Lack of adaptation of pancreatic colipase in rats and mice.

1977 ◽  
Vol 232 (2) ◽  
pp. E131
Author(s):  
M C Vandermeers-Piret ◽  
A Vandermeers ◽  
W Wijns ◽  
J Rathé ◽  
J Christophe
Keyword(s):  
Alloxan Diabetes ◽  
Specific Activity ◽  
Limiting Factor ◽  
High Fat ◽  
Rat Pancreas ◽  
High Starch ◽  
Rats And Mice ◽  
Moderate Diabetes

A new automated potentiometric method for the determination of colipase was developed, taking advantage of the reactivation of purified lipase, in the presence of bile salt and at pH 6.5. High-fat and high-starch diets induced an opposite regulation of lipase and amylase in the rat pancreas. At the same time, the level of colipase was not influenced by nutrition. During fasting and in alloxan diabetes, the specific activity of lipase almost doubled, that of amylase decreased sharply, and colipase was not affected in the rat pancreas. In obese-hyperglycemic mice, suffering from obesity, hyperinsulinism, and moderate diabetes, there was also no regulation of pancreatic colipase. Thus, at variance with a number of hydrolases, there was no dietary or hormonal adaptation of colipase. However, this was probably without any bearing on intraluminal lipolysis. Indeed, comparison of lipase and colipase activities in pancreas and in small intestine suggests that colipase concentration is not a limiting factor of intraluminal lipolysis. The molecular mechanism of this assumption is discussed on the basis of in vitro studies.

scholarly journals Quantification and characterization of the 5’exonuclease activity of the lysosomal nuclease PLD3 by a novel cell-based assay

2020 ◽  
pp. jbc.RA120.015867
Author(s):  
Cedric Cappel ◽  
Adriana Carolina Gonzalez ◽  
Markus Damme
Keyword(s):  
Lupus Erythematosus ◽  
Specific Activity ◽  
Nuclease Activity ◽  
Hydrolytic Activity ◽  
Proteolytic Processing ◽  
Exonuclease Activity ◽  
Fluorescence Quencher

Phospholipase D3 (PLD3) and phospholipase D4 (PLD4), the most recently described lysosomal nucleases, are associated with Alzheimer`s disease, spinocerebellar ataxia, and systemic lupus erythematosus. They exhibit 5’ exonuclease activity on single-stranded DNA, hydrolyzing it at the acidic pH associated with the lysosome. However, their full cellular function is inadequately understood. To examine these enzymes, we developed a robust and automatable cell-based assay based on fluorophore- and fluorescence-quencher coupled oligonucleotides for the quantitative determination of acidic 5’ exonuclease activity. We validated the assay under knockout and PLD-overexpression conditions, and then applied it to characterize PLD3 and PLD4 biochemically. Our experiments revealed PLD3 as the principal acid 5’ exonuclease in HeLa cells, where it showed a markedly higher specific activity compared to PLD4. We further used our newly developed assay to determine the substrate specificity and inhibitory profile of PLD3, and found that proteolytic processing of PLD3 is dispensable for its hydrolytic activity. We followed the expression, proteolytic processing, and intracellular distribution of genetic PLD3 variants previously associated with Alzheimer’s disease and investigated each variant's effect on the 5’ nuclease activity of PLD3, finding that some variants lead to reduced activity, but others not. The development of a PLD3/4-specific biochemical assay will be instrumental in understanding better both nucleases and their incompletely unknown roles in vitro and in vivo.

Rumen microbial degradation of full-fat and defatted palm oil sludge (POS) in a consecutive batch culture system

2001 ◽  
Vol 2001 ◽  
pp. 126-126
Author(s):  
Ismartoyo ◽  
B. Nohong
Keyword(s):  
Palm Oil ◽  
Microbial Degradation ◽  
Culture System ◽  
Nutritive Value ◽  
Fat Content ◽  
Limiting Factor ◽  
High Fat ◽  
Fast Development ◽  
Micro Organisms

In Indonesia the production of POS increased rapidly as the areas of palm oil plantation increased. The POS contains high protein (15%), fibre (18%), and fat (20%) and thus likely to have the potential to provide high levels of protein and carbohydrate supplements for ruminant. However, the fast development of POS production in Indonesia is not supported by good researches to examine its nutritive value. High fat content in the diet of steers (Mooreet al, 1986) has been found to depress the fibre digestibility; it is thought that high fat content in POS could be the limiting factor for the utilisation of the POS by ruminant. Therefore, an experimentin vitrowas conducted to examine the degradation and fermentation of full-fat and defatted POS by rumen micro-organisms.

Ontogeny of prolyl endopeptidase, pyroglutamyl peptidase I, TRH, and its metabolites in rat pancreas

1992 ◽  
Vol 262 (6) ◽  
pp. E845-E850
Author(s):  
P. Salers ◽  
L. H. Ouafik ◽  
P. Giraud ◽  
J. Y. Maltese ◽  
A. Dutour ◽  
...  
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Early Stage ◽  
Prolyl Endopeptidase ◽  
Neonatal Rat ◽  
Rat Pancreas ◽  
Low Levels ◽  
Intact Rats

We demonstrate that two enzymes, soluble unspecific pyroglutamyl peptidase I and prolyl endopeptidase, able to degrade thyrotropin-releasing hormone (TRH) in vitro were present in pancreas at the early stage of rat development. Specific particulate pyroglutamyl peptidase II remained undetectable during ontogenesis. Pyroglutamyl peptidase I specific activity increased until day 3 and decreased after day 5. Furthermore, prolyl endopeptidase specific activity rose slightly to a peak on postnatal day 20. A good correlation between immunoreactive TRH and deaminated TRH (TRH-OH) was found in the 1st wk after birth. However, His-Pro diketopiperazine (DKP) levels were stable and low during development. We show that hot acidic extraction conditions could artefactually generate His-Pro DKP. In vivo, active site-directed inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase enzymes do not show any TRH-deamidating and/or pyroglutamyl peptidase I pathways in neonatal rat pancreas. The data suggest that these two enzymes are not involved in intra- or extracellular control of TRH levels in neonatal rat pancreas and that pancreatic TRH content appears to be principally regulated by biosynthetic steps. Nevertheless, low levels of endogenous His-Pro DKP and TRH-OH identified in neonatal rat pancreas suggest that TRH or TRH-like peptides may be metabolized in this tissue in intact rats, albeit at low rates.

scholarly journals METHODS FOR ASSESSMENT OF THE SPECIFIC ACTIVITY OF DRUGS FOR PREVENTION OF HEPATITIS B

2017 ◽  
Vol 62 (5) ◽  
pp. 233-240
Author(s):  
E. L. Postnova ◽  
N. V. Shalunova ◽  
K. A. Sarkisyan ◽  
A. A. Movsesyants
Keyword(s):  
Hepatitis B ◽  
Enzyme Immunoassay ◽  
Specific Activity ◽  
Elisa Method ◽  
Normative Documents ◽  
Immunologic Activity

The immunologic activity (specific activity) is one of the main indicators of quality of vaccines for prophylaxis of hepatitis B, along with their safety. Retrospective analysis of the use of laboratory methods for assessment of specific (immunogenic) activity of modern vaccines against hepatitis B using indicators was carried out: in vitro method based on evaluation of HBsAg content and in vivo method based on evaluation of immunogenic activity in mice. Both methods are standardized and described in normative documents on the vaccines against hepatitis B of domestic production registered in the Russian Federation. Indicators of specific (immunogenic) activity of vaccines against hepatitis B were used to investigate more than 170 vaccine series using the ELISA method in the period from 2013 to 2015. The obtained control results confirmed the expediency and efficiency of enzyme immunoassay for determination of HBsAg content, as well as permissibility of use of ready sets of the Murex HBsAg Version 3 test systems for testing vaccines against hepatitis B by the ELISA method. Analysis of the results of laboratory control of series of vaccines against hepatitis B using a biological method for immunogenicity evaluation based on ED50 analysis confirms persistently high immunogenic activity of the Russian commercial vaccines intended for prophylaxis of hepatitis B. The confirmed comparability of methods allows the number of in vivo tests to be further reduced in favor of the enzyme immunoassay authentically characterizing the produced drug.

scholarly journals Development and validation of a method for the determination of the specific activity of recombinant monoclonal antibody eculizumab

2020 ◽  
Vol 15 (2) ◽  
pp. 77-85
Author(s):  
D. I. Zybin ◽  
A. S. Seregin ◽  
A. D. Askretkov ◽  
N. V. Orlova ◽  
Yu. A. Seregin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Pharmaceutical Analysis ◽  
Comparative Evaluation ◽  
Specific Activity ◽  
In Vitro Method ◽  
Recombinant Monoclonal Antibody ◽  
Development And Validation ◽  
First Time

Objectives. Developing reliable and accurate analytical methods is necessary for comparative pharmaceutical analysis using physicochemical, biological (in vitro), preclinical, and clinical trials. The main objective of this study was to develop and validate an in vitro method for determining the specific activity of the recombinant monoclonal antibody eculizumab.Methods. The method of indirect enzyme immunoassay was used in the study.Results. A method for determining the specific activity of the humanized recombinant monoclonal antibody eculizumab was described and validated for the first time. A comparative evaluation of the specific activity of Soliris® (Alexion Pharmaceuticals Inc., USA), and its biosimilar PRK-001 (Pharmapark, Russia) was performed using the developed method.Conclusions. The similarity of PRK-001 and the original Soliris® in relation to their specific activity, that is, binding to the human complement system C5 protein, was proved. 

Biochemical characterization a digestive trypsin in the midgut of large cabbage white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae)

2017 ◽  
Vol 108 (4) ◽  
pp. 501-509
Author(s):  
A. Sharifloo ◽  
A. Zibaee ◽  
J. Jalali Sendi ◽  
K. Talebi Jahroumi
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Pieris Brassicae ◽  
Larval Midgut ◽  
Posterior Midgut ◽  
Diethylaminoethyl Cellulose ◽  
Cabbage White Butterfly ◽  
Fast Flow

AbstractA comprehensive study on digestive trypsin was undertaken in the larval midgut of Pieris brassicae L. Results of enzymatic compartmentalization showed a significantly higher activity of crude trypsin in the anterior larval midgut rather than posterior-midgut. Using Diethylaminoethyl cellulose fast flow column chromatography a purified trypsin was obtained by specific activity of 21 U mg−1 protein, recovery of 22%, purification fold of 28-fold and molecular weight of 25 kDa. This purified enzyme showed the highest activity at pH 8 and the corresponding temperature of 40°C. However, the specific inhibitors used including 4-(2-Aminoethyl) benzenesulfonyl fluroride hydrochloride, N-p-Tosyl-L-lysine methyl ester hydrochloride and Soybean Trypsin Inhibitor significantly lowered the activity of the purified enzyme in vitro. Moreover, the activity of trypsin and likewise the nutritional indices were significantly altered in the larval midgut feeding upon the leaves treated by 1 mM concentration of each inhibitor in comparison with control. Determination of enzymatic characteristics of insect trypsins is crucial in paving the path for controlling pests by potential natural compounds via transgenic plants.

Determination of rumen microbial growth in vitro from32P-labelled phosphate incorporation

1977 ◽  
Vol 38 (1) ◽  
pp. 101-114 ◽  
Author(s):  
C. J. Van Nevel ◽  
D. I. Demeyer
Keyword(s):  
Microbial Growth ◽  
Specific Activity ◽  
Rumen Fluid ◽  
List Type ◽  
Substrate Protein ◽  
Isotope Method ◽  
Total Growth ◽  
N Incorporation

1.The extracellular phosphate pool in incubations of rumen fluid or washed cell suspensions of mixed rumen bacteria (WCS) was labelled with32P. From the constant extracellular phosphate pool specific activity and the amount of radioactivity incorporated during incubation, the amount of P incorporated in the microbial fraction was calculated. From the value for nitrogen: P determined in microbial matter, the amount of N incorporated was calculated as a measure of microbial growth.2.Incorporation of soluble non-protein-N in incubations devoid of substrate protein was 50 and 80 % of the values obtained using the isotope method for rumen fluid and WCS respectively. It is suggested that results obtained using the former method reflect 'net growth' of micro-organisms which is the result of simultaneous growth and degradation. The isotope method measures 'total growth', as isotope incorporation is not affected by degradation of non-growing cells.3.Incorporation of32P in P-containing microbial components (mainly nucleic acids) was compared with net synthesis of these components in incubations of WCS. The results showed different specific rates of synthesis and degradation for all components studied. It is concluded that the composition of microbial matter changed during growth.4.When N incorporation, calculated from results obtained using the isotope method in incubations with rumen fluid, was compared with the amount of carbohydrate substrate fermented and the type of fermentation, values between 18.3 and 44.6 g N incorporated/kg of organic matter fermented were obtained. Low values were associated with large proportions of the substrate being fermented to lactate and the use of glucose instead of disaccharides as substrate. Part of the variation could also be attributed to differences in incubation period, reflected in different proportions of polysaccharide formed.5.The use of isotopes for determination of rumen microbial growth in vitro is critically discussed.

THE CONVERSION OF PREGNENOLONE TO PROGESTERONE AND 16α-HYDROXYDEHYDROEPIANDROSTERONE IN VITRO BY ADRENAL TISSUE FROM A NEWBORN ANENCEPHALIC INFANT

1968 ◽  
Vol 40 (1) ◽  
pp. 29-35 ◽  
Author(s):  
M. M. SHAHWAN ◽  
R. E. OAKEY ◽  
S. R. STITCH
Keyword(s):  
Specific Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Enzymatic Conversion ◽  
Partition Chromatography ◽  
Adrenal Tissue ◽  
Enzyme Systems ◽  
Constant Specific Activity

SUMMARY Adrenal tissue, largely composed of the definitive zone, from a newborn anencephalic infant, contained the following enzyme systems: (1) a Δ5-3β-hydroxysteroid dehydrogenase for pregnenolone, demonstrated by the conversion of [14C]pregnenolone to [14C]progesterone; (2) a C(17)-C(20) desmolase, and (3) a steroid 16α-hydroxylase, demonstrated by the conversion of [14C]pregnenolone to [14C]3β, 16α-dihydroxyandrost-5-en-17-one. The metabolites could not be separated from carrier steroids during sequential partition chromatography. [14C]Progesterone was identified by recrystallization to constant specific activity. [14C]3β, 16α-Dihydroxyandrost-5-en-17-one was identified by enzymatic conversion to [14C]16α-hydroxyoestrone followed by reduction to oestriol and determination of the specific activity of the oestriol after partition chromatography. It is suggested that these enzymes may play some part in the production of cortisol by the newborn anencephalic infant, and in the provision of precursors for placental oestriol production.

scholarly journals Qualitative Indicators of Experimental Brucellosis Antigen Preparations Designed for Cellular Tests in vitro

2020 ◽  
pp. 83-88
Author(s):  
S. A. Kurcheva ◽  
D. A. Kovalev ◽  
D. G. Ponomarenko ◽  
Yu. V. Siritsa ◽  
M. V. Kostyuchenko ◽  
...  
Keyword(s):  
Specific Activity ◽  
Laboratory Diagnosis ◽  
Polysaccharide Composition ◽  
Methodological Approaches ◽  
Cellular Tests ◽  
Refractometric Detection ◽  
Experimental Preparation ◽  
Selection Of

In order to develop the most diagnostically informative methods for carrying out antigen-stimulated cellular tests in vitro a careful selection of stimulating agent (antigen) is required, possessing an adequate activating potential and providing specificity of the reaction.Objective of the study was to identify the qualitative indicators of experimental batches of brucellosis antigen preparations designed for cellular tests in vitro.Materials and methods. Initially we produced antigen complexes of brucellosis microbe on the basis of the vaccine strains of three epidemically significant Brucella species (B. abortus, B. melitensis, B. suis). Quantitative determination of WsAg and PPBC proteins of experimental preparation series was performed applying capillary electrophoresis. Qualitative composition was assessed through ion exchange liquid chromatography with refractometric detection.Results and discussion. We have specified physical-chemical features, investigated chromatographic profiles and composition of protein fractions, as well as tried the produced experimental batches of brucellosis antigen preparations. After analyzing the defined protein and polysaccharide composition of the obtained WsAg samples, one can conclude that WsAg preparation cannot be used for cellular tests as the probability of non-specific lymphocyte reaction manifestation in vitro was experimentally proven. By contrast, complex brucellosis antigen preparation PPBC has an expressed specific activity and specificity under in vitro conditions and the prospects to be used when developing methodological approaches for laboratory diagnosis of brucellosis and assessment of de facto immunity rate in risk contingents after vaccination. The obtained parameters will allow for proper quality provision when manufacturing the developed experimental PPBC preparation designed for cellular tests in vitro, taking into account modern validation and standardization regulations. 

Method for kinetic study of in vitro conversion of a C14-labeled substrate to CO2

1960 ◽  
Vol 15 (5) ◽  
pp. 949-952 ◽  
Author(s):  
Cecil L. Allweis ◽  
Harold Gainer ◽  
I. L. Chaikoff
Keyword(s):  
Gas Phase ◽  
Specific Activity ◽  
Brain Slices ◽  
Constant Concentration ◽  
Tissue Slices ◽  
Exogenous Substrate ◽  
Isotopic Equilibration ◽  
Periodic Replacement

A method is described for the determination of CO2 and C14O2 production rates by tissue slices incubated in the presence of a constant concentration and constant specific activity of a C14-labeled substrate. The constant conditions were maintained by periodic replacement of the incubation medium and gas phase. It is pointed out that, in order to determine the true rates of conversion to CO2 of an exogenous labeled substrate and of an endogenous, nonequilibrating CO2 precursor, measurements must be made after isotopic equilibration is attained. In the case of whole brain slices, such equilibration was observed after 3–4 hours of incubation. With glucose as the exogenous substrate, 30–40% of the CO2 produced by rat whole cerebrum slices was derived from endogenous sources for as long as 12 hours. Submitted on December 14, 1959

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