[Molecular mechanism of cell membrane lipids].
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Acylation↗
Biomedical subjects
Y Sakano
Publications and source records attributed to Y Sakano.
Molecular cloning and heterologous expression of the isopullulanase gene from Aspergillus niger A.T.C.C. 9642.
Isopullulanase (IPU) from Aspergillus niger A.T.C.C. (American Type Culture Collection) 9642 hydrolyses pullulan to isopanose. IPU is important for the production of isopanose and is used in the structural analysis of oligosaccharides with alpha-1,4 and alpha-1,6 glucosidic linkages. We have isolated the ipuA gene encoding IPU from the filamentous fungi A. niger A.T.C.C. 9642. The ipuA gene encodes an open reading frame of 1695 bp (564 amino acids). IPU contained a signal sequence of 19 amino acids, and the molecular mass of the mature form was calculated to be 59 kDa. IPU has no amino-acid-sequence similarity with the other pullulan-hydrolysing enzymes, which are pullulanase, neopullulanase and glucoamylase. However, IPU showed a high amino-acid-sequence similarity with dextranases from Penicillium minioluteum (61%) and Arthrobacter sp. (56%). When the ipuA gene was expressed in Aspergillus oryzae, the expressed protein (recombinant IPU) had IPU activity and was immunologically reactive with antibodies raised against native IPU. The substrate specificity, thermostability and pH profile of recombinant IPU were identical with those of the native enzyme, but recombinant IPU (90 kDa) was larger than the native enzyme (69-71 kDa). After deglycosylation with peptide-N-glycosidase F, the deglycosylated recombinant IPU had the same molecular mass as deglycosylated native enzyme (59 kDa). This result suggests that the carbohydrate chain of recombinant IPU differed from that of the native enzyme.
Amino Acid Sequence↗
A classification of dextran-hydrolysing enzymes based on amino-acid-sequence similarities.
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Amino Acid Sequence↗
The significance of tumor necrosis factor (TNF) levels for rejection of joint allograft.
This paper describes the efficacy of utilizing Tumor Necrosis Factor-alpha (TNF) as a detectable parameter of acute rejection after bone allografts. The authors used a bone-joint allograft model, transferring vascularized allograft knee joints across a major histocompatibility barrier, using three groups of rats divided into controls, non-immunosuppressed, and immunosuppressed with cyclosporine. The paper supplies preliminary information suggesting that TNF may be a marker for early bone-joint allograft rejection.
Animals↗
Two components of cell-bound isopullulanase from Aspergillus niger ATCC 9642--their purification and enzymatic properties.
Cell-bound isopullulanase (pullulan 4-glucanohydrolase: EC 3.2.1.57, IPU) from Aspergillus niger ATCC9642 [Y. Sakano et al., Denpun Kagaku, 37, 39-41 (1990)] was separated into two active components, IPU F1 (pI = 5.0) and IPU F2 (pI = 4.9), using a Mono-P HR 5/20 column. The substrate specificity on pullulan and panose, specific activity, optimum pH, pH stability, and susceptibility to certain chemical reagents were similar between IPU F1 and IPU F2. IPU F1 and F2 had an identical N-terminal amino acid sequence, A-V-T-A-D-N-S-Q-L-L. However, IPU F1 contained more total carbohydrate (15.3%) than IPU F2 (12.4%). SDS-polyacrylamide gel electrophoresis showed that the molecular weight of IPU F1 (71,000) was greater than that of IPU F2 (69,000). After deglycosylation of IPU F1 and F2 with peptide-N-glycosidase F, the molecular weights of IPU F1 and F2 became 59,000.
Amino Acid Sequence↗
Adolescent reinforcement survey schedule: summary of research and future directions.
Three completed studies with the Adolescent Reinforcement Survey Schedule are reviewed and further studies are discussed.
Adolescent↗
[Mesonephric adenocarcinoma arising in the female urethral diverticulum].
A forty four-year-old house-wife presented with gross hematuria and difficulty on urination of a year and 3 months duration. Transvaginal examination showed a hen egg-sized soft mass on the anterior vaginal wall. Urine cytology revealed many clusters of malignant cells suggestive of adenocarcinoma. Cystourethrography revealed two urethral diverticula, whose orifices were cystoscopically located at the proximal and distal side of urethral sphincter, respectively. By vaginal digital pressing, a soy-bean sized papillary tumor came out of the proximal diverticulum. Histopathological examination of the biopsied tumor suggested poorly differentiated transitional cell carcinoma with inverted growth. Under the diagnosis of carcinoma arising in the urethral diverticulum, anterior pelvic exenteration with formation of Indiana pouch was carried out. The tumor in the proximal diverticulum was histologically composed of a variety of adenocarcinomatous pattern, such as tubular, papillary and cystic structure with a distinctive pattern of tubules lined by a superficial layer of hobnail cells, leading to the diagnosis of mesonephric adenocarcinoma of urethral diverticulum. Postoperative radiation therapy was given because the diverticulum was adherent to the pubic bone, though lymph node metastasis was negative. She has been well with no evidence of the disease for 1 year and 4 months after the operation. Although the histogenesis of female urethral mesonephric adenocarcinoma was still controversial, this case seems to be the forty fourth case in the world literature.
Adult↗
Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing alpha-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47.
Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVA I, an extracellular enzyme, and TVA II, an intracellular enzyme. Both enzymes hydrolyze pullulan to produce panose, and also hydrolyze cyclodextrins. We cloned and sequenced the TVA I gene. The TVA I gene consisted of 1833 base pairs, and the deduced primary structure was composed of 611 amino-acid residues, including an N-terminal signal sequence consisting of 29 amino-acid residues. The similarity between the amino-acid sequence of mature TVA I with those of other pullulan/cyclodextrin-hydrolyzing enzymes, such as TVA II and Bacillus stearothermophilus neopullulanase, was only 30%, although that of TVA II with neopullulanase was 48%. TVA II prefers specific small oligosaccharides and alpha- and beta-cyclodextrins. Whereas kcat/Km values of TVA I for pullulan were larger than that of TVA II, and TVA II could not hydrolyze starch completely. TVA II was inhibited by maltose, the hydrolysate of starch, which seems to be the reason for inefficient hydrolysis of starch. These kinetic properties indicate that TVA I and TVA II have differential physiological roles in sugar metabolism extracellularly and intracellularly, respectively.
Amino Acid Sequence↗
Reddish Escherichia coli cells caused by overproduction of Bacillus stearothermophilus uroporphyrinogen III methylase: cloning, sequencing, and expression of the gene.
During shotgun cloning of an amylase gene, we found a transformant of Escherichia coli with a reddish color. The transformant produced highly water-soluble red pigments the molecular masses of which were less than 3000. The plasmid harbored by the transformant contained a DNA fragment derived from a strain of Bacillus stearothermophilus. Truncation of the insert DNA showed that an 1.1-kbp Sau 3A-SalI fragment was responsible for the reddish colony. An open reading frame was found in the nucleotide sequence of the 1.1-kbp DNA fragment. The production of the red pigment was accompanied by a colorless 28-kDa protein. The sequence of the 28-kDa protein was highly homologous to bacterial uroporphyrinogen III methylases participating in corrinoid biosynthesis. The 28-kDa protein was found to be a thermostable uroporphyrinogen III methylase.
Amino Acid Sequence↗
[Primary retroperitoneal pure choriocarcinoma].
A thirty-year-old clerk presented with a history of left supraclavicular lymph node swelling of a month duration and newly detected multiple lung and mediastinal tumors. Abdominal computed tomography showed two retroperitoneal masses, but no abnormality in both testes was found by physical and ultrasonographic examination. Biopsy of supraclavicular lymph nodes revealed pure choriocarcinoma. Furthermore, beta-subunit of human chorionic gonadotropin was elevated up to 700 ng/ml but alfafetoprotein was normal, resulting in the diagnosis of primary retroperitoneal pure choriocarcinoma. Two cycles of combination chemotherapy with etoposide (VP-16), ifosfamide and cisplatin (VIP) was given and followed by bleomycin, etoposide and cisplatin (BEP) (2 cycles) because of cystitis symptom due to ifosfamide. Since post chemotherapeutic evaluation revealed only partial response, we surgically removed all residual masses in the lung, mediastinum and retroperitoneum, which histologically proved to be necrotic fibrous tissue. He has remained disease-free 15 months after surgery without additional therapy. In order to improve prognosis of primary retroperitoneal pure choriocarcinoma, intense chemotherapeutic regimen like VIP should be given, regularly repeated and followed by aggressive surgical resection of residual masses.
Adult↗
[Leiomyosarcoma of the prostate: a case report of remission for 9 years by radiotherapy].
A 68-year-old-man with chief complaints of pollakisuria and lower abdominal discomfort was referred to our hospital on September 19, 1983. A histopathological study of the transrectal needle biopsy specimens revealed a malignant tumor of the prostate with spindle-shaped cells. The patient had been considerably improved by radiotherapy. However, 9 years later, the tumor recurred and the histopathological study showed the same findings as the initial biopsy and furthermore the recurrent tumor was diagnosed as a leiomyosarcoma of the prostate by immunohistochemical stain. He was unresponsive to chemotherapy and died 11 years after initial diagnosis.
Aged↗
A convenient enzymatic synthesis of 4(2)-alpha-isomaltosylisomaltose using Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II).
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Carbohydrate Conformation↗
Japanese high school and college students' responses to the Adolescent Reinforcement Survey Schedule.
Factor-analyzed correlations among items of the Japanese Adolescent Reinforcement Survey Schedule by Japanese high school students (N = 939) and college students (N = 500) were compared to investigate the changes in reinforcers between mid- and late adolescence, gender differences, and the specific groupings of reinforcers which suggest certain interventions for either or both groups of adolescents. The factor analysis yielded ten interpretable factors in both groups. These factors were similar and did not suggest a dramatic shift in reinforcers between mid- and late adolescence. Items related to heterosexual activities and antisocial behaviors were rated as more pleasurable by males and items related to interpersonal relationships and academic activities were rated as more pleasurable by females. Since both groups of students attach high reinforcement value to interpersonal interaction with peers and family members, interventions focusing on social skills development might be popular and well attended.
Adolescent↗
Active oxygen-induced modification alters properties of collagen as a substratum for fibroblasts.
Acid-soluble collagen from rat skin was modified by active oxygen in vitro, and properties of the modified collagen as a substratum for fibroblasts were studied. When collagen was treated with ascorbate-copper ion systems, cross-linking and a little degradation occurred rapidly. The cells attached but spread poorly on the modified collagen gel as compared with on the untreated collagen gel. On the other hand, when collagen was treated with H2O2-copper ion systems, only degradation of collagen molecule occurred rapidly. This treatment did not affect the attachment and spreading of the cells on the collagen gel, but when the incubation was continued for a long time, the cells migrated actively and gathered. Thymidine incorporation by the cells was suppressed on both modified collagen gels as compared with that on untreated collagen gel, and the extent of the suppression on the H2O2-copper-treated collagen was larger than that on the ascorbate-copper-treated collagen. These results indicate that the active oxygen-induced cross-linking and degradation significantly alter properties of collagen as a substratum for fibroblasts.
Animals↗
Interaction of human plasma fibronectin with alpha-elastin.
Because there are contradictory reports about the interaction of plasma fibronectin with elastin, we investigated the interaction in vitro. When human plasma was applied to an alpha-elastin-Sepharose column at 4 degrees C, the column-binding fraction contained fibronectin. When isolated plasma fibronectin was applied to the same column at 4 degrees C, most of the fibronectin bound to the column and was eluted with 1 M KBr. However, the binding affinity of plasma fibronectin to the alpha-elastin-Sepharose column was much weaker at 25 degrees C than at 4 degrees C. The elastin-plasma fibronectin interaction was further confirmed by demonstrating the binding of alpha-elastin to fibronectin on polyvinylidene difluoride membranes using an alpha-elastin specific antibody. The elevation of the surface hydrophobicity of plasma fibronectin at 4 degrees C was observed by hydrophobic chromatography, using alkyl-Sepharose columns. It seems that the binding of plasma fibronectin to alpha-elastin involves hydrophobic interaction, which is affected by temperature and possibly by other factors.
Chromatography, Affinity↗
A neopullulanase-type alpha-amylase gene from Thermoactinomyces vulgaris R-47.
Shimizu et al. and ourselves have reported some enzymatic properties of an alpha-amylase from Thermoactinomyces vulgaris R-47 that hydrolyzed pullulan to produce panose [M. Shimizu et al., Agric. Biol. Chem., 42, 1681-1688 (1978); Y. Sakano et al., Agric. Biol. Chem., 46, 1121-1129 (1982)]. In this study, we cloned a gene for an alpha-amylase, which was different from the one mentioned above but also hydrolyzed pullulan to produce panose, from T. vulgaris R-47, and analyzed the entire primary structure of the gene. We designated the previously reported enzyme as T. vulgaris alpha-amylase I (TVA I), and this novel enzyme as T. vulgaris alpha-amylase II (TVA II). The nucleotide sequence had an open reading frame of 1755 base pairs corresponding to a protein of 585 amino acid residues. Although this novel alpha-amylase, TVA II, hydrolyzed both pullulan and starch, the ratio of pullulan-hydrolyzing activity to starch-hydrolyzing activity of the enzyme was higher than that of TVA I, and the primary structure of the enzyme resembled neopullulanase, which scarcely hydrolyzed starch, rather than that of TVA I.
Amino Acid Sequence↗
Cloning and nucleotide sequence of the gene (citA) encoding a citrate carrier from Salmonella typhimurium.
A cryptic citrate transport gene (citA) from Salmonella typhimurium chromosome was cloned and its nucleotide sequence was determined. The cloned plasmid conferred citrate-utilizing ability on wild-type Escherichia coli, which cannot grow on citrate as the sole source of carbon. The resultant E. coli transformant was able to transport citrate. A 1,302-base-pair open reading frame with a preceding ribosomal binding site was found in the cloned DNA fragment. The 434-amino-acid protein that could be translated from this open reading frame is highly hydrophobic (69% nonpolar amino acid residues), consistent with the fact that the transport protein is an intrinsic membrane protein. The molecular weight of this protein was calculated to be 47,188. The gene sequence determined is highly homologous to those of Cit+ plasmid-mediated citrate transport gene, citA, from E. coli, the chromosomal citA gene from Citrobacter amalonaticus and the chromosomal cit+ gene from Klebsiella pneumoniae. The hydropathy profile of the deduced amino acid sequence suggests that this carrier has 12 hydrophobic segments, which may span the membrane lipid bilayer.
Amino Acid Sequence↗
Comparison of factor-analyzed Adolescent Reinforcement Survey Schedule (ARSS) responses from Japanese and American adolescents.
The Adolescent Reinforcement Survey Schedule (ARSS) was administered to a sample of male and female late adolescent college students from Japan (N = 500). The responses to the ARSS are factor analyzed using a principal component method. The results of the factor-analyzed ARSS from the Japanese sample are compared and contrasted with the results from a previous study (Holmes et al., 1987) in which ARSS was administered to a sample of American male and female college students (N = 231). Both the American and Japanese samples produced 10 interpretable factors. A recommendation is made to replicate the present study with groups of early and mid-adolescents in each culture in order to study shifts in reinforcers during different periods of adolescent development.
Adolescent↗