Modification of a single tryptophan of the inorganic pyrophosphatase from thermophilic bacterium PS-3: possible involvement in its substrate binding.

The effect of N-bromosuccinimide (NBS) on the activity of the inorganic pyrophosphatase (PPiase) from thermophilic bacterium PS-3 was studied. The enzyme was almost completely inactivated on chemical modification with NBS, depending upon the concentration of NBS. The presence of a complex of Mg2+ and a substrate analogue, imidodiphosphate (PNP), provided extensive protection against the inactivation, whereas Mg2+ or PNP alone showed no protective effect. Amino acid analysis of the NBS-modified enzyme after hydrolysis with 6 M HCl indicated no change in the amino acid composition. However, the magnetic circular dichroism (MCD) bands around 293 nm due to the tryptophan residue and the optical density at 280 nm, decreased concomitantly with modification by NBS. These results strongly suggested that the tryptophan residue at position 143, which is the only tryptophan residue per subunit in the thermophilic PPiase (Ichiba, T., Takenaka, O., Samejima, T. and Hachimori, A. (1990) J. Biochem. 108, 572-578), might be involved in the active site or be located in the vicinity of the active site. The circular dichroism (CD) spectrum in the far ultraviolet region showed no significant alteration during the modification, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modification considerably altered the CD bands in, the near ultraviolet region, indicating that a conformational change occurred in the vicinity of the active site in the enzyme molecule.

Nucleotide sequence of ornithine decarboxylase antizyme cDNA from Xenopus laevis.

An ornithine decarboxylase antizyme cDNA was obtained from Xenopus laevis liver and its sequence was determined. The cDNA consists of two major open reading frames as found in mammalian antizymes, which require +1 ribosomal frameshifting for its translation. Sequences important for frameshifting, namely the frameshift site and downstream stimulatory pseudoknot determined in the rat mRNA, are conserved.

Modifying effects of Terminalia catappa on azoxymethane-induced colon carcinogenesis in male F344 rats.

The modifying effects of dietary administration of an herb, Terminalia catappa (TC), were investigated on rat colon carcinogenesis induced by a carcinogen azoxymethane (AOM). The number of aberrant crypt foci (ACF) and beta-catenin accumulated crypts (BCACs) in the colon, and proliferating cell nuclear antigen (PCNA) labelling index in the colonic epithelium were examined in a total of 36 male F344 rats. All animals were randomly divided into five experimental groups (4-10 rats in each group). At 6 weeks of age, rats in groups 1, 2 and 3 were given s.c. injections of AOM once a week for 2 weeks at a concentration of 20 mg/kg body weight. One week before the first injection of AOM, rats in groups 2 and 3 were fed a diet containing 0.02 and 0.1% TC, respectively, throughout the experiment. Rats in group 4 were fed a diet containing 0.1% TC. Rats in group 5 were served as untreated controls. All animals were sacrificed at the experimental week 5 after the start of the experiment. Oral administration of TC at both doses significantly decreased the numbers of both ACF/colon/rat (P<0.05 for 0.02% TC, P<0.005 for 0.1% TC) and BCAC/cm/rat (P<0.05 for both 0.02 and 0.1% TC), when compared with the control group (group 1). Colonic PCNA labelling index in groups 2 and 3 was also significantly lower than that in group 1 (P<0.001 for 0.02% TC, P<0.005 for 0.1% TC). These results suggest that TC has a potent short-term chemopreventive effect on biomarkers of colon carcinogenesis and this effect may be associated with the inhibition of the development of ACF and BCACs.

Characterization of GFR, a novel guanine nucleotide exchange factor for Rap1.

Three groups of Rap1-specific guanine nucleotide exchange factors including C3G, CalDAG-GEFI, and Epac/cAMP-GEFI/II have been identified to date. In the present study, we report a new Rap1 guanine nucleotide exchange factor which we have named GFR (guanine nucleotide exchange factor for Rap1). GFR shows close sequence similarity to EPAC/cAMP-GEFI/II although GFR lacks a cAMP binding domain and contains a nuclear localization signal. We demonstrated that GFR can activate Rap1 but not H-Ras in 293T cells and that the cdc25 domain of GFR is required for the activation of Rap1. Northern blot analysis suggested that GFR mRNA is strongly expressed in the brain. In transfected HeLa cells, GFR has been found to be localized in the nuclei.

Enhancement of the thermostability of thermophilic bacterium PS-3 PPase on substitution of Ser-89 with carboxylic amino acids.

Serine 89 of the inorganic pyrophosphatase (PPase) subunit from thermophilic bacterium PS-3 (PS-3) was replaced with glycine, alanine, threonine, glutamic acid, or aspartic acid by the PCR-mutagenesis method with Mut-1 in order to determine the contribution of this serine residue to the thermostability and structural integrity of the enzyme molecule. S89G, S89A, and S89T showed reduced catalytic activity, whereas S89D and S89E showed increased enzyme activity. S89G, S89A, and S89T as well as the wild-type PPase were stable in the presence of 5 mM MgCl(2) at 70 degrees C for 1 h, but were inactivated rapidly with time at 80 degrees C. On the contrary, S89D and S89E were stable at 80 degrees C, showing more than 95% of the original activity after 1 h incubation. The wild-type PPase, S89D and S89E were each a hexamer before and after incubation at 80 degrees C for 1 h, while S89G and S89A comprised a mixture of a hexamer and a trimer both before and after incubation at 80 degrees C for 1 h. On the other hand, S89T was a mixture of a hexamer, a trimer and a monomer, and it was partially precipitated during heat treatment at 80 degrees C. The CD spectra of the recombinant enzymes in the far-ultraviolet region were the same as that of the wild-type PPase, whereas those of S89G, S89A, and S89T as well as the wild-type PPase were markedly different after heat treatment, although those of S89D and S89E did not change. The present study suggested that local small change(s) in the network of interactions among amino acid residues on replacement at position 89 led to the PS-3 PPase molecule being unable to form a hexamer from trimers or to dissociate into monomers in some cases without a significant change in the backbone conformation. It was also suggested that the partial disordering of the conformation of PS-3 PPase caused by heat depended on the degree of hydrophilicity in the vicinity of position 89.

Primary structure of the inorganic pyrophosphatase from thermophilic bacterium PS-3.

The complete amino acid sequence of the inorganic pyrophosphatase from thermophilic bacterium PS-3 was determined by automated Edman analysis of the intact protein and of peptides derived from digests obtained with lysylendopeptidase, Staphylococcus aureus strain V8 protease, and arginylendopeptidase. The monomer peptide chain comprises 164 amino acid residues and has a calculated molecular weight of 18,792. The sequence is identical at about 46% of the amino acid positions with that of the Escherichia coli enzymes.

Adrenergic control of the glucagon response to ammonia in the perfused rat pancreas.

The isolated perfused rat pancreas was used to investigate how adrenergic influences within the pancreas might mediate ammonia-induced glucagon secretion. The addition of 2 mM ammonia to the perfusate increased norepinephrine release and glucagon secretion in the effluent. Upon cessation of ammonia addition, a pronounced burst of glucagon release was observed. Alpha-adrenergic blockade with phentolamine (10 microM) blocked the glucagon response to ammonia. Beta-adrenergic blockade with propranolol (10 microM) had no significant effect on the amount of glucagon release induced by ammonia. Depletion of norepinephrine from sympathetic nerve terminals by pretreatment with 6-hydroxydopamine lowered the pancreatic norepinephrine content to less than 16% of the control value and diminished the glucagon and norepinephrine response to ammonia almost completely. The burst of glucagon release after the removal of ammonia was inhibited to 2% of the control value by phentolamine and to 57% by propranolol. Pretreatment with 6-hydroxydopamine reduced the burst of glucagon secretion to 28% of the control value. Neither phentolamine nor propranolol reduced the magnitude of the ammonia-induced suppression of insulin secretion. We conclude that the effect of ammonia on glucagon release from the isolated rat pancreas is mediated by intrapancreatic adrenergic control.

CJ-15,801, a novel antibiotic from a fungus, Seimatosporium sp.

A novel antibiotic, CJ-15,801 (I), was isolated from the fermentation broth of a fungus, Seimatosporium sp. CL28611. The structure was determined to be a pantothenic acid analog having an alpha,beta-unsaturated carboxylic acid moiety by spectroscopic analyses. The compound inhibits the growth of multi-drug resistant (MDR) Staphylococcus aureus strains with MIC ranging from 6.25 to 50 microg/ml.

Atrial blood cyst in an adult with regurgitation of mitral and tricuspid valves.

Blood cysts of the heart are extremely rare in adults and usually involve valves or the left ventricle. Although two cases of blood cysts in the right atrium in adults have been reported, a cyst combined with a disorder of the valves has never been reported. We report a 52-year-old woman with a blood cyst that generated from the right atrial septum. Furthermore, the patient had regurgitation of both the mitral and tricuspid valves and then underwent surgical excision of the blood cyst, mitral valve plasty and tricuspid valve annuloplasty. We believe that it is possible to diagnose blood cysts with echocardiography, CT and magnetic resonance imaging. Echocardiography showed the cyst as a circle without a complete inner free-echo. CT and magnetic resonance imaging showed a mass with a non-enhanced inner structure. Furthermore, the latter showed a cyst that was enhanced by T1- but not T2-weighted images, indicating that the content of the cyst was a persistent substance such as blood. Concerning the generation of blood cysts, we hypothesize that heteroplastic growth arising from primitive pericardial mesothelium causes disorders of valves and blood cysts.

[A case of long thoracic nerve palsy, with winged scapula, as a result of prolonged exertion on practicing archery].

Reports of isolated long thoracic nerve palsy are rare in Japan. We reported a case of isolated long thoracic nerve palsy, resulted from recurrent injury to the nerve. Muscle CT and electrodiagnostic study were useful for confirming diagnosis of this cases. This patient was a student aged 20 years, with nothing of importance in his family or past history. After he started practicing archery, winging of left scapula was gradually developed. Physical examination revealed weakness and atrophy of left serratus anterior muscle. There was no wasting and weakness of other should girdle muscles. Hematochemical tests were normal, except slight hyperthyroidism. Radiography and myelography of the cervical spine were normal. Muscle CT of upper thoracic level demonstrated atrophy of left serratus anterior muscle, and no abnormality were found in other muscles. Electromyogram of the left serratus anterior revealed discrete activity of reduced amplitude, and fibrillation potentials and positive sharp waves. Conduction time for left long thoracic nerve was prolonged, and amplitude of the evoked response was small and there were temporal dispersion. Muscle CT and electrodiagnostic studies were suggestive of neuroapraxia of left long thoracic nerve. Over stretching or compression during exercises may be responsible for the damage to the long thoracic nerve.

[Neuroleptic malignant syndrome after aortic valve replacement; report of a case].

A 64-year-old male with treated Parkinson's disease underwent mechanical valve replacement for aortic valve regurgitation. The antiparkinsonian drugs for internal use were interrupted on the morning of the operative day. After the operation, the patient developed fervescence, muscle rigidity, hidropoiesis and a rise in creatine kinase. The patient was diagnosed as neuroleptic malignant syndrome and given medication dantrolene sodium and antiparkinsonian drugs on the 5th postoperative day. The symptom of neuroleptic malignant syndrome disappeared on 12 postoperative days. As the stress of open heart surgery with extracorporeal circulation trigger off neuroleptic malignant syndrome, the patient with Parkinson's disease need early beginning of antiparkinsonian drugs on account of prevention of neuroleptic malignant syndrome after operation.

[A case of constrictive pericarditis reoperated 13 years after pericardiectomy by left thoracotomy approach].

A 48-year-old male with constrictive pericarditis was reoperated through median sternotomy 13 years after pericardiectomy through left thoracotomy. Extensive pericardiectomy is the most important point for constrictive pericarditis. Therefore, we have recently chosen the median sternotomy approach which has advantages over the left thoracotomy approach. Namely, the cardiopulmonary bypass is performed in the former approach immediately if the posterior pericardiectomy is difficult or the coronary arterial injury happens during decortication.

[Coronary artery bypass grafting for a patient with bronchial asthma seceeded from cardiopulmonary bypass by additional bypass for spasm of radial artery graft: a case report].

A 78-year-old male who had a bronchial asthma underwent coronary artery bypass grafting (CABG) using the left internal thoracic artery and the radial artery. The patient could not be weaned from the cardiopulmonary bypass because the radial artery which anastomosed to the obtuse marginal artery (OM) had a spasm after CABG. An additional bypass using a long saphenous vein to OM was carried out immediately. It brought a weaning from cardiopulmonary bypass. If the cardiac function after CABG is insufficient in patients with bronchial asthma, CABG must be re-done immediately, considering that they cause the arterial spasm more than patients without bronchial asthma.

Antioxidant and hepatoprotective actions of medicinal herb, Terminalia catappa L. from Okinawa Island and its tannin corilagin.

The antioxidant and hepatoprotective actions of Terminalia catappa L. collected from Okinawa Island were evaluated in vitro and in vivo using leaves extract and isolated antioxidants. A water extract of the leaves of T. catappa showed a strong radical scavenging action for 1,1-diphenyl-2-picrylhydrazyl and superoxide (O(2)(.-)) anion. Chebulagic acid and corilagin were isolated as the active components from T. catappa. Both antioxidants showed a strong scavenging action for O(2)(.-) and peroxyl radicals and also inhibited reactive oxygen species production from leukocytes stimulated by phorbol-12-myristate acetate. Galactosamine (GalN, 600 mg/kg, s.c.,) and lipopolysaccharide (LPS, 0.5 microg/kg, i.p.)-induced hepatotoxicity of rats as seen by an elevation of serum alanine aminotransferase, aspartate aminotransferase and glutathione S-transferase (GST) activities was significantly reduced when the herb extract or corilagin was given intraperitoneally to rats prior to GalN/LPS treatment. Increase of free radical formation and lipid peroxidation in mitochondria caused by GalN/LPS treatment were also decreased by pretreatment with the herb/corilagin. In addition, apoptotic events such as DNA fragmentation and the increase in caspase-3 activity in the liver observed with GalN/LPS treatment were prevented by the pretreatment with the herb/corilagin. These results show that the extract of T. catappa and its antioxidant, corilagin are protective against GalN/LPS-induced liver injury through suppression of oxidative stress and apoptosis.

The role of histidine-118 of inorganic pyrophosphatase from thermophilic bacterium PS-3.

Treatment of the inorganic pyrophosphatase from thermophilic bacterium PS-3 with diethyl pyrocarbonate resulted in the almost complete loss of its activity, which followed pseudo-first-order kinetics. The presence of Mg2+ prevented the inactivation. Enzyme inactivated with diethyl pyrocarbonate was re-activated by hydroxylamine. The inactivation parallelled the amount of modified histidine residue, and a plot of the activity remaining against the amount of modified histidine residue suggested that the modification of one of two histidine residues totally inactivated the enzyme. The site involved was found to be located in a single lysyl endopeptidase-digest peptide derived from the ethoxy[14C]carbonylated enzyme. Amino acid analysis and sequence analysis of the peptide revealed that it comprised residues 96-119 of the inorganic pyrophosphatase from thermophilic bacterium PS-3. These results, when compared with those reported for the Escherichia coli and yeast enzymes, imply that His-118 of the inorganic pyrophosphatase from thermophilic bacterium PS-3 is located near the Mg(2+)-binding site and thus affects the binding of Mg2+.

Antioxidant activity of medicinal herb Rhodococcum vitis-idaea on galactosamine-induced liver injury in rats.

Aim of the study was to evaluate in vivo antioxidant action of medicinal herb Rhodococcum vitis-idaea (Rh.v) on galactosamine (GalN)-induced rat liver toxicity. The results showed that the hepatotoxicity and oxidative stress induced by GalN (700 mg/kg, s.c.) after 24 h evidenced by an increase in serum alanine aminotransferase and glutathione (GSH) S-transferase activities, and lipid peroxidation in liver homogenate were significantly inhibited, when 10 times diluted Rh.v. extract (5 ml/kg, i.p.) was given to rats 12 and 1 h before GalN treatment demonstrating that the extract of Rh.v is a potent antioxidant and protective against GalN-induced hepatotoxicity. The main antioxidant compound of the herb water extract used in the experiment was determined as arbutin, which possess 8% of dry weight of the herb. The electron spin resonance (ESR) spectrometer analysis revealed that the arbutin isolated from Rh.v exhibited strong superoxide and hydroxyl radical scavenging ability.

Modification of tryptophan 149 of inorganic pyrophosphatase from Escherichia coli.

1. The inorganic pyrophosphatase from Escherichia coli was almost completely inactivated on chemical modification of Trp-149 with N-bromosuccinimide (NBS). 2. The presence of a complex of Mg2+ and a substrate analogue, iminodiphosphate (PNP), provided considerable protection against the inactivation, whereas Mg2+ or PNP alone afforded only slight protection.

Cloning of antizyme inhibitor, a highly homologous protein to ornithine decarboxylase.

The degradation of ornithine decarboxylase (ODC) catalyzed by the 26 S proteasome is accelerated by antizyme, an ODC inhibitory protein induced by polyamines. Previously, we have found another possible regulatory protein of ODC degradation, antizyme inhibitor. Antizyme inhibitor binds to the antizyme with a higher affinity than that of ODC, releasing ODC from ODC-antizyme complex. We report here the cDNA sequence of rat heart antizyme inhibitor. The deduced sequence of the protein is highly similar to, but distinct from, sequences of ODCs from various species. Antizyme inhibitor contains amino acid residues required for formation of active sites of ODC, but it completely lacks ODC activity. Antizyme inhibitor has no homology with peptide sequence in the mammalian ODC carboxyl terminus, which is needed for rapid turnover of ODC. It inhibits antizyme-dependent ODC degradation, but, unlike ODC, its degradation is not accelerated by antizyme.

Cation-induced thermostability of yeast and Escherichia coli pyrophosphatases.

Inorganic pyrophosphatases (PPiases) from both yeast and Escherichia coli were found to be stable against heat denaturation in the presence of Mg2+, as previously observed with the enzymes from thermophilic bacteria. No loss of activity was observed after 1 h of incubation at 50 degrees C and pHs between 6 and 9 in the yeast enzyme, and at 60 degrees C and pHs between 7.2 and 9.2 in the E. coli enzyme. Such an induced thermostability of the E. coli enzyme was detected when Mn2+, Co2+, Ca2+, Cd2+, and Zn2+ were added in place of Mg2+. On the other hand, the degree of induced thermostability of the yeast enzyme was dependent upon the divalent cations used, and Ni2+ and Cu2+ accelerated the heat inactivation. On adding the divalent cations, the difference spectra of the E. coli enzyme always showed negative peaks in the ultraviolet region, but those of the yeast enzyme changed again depending upon the divalent cations. The circular dichroism spectra in the near ultraviolet region of both enzymes greatly differed from each other, but both were not affected so much by adding the divalent cations unlike the thermophilic enzymes from Bacillus stearothermophilus and thermophilic bacterium PS-3. Yeast and E. coli PPiases did not cross-link with the anti-immunoglobulin G's from the thermophilic enzymes, but the thermophilic enzymes did with each other's antisera. The results in the present study indicated that the conformation of PPiase, in which the aromatic amino acid residues were buried in the interior of the protein molecule, was very important for the thermostability and also that the protein structures of PPiases from B. stearothermophilus and thermophilic bacterium PS-3 were very similar to each other, but were very different from those of the mesophilic enzymes.

Mucosa-associated lymphoid tissue lymphoma of the rectum that regressed spontaneously.

We report a case of mucosa-associated lymphoid tissue (MALT) lymphoma of the rectum that regressed spontaneously. A 76-year-old man visited our hospital because of positive faecal occult blood testing. Colonoscopic examination revealed a slightly yellowish protruded lesion with a grooved depression in the lower rectum and two flat elevations in the upper rectum. Microscopic and immunohistological studies led to a diagnosis of MALT lymphoma. As the patient exhibited severe renal dysfunction and angina pectoris, the lesions were left untreated. Three months later, the protruded lesion became flat and the other lesions became unclear. He was followed up closely with endoscopy, but no relapse of these lesions was detected 19 months after the diagnosis.