Can cell-free enzymes replace rumen micro-organisms to model energy and protein suppty?

1998 ◽  
Vol 22 ◽  
pp. 99-114
Author(s):  
G. A. Broderick
Keyword(s):  
Protein Degradation ◽  
Trichoderma Viride ◽  
Streptomyces Griseus ◽  
Food Samples ◽  
Detergent Solution ◽  
Micro Organisms ◽  
Ruminal Digestion

AbstractNew ruminant feeding systems being developed in Europe and North America have greatly increased the need for rapid measurement of rate and extent of ruminal digestion of complex carbohydrates and proteins in individual foods. Application of these systems is lagging because of a lack of reliable kinetic digestion data. This has increased interest in using cell-free enzymes to assess ruminal breakdown of carbohydrates and proteins. The simplest kinetic model describing ruminal digestion divides carbohydrates and proteins into three fractions: A (that already digested and/ or that soluble), B (that digestible) and C (that completely indigestible). Fractions A, B and C sum to 1·0; proportion of digestible fraction B that is degraded is given by: [kd/ (kd+ kp], where kpand kdare, respectively, rates of ruminal passage and digestion. Some models divide fraction B into two or three subfractions while others include a digestion lag. Fully successful, cell-free enzyme systems would yield accurate estimates of digestion fractions and rates and, where appropriate, lag time. As a minimum, results with enzymes should be well correlated to in situ extents of digestion. In vivo total tract digestibility of organic matter (OM) and energy was predicted accurately when food samples were pre-treated with acid-pepsin or neutral-detergent solution, followed by treatment with cellulase from Trichoderma viride (T. reesei). Pepsin-cellulase and neutral detergent-cellulase methods have predicted in vivo digestibility more precisely than Tilley and Terry methods in some cases. Two-stage cellulase assays have given good results with many forages and, when including an amylase treatment, with mixed foods; OM digestibility of straws was not well predicted. Many different cell-free proteases have been tested to estimate ruminal protein degradability. Generally, effectiveness of proteases was assessed by correlating proportions of food nitrogen solubilized after specific incubation times with extents of in situ protein degradation. The broad specificity protease fromStreptomyces griseushas been used most extensively for this purpose in incubations at pH 8·0. However, somewhat better correlations have been reported for ficin, bromelain, papain and neutral proteases of fungal and bacterial origin in incubations at pH more similar to the rumen. Prior treatment with amylases improved correlations for concentrate foodstuffs. As yet, cell-free proteases have not accurately predicted rates and extents of protein degradation observed in ruminal in vitro and in situ systems.

A history ofin vitrotechniques

1998 ◽  
Vol 22 ◽  
pp. 13-19
Author(s):  
D. J. Minson
Keyword(s):  
Continuous Fermentation ◽  
In Vitro Digestion ◽  
Food Samples ◽  
Gas Production ◽  
In Vitro Techniques ◽  
Dry Matter Digestibility ◽  
Calibration Methods ◽  
Micro Organisms

AbstractRegressions relating in vivo digestibility to chemical composition of the food have residual standard deviations that are unacceptably high. The development of the two-stage in vitro technique inoculated with rumen liquor (Tilley and Terry, 1963) allowed dry-matter digestibility to be predicted with greater accuracy. This success was followed by a series of developments which replaced rumen liquor with inoculum produced from fresh or preserved faeces collected from sheep or cattle. Other methods used inoculum from a continuous fermentation containing rumen micro-organisms and enzymes produced by fungi. Another modification was to use gas production as a measure of in vitro digestion. The range of nutritional problems that could be measured by in vitro techniques was extended to include the estimation of voluntary food intake and protein degradation. All these in vitro techniques require standardization using food samples that have previously been analysed in vitro or offered as the sole diet to animals. The relative merits of these two calibration methods are discussed. Special facilities are required for storing and distributing these standard foods.

scholarly journals Digestibility and ruminal digestion kinetics of corn silage

2005 ◽  
Vol 57 (2) ◽  
pp. 223-228 ◽  
Author(s):  
O.N. Di Marco ◽  
M.S. Aello ◽  
S. Arias
Keyword(s):  
Fixed Effects ◽  
Exponential Model ◽  
In Vitro Digestibility ◽  
Passage Rate ◽  
Digestion Kinetics ◽  
Ruminal Digestion ◽  
Kinetics Of

The in situ dry matter (DM) disappearance of corn silages in two maturity stages (milk grain and half milk line) of known in vivo and in vitro digestibility was determined, with the main purpose of comparing digestibility values with the ruminal disappearance at 24 and 48h of incubation. A secondary goal was the description of their ruminal digestion kinetics, from which the effective degradability was calculated at an assumed passage rate of 4%/h. Data of in vivo, in vitro and in situ degradability at 24 and 48-h were analyzed with a linear model that included as fixed effects the maturity and the methodology of evaluation, and the kinetic data were described by the exponential model of McDonald. There was a significant effect (P<0.05) of methodology in the estimation of digestibility, but not of maturity or interaction maturity × methodology. The in vivo digestibility (52.9%) was not different from the 24-h in situ degradability (55.6%) with numerical values in the range of the effective degradability. The in vitro digestibility (61.6%) was not different from the 48-h in situ degradability (61.9%), being both estimates higher than the in vivo digestibility. The 24-h in situ degradability was a closer estimator of the in vivo digestibility and the 48-h in situ degradability and the in vitro digestibility overestimated the in vivo parameter by 15-20%.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Evaluation of Structure-Controlled Silk Fibroin Coatings for Orthopedic Infection and In-Situ Osteogenesis: In Vitro and In Vivo

2020 ◽  
Author(s):  
Wenhao Zhou ◽  
Teng Zhang ◽  
Jianglong Yan ◽  
QiYao Li ◽  
Panpan Xiong ◽  
...  
Keyword(s):  
Silk Fibroin ◽  
Orthopedic Infection

scholarly journals Development of In Situ Self-Assembly Nanoparticles to Encapsulate Lopinavir and Ritonavir for Long-Acting Subcutaneous Injection

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 904
Author(s):  
Irin Tanaudommongkon ◽  
Asama Tanaudommongkon ◽  
Xiaowei Dong
Keyword(s):  
Sustained Release ◽  
Trough Concentration ◽  
Self Assembly ◽  
Subcutaneous Injection ◽  
Drug Loading ◽  
Entrapment Efficiency ◽  
Long Acting

Most antiretroviral medications for human immunodeficiency virus treatment and prevention require high levels of patient adherence, such that medications need to be administered daily without missing doses. Here, a long-acting subcutaneous injection of lopinavir (LPV) in combination with ritonavir (RTV) using in situ self-assembly nanoparticles (ISNPs) was developed to potentially overcome adherence barriers. The ISNP approach can improve the pharmacokinetic profiles of the drugs. The ISNPs were characterized in terms of particle size, drug entrapment efficiency, drug loading, in vitro release study, and in vivo pharmacokinetic study. LPV/RTV ISNPs were 167.8 nm in size, with a polydispersity index of less than 0.35. The entrapment efficiency was over 98% for both LPV and RTV, with drug loadings of 25% LPV and 6.3% RTV. A slow release rate of LPV was observed at about 20% on day 5, followed by a sustained release beyond 14 days. RTV released faster than LPV in the first 5 days and slower than LPV thereafter. LPV trough concentration remained above 160 ng/mL and RTV trough concentration was above 50 ng/mL after 6 days with one subcutaneous injection. Overall, the ISNP-based LPV/RTV injection showed sustained release profiles in both in vitro and in vivo studies.

scholarly journals Establishment of MHHi001-A-5, a GCaMP6f and RedStar dual reporter human iPSC line for in vitro and in vivo characterization and in situ tracing of iPSC derivatives

2021 ◽  
Vol 52 ◽  
pp. 102206
Author(s):  
Alexandra Haase ◽  
Tim Kohrn ◽  
Veronika Fricke ◽  
Maria Elena Ricci Signorini ◽  
Merlin Witte ◽  
...  
Keyword(s):  
Ipsc Line ◽  
Dual Reporter ◽  
In Vivo Characterization ◽  
Human Ipsc

scholarly journals Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick—Pathogen Interactions

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 70
Author(s):  
Lourdes Mateos-Hernández ◽  
Natália Pipová ◽  
Eléonore Allain ◽  
Céline Henry ◽  
Clotilde Rouxel ◽  
...  
Keyword(s):  
Cell Line ◽  
Peripheral Nerves ◽  
Ixodes Scapularis ◽  
Embryonic Cell ◽  
Cell Subpopulation ◽  
In Vivo Experiments

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick–pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick–pathogen interactions.

scholarly journals TMOD-24. GENERATION AND CHARACTERIZATION OF A NOVEL TRANSGENIC IDO REPORTER MOUSE FOR IDO POSTTRANSLATIONAL MODIFICATION ANALYSIS IN SITU

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii233-ii233
Author(s):  
April Bell ◽  
Lijie Zhai ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
Lakshmi Bollu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Cell ◽  
Central Nervous System Tumor ◽  
Post Translational Modifications ◽  
Reporter Mouse ◽  
C Terminus ◽  
Human Gbm

Abstract Glioblastoma (GBM) is the most common and aggressive primary central nervous system tumor in adults with a median survival of 14.6 months. GBM is a potently immunosuppressive cancer due in-part to the prolific expression of immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO). Tumor cell IDO facilitates the intratumoral accumulation of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Although immunosuppressive IDO activity is canonically characterized by the conversion of tryptophan into kynurenine, we have utilized transgenic and syngeneic mouse models and mutant glioma lines to demonstrate that tumor cell IDO increases Treg accumulation independent of tryptophan metabolism. Here, we address the gap in our understanding of IDO signaling activity in vivo. Subcutaneously-engrafted human GBM expressing human IDO-GFP cDNA was isolated from immunodeficient humanized NSG-SGM3 mice. The tumor was immunoprecipitated for the GFP tag using GFP-TRAP followed by mass spectrometry which revealed a novel methylation site on a lysine residue at amino acid 373 in the IDO C-terminus region. Western blot analysis of IDO protein also revealed the presence of tyrosine phosphorylation. Additionally, we recently created a new transgenic IDO reporter mouse model whereby endogenous IDO is fused to GFP via a T2A linker (IDO→GFP). This model allows for the isolation of IDO+ cells in real-time and without causing cell death, thereby creating the opportunity for downstream molecular analysis of in situ-isolated GFP+ cells. Collectively, our work suggests that IDO non-enzyme activity may involve the post-translational modifications we recently identified. As IDO activity may differ between in vitro and in vivo modeling systems, we will use the new IDO→GFP reporter mouse model for an improved mechanistic understanding of how immunosuppressive IDO facilitates Treg accumulation in vivo.

Turnover of total proteins and ornithine aminotransferase during liver regeneration in rats

1980 ◽  
Vol 238 (1) ◽  
pp. E46-E52
Author(s):  
S. L. Augustine ◽  
R. W. Swick
Keyword(s):  
Amino Acids ◽  
Liver Regeneration ◽  
Protein Degradation ◽  
Partial Hepatectomy ◽  
Free Amino ◽  
Liver Protein ◽  
Ornithine Aminotransferase ◽  
Fractional Rate

The recovery of approximately 40% of the total liver protein during the first day after partial hepatectomy was shown to be due to the near cessation of protein breakdown rather than to an increase in protein synthesis. The decrease in degradation of total protein was less if rats were adrenalectomized or protein-depleted prior to partial hepatectomy. The effect of these treatments originally suggested that changes in free amino acid levels in liver might be related to the rate of protein degradation. However, no correlation was found between levels of total free amino acids and rates of breakdown. Measurements of individual amino acids during liver regeneration suggested that levels of free methionine and phenylalanine, amino acids that have been found to lower rates of protein degradation in vitro, are not correlated with rates of breakdown in vivo. The difference between the fractional rate of ornithine aminotransferase degradation (0.68/day and 0.28/day in sham-hepatectomized and partially hepatectomized rats, respectively) was sufficient to account for the higher level of this protein 3 days after surgery in the latter group.

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