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PubMed2006

C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests.

A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.

Cathepsin A↗

PubMed2005

Anaerobic digestion of animal waste: waste strength versus impact of mixing.

We studied the effect of mode of mixing (biogas recirculation, impeller mixing, and slurry recirculation) and waste strength on the performance of laboratory scale digesters. The digesters were fed with 5% and 10% manure slurry, at a constant energy supply per unit volume (8 W/m3). The experiments were conducted in eight laboratory scale digesters, each having a working volume of 3.73 L, at a controlled temperature of 35+/-2 degrees C. Hydraulic retention time (HRT) was kept constant at 16.2 days, resulting in a total solids (TS) loading rate of 3.08 g/Ld and 6.2 g/Ld for 5% and 10% manure slurry feeds, respectively. Results showed that the unmixed and mixed digesters performed quite similarly when fed with 5% manure slurry and produced biogas at a rate of 0.84-0.94 L/Ld with a methane yield of 0.26-0.31 L CH4/g volatile solids (VS) loaded. This was possibly because of the low solids concentration in the case of 5% manure slurry, where mixing created by the naturally produced gas might be sufficient to provide adequate mixing. However, the effect of mixing and the mode of mixing became prominent in the case of the digesters fed with thicker manure slurry (10%). Digesters fed with 10% manure slurry and mixed by slurry recirculation, impeller, and biogas recirculation produced approximately 29%, 22% and 15% more biogas than unmixed digester, respectively. Deposition of solids inside the digesters was not observed in the case of 5% manure slurry, but it became significant in the case of 10% manure slurry. Therefore, mixing issue becomes more critical with thicker manure slurry.

Bacteria, Anaerobic↗

PubMed2004

Enzymatic digestion and mass spectrometry in the study of advanced glycation end products/peptides.

An extensive study was carried out on HSA and non-enzymatically glycated HSA by enzymatic digestion with trypsin and endoproteinase Lys-C, with the aim of identifying specific glycated peptides deriving from enzymatic digestion of glycated HSA. They may be considered, in pectore, as advanced glycation end products/peptides. These compounds, important at a systemic level in diabetic and nephropathic subjects, are produced by enzymatic digestion of in vivo glycated proteins: They are related to the pathological state of patients and have been invoked as responsible for tissue modifications. The digested mixtures obtained by the two enzymes were analyzed by MALDI/MS and LC/ESI/MSn, and clear cut differences were found. First of all, the digestion products of glycated HSA are generally less abundant than those observed in the case of unglycated HSA, accounting for the lower proclivity of the former to enzymatic digestion. MS/MS experiments on doubly charged ions, comparisons with a protein database, and molecular modeling to identify the lysine NH2 groups most exposed to glycation, identified some glycated peptides in digestion mixtures obtained from both types of enzymatic digestion. Residues 233K, 276K, 378K, 545K, and 525K seem to be privileged glycation sites, in agreement with the fractional solvent accessible surface values calculated by molecular modeling.

Amino Acid Sequence↗

PubMed2005

Anaerobic digestion of animal waste: effect of mode of mixing.

Laboratory-scale digesters were operated to study the effect of mixing (via biogas recirculation, impeller mixing, and slurry recirculation) on biogas production. Three sets of experiments were performed using cow manure slurry feed with either 50, 100, or 150 g/L total solids (TS) concentrations (referred in the text as 5%, 10%, and 15% manure slurry). The experiments were conducted at a controlled temperature of 35 degrees C and a hydraulic retention time of 16.2 days, resulting in TS loadings of 3.1, 6.2, and 9.3g/Ld for 5%, 10%, and 15% manure slurry feeds, respectively. Results showed that the unmixed and mixed digesters performed quite similarly when fed with 5% manure slurry and produced biogas at a rate of 0.84-0.94 L/Ld. The methane yield was found to be 0.26-0.28 L CH4/g volatile solids loaded. However, the effect of mixing and the mode of mixing became important when the digesters were fed thick manure slurry feeds (10% and 15%). Digesters fed with 10% and 15% manure slurry and equipped with external mixing produced about 10-30% more biogas than the unmixed digester. While the mixed digesters produced more biogas than unmixed digesters, digester mixing during start-up was not beneficial, as it resulted in lower pH, performance instability and prolonged start-up time. Mixing using biogas recirculation system was found not to be effective in the case of 15% manure slurry feed under the experimental conditions studied.

Animals↗

PubMed1999

Polyethylene glycol augments thyroid cAMP responses by fragments from protease-digested TSAb or TSBAb-IgG.

In the previous reports, we have demonstrated (1) that polyethylene glycol (PEG)(5%) augmented TSAb (thyroid stimulating antibody)-stimulated cAMP responses of porcine thyroid cells, and (2) that fragments from papain-digested TSBAb (thyroid stimulation blocking antibody) could stimulate thyroid cAMP synthesis. Thus, we studied the effect of 5% PEG on cAMP responses stimulated by the protease-digested TSAb- or TSBAb-fragments. Stimulatory effect of 5% PEG on cAMP production by Fab fragment (Mr 50 KDa) and the retarded fraction (Mr 20 KDa) from the gel-filtration on Sephadex G-100 using papain-digested TSAb-IgG unbound to Protein A-Sepharose was observed. Similar stimulatory effect of 5% PEG on the second fraction (Fc with trace amounts of Fab) in the gel-filtration on Sephadex G-100 using papain digested TSAb-IgG bound to Protein A-Sepharose was observed. Stimulatory effect of PEG on the second fraction was derived from Fab fragment. PEG (5%) also showed stimulatory effect on cAMP production by F(ab')2 fragment (Mr 100 KDa) from the gel-filtration on Sephadex G-100 using pepsin-digested TSAb-IgG unbound to Protein A-Sepharose. PEG (5%) augmented cAMP responses by both Fab and the retarded fractions from the gel-filtration using papain-digested TSBAb-IgG unbound to Protein A when these fractions could stimulate cAMP synthesis. In conclusion, PEG (5%) augments cAMP responses stimulated by F(ab')2, Fab and the smaller molecular components (Mr 20 KDa) separated from protease-digested TSAb-IgG. PEG also augments cAMP responses stimulated by Fab and the smaller molecular components with thyroid stimulating activity separated from papain-digested TSBAb-IgG.

Autoantibodies↗

PubMed2001

Anaerobic codigestion of municipal solid waste and biosolids under various mixing conditions--I. Digester performance.

The feasibility of codigestion of the organic fraction of municipal solid waste, primary sludge, and waste activated sludge was evaluated in mesophilic (37 degrees C), laboratory-scale digesters. In a first experiment, different startup strategies were compared using four digesters, operated under continuously mixed conditions. After two weeks, the experiment was continued under minimally mixed conditions. Results demonstrated that reducing the level of mixing improved digester performance. Therefore, in a second experiment, six digesters were operated to compare performance under continuous mixing and reduced mixing levels at various loading rates and solids levels. The continuously mixed digesters exhibited unstable performance at the higher loading rates, while the minimally mixed digesters performed well for all loading rates evaluated. In a third experiment, it was demonstrated that an unstable, continuously mixed digester was quickly stabilized by reducing the mixing level. These experiments confirmed that continuous mixing was not necessary for good performance and was inhibitory at higher loading rates. In addition, reduction of mixing levels may be used as an operational tool to stabilize unstable digesters.

Oxygen↗

PubMed2003

Mesophilic and thermophilic anaerobic digestion of primary and secondary sludge. Effect of pre-treatment at elevated temperature.

Anaerobic digestion is an appropriate technique for the treatment of sludge before final disposal and it is employed worldwide as the oldest and most important process for sludge stabilization. In general, mesophilic anaerobic digestion of sewage sludge is more widely used compared to thermophilic digestion. Furthermore, thermal pre-treatment is suitable for the improvement of stabilization, enhancement of dewatering of the sludge, reduction of the numbers of pathogens and could be realized at relatively low cost especially at low temperatures. The present study investigates (a) the differences between mesophilic and thermophilic anaerobic digestion of sludge and (b) the effect of the pre-treatment at 70 degrees C on mesophilic and thermophilic anaerobic digestion of primary and secondary sludge. The pre-treatment step showed very positive effect on the methane potential and production rate upon subsequent thermophilic digestion of primary sludge. The methane production rate was mostly influenced by the pre-treatment of secondary sludge followed by mesophilic and thermophilic digestion whereas the methane potential only was positively influenced when mesophilic digestion followed. Our results suggest that the selection of the pre-treatment duration as well as the temperature of the subsequent anaerobic step for sludge stabilization should depend on the ratio of primary to secondary sludge.

Bacteria, Anaerobic↗

PubMed1999

Pepsin-digested peanut contains T-cell epitopes but no IgE epitopes.

BACKGROUND: Peanuts are a common cause of food-induced anaphylaxis and fatalities. Previous studies have demonstrated that rush immunotherapy to crude peanut extract reduces clinical symptoms triggered by oral peanut challenges, but the immunotherapy was associated with an unacceptably high incidence of systemic allergic reactions. One approach to reduce the frequency of allergic reactions would be to use a modified peanut antigen with low allergenic properties. OBJECTIVE: We sought to determine the immunologic characteristics of crude intact peanut extract before and after pepsin digestion. METHODS: We used IgE immunoblotting and assessment of T-lymphocyte responses to intact and peptic digests of peanut extracts. RESULTS: Western blot analysis of sera from 5 subjects with peanut allergy showed multiple IgE-reactive proteins in crude intact peanut extract that were eliminated after pepsin treatment of the peanut extract. In contrast, pepsin-digested peanut induced significant T-cell proliferation responses (stimulation index = 30) in vitro in PBMCs from 7 subjects with peanut allergy, albeit at lower levels than that induced by intact peanut (stimulation index = 66). Furthermore, IFN-gamma production was induced by intact peanut and pepsin-digested peanut in a concentration-dependent manner. Importantly, T-cell lines generated in response to intact peanut also reacted to pepsin-digested peanut, indicating cross-reactive T-cell epitopes in intact and pepsin-digested peanut. CONCLUSION: These findings suggest that pepsin-digested peanut may be useful in peanut immunotherapy because pepsin digestion eliminates IgE reactivity but maintains T-cell reactivity.

Adult↗

PubMed2002

The use of ultrasound and gamma-irradiation as pre-treatments for the anaerobic digestion of waste activated sludge at mesophilic and thermophilic temperatures.

The effect of ultrasound and gamma-irradiation used as pre-treatments for the anaerobic digestion of waste activated sludge at both mesophilic and thermophilic temperatures was examined. Untreated activated sludge was also subjected to anaerobic digestion at these temperatures as a control. The sonication time was 90 s using a Soniprep 150 (MSE Scientific Instruments) which operated at 23 kHz and had been adjusted to give an output of 47 W and the gamma-irradiation dose was 500 krad. The digesters were operated in a semi-continuous mode, being fed with fresh sludge every 24 h at hydraulic retention times (HRT) of 8, 10 and 12 days. Over the 24 h period the differences between the digesters, in terms of volatile solids (VS) reductions and biogas production, were not statistically significant for any particular set of conditions. Thermophilic digestion performed better than mesophilic digestion in terms of biogas production, VS reductions (except at HRT of 8 days) and specific methane yields and the optimum retention time was 10 days, at both temperatures. When gas production over the initial eight hours (probably the hydrolytic stage) was examined, it was found that the gas production rates for pre-treated sludges were higher than those for untreated sludges. This was most pronounced at thermophilic temperatures and a HRT of 10 days. Sonication did not affect the numbers of faecal coliforms in the sludge. However, gamma-radiation caused a 3-log reduction and, when coupled with mesophilic digestion, gave a product which contained < 100 g(-1) TS. Thermophilic anaerobic digestion produced sludges which contained < 1 g(-1) TS irrespective of any pre-treatment.

Anaerobiosis↗

PubMed2002

Super blue box recycling (SUBBOR) enhanced two-stage anaerobic digestion process for recycling municipal solid waste: laboratory pilot studies.

The super blue box recycling (SUBBOR) process is an enhanced, multi-stage anaerobic digestion process for mixed municipal solid waste (MSW) and other biomass feedstock materials. The technology centers on enhanced high solids, thermophilic digestion after steam-pressure disruption of the ligno-cellulosic fiber components that are recalcitrant to conventional anaerobic digestion. Mixed MSW, rich in organic components but also containing inorganic materials, such as glass, aluminum and steel, as well as non-digestible plastic materials, has been laboratory pilot tested with a fully integrated process train designed to treat and recycle all of the MSW components. Methane yields from the MSW were 0.36 m3 CH4/kg volatile solids (VS) representing a 40% increase over the yield obtained from conventional single stage digestion. The secondary digestion step after steam pressure disruption also provided a 40% improvement in total solids and VS reduction. The residual organic fraction following two-stage digestion was fine in texture and was recovered as a clean peat fraction with reduced contents of heavy metal and other fugitive non-digested contaminants. Mass and energy balance determinations indicated a high degree of MSW diversion from landfill disposal (>80%) was achievable by the SUBBOR process as well as substantial net electrical and thermal energy production. Continuous long-term trials of the SUBBOR process at 25,000 tonnes/year are underway.

Anaerobiosis↗

PubMed2006

Slow digestion property of native cereal starches.

The slow digestion property of native cereal starches, represented by normal maize starch, was investigated. The in vitro Englyst test showed that 53.0% of the maize starch is slowly digestible starch (SDS), and scanning electron microscopy (SEM) revealed that SDS starts from an increase of pore size until almost complete fragmentation of starch granules. However, similar amounts of SDS ( approximately 50%) were shown for partially digested fragmented starch residuals, which would normally be considered resistant to digestion based on the Englyst assay. Molecularly, both amylopectin (AP) and amylose (AM) contributed to the amount of SDS as evidenced by a similar ratio of AP to AM at different digestion times. Consistently, similar degrees of crystallinity, comparable gelatinization behavior, and similar debranched profiles of starch residuals following different digestion times indicated that the crystalline and amorphous regions of starch granules were evenly digested through a mechanism of side-by-side digestion of concentric layers of semicrystalline shells of native starch granules.

Crystallization↗

PubMed2005

Angiotensin I converting enzyme inhibitory peptides from in vitro pepsin-pancreatin digestion of soy protein.

Angiotensin I converting enzyme (ACE) inhibitory activity was determined in the soy protein isolate (SPI) digest produced by in vitro pepsin-pancreatin sequential digestion. The inhibitory activity was highest within the first 20 min of pepsin digestion and decreased upon subsequent digestion with pancreatin. An IC(50) value of 0.28 +/- 0.04 mg/mL was determined after 180 min of digestion, while no ACE inhibitory activity was measured for the undigested SPI at 0.73 mg/mL. Chromatographic fractionation of the SPI digest resulted in IC(50) values of active fractions ranging from 0.13 +/- 0.03 to 0.93 +/- 0.08 mg/mL. Although many of the fractions showed ACE inhibition, peptides with lower molecular masses and higher hydrophobicities were most active. The findings show that many different peptides with ACE inhibitory activities were produced after in vitro pepsin-pancreatin digestion of SPI and lead to the speculation that physiological gastrointestinal digestion could also yield ACE inhibitory peptides from SPI.

Amino Acids↗

PubMed2006

Thermophilic anaerobic digestion of industrial orange waste.

Thermophilic anaerobic digestion of industrial orange waste (pulp and peel) with subsequent aerobic post-treatment of the digestate was evaluated. Methane production potential was first determined in batch assays and the effects of operational parameters such as hydraulic retention times (HRT) and organic loading rates (OLR) on process performance were studied through semi-continuous digestion. In batch assays, methane production potential of about 0.49 m(3) kg(-1) volatile solids (VS)(added waste) was achieved. In semi-continuous digestion, loading at 2.8 kgVS m(-3) d(-1) (2.9 kg total solids (TS) m(-3) d(-1)) and HRT of 26 d produced specific methane yields of 0.6 m(3) kg(-1) VS (added waste) (0.63 m(3) kg(-1) VS(added waste)). Operating at a higher OLR of 4.2 kgVS m(-3) d(-1) (4.4 kg TS m(-3) d(-1)) and 40 d HRT produced 0.5 m(3) of methane kg(-1) VS (added waste) (0.63-0.52 m(3) kg(-1) TS (added waste). Up to 70% of TS of industrial orange waste (11.6% TS) was methanised. Further increase in OLR to 5.6 kg VS m(-3) d(-1) (5.9 kg TS m(-3) d(-1); HRT of 20 d) resulted in an unstable and non-functional digester process shown directly through complete cessation of methanogenesis, drop in methane content, reduced pH and increase in volatile fatty acid (VFA) concentrations, especially acetate and soluble chemical oxygen demand. A pH adjustment (from an initial 3.2 to ca. 8) for the low pH orange waste was necessary and was found to be a crucial factor for stable digester operation as the process showed a tendency to be inhibited due to accumulation of VFAs and decrease in digester pH. Aerobic post-treatment of digestate resulted in removal of ammonia and VFAs.

Anaerobiosis↗

PubMed2005

Rapid determination of total Kjeldahl nitrogen using microwave digestion.

A closed-vessel microwave digestion process for the determination of total Kjeldahl nitrogen (TKN) has been developed for sewage and wastewater. TKN values obtained from the microwave digestion method were in excellent agreement with those of the thermal digestion method. The accuracy of both analytical methods is comparable. In comparison to the conventional thermal digestion, the microwave method shortened the time required for complete digestion from 4 h to 25 min, and also decreased the maximum digestion temperature from 380 degrees C to 200 degrees C. This developed method may contribute to a significant reduction in sample digestion time, resulting in an increase in analytical throughput. The microwave digestion method developed in this study could be a rapid and efficient means for TKN determination for sewage wastewater and sludge.

Hot Temperature↗

PubMed1984

The binding of dietary protein by sorghum tannins in the digestive tract of pigs.

The effects of reconstitution of sorghums on dry matter, energy, protein and amino acid digestibilities, and the nature of protein binding by sorghum tannins during digestion were investigated. Grains from a high and a low tannin sorghum were reconstituted by adding 30% (wt/wt) distilled water to the grain and stored at 25 degrees C for 20 days with an acetic-propionic acid mixture added to deter fungal growth. Another lot of grain from the same sources was untreated and used as control. The sorghum grains were then incorporated at an 85% level in diets for a digestibility trial with pigs cannulated at the terminal ileum. The digestibilities of dry matter, protein and amino acids at the terminal ileum were lower than the corresponding values measured over the total digestive tract. Reconstitution improved the digestibilities of dry matter, energy, protein and amino acids in the high but not low tannin sorghum diets. The improvement in digestibility of individual amino acids ranged from 7.5 to 23.5%. The tannin-associated proteins were more hydrophobic than the dietary protein. The results suggest that hydrophobic bonding is important in the formation of tannin-protein complexes in the digestive tract of pigs.

Amino Acids↗

PubMed1985

Effect of regulatory light chain on chymotryptic digestion of scallop adductor myosin.

Chymotryptic digestability of scallop myosin was studied by measuring (a) changes in the gel electrophoretic pattern and (b) production of the soluble fraction obtained by centrifugation. Chymotryptic digestion of essential light chain (SH-LC) was strongly inhibited by association of regulatory light chain (R-LC) with myosin. This is in agreement with the observation of Stafford et al. (Biochemistry 18, 5273 (1979]. SH-LC and R-LC were both more resistant to the chymotryptic digestion when R-LCs were associated with myosin in the presence of calcium than when they dissociated from myosin in the presence of EDTA. In contrast, heavy chains of scallop myosin were digested more quickly in the presence of calcium than EDTA. This suggests that association of R-LC induces reversible changes in the heavy chain conformation, which lead to an increase in the chymotryptic digestability of heavy chains. The chymotryptic digestability of scallop myosin increased in two distinct phases as the calcium concentration in the digestion medium was increased, but monophasically as the magnesium concentration was increased. The magnesium increased the digestability by approximately half as much as did calcium. These findings suggest two types of attachment between regulatory light chains and desensitized myosin: one mediated specifically by low concentrations of calcium ions, the second by higher concentrations of either calcium or magnesium.

Animals↗

PubMed2003

Nutrient digestibility of broiler feeds containing different levels of variously processed rice bran stored for different periods.

Nutrient digestibility of broiler feeds containing different levels of variously processed rice bran stored for varying periods was determined. A total of 444 Hubbard male chicks were used to conduct four trials. Each trial was carried out on 111 chicks to determine digestibility of 36 different feeds. Chicks of 5 wk age were fed feeds containing raw, roasted, and extruded rice bran treated with antioxidant, Bianox Dry (0, 125, 250 g/ton), stored for a periods of 0, 4, 8, and 12 mo and used at levels of 0, 10, 20, and 30% in feeds. Digestibility coefficients for fat and fiber of feeds were determined. Increasing storage periods of rice bran significantly reduced the fat digestibility of feed, whereas no difference in fiber digestibility was observed. Processing of rice bran by extrusion cooking significantly increased digestibility of fat even used at higher levels in broiler feeds. Interaction of storage, processing, and levels was significant for fat digestibility. Treatments of rice bran by different levels of antioxidant had no effect on digestibility of fat and fiber when incorporated in broiler feed.

Animal Feed↗

PubMed2000

Improved quality and yield of islets isolated from human pancreata using a two-step digestion method.

A new approach, involving a two-step digestion process and Los Angeles preservation solution #1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty-six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age, cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5% (43 of 46 cases) in group 1, and 56.5% (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 +/- 36,244 islets equivalent to 150 microm (IEQ) and 6,233 +/- 681 IEQ/g of pancreas with 83 +/- 2.5% purity, whereas the old method yielded a total of 195,587 +/- 25,242 IEQ and 3,763 +/- 5,509 IEQ/g with 69.2 +/- 4.7% purity. Isolated islets in group 1 maintained a good three-dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transplantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a single pancreas.

Adolescent↗