phasic contraction, but slow waves persisted against adrenaline. Propranolol depressed this inhibitory action produced by adrenaline, while it did not block the inhibitory action by pentagastrin. The inhibitory effect of pentagastrin was not influenced by caffeine or theophylline. Imidazole depressed slightly the inhibitory action of pentagastrin. Verapamil inhibited the spike activity. Additional application of pentagastrin after verapamil produced further inhibition of both slow wave generation and phasic contraction. These results suggest that the inhibitory action produced by pentagastrin on the electrical and mechanical activities is directly on the smooth muscle cell membrane mediated through a mechanism other than via beta-receptors. It is also suggested that the primary action of pentagastrin is to inhibit the generation of slow waves of the intestinal smooth muscle.

We have examined the induction of drug metabolizing enzymes in rat liver microsomes by azo dye, 1-(p-phenylazophenylazo)-2-naphthol (Sudan III). Marked increases were observed in the levels of cytochrome P-448 as well as in p-nitroanisole O-demethylase (p-NAD), amaranth (AR) and neoprontosil reductases (NPR) and 7-ethoxycoumarin O-deethylase (ECD) activities. On the other hand, aminopyrene N-demethylase activity was not significantly increased. Further, induced ECD activity was inhibited 90% by a specific antibody against cytochrome P-448 while the inhibition observed with an antibody against cytochrome P-450 was less than 25%. Simultaneous administration of Sudan III and 3-methylcholanthene (3-MC) induced cytochrome P-448 up to a level brought about by either Sudan III or 3-MC treatment alone. In contrast, Sudan III did not induce cytochrome P-448 in the 3-MC insensitive DBA/2 mouse. Solubilized microsomes from Sudan III-treated rats showed an identical sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) pattern with those from 3-MC-treated animals. It is concluded that the cytochrome P-448 induced in liver by Sudan III is very similar to that induced by 3-MC. Sudan III also induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. It did not induce NADPH-cytochrome c reductase, nor any of the enzymes which constitute the microsomal electron transport chain except for cytochrome P-448.

The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.

The effects of 4-aminopyridine (4-AP) on the non-adrenergic inhibitory potential (i.p.) in the longitudinal and circular smooth muscle cells of the guinea-pig duodenum were investigated using intracellular microelectrodes. The membrane potential of both smooth muscles was decreased by 4-AP and the amplitude of the evoked i.p.s. was increased. In a low-calcium solution (0.25 mM), the amplitude of the i.p.s. was reduced but the additional application of 4-AP increased the amplitude. In the 4-AP containing low-calcium solution, the i.p. was inhibited by verapamil and EGTA. The inhibitory effect of verapamil on the i.p. evoked in a low-calcium solution was less in the presence of 4-AP than that in its absence. In a high-calcium solution (5 mM), the amplitude of the i.p. increased and the additional application of 4-AP enhanced the i.p. further. The amplitude of the i.p. elicited in high-potassium solutions was not changed by 4-AP. The i.p. was not potentiated by 2-aminopyridine or 2,5-diaminopyridine. Tetraethylammonium ions enhanced the amplitude of the i.p. at low concentration but decreased it in high concentration. In a few longitudinal smooth muscle cells, the evoked excitatory potential (e.p.) was observed. Potentiation of the e.p. by 4-AP was completely blocked by atropine. From the results obtained, it is suggested that the release of the non-adrenergic inhibitory neurotransmitter is increased by 4-AP due to increase calcium influx in the nerve terminals. The release of the cholinergic excitatory neurotransmitter seems to be increased by 4-AP.

Effects of gastrointestinal hormones on the electrical and mechanical activities of the smooth muscle of the cat small intestine were examined. Both spontaneous electrical and mechanical activities of the intestinal smooth muscle were inhibited by tetragastrin, pentagastrin and pancreozymin. The spike activity and the phasic contraction of the smooth muscle were depressed by these hormones while the slow waves were maintaned. On the other hand, secretin showed an excitatory action on the mechanical activity of the isolated preparation of smooth muscle. Spike activity disappeared and the level of tone was increased gradually. The inhibitory and excitatory actions of each hormone on the mechanical activity were not abolished by the application of atropine and tetrodotoxin. Furthermore, the inhibitory effects of pentagastin and pancreozymin on the smooth muscle were not antagonized with phenoxybenzamine or propranolol. These results suggest that the inhibitory actions of pentagastrin and pancreozymin on the smooth muscle were not mediated by alpha or beta receptors and not due to the stimulation of an inhibitory nervous system in the gut. Antagonistic relations between secretin and pentagastrin and also between secretin and pancreozymin were observ ed. The mechanisms of the actions of gastrointestinal hormones are discussed.

A laboratory model of renal osteodystrophy was developed in rats by a single injection of glycopeptide isolated from renal cortical tissues of rats according to the method used by Shibata et al. to induce glomerulonephritis. Approximately 60-70 days after injection, severe proteinuria appeared and continued for at least 170 days at a rate of more than 1 g/day. Morphological changes in the kidney were typical of chronic glomerulonephritis. The plasma calcium concentration was lowered transiently by the 96th day after injection, but was restored to the normal range thereafter. Plasma parathyroid hormone levels, however, continued to rise in parallel with the degree of proteinuria. Marked secondary hyperparathyroidism was induced which led to severe bone atrophy. Histological examinations showed a marked increase of resorbing cavities, with a quantitatively larger number of osteoclasts in cortical bone tissues compared with the control animals. No spontaneous remission was observed. It is emphasized that all of the biochemical and morphological changes reported here were induced by a single injection of homologous renal glycopeptide, and they were highly reproducible.

The following conditions must be observed if the actual pressure in the alimentary tract is measured and recorded with the aid of an open probe: 1. The probe must have a laterally placed opening, rather than a terminal one; 2. The opening must be as large as or larger than the internal diameter of the probe; 3. The internal diameter of the probe must be at least 1.2 mm or larger; 4. The pressure should not be measured with a nonperfusion technique if exact measurements of the intraluminal pressure are to obtained. One always has to use a perfusion, and the measured pressure depends on the speed of perfusion; 5. In order to be certain that the measured pressure corresponds to the actual pressure, one must ascertain whether the pressure will not change although the speed of perfusion is increased.

An enzyme hydrolyzing sphingomyelin was purified from extracts of solid cultures of Aspergillus saitoi 7041 by fractionation with isopropanol followed by successive column chromatographies on DEAE-Sepharose CL-6B, butyl-Toyopearl 650 M, and phenyl-Sepharose CL-4B. The preparation of purified enzyme was homogeneous and had an activity increased 81-fold over that of the isopropanol fraction. The yield was about 65%. The molecular weight was estimated to be 54,000 by sodium dodecyl sulfate-gel electrophoresis. The enzyme solution had a violet color and contained iron atoms. The enzyme catalyzed the hydrolysis of sphingomyelin to N-acylsphingosine and phosphorylcholine. The optimum pH for hydrolytic activity was around 3.5. The Km values for sphingomyelin and 2-hexadecanoylamino-4-nitrophenylphosphorylcholine were 0.11 and 0.33 mM, respectively. The enzyme also catalyzed the hydrolysis of other phospholipids; the order of its hydrolytic activity at a substrate concentration of 2.5 mM was phosphatidylcholine greater than or equal to sphingomyelin = phosphatidylethanolamine = lysophosphatidylethanolamine greater than phosphatidyl DL-glycerol = phosphatidyl L-serine greater than phosphatidylinositol. From these results, this enzyme appears to be a new type of phospholipase C(phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3).

Rat cytochrome P-450MC cDNA was expressed in Saccharomyces cerevisiae AH22, SHY3 and NA87-11A cells under the control of the yeast ADH1 promoter and terminator. Although the three yeast strains transformed with the constructed expression plasmid, pAMC1, contained approximately three copies of the plasmid, the levels of both P-450MC mRNA and the corresponding protein in the AH22 cells carrying plasmid pAMC1 were 1.4- to 1.7-fold and 2-fold higher than in the other two strains, respectively. The P-450MC protein was purified from the microsomal fraction of AH22 cells carrying pAMC1 by a rapid purification method. The apparent molecular weight, chromatographic behavior, spectral properties, substrate specificity and immunochemical properties of the purified P-450MC protein were indistinguishable from those of rat liver P-450MC-I and P-450MC-II (Sasaki, T., et al. (1984) J. Biochem. 96, 117-126). The NH2-terminal amino acid sequence of the purified protein up to 10 residues was the same as those of P-450MC-I and P-450MC-II. In addition, HPLC analysis of the microsomal fraction of AH22 cells containing pAMC1 indicated that the synthesized P-450MC protein corresponds to P-450MC-II, but not P-450MC-I. With another purification method, we obtained the cleaved P-450MC protein which lacked the NH2-terminal 30 amino acids of intact P-450MC. The spectral properties and monooxygenase activities towards benzo(a)pyrene and 7-ethoxycoumarin of the cleaved P-450MC were nearly the same as those of intact P-450MC.

Effects of externally applied cyclic AMP and other adrenergic stimulants on the electrical and mechanical activities of the cat small intestine were observed by using pressure electrodes. The electrical and mechanical activities were suppressed by cyclic AMP and beta-stimulants. Those inhibitory actions of cyclic AMP and beta-stimulants were potentiated under the treatment with caffeine, theophylline and papaverine which inhibits the phosphodiesterase activity. On the other hand, the inhibitory action of cyclic AMP and beta-stimulants was decreased in imidazole, an agent that increases phosphodiesterase activity. Exogenous applied concanavalin A, an agent that inhibits the adenyl cyclase activity, showed no observable changes in both activities but the effects of beta-stimulants were decreased after treatment with concanavalin A. No obvious changes on both activities were obtained in cyclic GMP and dibutyryl cyclic GMP. These findings tentatively support the hypothesis that cyclic AMP is a second messenger in the inhibitory responses to beta-stimulants on the intestinal smooth muscle. However, it is also concluded that the inhibition of mechanical activity caused by cyclic AMP is partially due to suppression of the membrane activity.