The possibilities of novel catalytic scanning probe lithography (cSPL) on starch using α-amylase was investigated. For this thin homogeneous layers of starch with good coverage were prepared by spin coating a starch solution on a silicon base. Amylase immobilized to an atomic force microscopy (AFM) cantilever tip were prepared and dragged along a spin coated starch surface. This after verifying the enzyme immobilization method using (3-Aminopropyl)triethoxysilane (APTES) on a silicon surface. In addition an unmodified cantilever tip were dipped in amylase solution and were dragged along a starch surface to investigate possibilities of dip-pen nanolithography (DPN). The preliminary experiments with AFM based enzymatic lithography were promising but non-conclusive. There are still many parameters not fully explored such as water availability, activity and reach of the amylase, speed of the enzymatic process and difference in structure between the starch and the shorter saccharides that are left after the hydrolysis

Ovarian cancer (OC) accounts for 4% of all cancer cases and 4.2% of all cancer deaths worldwide. OC is the most lethal gynecological cancer because it lacks early disease symptoms and does not have a specific diagnostic marker. As a result, more than 70% of OC patients are diagnosed in later stages when the disease has already metastasized and the 5-year survival rate has decreased to less than 20% compared with approximately 90% survival for women diagnosed with early stage disease. Therefore, I initiated my studies with a computational analysis of the 27 most commonly reported literature-derived ovarian cancer (LDOC) protein biomarkers. I found that LDOC protein biomarkers share many biochemical features including a preponderance for a stable protein structure, the ability to be secreted, and functionality related to extracellular matrix (ECM) modification, immune response and/or energy production. Subsequently, I analyzed the human proteome to identify proteins that also share these biochemical features. Of the 70,616 proteins in the human proteome, 683 proteins were found to have similar biochemical features to the 27 LDOC proteins. I also identified a subset of 21 potential additional protein regulators of ovarian cancer (APROC) that interact with LDOCs. Three of the APROCs identified were amylase proteins AMY1A, AMY2A, and AMY2B which cleaves alpha 1, 4-glycosidic bonds in polysaccharides. Amylase is reportedly overexpressed in and secreted by ovarian tumors but its functional contribution to OC remains unknown[1]. In this thesis, I posit that amylase contributes to OC invasion. I initiated my studies by computational characterizing the different amylase isozymes to predict which amylase isozyme(s) is most likely overexpressed in and contributory to OC invasion. I found that AMY1 and AMY2B have unique regions of disorder and unique phosphorylation sites indicating that AMY1 and AMY2B would be more likely to interact with other proteins, and to be easily secreted. Using OC patient serum samples, I was able to validate AMY1 and AMY2B overexpression by western immunoblotting. I then developed an in vitro model system to study the molecular contribution of amylase to OC invasion using normal ovarian surface epithelial (IOSE) and OC cell lines. I showed that OC cells generally overexpress and secrete metabolically active amylase isozymes AMY1 and AMY2B. Abrogating amylase activity using siRNA silencing technology decreased the capacity of OC cells to invade collagen coated Boyden chambers and increased sulfated glycosaminoglycans (sGAG) production. Since a survey of OC cell lines indicated that cancer cells have a bulkier glycocalyx compared to IOSE cells and immunogold labeling studies indicated the presence of amylase within the immediate OC microenvironment, my data suggest that, by cleaving alpha 1, 4-glycosidic bonds in glycoconjugates present within ECM, amylase may remodel the ECM to promote an invasive cancer phenotype. Amylase is therefore a target for therapeutic intervention in OC patients with hyperamylasemia. I established Spirulina, a dietary supplement, as a novel transcriptional inhibitor of amylase. Spirulina inhibited amylase expression in OC cell lines at both the message and protein levels. Spirulina reduced OC cell invasion and migration in vitro, putatively by decreasing amylase expression.

<p>For this dissertation, four experiments were conducted to evaluate the effect of dietary addition of exogenous protease and amylase enzymes on growth performance and nutrient utilization in broiler chickens. An additional fifth experiment was designed to determine the role of central and peripheral appetite regulators in birds fed diets deficient in dietary phosphorus (P). This arose from consistent reports in literature of a direct effect of dietary P concentration on feeding response in broiler chickens. </p><p>Experiment 1 examined the growth performance and protein utilization responses of broiler chickens to purified trypsin inhibitors (TI) and exogenous protease additions. Experimental diets were arranged as a 2 × 2 factorial with factors being dietary TI (1,033 or 10,033 TIU/g) and exogenous protease (0 or 15,000 PROT/kg). Protease supplementation improved BW gain (<i>P < </i>0.01) and gain to feed ratio (<i>P < </i>0.05) of birds. The relative weight of pancreas increased (<i>P < </i>0.05) with added TI on d 14 and 21 but was reduced (<i>P < </i>0.001) with protease supplementation. Apparent ileal digestibility (AID) of all amino acids (AA), except methionine, decreased (<i>P < </i>0.001) with added TI, but increased (<i>P < </i>0.05) with protease supplementation. Duodenal trypsin and chymotrypsin activities were reduced (<i>P < </i>0.05) with added TI but increased <i>(P < </i>0.01) with protease supplementation. It was concluded that dietary addition of purified TI negatively affects nutrient utilization by broiler chickens and that the efficacy of the exogenous protease might be independent of dietary TI concentration. A follow-up experiment was conducted (Experiment 2) to evaluate the impact of TI and exogenous protease supplementation on endogenous AA loss in broiler chickens. Four diets were arranged as a 2 × 2 factorial with factors being dietary TI (0 or 8,000 TIU/g) and exogenous protease (0 or 15,000 PROT/kg). There was no effect of TI, exogenous protease, or their interaction on growth performance of birds. Endogenous nitrogen (N) loss and all AA (except Cys) increased (P < 0.05) due to added dietary TI. Exogenous protease had no effect on endogenous loss of N and all AA. The AID of Ca, Fe, Mg, Mn, and Cu was reduced (P < 0.05) by added dietary TI. Protease supplementation improved the AID of Cu (P < 0.01) and K (P < 0.05). Secretion of crude mucin and sialic acid (g/kg DM intake) increased (P < 0.05) with increased dietary TI and was not recovered by protease supplementation. It was concluded from this study that TI increases the endogenous loss of AA, reduces the digestibility of minerals in broiler chickens, and that exogenous protease had no effect on endogenous AA flow, irrespective of added dietary TI. </p><p>In Experiment 3, the responses of broiler chickens fed corn-soybean meal-based diets to dietary α-amylase supplementation during 4 growth phases were evaluated. Birds were assigned to 8 treatment diet in a 2 × 4 factorial arrangement of 2 dietary levels of α-amylase supplementation (0 or 80 kilo-Novo alpha amylase units (KNU) per kg diet) and 4 post hatching growth phases (d 0 to 11, d 11 to 21, d 21 to 42, or d 42 to 56). Body weight gain and feed efficiency of birds improved (<i>P</i> < 0.01) with α-amylase supplementation. There were main effects of α-amylase, growth phase and interaction (<i>P</i> < 0.01) on AID of starch. The total tract retention (TTR) of starch increased (<i>P</i> < 0.05) with amylase supplementation but was not different across growth phases. Amylase supplementation improved (<i>P</i> < 0.05) gross energy utilization in birds, and specifically, during d 11 to 21 post hatching, the viscosity of jejunal digesta and pancreatic amylase activity increased (<i>P</i> < 0.01) with amylase supplementation. The conclusion from the study was that the growth phase of birds may affect the response to exogenous amylase. Following the result of this study, Experiment 4 was conducted to evaluate the effect of amylase supplementation on starch and energy digestibility at various intestinal sites in broiler chickens. Experimental diets comprised 3 concentrations of α-amylase supplementation (0, 80, or 160 KNU/kg diet) and sampling was done on 4 intestinal sites: anterior jejunum (AJ), posterior jejunum (PJ), anterior ileum (AI) and posterior ileum (PI). There were linear and quadratic (<i>P</i> < 0.01) responses of increasing α- amylase supplementation on starch and energy digestibility at the PJ and AI, with only linear effects on TTR of starch (<i>P</i> < 0.05). A linear increase in starch disappearance and digestible energy (kcal/kg) was observed (<i>P</i> < 0.01) with digesta flow from AJ to PJ with increasing amylase supplementation, which may be related to the observed decrease in the viscosity of the jejunal digesta (<i>P</i> < 0.05). Results from this experiment demonstrate the efficacy of exogenous amylase to improve starch, and energy digestibility in broiler chickens, with the highest impact observed in the posterior jejunum.</p><p>A final study (Experiment 5) was conducted to evaluate the impact of dietary phosphorus (P) concentration on hypothalamic molecular regulation of appetite by broiler chickens. Birds were randomly assigned to 3 experimental diets which contained 1.2 (P-deficient), 2.8 (P-marginal) or 4.4 (P-adequate) g/kg non-phytate P (nPP). A decrease in feed intake and BW gain was observed (P < 0.001) in birds fed the P-deficient diet. There was upregulation (P < 0.05) in the mRNA expression of Sodium-phosphate cotransporter (NaPi-IIb), anorexia-related hypothalamic cholecystokinin receptor (CCKAR) and melanocortin receptors (MC3R and MC4R) in birds fed P-deficient diets, whereas cholecystokinin (CCK) mRNA was downregulated (P < 0.01). It may be concluded that a deficiency in dietary P decreases feed intake in broiler chickens by altering the expression of anorexigenic genes in the gut and hypothalamus.</p>

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18315号 / 農博第2040号 / 新制||農||1020(附属図書館) / 学位論文||H26||N4822(農学部図書室) / 31173 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 安達 修二, 教授 入江 一浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Antioxidant-rich plant foods can inhibit starch and lipid digestion that are relevant to the management of type-II diabetes. Our objective was to compare the three phenolic substances (total phenolic, total flavonoids, and condensed tannin content) in crude, semi-purified extracts from eight types of foods (purified by XAD-7 column), five fractions (semi-purified extracts fractionated by Sephadex LH-20 column) from black legumes, and to compare their antioxidant capacities. The IC50 values of these crude extracts, semi-purified extracts and fractions against alpha-amylase, alpha-glucosidase and lipase were measured. Results showed that Fraction V from black soybean had the lowest IC50 value (0.25 mg/mL) against alpha-amylase; Fraction V from black bean had the lowest IC50 value (0.25 micro gram/mL) against alpha-glucosidase; Fraction IV of black bean had the lowest IC50 value (76 micro gram/mL) against lipase; myricetin showed the lowest IC50 value against alpha-amylase, alpha-glucosidase and lipase.

Acute pancreatitis is the most common serious complication of endoscopic retrograde cholangiopancreatography (ERCP) resulting in significant morbidity and occasional mortality. Post-ERCP pancreatitis (PEP) has been recognized since ERCP was first performed, and many studies have shown a consistent risk that must be balanced against the many benefits of this procedure. This review will discuss the pathogenesis, epidemiology, potential risk factors, and clinical presentation of PEP. Moreover, it will discuss in detail the most recent updates of PEP prevention and management.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24247号 / 農博第2526号 / 新制||農||1094(附属図書館) / 学位論文||R4||N5418(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 井上 和生, 教授 谷 史人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Utilization of a fat substitute (100% replacement) with and without added fungal protease, fungal amylase, and bacterial amylase in a muffin was compared to a full fat counterpart. The enzymes were evaluated independently and in combination with each other. Physical and sensory data were reported with a p<0.05 significance level. The physical tests indicated that there were no significant differences (p>0.05) among any of the variations in volume, water activity (freshly baked, and after 24 and 48 hours storage), crumb L values and crust Land b values. The full fat muffin (control) was significantly (p<0.05) more tender than all formulations. In addition, the control had a significantly (p<0.05) lower moisture content and a significantly (p<0.05) more yellow crumb color than all the other variations. The 100% fat substituted muffins with enzymes, generally, had lower moisture contents, lower volumes, decreased staling rates, and an increased crumb tenderness when compared to the 100% fat substituted muffin without any enzymes. The 100% fat substituted muffins containing bacterial amylase or fungal protease alone had a significantly (p<0.05) lower staling rate than a 100% fat substituted muffin with a combination of bacterial amylase and fungal protease. / Master of Science

O gene A1 da alfa amilase foi isolado de uma biblioteca genômica em lambda EMBL3, feita com DNA do díptero primitivo Anopheles merus (Díptera, Nematocera, Culicoidea). Ele foi parcialmente seqüenciado, caracterizado pelo padrão da clivagem das enzimas restritivas e clonado no plasmídeo pIBI24 por Pernasetti (1991 ). No presente trabalho relata-se sua expressão em: bactéria (Escherichia coli AD494), células de inseto ( Spodoptera frugiperda) e em levedura (Pichia pastoris), tendo-se observado que somente nas células de Spodoptera frugiperda a enzima é expressa com atividade. A expressão extracelular do gene A1 foi ensaiada em E.coli AD494 usando o vetor pRSETc. Para este fim a seqüência do gene contendo seu próprio peptídeo sinal, foi inserida em fase de leitura (\"frame\") nesse vetor. A expressão sem peptídeo sinal foi executada usando o vetor pAE2, uma construção derivada- do pRSETc. Numa outra construção, o gene A1 foi inserido em fase de leitura após a seqüência do promotor e do peptídeo sinal do gene A2 (alfa amilase de Bacillus stearothermophilus) utilizando-se o vetor pIBI24-Bst. As células de E.coli AD494 transformadas com estes construções expressaram a enzima sem atividade. A amilase A1 não foi secretada nem com seu peptídeo sinal nem com o da amilase A2. A expressão do gene A1 em levedura (Pichia pastoris) foi feita usando o vetor pPic9, que permite a expressão tanto intracelular quanto a secreção da proteína. O gene foi amplificado por PCR usando \"primers forward\" com o intuito de obter o gene com ou sem peptídeo sinal. O gene contendo o peptídeo sinal foi construído com diferentes seqüências tipo Kozak precedendo o códon ATG. Para a expressão extracelular usou-se o peptídeo sinal do fator alfa do pPic9. Os resultados mostraram que a enzima é expressa mas que permanece dentro da célula sem atividade. As mesmas construções do gene A1 feitas para Pichia foram inseridas em Baculovírus para transfectar células de Spodoptera frugiperda. Neste sistema a amilase foi expressa com atividade principalmente no sobrenadante das culturas transformadas com construções usando o gene contendo o seu próprio peptídeo sinal assim com o peptídeo sinal da amilase de Zabrotes subfasciatus. O gene da alfa amilase de Bacillus stearothermophilus foi expresso no sistema Baculovírus-Spodoptera e na levedura Pichia, usando o gene contendo o peptídeo sinal da amilase de Z. subfasciatu. A proteína foi secretada e tinha atividade em ambos os sistemas. / The alpha amylase A1 gene was isolated trom a genomic library of lambda EMBL3 made with DNA from the primitve Díptera Anopheles merus (Díptera, Nematocera, Cuclicoidea). This gene was partialy sequenced, characterized by standard restriction enzyme cleavage, and cloned into the pIBI24 plasmid by Pernasetti (1991). Here we present the expression of this gene in: bacteria (Escherichia coli AD494), insect cell (Spodoptera frugiperda) and yeast (Pichia pastoris). It was shown that only in Spodoptera frugiperda cells the protein display enzymatic activity. The presence of the A1 product in the extracelullar culture media was tested in Escherichia coli AD494 using the vector pRSETc. To this end, the gene sequence containing its own signal peptide was inserted in frame into the vector. Expression without a signal peptide was conducted using the pAE2 vector, a construct derived from pRSETc. In another construct, the A1 gene was inserted in frame after the promoter sequence and the signal peptide of the A2 gene (alpha amylase Bacillus stearothermophilus) using the pIBI24-Bst vector. Although the E.coli AD494 cells transformed with these constructs expressed the enzyme, no enzyme activity was detected. A1 was not secreted, either with its own signal peptide or with that of amylase A2. Expression of the A1 gene in yeast (Pichia pastoris) was conducted using the pPic9 vector, wich allows intracellular expression or secretion of this protein. The gene was amplified by PCR with forward primers, so as to amplyfy the gene sequence with or without the signal peptide. The gene containing the signal peptide was constructed with different Kozak sequences preceeding the ATG codon. For extracellular expression, the signal peptide of the pPic9 alpha factor was used. The results showed that the enzyme is expressed , but remained within the cell and do not display activity. The same constructs of the A1 gene made for P. pastoris were inserted in Baculovirus to infect Spodopera frugiperda cells. In this system, the amylase was successfully expressed and display enzymatic activity, mainly in the extracellular supernatant, using constructs of the gene with own signal peptide or with the signal peptide for amylase of Zabrotes subfasciatus. The gene A2 was expressed in the Baculovirus/Spodoptera system and the yeast P. pastoris using constructs containing the signal peptide of alpha amylase of Z. subfasciatus, the protein was secreted with activity.

Inibidores de alfa amilase de feijão (Phaseolus vulgaris) foram caracterizados. O inibidor da variedade Jalo apresentou peso molecular de 50kDa (por filtração em gel), ponto isoelétrico de 4,75 e 9,6% de carboidratos. O inibidor da variedade Argentino apresentou peso molecular de 48kDa, ponto isoelétrico de 4,90 e 7,6% de carboidratos. Ambos inibidores apresentaram pH ótimo de interação com alfa amilase pancreática de porco de 5,0 e, a pH 6,9, a complexação com a mesma enzima se deu na proporção de 1:1. Esses resultados, mais a composição de aminoácidos, indicaram que as características desses inibidores são semelhantes às de outros já purificados de diferentes variedades de feijão. As principais diferenças entre o IA do Jalo e do Argentino, foram observadas através de termogramas, do teor de carboidratos e dos ensaios imunológicos. Além disso, são sintetizados entre 20 e 30 dias após a floração, sendo que o processo ocorre simultaneamente ao da síntese das proteínas de reserva do grão. Inibidores purificados das variedades Jalo, Argentino e Rico 23, em diferentes fases do desenvolvimento, indicaram ausência de grandes alterações estruturais durante esse processo. / &#945;-amylase inhibitors of bean (Phaseolus vulgaris) were characterized. The amylase inhibitor from Jalo had molecular weight of 50 kDa (by gel filtration), an isoeletric point of 4.75 and a carbohydrate content of 9.6%. Argentino presented molecular weight of 48 kDa, an isoeletric point of 4.90 and a carbohydrate content of 7.6%. The optimum pH for inhibition of porcine pancreatic &#945;-amylase for both inhibitors was about 5.0 and they formed 1:1 stoichiometric complex. These results and the amino acid composition indicated that these inhibitors have characteristics similar to others already purified from beans. They were synthesized between 20 and 30 days after anthesis and this process occurred simultaneously to the storage proteins synthesis. Purified inhibitors from seeds in different phases of development indicated lack of great structural changes during this process.