Biomedical subjects

R Greiner

Publications and source records attributed to R Greiner.

At least 19 recordsLinked to original sources

PubMed2005

Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity.

Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg(-1) protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg(-1) of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent.

6-Phytase↗

PubMed2005

A multi-criteria approach to Great Barrier Reef catchment (Queensland, Australia) diffuse-source pollution problem.

This paper presents a multi-criteria based tool for assessing the relative impact of diffuse-source pollution to the Great Barrier Reef (GBR) from the river basins draining into the GBR lagoon. The assessment integrates biophysical and ecological data of water quality and pollutant concentrations with socio-economic information pertaining to non-point source pollution and (potential) pollutant impact. The tool generates scores for each river basin against four criteria, thus profiling the basins and enabling prioritization of management alternatives between and within basins. The results support policy development for pollution control through community participation, scientific data integration and expert knowledge contributed by people from across the catchment. The results specifically provided support for the Reef Water Quality Protection Plan, released in October 2003. The aim of the plan is to provide a framework for reducing discharge of sediment, nutrient and other diffuse-source loads and (potential) impact of that discharge and for prioritising management actions both between and within river basins.

Agriculture↗

PubMed2004

Predicting subcellular localization of proteins using machine-learned classifiers.

MOTIVATION: Identifying the destination or localization of proteins is key to understanding their function and facilitating their purification. A number of existing computational prediction methods are based on sequence analysis. However, these methods are limited in scope, accuracy and most particularly breadth of coverage. Rather than using sequence information alone, we have explored the use of database text annotations from homologs and machine learning to substantially improve the prediction of subcellular location. RESULTS: We have constructed five machine-learning classifiers for predicting subcellular localization of proteins from animals, plants, fungi, Gram-negative bacteria and Gram-positive bacteria, which are 81% accurate for fungi and 92-94% accurate for the other four categories. These are the most accurate subcellular predictors across the widest set of organisms ever published. Our predictors are part of the Proteome Analyst web-service.

Algorithms↗

PubMed2004

Molecular and physiological characterisation of a 3-phytase from soil bacterium Klebsiella sp. ASR1.

Klebsiella sp. strain ASR1 isolated from an Indonesian rice field is able to hydrolyse myo-inositol hexakis phosphate (phytate). The phytase protein was purified and characterised as a 42 kDa protein accepting phytate, NADP and sugar phosphates as substrates. The corresponding gene (phyK) was cloned from chromosomal DNA using a combined approach of protein and genome analysis, and expressed in Escherichia coli. The recombinant enzyme was identified as a 3-phytase yielding myo-inositol monophosphate, Ins(2)P, as the final product of enzymatic phytate hydrolysis. Based on its amino acid sequence, PhyK appears to be a member of a hitherto unknown subfamily of histidine acid phytate-degrading enzymes with the active site RHGXRXP and HD sequence motifs, and is different from other general phosphatases and phytases. Due to its ability to degrade sodium phytate to the mono phosphate ester, the phyK gene product is an interesting candidate for industrial and agricultural applications to make phytate phosphorous available for plant and animal nutrition.

Klebsiella↗

PubMed2001

Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli.

Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established. High-performance ion chromatography revealed that the phytate-degrading enzyme P2 of E. coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via D/L-Ins(1,2,3,4,5)P(5), D/L-Ins(2,3,4,5)P(4), D/L-Ins(2,4,5)P(3) or D/L-Ins(1,2,4)P(3), D/L-Ins(1,2)P(2) or Ins(2, 5)P(2) or D/L-Ins(4,5)P(2) to finally Ins(2)P or Ins(5)P. Kinetic parameters for myo-inositol pentakisphosphate hydrolysis by E. coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E. coli are not identical. The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E. coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the D-Ins(1, 2,3,5,6)P(5) isomer (Lim, P.E., Tate, M.E., 1973. The phytases: II. Properties of phytase fraction F(1) and F(2) from wheat bran and the myo-inositol phosphates produced by fraction F(2). Biochim. Biophys. Acta 302, 326-328). The data demonstrate that the phytate-degrading enzyme P2 of E. coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,3,4,5)P(5), D-Ins(2,3,4,5)P(4), D-Ins(2,4,5)P(3), Ins(2,5)P(2) to finally Ins(2)P (notation 6/1/3/4/5).

6-Phytase↗

PubMed2001

Modeling the effect of temperature and pH on activity of enzymes: the case of phytases.

In this study, the behavior of enzyme activity as a function of pH and temperature is modeled on the basis of fundamental considerations. A formulation is developed that includes the activation of enzymes with increasing temperatures and the deactivation of enzymes at higher temperature, together with the effect of protonation and hydroxylation on activity at various constant pH levels. The model is calibrated and validated against an extensive set of experimental data on phytases from seven different origins. The percentage variance accounted for (R(2)(adj)), obtained by statistical nonlinear regression analysis on all data sets, was shown to range from 97.6% to 99.5%. The equilibrium constant of protonation and hydroxylation proved to be independent of temperature.

6-Phytase↗

PubMed2001

Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of baker's yeast.

During food processing such as baking, phytate is dephosphorylated to produce degradation products, such as myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphates. Certain myo-inositol phosphates have been proposed to have positive effects on human health. The position of the phosphate groups on the myo-inositol ring is thereby of great significance for their physiological functions. Using a combination of high-performance ion chromatography analysis and kinetic studies the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme from baker's yeast (Saccharomyces cerevisiae) was established. The data demonstrate that the phytate-degrading enzyme from baker's yeast dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), D-Ins(1,2)P(2), to finally Ins(2)P (notation 3/4/5/6/1). Knowledge of the absolute stereochemical specificity of the baker's yeast phytase allows use of the enzyme to produce defined myo-inositol phosphates for kinetic and physiological studies.

6-Phytase↗

PubMed2001

Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba Var. Alameda).

A phytate-degrading enzyme was purified approximately 2190-fold from germinated 4-day-old faba bean seedlings to apparent homogeneity with a recovery of 6% referred to the phytase activity in the crude extract. It behaves as a monomeric protein of a molecular mass of approximately 65 kDa. The phytate-degrading enzyme belongs to the acidic phytases. It exhibits a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 148 micromol L(-1) and k(cat) = 704 s(-1) at 35 degrees C and pH 5.0. The faba bean phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyzes phytate in a stepwise manner. The first hydrolysis product was identified as D/L-myo-inositol(1,2,3,4,5)pentakisphosphate.

6-Phytase↗

PubMed2000

n-3 fatty acid deficiency decreases phosphatidylserine accumulation selectively in neuronal tissues.

We have previously shown that the docosahexaenoate (22:6n-3) status in membrane phospholipids influences the biosynthesis and accumulation of phosphatidylserine (PS) in brain microsomes and C6 glioma cells. In the present study, we investigated whether the observed effect of membrane docosahexaenoic acid status on PS accumulation is universal or occurs specifically in neuronal tissues. We observed that rat brain cortex, brain mitochondria, and olfactory bulb, where 22:6n-3 is highly concentrated, contain significantly higher levels of PS in comparison to liver and adrenal, where 22:6n-3 is a rather minor component. Phospholipid molecular species analysis revealed that in brain cortex, mitochondria, and olfactory bulb 18:0,22:6n-3 was the most abundant species representing 45-65% of total PS. In nonneuronal tissues such as liver and adrenal, 18:0,20:4n-6 was the major PS species. Dietary depletion of n-3 fatty acids during prenatal and postnatal developmental periods decreased the brain 22:6n-3 content by more than 80%, with a concomitant increase in 22:5n-6 in all tissues. Under these conditions, an approximately 30-35% reduction in total PS in rat brain cortex, brain mitochondria, and olfactory bulb was observed, while PS levels in liver and adrenal were unchanged. The observed reduction of PS content in neuronal membranes appears to be due to a dramatic reduction of 18:0,22:6n-3-PS without complete replacement by 18:0,22:5n-6-PS. These results establish that variations in membrane 22:6n-3 fatty acid composition have a profound influence on PS accumulation in neuronal tissues where 22:6n-3 is abundant. These data have implications in neuronal signaling events where PS is believed to play an important role.

Adrenal Glands↗

PubMed2000

Health care resources consumed to treat postoperative infections: cost saving by perioperative immunonutrition.

The objectives of the study were to calculate the costs of postoperative complications and to evaluate whether the use of perioperative enteral immunonutrition, may lead to a saving in health care resources consumed. The economic analysis was based on data from a randomized double-blind trial that include 206 cancer patients who received perioperatively either enteral immunonutrition (treatment group, n = 102) or a standard enteral diet (control group, n = 104). Estimates of costs were based on resource use for treatment of complications, which were valued according to the National List of Sanitary Costs of the Italian Ministry of Health and on the medical Diagnosis-Related-Group (DRG) reimbursement rates. Costs of nutrition were also calculated. Cost comparison and cost effectiveness analyses were then carried out. Intent-to-treat analysis showed that the total costs of 52 postoperative complications were 322,218 euros, with a consumption of the DRG reimbursement rate of 15.4%. The costs of nutrition were 35,437 euros in the treatment group versus 10,768 euros in the control group. The total costs (nutrition plus treating complications) amounted to 113,778 euros in the treatment group versus 254,450 euros in the control group. The mean total costs per patient were 1,115 euros in the treatment group versus 2,447 euros in the control group (P = 0.04). Effectiveness was 83.3% in the treatment group versus 68.3% in the control group (P = 0.009). Cost effectiveness analysis showed a net saving of 2,386 euros per complication-free patient in favor of the treatment group. In conclusion, the perioperative use of immunonutrition appears cost effective due to a substantial saving of resources used to treat postoperative complications.

Aged↗

PubMed1999

Accelerated radiotherapy, carbogen, and nicotinamide in glioblastoma multiforme: report of European Organization for Research and Treatment of Cancer trial 22933.

PURPOSE: A three-step phase I/II trial associating accelerated radiotherapy with carbogen (step 1, ARCO), with nicotinamide (step 2, ARN), or with both (step 3, ARCON) was conducted, the aim of which was to overcome the effects of proliferation and hypoxia as potential causes of tumor radioresistance in glioblastoma multiforme. PATIENTS AND METHODS: Radiotherapy consisted of 60 Gy delivered over 4 weeks in 1.5-Gy fractions twice daily, 5 days a week. Carbogen breathing was started 5 minutes before each fraction and continued until the end of each treatment session. Nicotinamide was given daily as a single oral dose of 85 mg/kg. RESULTS: A total of 115 patients with a median age of 55 years were registered. Of 107 eligible patients, 23 were registered in step 1, 28 in step 2, and 56 in step 3. The planned treatment was administered without any interruption in 72% of patients (86% in ARCO but 68% in ARN and ARCON). The incidence and severity of acute skin and mucous membrane toxicity were higher in patients who received nicotinamide (ie, the ARN and ARCON groups). Grade 1 to 2 gastrointestinal toxicity was observed in 44% of patients in the ARN group and 32% of patients in the ARCON group, but only in 8% of patients in the ARCO group. Eight percent of evaluated patients presented with abnormal liver test results at treatment completion. The dose of corticosteroids had to be increased in 44% of patients. Late neurologic side effects were similar in all treatment steps and were observed mostly in patients with disease progression. Median survival times for patients treated with ARCO, ARN, and ARCON were 10.1, 9.7, and 11.1 months, respectively. CONCLUSION: Feasibility of ARCO treatment was good but that of ARN and ARCON was only fair. This probably reflected the higher acute toxicity rate, particularly gastrointestinal, for patients receiving nicotinamide. The dose of corticosteroids had to be increased frequently during treatment, suggesting a higher than expected acute neurologic toxicity. Overall survival was similar in the three treatment steps and not different when compared with results of other series that used radiotherapy alone.

Administration, Inhalation↗

PubMed1998

Combined intravenous and intraperitoneal chemotherapy with fluorouracil + leucovorin vs fluorouracil + levamisole for adjuvant therapy of resected colon carcinoma.

Adjuvant chemotherapy with fluorouracil (FU) and levamisole or FU/leucovorin (LV) has been established as effective adjuvant treatment for patients with stage III colon cancer. Among several other promising treatment strategies in resected colon cancer, intraperitoneal anti-cancer drug administration with its appealing rationale of counteracting microscopic residual disease on peritoneal surfaces and occult metachronous liver metastases by achieving high intraportal drug concentrations has not yet undergone sufficient clinical evaluation. To determine whether a combination of this locoregional therapeutic concept with systemic intravenous administration of FU/LV would yield better results than conventional adjuvant chemoimmunotherapy with FU/levamisole, the present randomized study was initiated. A total of 241 patients with resected stage III or high-risk stage II (T4N0M0) colon cancer were randomly assigned to 'standard therapy' with FU and levamisole, given for a duration of 6 months, or to an investigational arm, consisting of LV 200 mg m(-2) plus FU 350 mg m(-2), both administered intravenously (days 1-4) and intraperitoneally (days 1 and 3) every 4 weeks for a total of six courses. In patients with stage II disease, no significant difference was noted between the two arms after a median follow-up time of 4 years (range 2.5-6 years). Among 196 eligible patients with stage III disease, however, a comparative analysis of the two treatment groups suggested both an improvement in disease-free survival (P = 0.0014) and a survival advantage (P = 0.0005), with an estimated 43% reduction in mortality rate (95% confidence interval 26-70%) in favour of the investigational arm. In agreement with its theoretical rationale, combined intraperitoneal and intravenous FU/LV was particularly effective in reducing locoregional tumour recurrences with or without liver or other organ site involvement (9 vs 25 patients in the FU/levamisole arm; P = 0.005). Treatment-associated side-effects were infrequent and generally mild in both arms, although a lower rate of severe (WHO grade 3) adverse reactions was noted in patients receiving locoregional plus intravenous chemotherapy (3% vs 12%; P = 0.01). The results of this trial suggest that combined intraperitoneal plus systemic intravenous chemotherapy with FU/LV is a promising adjuvant treatment strategy in patients with surgically resected stage III colon carcinoma.

Adenocarcinoma↗

PubMed1998

[Invasive carcinomas of the hypopharynx with esophageal involvement--multidisciplinary treatment concept].

In UICC stage IV hypopharnygeal cancer, a radical approach with circular laryngo-pharyngo-esophagectomy with ipsilateral radical neck dissection and contralateral modified radical neck dissection, reconstruction of the pharynx defect with a fundus-rotation-gastroplasty, and postoperative high-dose cervical radiotherapy improves long term survival and maintains a good quality of life.

Aged↗

PubMed1997

Purification and characterization of a phytase from Klebsiella terrigena.

A cytoplasmatic phytase was purified about 410-fold to apparent homogeneity with a recovery of 28%. The enzyme is induceable under carbon limitation in the presence of phytate. It behaves as a monomeric protein of a molecular mass of about 40 kDa. The phytase is rather specific for phytate and exhibits optimal conditions for phytate degradation at pH 5.0 and 58 degrees C. Kinetic parameters for the hydrolysis of Na phytate are KM 300 microM and kcat 180 s-1 at 35 degrees C and pH 5.0. Phytate is hydrolyzed in a stepwise manner; the penta- and tetrakisphosphate were identified as I(1,2,4,5,6)P5 and I(1,2,5,6)P4. Consequently, this enzyme is a 3-phytase (EC 3.1.3.8).

6-Phytase↗

PubMed1997

Is there any possibility of detecting the use of genetic engineering in processed foods?

To elucidate if there is any possibility to identify highly processed foods as produced through genetic engineering, beer, soya bean oil, processed tomato (ketch-up, paste, pizza tomatoes, peeled tomatoes, soup) and potato (french fries, crisps, mashed potatoes, flour, starch, fried potatoes) products as well as an enzyme preparation (Natuphos) were investigated by PCR. In pizza tomatoes, peeled tomatoes, french fries, fried potatoes, potato flour and potato crisps DNA suitable for PCR was found. Therefore, it is possible to identify these products as produced through genetic engineering. Such an identification is impossible in certain beers (pilsener, export, Nutfield lyte), soya bean oil, tomato soup, potato starch, mashed potatoes and Natuphos since PCR-analysis gave no indication of the presence of DNA in these products. As it was shown by adding Escherichia coli DNA the used method is, in principle, capable of detecting specifically small amounts of DNA in such products.

DNA Primers↗

PubMed1997

Undifferentiated carcinoma of nasopharyngeal type of the laryngopharyngeal region.

Undifferentiated carcinoma of nasopharyngeal type (lymphoepithelioma) is an extremely rare malignancy in the laryngohypopharyngeal region. We found reports of only 13 such tumors in the English language literature. We present the findings of four additional cases, one hypopharyngeal and three laryngeal in origin. The three laryngeal tumors were characterized by submucosal spread. The tumors were classified T3 (2x) and T4 (2x) with cervical lymph node metastases at initial presentation in all cases. In three of our four cases the Epstein-Barr virus was demonstrated by the Epstein-Barr virus-encoded RNAs in situ hybridization.

Aged↗

PubMed1997

Fluorouracil plus racemic leucovorin versus fluorouracil combined with the pure l-isomer of leucovorin for the treatment of advanced colorectal cancer: a randomized phase III study.

PURPOSE: To compare the efficacy and toxicity of fluorouracil (FU) and racemic leucovorin (d,l-LV) versus FU combined with the l-isomer of leucovorin (l-LV) in the treatment of advanced colorectal cancer. PATIENTS AND METHODS: A total of 248 patients with advanced measurable colorectal cancer previously unexposed to chemotherapy were randomly assigned to treatment with either FU (400 mg/m2/d by intravenous [I.V.] infusion for 2 hours) and racemic LV (100 mg/m2/d by I.V. bolus injection) given for 5 consecutive days, or the combination of FU and the pure l-isomer of LV using the same dose schedule. In both treatment arms, courses were administered every 28 days if toxicity allowed for a total of 6 months, unless evidence of tumor progression was documented earlier. RESULTS: There were no significant differences between the FU/racemic LV and the FU/l-LV arm in the overall response rate (25% v 32%), duration of response (7.2 v 8.0 months), median time to progression or death (6.25 v 8.0 months), or median overall survival time (14.5 v 15.0 months). Except for minor myeloid toxic effects associated with FU/l-LV, there was also no significant difference in terms of adverse reactions. Gastrointestinal symptoms, specifically mucasitis and diarrhea, were less frequent and less severe in both treatment arms compared with other trials with FU/racemic LV reported in the literature, which might be because of the prolonged administration of FU used in both arms. CONCLUSION: The combination of FU/l-LV produced response rates, response durations, and survival times similar to those with FU/d,l-LV. Biochemical modulation of FU by either pure l-LV or racemic LV thus appears to result in equivalent clinical efficacy.

Adult↗

PubMed1996

Construction of a bioreactor to produce special breakdown products of phytate.

Escherichia coli phytase was covalently immobilized on NHS-activated Sepharose High Performance. The pH dependence of the phytase activity was not influenced by immobilization, whereas stability against heat treatment was enhanced as a consequence of immobilization. Compared to the free phytase the immobilized enzyme exhibits the same excellent substrate specifity, but showed an increased Km-value. Using the immobilized phytase in a packed-bed bioreactor makes special isomers of the lower myo-inositol phosphate esters available. The major isomers formed were identified as I(1,2,3,4,5)P5, I(2,3,4,5)P4, I(2,4,5)P3 and I(2,5)P2.

6-Phytase↗