Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

Use of fibrolytic enzymes to improve the nutritive value of ruminant diets - A biochemical and in vitro rumen degradation assessment

Two commercial enzyme products, Depol 40 (D) and Liquicell 2500 (L), were characterised from a biochemical standpoint and their potential to improve rumen degradation of forages was evaluated in vitro. Enzyme activities were determined at pH 5.5 and 39 degreesC. Analysis of the enzyme activities indicated that L contained higher xylanase and endoglucanase, but lower exoglucanase, pectinase and alpha-amylase activities than D. The Reading Pressure Technique (RPT) was used to investigate the effect of enzyme addition on the in vitro gas production (GP) and organic matter degradation (OMD) of alfalfa (Medicago sativa L.) stems and leaves. A completely randomised design with factorial arrangement of treatments was used. Both alfalfa fractions were untreated or treated with each enzyme at four levels, 20 h before incubation with rumen fluid. Each level of enzyme provided similar amounts of filter paper (D1, L1), endoglucanase (D2, L2), alpha-L-arabinofuranosidase (D3, L3) and xylanase units (D4, L4) per gram forage DM. Enzymes increased the initial OMD in both fractions, with improvements of up to 15% in leaves (D4) and 8% in stems (L2) after 12 h incubation. All enzyme treatments increased the extent of degradation (96 h incubation) in the leaf fractions, but only L2 increased final OMD in the stems. Direct hydrolysis of forage fractions during the pre-treatment period did not fully account for the magnitude of the increases in OMD, suggesting that the increase in rate of degradation was achieved through a combined effect of direct enzyme hydrolysis and synergistic action between the exogenous (applied) and endogenous (rumen) enzymes. (C) 2003 Elsevier Science B.V. All rights reserved.

Influence of exogenous fibrolytic enzyme level and incubation pH on the in vitro ruminal fermentation of alfalfa stems

A series of in vitro experiments was carried out to examine the impact of enzyme application rate and incubation medium pH on the rate and extent of fermentation of alfalfa stems. In Experiment 1, a commercial enzyme product (Liquicell 2500, Specialty Enzyme and Biochemicals, Fresno, CA, USA) was added to alfalfa stems at six levels: 0, 0.51, 1.02, 2.55, 5.1, and 25.5 mu l/g (control and L1-L5, respectively) to forage DM in a completely randomized design, with a factorial arrangement of treatments. Rate and extent of fermentation and apparent organic matter degradation (OMD) were determined in vitro, using a gas production technique. Addition of enzyme linearly increased (P < 0.01) gas production for up to 12 h (68.9, 70.9, 67.6, 67.9, 71.9, and 74.9 ml/g OM for control, L1-L5, respectively) and OMD for up to 19 h incubation (0.425, 0.444, 0.433, 0.446, 0.443, and 0.451 for control, L1-L5, respectively), but no increases (P > 0.05) were detected thereafter. In Experiment 2, the effect of the same enzyme as used previously (added at 0.51 mu l/g forage DM, directly into the incubation medium), and buffer pH were examined using the ANKOM system, in a completely randomized design. Incubation medium pH was altered using 1 M citric acid, in order to obtain target initial pH values of 6.8 (control, no citric acid added), 6.2, 5.8, and 5.4. Actual initial pH values achieved were 6.72, 6.50, 6.20, and 5.72. Lowering the pH decreased (P < 0.01) dry matter disappearance (DMD) at 18 h incubation (0.339, 0.341, 0.314, and 0.291 for 6.72, 6.50, 6.20, and 5.72, respectively), whereas enzyme addition increased (P < 0.05) DMD at 24 h (0.363 versus 0.387 for control and enzyme-treated, respectively). Addition of enzyme increased (P < 0.05) neutral detergent fibre (NDF), acid detergent fibre (ADF), and hemicellulose (HC) degradation at pH 6.50 (0.077 versus 0.117; 0.020 versus 0.051; 0.217 versus 0.270 for control and enzyme-treated NDF, ADF and hemicellulose degradation, respectively) and 6.72 (0.091 versus 0.134; 0.041 versus 0.079; 0.205 versus 0.261 for control and enzyme-treated NDF, ADF and HC degradation, respectively). It is concluded that the positive effects of this enzyme product were independent of the pre-treatment period, but pH influenced the responses to enzyme supplementation. Under the conditions of this experiment, exogenous fibrolytic enzymes seemed to work better at close to neutrality ruminal pH conditions. (C) 2006 Elsevier B.V. All rights reserved.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - I. Effects of ensiling temperature, enzyme source and addition level

A series of experiments was completed to investigate the impact of addition of enzymes at ensiling on in vitro rumen degradation of maize silage. Two commercial products, Depot 40 (D, Biocatalysts Ltd., Pontypridd, UK) and Liquicell 2500 (L, Specialty Enzymes and Biochemicals, Fresno, CA, USA), were used. In experiment 1, the pH optima over a pH range 4.0-6.8 and the stability of D and L under changing pH (4.0, 5.6, 6.8) and temperature (15 and 39 degreesC) conditions were determined. In experiment 2, D and L were applied at three levels to whole crop maize at ensiling, using triplicate 0.5 kg capacity laboratory minisilos. A completely randomized design with a factorial arrangement of treatments was used. One set of treatments was stored at room temperature, whereas another set was stored at 40 degreesC during the first 3 weeks of fermentation, and then stored at room temperature. Silages were opened after 120 days. Results from experiment I indicated that the xylanase activity of both products showed an optimal pH of about 5.6, but the response differed according to the enzyme, whereas the endoglucanase activity was inversely related to pH. Both products retained at least 70% of their xylanase activity after 48 h incubation at 15 or 39 degreesC. In experiment 2, enzymes reduced (P < 0.05) silage pH, regardless of storage temperature and enzyme level. Depol 40 reduced (P < 0.05) the starch contents of the silages, due to its high alpha-amylase activity. This effect was more noticeable in the silages stored at room temperature. Addition of L reduced (P < 0.05) neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents. In vitro rumen degradation, assessed using the Reading Pressure Technique (RPT), showed that L increased (P < 0.05) the initial 6 h gas production (GP) and organic matter degradability (OMD), but did not affect (P > 0.05) the final extent of OMD, indicating that this preparation acted on the rumen degradable material. In contrast, silages treated with D had reduced (P < 0.05) rates of gas production and OMD. These enzymes, regardless of ensiling temperature, can be effective in improving the nutritive quality of maize silage when applied at ensiling. However, the biochemical properties of enzymes (i.e., enzymic activities, optimum pH) may have a crucial role in dictating the nature of the responses. (C) 2003 Elsevier B.V. All rights reserved.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - II. Effects on rate of acidification, fibre degradation during ensiling and rumen fermentation

A study was carried out to determine the influence of fibrolytic enzymes derived from mesophilic or thermophilic fungal sources, added at ensiling, on time-course fermentation characteristics and in vitro rumen degradation of maize silage. The mesophilic enzyme was a commercial product derived from Trichodenna reesei (L), whereas the thermophilic enzyme was a crude extract produced from Thermoascus aurantiacus (Ta) in this laboratory. The fungus was cultured using maize cobs as a carbon source. The resulting fermentation extract was deionised to remove sugars and characterised for its protein concentration, main and side enzymic activities, optimal pH, protein molecular mass and isoelectric point. In an additional study, both enzymes were added to maize forage (333.5 g DM/kg, 70.0, 469.8, 227.1 and 307.5 g/kg DM of CP, NDF, ADF and starch, respectively) at two levels each, normalized according to xylanase activity, and ensiled in 0.5 kg capacity laboratory minisilos. Duplicate silos were opened at 2, 4, 8, 15, and 60 days after ensiling, and analysed for chemical characteristics. Silages from 60 days were bulked and in vitro gas production (GP) and organic matter degradability (OMD) profiles evaluated using the Reading Pressure Technique (RPT), in a completely randomised design. The crude enzyme extract contained mainly xylanase and endoglucanase activities, with very low levels of exoglucanase, which probably limited hydrolysis of filter paper. The extract contained three major protein bands of between 29 and 55 kDa, with mainly acidic isoelectric points. Ensiling maize with enzymes lowered (P < 0.05) the final silage pH, with this effect being observed throughout the ensiling process. All enzyme treatments reduced (P < 0.05) ADF contents. Treatments including Ta produced more gas (P < 0.05) than the controls after 24 h incubation in vitro, whereas end point gas production at 96 h was not affected. Addition of Ta increased (P < 0.01) OMD after 12 h (410 and 416 g/kg versus 373 g/kg), whereas both L and Ta increased (P < 0.05) OMD after 24 h. Addition of enzymes from mesophilic or thermophilic sources to maize forage at ensiling increased the rate of acidification of the silages and improved in vitro degradation kinetics, suggesting an improvement in the nutritive quality. (C) 2003 Elsevier B.V All rights reserved.

Degradation products of clavulanic acid promote clavulanic acid production in cultures of Streptomyces clavuligerus

The role of clavulanic acid, an unstable antibiotic produced by Streptomyces clavuligerus, in biomass accumulation and production of clavulanic acid in batch cultures of the organism was examined. The organism was grown in a medium containing either 20 g/l lysine, 1 g/l lysine or 1 g/l lysine supplemented with degraded clavulanic acid as nitrogen sources. Biomass accumulation was highest in cultures grown with supplemented degraded clavulanic acid and reached a maximum of 2.2 g/l, compared with 1.5 g/l when lysine only was used. The yield coefficient for clavulanic acid production was again highest in cultures grown with supplemented degraded clavulanic acid, with a Y-p/x, value of 2 mg/g compared with Y-p/x value of 1.5 mg/g in 20 g/l lysine. No clavulanic acid was produced in cultures containing non-supplemented 1 g/l lysine. Non-degraded clavulanic, acid was added at 60 h to non-producing cultures of the organism containing 1 g/l lysine only. Clavulanic acid concentration immediately decreased on addition from 0.04 g/l over a period of 20 h, then remained constant at 0.02 g/l for a further 30 h until the end of the cultivation. This suggests that the rate of degradation was equivalent to the rate of production of clavulanic acid following a period of initial additive degradation. These results indicate that clavulanic acid is both produced and degraded in cultures of S. clavuligerus and that the products of degradation are used by the organism, resulting in further production of the antibiotic. (C) 2003 Elsevier Inc. All rights reserved.

Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones

Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by beta-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by beta-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK(1)R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nM, 10 min) induced interaction of NK(1)R and beta-arrestin at the plasma membrane, and the SP-NK(1)R-beta-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK(1)R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H(+)ATPase inhibitor bafilomycin A(1), which prevent endosomal SP degradation, suppressed NK(1)R recycling by >50%. Preincubation of neurones with SP (10 nM, 5 min) desensitized Ca(2+) transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK(1)R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK(1)R-beta-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK(1)R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating beta-arrestin-mediated endosomal signalling.

Endothelin-converting enzyme-1 regulates endosomal sorting of calcitonin receptor-like receptor and beta-arrestins

Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a-d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), beta-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B(2) receptor, which transiently interacts with beta-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/beta-arrestin complex, freeing internalized receptors from beta-arrestins and promoting recycling and resensitization.