Sequence and localization of an ultraviolet (sws1) opsin in the retina of the Japanese sardine Sardinops melanostictus (Teleostei: Clupeiformes).

A full-length complementary (c)DNA encoding ultraviolet (UV)-sensitive opsin (sws1) was isolated from the retina of the Japanese sardine Sardinops melanostictus. The sws1 phylogenetic tree showed a sister group relationship with the Cypriniformes, following the ray-finned fish phylogeny. By expressing reconstituted opsin in vitro, it was determined that the maximum absorbance spectrum (λmax ) of sws1 is around 382 nm, being intermediate in position between two subtypes of sws1 pigment that are UV sensitive (λmax = 355-380 nm) and violet sensitive (λmax = 388-455 nm), which have been reported to date. The ocular media transmitted >20% transmittance of light in the range of 360-600 nm. In situ hybridization analyses revealed that sws1 messenger (m)RNA is localized in a central single cone surrounded by four double cones in a square mosaic. The square mosaic occupies the ventro-temporal quadrant of the retina and the in situ hybridization signals were dominant in this area suggesting that the fish may use UV vision when looking upward. Based on these results, considerable significances of potential UV sensitivity, in relation to characteristic habits of S. melanostictus, are discussed.

Purification and some properties of cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus.

An intracellular cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus E-244 isolated from soil was purified to a homogeneous state by means of Triton X-100 extraction, DEAE-Sepharose column chromatography, hydrophobic and molecular-sieve HPLC. The enzyme was estimated to have an Mr of 72,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 144,000 by HPLC gel filtration on TSK gel G 3000 SW. It had a pH optimum of 8.0, and the enzyme, stable at 25 degrees C and pH 5.5-9.5 for 24 h, was inactivated at 50 degrees C for 10 min. The enzyme hydrolyzed beta-cyclodextrin more effectively than linear maltooligosaccharides such as maltopentaose, maltohexaose and maltoheptaose or polysaccharides such as starch, amylopectin, amylose and pullulan.

Cloning and sequencing of the gene encoding tannase and a structural study of the tannase subunit from Aspergillus oryzae.

We cloned the Aspergillus oryzae tannase gene using three oligodeoxyribonucleotide (oligo) probes synthesized according to the tannase N-terminal and an internal amino acid (aa) sequence. The nucleotide (nt) sequence of the tannase gene was determined and compared with that of a tannase DNA complementary to RNA (cDNA) by means of reverse transcriptase PCR. The results indicated that there was no intron in the tannase gene and that it coded for 588 aa with a molecular weight of about 64,000. The tannase low-producing strain A. oryzae AO1 was transformed with the plasmid pT1 which contained the tannase gene, and tannase activities of the transformants increased in proportion to the number of copies. Tannase consisted of two kinds of subunits, linked by a disulfide bond(s) with molecular weights of about 30,000 and 33,000, respectively. We purified these subunits and determined their N-terminal aa sequences. The large and small subunits of tannase were encoded by the first and second halves, respectively. Judging from the above results, the tannase gene product is translated as a single polypeptide that is cleaved by post-translational modification into two tannase subunits linked by a disulfide bond(s). We concluded that native tannase consisted of four pairs of the two subunits, forming a hetero-octamer with a molecular weight of about 300,000.

Hydrolysis of branched cyclodextrins by a cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus E-244.

The action of a cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus E-244 on branched alpha- and beta-cyclodextrins was investigated. Glucosyl-alpha-cyclodextrin (6-O-alpha-D-glucosylcyclomaltohexaose) and maltosyl-alpha-cyclodextrin (6-O-alpha-D-maltosylcyclomaltohexaose) were hydrolyzed to 6(3)-O-alpha-D-glucosylmaltohexaose and 6(3)-O-alpha-D-maltosylmaltohexaose, respectively. Glucosyl-beta-cyclodextrin (6-O-alpha-D-glucosylcyclomaltoheptose) and maltosyl-beta-cyclodextrin (6-O-alpha-D-maltosyclomaltohepatose) were also mainly transformed to 6(4)-O-alpha-D-glucosylmaltoheptaose and 6(4)-O-alpha-D-maltosylmaltoheptaose, respectively. These results suggest that the cyclodextrin-hydrolyzing enzyme cleaves branched alpha- and beta-cyclodextrins at an alpha-1,4 linkage which is located furthest from the branching point on the cyclodextrin ring.

Photic regulation of arylalkylamine N-acetyltransferase 1 mRNA in trout retina.

Melatonin production in the pineal organ and retina is controlled by both light-dark cycles and a circadian clock via the oscillating activity of arylalkylamine N-acetyltransferase (AANAT) in most vertebrates. However, this clock regulation is absent in the rainbow trout (Oncorhynchus mykiss) pineal organ: the trout has two different AANAT genes (AANAT1 and AANAT2), and AANAT2 mRNA levels in the pineal organ did not exhibit circadian oscillation In this study, we confirmed by RT-PCR analysis that AANAT1 is expressed only in the retina, while AANAT2 is expressed in the pineal organ and brain. Real-time quantitative PCR analysis demonstrated that AANAT1 mRNA levels in the retina exhibited daily variations with high levels during the dark phase under light-dark cycles, but kept high and low titers under constant darkness and constant light, respectively. Thus, AANAT1 gene expression in the trout retina is regulated not by a circadian clock but by lighting conditions.

Inhibition of RNA synthesis differentially affects in vitro melatonin release from the pineal organs of ayu (Plecoglossus altivelis) and rainbow trout (Oncorhynchus mykiss).

The effects of actinomycin D (RNA synthesis inhibitor) and cycloheximide (protein synthesis inhibitor) on melatonin release from the cultured pineal organ of two teleosts with or without the circadian regulation of melatonin production (ayu Plecoglossus altivelis and rainbow trout Oncorynchus mykiss, respectively) were investigated. Actinomycin D decreased melatonin release from the pineal organ during the dark phase but there was a significant difference between the two species (22.2% for ayu and 59.1% for trout as compared with the respective control). This difference might be due to whether the circadian regulation via gene transcription of melatonin synthesis exists or not. On the other hand, cycloheximide decreased melatonin release to approximately 1% in both species, indicating that the fish pineal organ requires de novo protein synthesis to maintain rhythmic melatonin release.

A new enzymatic assay of urinary guanidinoacetic acid.

We describe a new enzymatic determination of urinary guanidinoacetic acid (GAA) with guanidinoacetate kinase (ATP: guanidinoacetate N-phosphotransferase, EC 2.7.3.1), which does not require a blank to correct for endogenous constituents (ADP and pyruvate). In the first step, pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) were used to eliminate endogenous constituents (ADP and pyruvate) in the presence of phosphoenolpyruvate and NADH. In the second step, urinary GAA was phosphorylated in the presence of ATP by guanidinoacetate kinase to form phosphoguanidinoacetate and ADP. The resultant ADP was sequentially measured at 340 nm in a coupled reaction catalyzed by pyruvate kinase and lactate dehydrogenase. The standard curve was linear up to 20 mg/dl for standard solutions of GAA. Analytical recovery of GAA added to normal urines ranged from 97.0 to 103.2% (mean 100.7%). The within-run and between-run studies gave CV values of less than or equal to 3.6% and less than or equal to 4.8%, respectively. No significant interference by endogenous urinary compounds were observed with the proposed method under this study. The results obtained by the present method correlated well with those obtained by a high-performance liquid chromatographic method. This method is accurate and simple, and less time-consuming than those previously reported. We determined the concentrations of GAA in 24-h urine samples by the proposed method, and observed that the urinary excretion of GAA decreased markedly in patients with renal failure.

Molecular Cloning and Characterization of a cDNA Encoding the Retinal Arylalkylamine N-Acetyltransferase of the Rainbow Trout, Oncorhynchus mykiss.

Melatonin synthesis in the retina as well as in the pineal gland exhibits daily variations with higher levels during the dark phase of light-dark cycles. To analyze the molecular mechanism of melatonin synthesis in the retina, we have cloned, sequenced and characterized a putative cDNA for arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87), a rate-limiting enzyme in melatonin production, from the retina of the rainbow trout (Oncorhynchus mykiss). The trout AANAT cDNA (1,585 bp) contains an open reading frame encoding 240 amino acid protein (predicted molecular weight, 27,420) that is 51-65% identical to avian and mammalian AANAT. The trout retinal AANAT protein contains motifs A and B that are conserved among the N-acetyltransferase superfamily and eight potential phosphorylation sites. Southern blot analysis demonstrated that the protein is expressed by a single copy gene. A single AANAT transcript (1.6 kb) was detected in the retina but not in the liver by Northern blot analysis. The levels of AANAT mRNA in the retina exhibited day-night changes with 3.3-fold increase at night. These results indicate that in the rainbow trout retina, the activity of AANAT and thus melatonin synthesis are regulated at least in part at the transcriptional level.

Safety evaluation of tannase enzyme preparation derived from Aspergillus oryzae.

Tannase is an acylhydrolase enzyme preparation from Aspergillus oryzae that can be used as a processing aid for the manufacture of cold water-soluble tea beverages. A 91-day oral toxicity study in the rat and a gene mutation study in Salmonella typhimurium were performed to establish the safety of the enzyme preparation for the consumer. General toxicity was low, with no adverse effects observed at the highest dose tested, 1% in the diet. There was no evidence of mutagenic potential with or without metabolic activation. These results, together with knowledge of the production organism and the chemical and microbiological characterization of the enzyme preparation, indicate that tannase can be regarded as safe for its intended use in processing tea.

Long-term postoperative analysis of nutritional status after limited gastrectomy for early gastric cancer.

BACKGROUND/AIMS: An examination was made of the postoperative long-term effects of limited gastrectomy (LG) on nutritional status, as one of the factors that influence quality of life. METHODOLOGY: Nutritional status in 33 patients who underwent LG for early gastric cancer, was compared with that of 36 patients who underwent standard gastrectomy (SG). Nutritional conditions were assessed preoperatively, and then 1 and 2 weeks, 1 and 6 months, and 1, 2 and 3 years after surgery. RESULTS: Postoperative recovery of both body weight and body fat mass was greater in LG than SG, and there were significant differences between the two groups of patients from six months after surgery until three years had elapsed. CONCLUSION: LG minimized the extent of nutritional impairment during long-term recovery from surgery, as compared with SG. LG would seem to be a suitable operative procedure for some patients with mucosal gastric cancer, without reducing radicality for cancer.

Chronic Sarcocystis infections in slaughtered cattle.

Parasitological, histopathological and immunohistochemical examinations were carried out on three slaughtered cattle with many nodules in all the striated muscles. At necropsy, many yellowish green rice-grain sized nodules including cheesy contents were observed in all the striated muscles. Histopathologically the nodules were granuloma principally consisting of eosinophiles. No Sarcocystis cysts nor bradyzoites were found in the nodules, but intact sarcocysts were found in the normal tissues surrounding the nodules. The central necrotic focus of nodules showed intense positive responses against anti-Sarcocystis cruzi rabbit serum by immunohistochemical examination. From the above findings the slaughtered cattle were diagnosed as chronic sarcocystiasis.

Comparative studies of color fields, visual acuity fields, and movement perception limits among varsity athletes and non-varsity groups.

This paper examined effects of sports practice on patterns of color fields, limits of peripheral movement perception, and visual acuity field by comparing varsity ball players and non-varsity control groups. The first study measured extent of color fields and limits of horizontal and vertical meridians for peripheral movement perception of 139 college students. The second study tested visual acuity fields of female and male basketball players and female and male controls. The first study indicated that athletes had wider limits for horizontal movement perception, while the non-athletes had better vertical movement perception limits. Basketball players demonstrated color fields and limits for peripheral movement perception superior to those of soccer players. In the second study, athletes did not have any wider visual acuity fields than non-athletes, but their movement-perception limits were significantly wider than those of non-athletes.

Production of thermostable alkaline proteases by thermophilic Streptomyces.

Conditions for the production of thermostable proteases (alkaline proteinase and carboxypeptidase) by a thermophilic streptomycete (Streptomyces rectus var. proteolyticus) were investigated in 20-liter volumes. Proteinase production was affected by the concentration of defatted soybean powder, its optimum being 1.2% in medium containing 2.0% soluble starch. Relatively high concentration of phosphate (0.3 to 0.4% K(2)HPO(4)) was needed for the maximum enzyme production. A large inoculum size (5 to 10%) was favorable, but the inoculum age did not significantly influence the production. The yield increase of 20 to 30% was obtained by feeding of medium during fermentation. The optimal temperature for proteinase production was 50 C, at which the maximal rate of production was 66.2 proteinase units per ml per hr, whereas at 40 C it was 9.0. Production at 50 C reached the maximum within 12 to 16 hr. The optimal agitation rate was different for the production of proteinase and carboxypeptidase, 400 rev/min for the former and 500 rev/min for the latter. The optimal aeration for proteinase production was 20 to 30 liters/min at 400 rev/min, whereas carboxypeptidase production was not markedly affected by aeration rate. The possibility that carboxypeptidase production was correlated with the shear of mycelium was discussed.

Induction of diffuse necrotizing enterocolitis by anticancer chemotherapy.

Fulminant, necrotizing colitis is a frequent, and generally fatal, complication of severe granulocytopenia, occurring during the treatment of hematological malignancies. In these cases, the patient complains of severe peritonitis, including nausea, vomiting, abdominal pain, diarrhea or melena, and a high temperature. Here, a rare case of anticancer chemotherapy-induced diffuse necrotizing enterocolitis throughout the entire intestinal tract is presented, which developed in a patient who did not have a hematologic malignancy but who had colon cancer, the only clinical symptom of which was watery stools, without any evidence of peritoneal irritation. Full attention should be paid to progressive diarrhea in patients with malignancies during anticancer chemotherapy.

The argyrophilic nucleolar organizer region (Ag-NOR) score in invasive ductal carcinoma of the pancreas.

The biological malignancy of pancreatic carcinoma was evaluated by determining the score of the argyrophilic nucleolar organizer region (Ag-NOR) in resected specimens, and comparing it with the various clinicopathological factors and long-term results of 38 patients who underwent surgical resection for invasive ductal carcinoma of the pancreas between 1977 and 1992, in whom the Ag-NOR could be stained. The Ag-NOR analysis of pancreatic carcinomas from the 38 patients resulted in a mean Ag-NOR score of 3.82 +/- 0.62, which was significantly (P < 0.01) higher than the mean score of 1.72 +/- 0.28 observed in normal pancreatic tissues obtained from 20 of these patients. The mean Ag-NOR score significantly increased in patients with anterior capsular infiltration (s) (P < 0.01), posterior tissue infiltration (rp) (P < 0.01), and lymph node metastasis (n) (P < 0.05) compared to those without these factors. The rate of curability A or B was only 13.0% in patients with a high Ag-NOR score of > or = 3.80 in comparison to 66.7% in those with a low Ag-NOR score of < 3.80. The 3-year survival rate was significantly (P < 0.05) higher in the low Ag-NOR score group than in the high Ag-NOR score group. These results suggest that the Ag-NOR score can serve as an indicator of the biological malignancy of pancreatic carcinoma, and of the patients' prognosis.

Negative correlation between cholecystectomy and the subsequent development of large bowel carcinoma in a low-risk Japanese population.

The incidence of previous cholecystectomy in a series of 541 patients with colorectal cancer and 1832 patients with stomach cancer was studied. Five patients (0.92 percent) with colorectal cancer and eight (0.44 percent) with stomach cancer had undergone previous cholecystectomy. To avoid biases in the two groups of patients, 416 pairs of patients, comparable in sex, age, and time of admission for cancer treatment, were matched from each group to compare the number of patients who had undergone previous cholecystectomy. Within these matched pairs, three patients with colorectal cancer and two with stomach cancer had histories of cholecystectomy. Hence, no substantial difference was noted between the two groups. In a follow-up study of 461 patients who had undergone cholecystectomy for gallstones, large bowel carcinoma and stomach carcinoma developed in one and six patients, respectively, during an observation period of four to 36 years. The ratio of patients with large bowel cancer to those with stomach cancer observed in this survey was almost equal to the value estimated for the population of Tottori Prefecture, where the majority of the patients reside. The incidence of large bowel carcinoma is not increased among cholecystectomized patients in a low-risk Japanese population.

Effective TAE therapy using Lipiodol with epirubicin for liver metastases of nonfunctioning islet cell carcinoma of the pancreas.

We report the response of two patients with advanced nonfunctioning islet cell carcinoma of the pancreas with liver metastases treated with a combination of surgi-cal resection and transarterial embolization (TAE), using Lipiodol with epirubicin. After pretreatment evaluation, the two patients were diagnosed with nonfunctioning islet cell carcinoma of the pancreas with liver metastases. Preoperatively, in both patients, TAE was performed through the hepatic arteries, using Lipiodol and sponzel plus epirubicin. Surgical resection of the primary tumor (radical distal pancreatectomy and pancreaticoduodenectomy) was performed. After surgical resection and evaluation of the malignant histopathological features of the neoplasms, chemotherapy, which included oral 5-fluorouracil (FU), and transarterial infusion therapy, using Lipiodol with epirubicin, was administered to the patients. Follow-up evaluation of the two patients by computerized tomography (CT) scan showed a reduction in the size of the metastatic hepatic masses after several chemoembolizations through the hepatic arteries. This combined treatment modality may be an effective therapeutic strategy for improved management of patients with advanced nonfunctioning islet cell carcinoma of the pancreas with liver metastases.

Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity.

A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding.