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Biomedical subjects

R F Power

Publications and source records attributed to R F Power.

At least 19 recordsLinked to original sources
PubMed2005

Effects of Natustat supplementation on performance, feed efficiency and intestinal lesion scores in broiler chickens challenged with Eimeria acervulina, Eimeria maxima and Eimeria tenella.

The effects of dietary supplementation of Natustat, a proprietary plant derived product (Alltech Inc., KY, USA) and Salinomycin, on performance, feed efficiency and intestinal lesion scores were observed during two Eimeria challenge trials in broiler chickens. In the first trial chickens were challenged with Eimeria sp. via infecting the litter with a known amount of Eimeria oocysts. In the second trial the source of the Eimeria challenge was the litter from the first trial and the same treatment groups were assigned to the same pens as in the initial trial. Birds were placed 55 per pen with seven pens per treatment. Performance parameters were recorded on days 21 and 42 during both trials. Intestinal lesion scores were assessed on days 14 and 21 during Trial 1 and on day 21 during Trial 2. Average weight gain and feed conversion ratios were significantly improved in the Natustat and Salinomycin treatment groups when compared to the non-supplemented infected group. Furthermore, lesion scores were lower on all sampling days in the Natustat and Salinomycin groups when compared to the non-supplemented group. However, only lesions associated with Eimeria tenella were significantly lowered by Natustat and Salinomycin supplementation. Natustat and Salinomycin were equivalent in alleviating the negative performance effects associated with coccidiosis challenge. In summary, Natustat has the potential to be used as a natural alternative to chemotherapeutic drugs for Eimeria control.

Animals↗
PubMed2005

Evaluation of dietary Natustat for control of Histomonas meleagridis in male turkeys on infected litter.

Histomoniasis (histomonosis, infectious enterohepatitis, or blackhead) is a disease of turkeys on litter or range caused by the protozoan Histomonas meleagridis, a parasite of worms, primarily spread in feces, in Heterakis gallinarum (cecal worm) eggs, or in Eisenia foetida (earthworms). In this trial, Natustat (Alltech, Inc., Nicholasville, KY), a proprietary plant-derived product, was used at 1.925 kg/tonne and compared with nitarsone in male hybrid turkey diets to 42 days of age on histomonad infected litter (day 7) from broiler breeders. Infected nonsupplemented and uninfected nonsupplemented control groups were also included. Natustat and nitarsone significantly improved 28- and 42-day feed conversion ratios and lowered 28- and 35-day cecal and liver lesion scores compared with infected nonsupplemented turkeys. The body weight at 42 days was greater in the Natustat and nitarsone supplemented groups than in the infected nonsupplemented group.

Animals↗
PubMed2004

Quantification of supplemental enzymes in animal feedingstuffs by radial enzyme diffusion.

Methods are described which facilitate quantification of supplemental cellulase, protease and alpha-amylase when added to animal feedingstuffs at normal industrial inclusion levels. The methods entail extraction of the enzymes from the feedingstuffs by agitation in buffer followed by quantification of extract activity using radial diffusion techniques. A linear relationship between the diameter of the zone of hydrolyzed substrate and the log of the enzyme activity applied is observed over a broad activity range. Assay of a feedingstuff supplemented with 1 kg t(-1) cellulase, protease and alpha-amylase yielded net supplemental activity recoveries of 104+/-11.7%, 91.3+/-6.74% and 126+/-29.5%, respectively. A similar assay method did not prove sufficiently sensitive to facilitate detection of xylanase at typical in-feed inclusion levels. The levels of endogenous cellulase, protease and alpha-amylase activity detected in the unsupplemented feedingstuffs were equivalent to 6.4+/-0.47%, 6.6+/-0.82% and 29.0+/-14.1%, respectively, of a 1 kg t(-1) supplement. The methods are technically straightforward and will facilitate determination of enzyme stabilities during processes such as high-temperature pelleting of feedingstuffs, as well as allowing more rigorous quality control related to enzyme-supplemented animal feedingstuffs.

Animal Feed↗
PubMed2001

An inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite prevents diabetes development in NOD mice.

Peroxynitrite (ONOO(-)) is a highly reactive oxidant produced by the interaction of the free radicals superoxide (O*-2) and nitric oxide (NO(*)). In a previous study, we found that peroxynitrite is formed in islet beta-cells of nonobese diabetic (NOD) mice. Here, we report that guanidinoethyldisulphide (GED), a selective inhibitor of inducible nitric oxide synthase (iNOS) and scavenger of peroxynitrite prevents diabetes in NOD mice. GED treatment of female NOD mice, starting at age 5 weeks, delayed diabetes onset (from age 12 to 22 weeks) and significantly decreased diabetes incidence at 30 weeks (from 80% to 17%). GED did not prevent pancreatic islet infiltration by leukocytes; however, beta-cells that stained positive for nitrotyrosine (a marker of peroxynitrite) were significantly decreased in islets of GED-treated mice (1+/-1%) compared with vehicle-treated mice (30+/-9%). In addition, GED significantly inhibited nitric oxide and nitrotyrosine formation and decreased destruction of beta-cells in NOD mouse islets incubated in vitro with the combination of proinflammatory cytokines interleukin 1-beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). These findings indicate that both superoxide and nitric oxide radicals contribute to islet beta-cell destruction in autoimmune diabetes via peroxynitrite formation in the beta-cells.

Animals↗
PubMed2001

Effects of dietary supplementation with Yucca schidigera Roezl ex Ortgies and its saponin and non-saponin fractions on rat metabolism.

Yucca schidigera Roezl ex Ortgies, family Lillaceae, was fractionated with butan-1-ol to yield a butanol extractable fraction (BE; saponin fraction) and a non-butanol fraction (NBE; non-saponin fraction). Four groups of eight male rats were allowed ad libitum access to diets supplemented with water (control) or 200 mg x kg(-1) total Y. schidigera (TOT) or 200 mg x kg(-1) of each of the fractions (NBE or BE). The effects of dietary supplementation with the fractions and their interactions in TOT were analyzed according to the factorial experimental design by two-way analysis of variance. All three supplementation groups displayed significantly reduced serum urea levels (P < 0.05). The TOT and NBE fractions were found to significantly increase serum insulin levels (P < 0.01) in the absence of any fluctuations in serum glucose levels. Urea cycle enzyme activities, namely, arginase (EC 3.5.3.1) and argininosuccinate lyase (EC 4.3.2.1), were significantly decreased (P < 0.05) in vivo, although no effect was observed in vitro. Both fractions displayed effects, indicating that the active constituents are present in both fractions.

Animal Feed↗
PubMed2000

Testicular Sertoli cells exert both protective and destructive effects on syngeneic islet grafts in non-obese diabetic mice.

AIMS/HYPOTHESIS: Testicular Sertoli cells protect allogeneic islet grafts from rejection after transplantation into animals with chemically induced diabetes. The aims of this study were to determine whether Sertoli cells can protect syngeneic islets from autoimmune destruction after transplantation into non-obese diabetic (NOD) mice and, if so, whether protection is due to Sertoli cell expression of Fas ligand (FasL), believed to be the mechanism that protects against allograft rejection. METHODS: We compared the survival of syngeneic islets transplanted under the renal capsule of nonobese diabetic mice, alone and together with purified Sertoli cells prepared from testes of newborn nonobese diabetic mice. Additionally, we examined the composition of the islet and Sertoli cell co-transplants by immunohistochemistry to determine whether islet graft survival correlated with Sertoli cell expression of Fas ligand. RESULTS: Sertoli cell doses of 1, 2 and 4 x 10(6) cells produced a dose-dependent prolongation of median islet graft survival from 11 days (islets alone) to 32 days (islets + 4 x 10(6) Sertoli cells); addition of 8 x 10(6) Sertoli cells to the islet grafts decreased, however, median survival to 8 days. Immunohistochemical analysis of the islet and Sertoli cell co-transplants showed a correlation between Fas ligand expression by Sertoli cells and graft infiltration by neutrophilic leucocytes, leading to islet beta-cell destruction and diabetes recurrence. CONCLUSION/INTERPRETATION: Sertoli cells exert opposing effects on survival of syngeneic islet grafts in nonobese diabetic mice: Fas ligand-dependent neutrophil infiltration and graft destruction, and Fas ligand-independent protection of the graft from autoimmune destruction.

Animals↗
PubMed2000

Fas ligand-mediated mechanisms are involved in autoimmune destruction of islet beta cells in non-obese diabetic mice.

AIMS/HYPOTHESIS: A mechanism implicated in pancreatic islet beta-cell destruction in autoimmune diabetes is the binding of the Fas ligand (FasL) on T cells to Fas receptors on beta cells, causing their destruction. Evidence for this mechanism is, however, controversial. The aim of this study was to find whether the Fas ligand contributes to beta-cell death in autoimmune diabetes. METHODS: We transplanted syngeneic islets under the renal capsule in non-obese diabetic (NOD) mice and treated the mice with a neutralizing monoclonal antibody to the Fas ligand. Survival of beta cells in islet grafts and phenotypes of graft-infiltrating cells were investigated. RESULTS: We found 58% (7 of 12) of mice treated with anti-Fas ligand antibody were normoglycaemic at 30 days after islet transplantation compared with none (0 of 9) of the mice treated with control antibody. Immunohistochemical analysis of islet grafts showed that infiltration of leucocytes (CD4+ T cells, CD8+ T cells, macrophages and neutrophils) and apoptosis of beta cells in the grafts was significantly decreased in mice treated with anti-Fas ligand antibody. Expression of proinflammatory cytokines (interleukin 1 alpha, tumour necrosis factor alpha and interferon gamma) was not different in islet grafts of mice treated with anti-Fas ligand and control antibodies. CONCLUSION/INTERPRETATION: These findings indicate that Fas ligand-mediated mechanisms play a major part in promoting leucocytic infiltration of islets and beta-cell destruction in autoimmune diabetes.

Animals↗
PubMed1999

Both CD4(+)and CD8(+)T cells are required for IFN-gamma gene expression in pancreatic islets and autoimmune diabetes development in biobreeding rats.

To study the relative roles of CD4(+)and CD8(+)T cells and their cytokine products in autoimmune diabetes development, we selectively depleted CD4(+)and CD8(+)T cells in autoimmune diabetes-prone (DP) biobreeding (BB) rats, by administrations of anti-CD2 and anti-CD8 monoclonal antibody (mAb) respectively. We then analysed cytokine mRNA expression, by PCR assay, in mononuclear leukocytes isolated from islets and spleens of control and mAb-treated DP-BB rats. Depletion of CD4(+)T cells (by anti-CD2 mAb) in blood, spleen and islets prevented diabetes development in DP-BB rats, and depletion of CD8(+)T cells (by anti-CD8 mAb) delayed and significantly decreased diabetes incidence. Depletion of either CD4(+)or CD8(+)T cells completely prevented IFN-gamma mRNA upregulation in islets of DP-BB rats above the low level expressed in islets of diabetes-resistant (DR) BB rats. Also, IL-10 mRNA levels in islets of DP-BB rats were significantly decreased by depletion of either CD4(+)or CD8(+)T cells, whereas the effects of the anti-T cell mAb on mRNA levels of other cytokines in islets (IL-2, IL-4, IL-12p40, and TNF-alpha) were discordant. In contrast, both mAb treatments significantly upregulated IL-4 and TNF-alpha mRNA levels in spleens of DP-BB rats. These results demonstrate that islet infiltration by both CD4(+)and CD8(+)T cells is required for IFN-gamma and IL-10 production in islets and beta-cell destruction. Depletion of either CD4(+)or CD8(+)T cells may prevent beta-cell destruction by decreasing IFN-gamma and IL-10 production in islets and increasing IL-4 and TNF-alpha production systemically.

Animals↗
PubMed1997

Combination therapy with cyclosporine and interleukin-4 or interleukin-10 prolongs survival of synergeneic pancreatic islet grafts in nonobese diabetic mice: islet graft survival does not correlate with mRNA levels of type 1 or type 2 cytokines, or transforming growth factor-beta in the islet grafts.

BACKGROUND: The recurrent autoimmune response to syngeneic pancreatic islet grafts transplanted into nonobese diabetic (NOD) mice is cell-mediated and relatively resistant to cyclosporine (CsA) therapy. Therefore, we asked whether interleukin (IL)-4 and IL-10, cytokines that inhibit cell-mediated immunity, might improve the therapeutic effect of CsA. METHODS: We compared the survival of syngeneic islet grafts transplanted into diabetic NOD mice treated with IL-4, IL-10, and CsA, administered as single agents and in combinations. Additionally, we measured mRNA levels of type 1 cytokines (interferon-gamma [IFN-gamma], IL-2, and IL-12), type 2 cytokines (IL-4 and IL-10), and transforming growth factor-beta (TGF-beta) to determine whether graft rejection or survival might correlate with expression of these cytokines in the grafts. RESULTS: CsA (20 mg/kg/day) significantly prolonged islet graft survival (median: 20 days vs. 10 days for vehicle-treated mice). Neither IL-4 (2.5 microg, twice daily), nor IL-10 (10 microg, twice daily) significantly prolonged islet graft survival. By contrast, combination therapy with CsA and IL-10 significantly prolonged islet graft survival (median: 34 days) compared with vehicle-treated mice (median: 10 days), and combination therapy with CsA and IL-4 significantly prolonged islet graft survival (median: 59 days) compared with both vehicle-treated mice (median: 10 days) and mice treated with CsA alone (median: 20 days). Islet grafts from normoglycemic mice treated with CsA plus IL-10, and with CsA plus IL-4, were surrounded but not infiltrated by mononuclear leukocytes and beta cells were intact, whereas islet grafts from mice treated with vehicle, IL-4, IL-10, and CsA (as single agents) were infiltrated by mononuclear leukocytes and fewer beta cells were detected. Polymerase chain reaction analysis of cytokine mRNA expression in islet grafts at 8-12 days after transplantation revealed that CsA decreased mRNA levels of type 1 cytokines (IFN-gamma and IL-12p40), whereas CsA plus IL-10 did not, and CsA plus IL-4 increased mRNA levels of IFN-gamma, IL-12p40, and TGF-beta. CONCLUSIONS: These results demonstrate that IL-4, and to a lesser extent IL-10, improves the ability of CsA to prevent autoimmune destruction of beta cells in syngeneic islets transplanted into diabetic NOD mice; however, there is no simple correlation between the protective effects of the different treatment regimens (CsA, CsA plus IL-4, and CsA plus IL-10) and mRNA levels of type 1 cytokines (IFN-gamma, IL-2, and IL-12), type 2 cytokines (IL-4 and IL-10), or TGF-beta in the islet grafts.

Animals↗
PubMed1997

TNF-alpha down-regulates type 1 cytokines and prolongs survival of syngeneic islet grafts in nonobese diabetic mice.

Administration of TNF-alpha to autoimmune diabetes-prone nonobese diabetic mice and biobreeding rats inhibits diabetes development; however, the mechanism(s) of diabetes prevention by TNF-alpha has not been established. We used the model of syngeneic islet transplantation into diabetic nonobese diabetic mice to study the effects of TNF-alpha administration on the types of mononuclear cells and cytokines expressed in the islet grafts and on autoimmune diabetes recurrence. Twice daily i.p. injections of TNF-alpha (20 microg/day) from day 1 to day 30 after islet transplantation significantly prolonged islet graft survival; thus, 70% (16 of 23) of mice treated with TNF-alpha were normoglycemic at 30 days after islet transplantation compared with none (0 of 14) of vehicle-treated control mice. Islet grafts and spleens from TNF-alpha-treated mice at 10 days after islet transplantation contained significantly fewer CD4+ and CD8+ T cells, and significantly decreased mRNA levels of type 1 cytokines (IFN-gamma, IL-2, and TNF-beta) than islet grafts and spleens from control mice. Regarding type 2 cytokines, IL-4 mRNA levels were not changed significantly in islet grafts or spleens of TNF-alpha-treated mice, whereas IL-10 mRNA levels were decreased significantly in islet grafts of TNF-alpha-treated mice and not significantly changed in spleens. TGF-beta mRNA levels in islet grafts and spleens were similar in TNF-alpha-treated and control mice. These results suggest that TNF-alpha partially protects beta cells in syngeneic islet grafts from recurrent autoimmune destruction by reducing CD4+ and CD8+ T cells and down-regulating type 1 cytokines, both systemically and locally in the islet graft.

Animals↗
PubMed1996

Fine-needle aspiration cytology of bronchocentric granulomatosis: a potential diagnostic pitfall.

Bronchocentric granulomatosis (BCTG) is a rare disease associated with bronchial asthma and bronchopulmonary aspergillosis. Idiopathic forms are rarely encountered. We report on a case of BCTG showing clinical, radiological, and cytological evidence suggestive of adenocarcinoma of the lung. The patient is a 69-yr-old female, lifetime nonsmoker with multiple sclerosis who was admitted with a history of ascending cholangitis. Admission chest X-ray documented a 1.5-cm nodule in the left upper lobe of the lung. This was confirmed by CT scan. The lesion was slowly growing. Bronchoscopic examination was normal. Bronchial brushings were inconclusive. A transthoracic fine-needle aspiration showed sheets of highly atypical epithelium with occasional small dyshesive clusters. There was an inflammatory background that was believed to represent tumor diathesis. The cytological interpretation was "suspicious for adenocarcinoma." The patient underwent left upper lobectomy. The lung showed multiple peribronchial granulomas with intense peribronchial lymphoid infiltrate extending into the bronchial mucosa, causing cytological atypia and focal ulceration. Special stains for microorganisms were negative. The patient recovered from surgery and shows no signs of infection. We conclude that BCTG and related lesions can give cytological features that are suggestive of malignancy. Cytological material obtained from these lesions should be interpreted with caution.

Aged↗
PubMed1996

Cytokine gene expression in pancreatic islet-infiltrating leukocytes of BB rats: expression of Th1 cytokines correlates with beta-cell destructive insulitis and IDDM.

Cytokines produced by islet-infiltrating mononuclear leukocytes may be involved in islet beta-cell destruction and IDDM. To determine which cytokine(s) might be involved in islet beta-cell destruction, we used a reverse transcriptase-polymerase chain reaction assay to compare levels of cytokine mRNA expression in mononuclear leukocytes freshly isolated from islets of four groups of BB rats aged 60-75 days: diabetes-prone (DP) rats, DP rats protected from diabetes by injection of complete Freund's adjuvant (CFA) at age 25 days, acutely diabetic rats, and diabetes-resistant (DR) rats. We found that islet mononuclear leukocyte levels of gamma-interferon (IFN-gamma) mRNA were significantly higher in DP and diabetic rats than in DR rats, whereas CFA-treated DP rats had similar IFN-gamma mRNA levels to DR rats. Also, interleukin (IL)-2 mRNA levels tended to be higher in islet leukocytes from DP and diabetic rats than from DR rats. Tumor necrosis factor-alpha, IL-4, and IL-10 mRNA levels were not significantly different in islet leukocytes from the four groups of rats. These findings suggest that production of T-helper 1 (Th1)-type cytokines, IFN-gamma and IL-2, by islet-infiltrating cells in BB rats is associated with beta-cell destruction and IDDM development.

Animals↗
PubMed1995

Combined therapy with interleukin-4 and interleukin-10 inhibits autoimmune diabetes recurrence in syngeneic islet-transplanted nonobese diabetic mice. Analysis of cytokine mRNA expression in the graft.

Syngeneic pancreatic islet grafts in nonobese diabetic (NOD) mice elicit a cell-mediated autoimmune response that destroys the insulin-producing beta cells in the islet graft. IL-4 and IL-10 are cytokines that inhibit cell-mediated immunity. In this study, we evaluated the effects of IL-4 and IL-10 on the survival of syngeneic pancreatic islets transplanted into diabetic NOD mice. Islet grafts survived beyond 18 days and normoglycemia was maintained in 67% (10 of 15) of mice treated with IL-4 plus IL-10, but in none (0 of 20) of vehicle-injected (control) mice. Also, 40% (6 of 15) of the mice treated with IL-4 plus IL-10 were normoglycemic at 30 days after transplantation, compared with 14% (1 of 7) of the mice treated with IL-4 alone, 8% (1 of 13) of the mice treated with IL-10 alone, and none (0 of 20) of the control mice. Histological examination of grafts at 10 days after transplantation revealed peri-islet accumulations of mononuclear leukocytes and intact islet beta cells in grafts from IL-4 plus IL-10-treated mice, whereas islets were infiltrated by leukocytes and the beta cell mass was greatly reduced in grafts from control mice. Polymerase chain reaction (PCR) analysis of cytokine mRNA expression in the grafts revealed higher levels of IL-2, IFN gamma, and IL-10 mRNA in grafts of diabetic compared with normoglycemic control mice, whereas IFN gamma and TNF alpha mRNA levels were significantly decreased in grafts of IL-4 plus IL-10-treated mice compared with either normoglycemic or diabetic control mice. These results suggest that T helper (Th)1 cells and their cytokine products (IL-2, IFN gamma, and TNF alpha) may promote islet beta cell destructive insulitis and autoimmune diabetes recurrence in syngeneic islet-transplanted NOD mice, and that administration of IL-4 plus IL-10 may inhibit diabetes recurrence by suppressing Th1 cytokine production in the islet grafts.

Animals↗
PubMed1995

IFN-gamma gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice.

Insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice results from selective destruction of pancreatic islet beta-cells following islet infiltration by mononuclear leukocytes. Cytokines produced by islet-infiltrating mononuclear cells may be involved in beta-cell destruction. Therefore, we analyzed cytokine mRNA expression, by reverse-transcriptase PCR (RT-PCR) assay, in mononuclear leukocytes isolated from pancreatic islets of four groups of mice: diabetes-prone female NOD mice; female NOD mice protected from diabetes by injection of CFA at an early age; male NOD mice with a low diabetes incidence; and female BALB/c mice that do not develop diabetes. We found that mRNA levels of IL-1 beta, IL-2, IL-4, IL-10, and IFN-gamma in mononuclear cells from islets of diabetes-prone female NOD mice increased progressively as these cells infiltrated the islets from age 5 wk to diabetes onset (> 13 wk). However, only IFN-gamma mRNA levels were significantly higher in islet mononuclear cells from 12-wk-old diabetes-prone female NOD mice than from less diabetes-prone NOD mice (CFA-treated females, and males) and normal mice (BALB/c). In contrast, IL-4 mRNA levels were lower in islet mononuclear cells from diabetes-prone female NOD mice than from NOD mice with low diabetes incidence (CFA-treated females and males). Splenic cell mRNA levels of IFN-gamma and IL-4 were not different in the four groups of mice. These results suggest that islet beta-cell destruction and diabetes in female NOD mice are dependent upon intra-islet IFN-gamma production by mononuclear cells, and that CFA-treated female NOD mice and male NOD mice may be protected from diabetes development by down-regulation of IFN-gamma production in the islets.

Aging↗
PubMed1995

Tumor necrosis factor mediates the protective effect of Freund's adjuvant against autoimmune diabetes in BB rats.

In previous studies we reported that a single injection of complete Freund's adjuvant (CFA) in early life inhibits the development of autoimmune diabetes in BB diabetes-prone (DP) rats, and that CFA upregulates tumor necrosis factor-alpha (TNF alpha) production by macrophages of DP rats. In this study, we asked if CFA-induced prevention of diabetes in DP rats might be mediated by TNF alpha. Chronic intraperitoneal injection of TNF alpha, 20 micrograms daily from age 50 to 110 days, significantly decreased diabetes incidence in DP rats from 83% (15 of 18) to 37% (seven of 19) at age 110 days. Similarly, a single intraperitoneal injection of CFA, 100 microliters at age 26 days, significantly decreased diabetes incidence from 80% (16 of 20) to 50% (10 of 20) at age 110 days. Prevention by either TNF alpha or CFA was associated with decreased insulitis and preservation of islet beta-cells. By contrast, treatment of DP rats with CFA, plus antiserum to TNF alpha three times a week from age 26 to 110 days, obviated CFA-induced protection against beta-cell destructive insulitis and diabetes. Thus, diabetes incidence was 75% (18 of 24) in DP rats treated with CFA plus anti-TNF alpha antiserum, similar to that in untreated DP rats (80%), but only 44% (eight of 18) in DP rats treated with CFA plus control serum, similar to that in DP rats with CFA alone (50%). These results suggest that TNF alpha may be a mediator of the protective effects of CFA against autoimmune diabetes development in BB rats.

Animals↗
PubMed1995

Molecular cloning, expression and evaluation of phosphohydrolases for phytate-degrading activity.

Four acid phosphatase (phosphomonoesterase E.C.3.1.3.2) genes were cloned by polymerase chain reaction (PCR). These were pho3, pho5 and pho11 from Saccharomyces cerevisiae and the gene for a phosphate-respressible acid phosphatase from Aspergillus niger. The individual genes were subcloned into an A. oryzae expression vector downstream from a starch-inducible alpha-amylase promoter and the resulting expression constructs were transformed into a mutant strain of A. oryzae, AO7. Southern hybridization analysis confirmed that the acid phosphatase genes had been integrated into the host genome with estimates of integrated copy numbers ranging from 2 to 20 for individual transformants. Northern hybridization analysis of total RNA from individual transformants revealed the presence of a single transcript of the expected size of 1.8 kb. Production of recombinant protein was induced by the addition of 30 g L-1 of soluble starch in the fermentation media. Active acid phosphatases, not present in control cultures, were detected in the supernatant fractions of transformant cultures by acid phosphatase activity staining of non-denaturing polyacrylamide gels. The ability of the recombinant acid phosphatases to hydrolyze phytate was assessed by referenced phytase (myoinositol hexakisphosphate phosphohydrolase E.C. 3.1.3.8) activity assay procedures. A two- to six-fold increase in phytase activity was measured in transformants compared to control, untransformed A. oryzae. Sufficient quantities of A. niger and pho5 recombinant acid phosphatases were generated from large-scale fermentations to assess the efficacy of these enzymes as phytate-degrading enzymes when included in poultry diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗