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PubMed1982

A radioimmunoassay method for quantification of alpha-tropomyosin in heart homogenates.

A new, extremely sensitive, solid phase radioimmunoassay has been developed to quantify tropomyosin levels in heart tissue homogenates. Specific antibody was coupled to Sepharose 4B and saturation levels of [125I]tropomyosin bound. Release of radiolabel into the supernatant portion occurred when heart homogenates, authentic tropomyosin or tropomyosin in the presence of homogenate were added to these immunobeads. Quantification of the amount of tropomyosin was based on the level of release effected by standard tropomyosin with and without homogenate. The assay was determined to be highly specific and sensitive for tropomyosin. Picomolar quantities of the protein were readily detectable. Linearity extended well over the range of 5-200 ng tropomyosin in the homogenate. The method can be applied to other proteins for quantification during embryonic development.

Amino Acids↗

PubMedDate not supplied

Corticosterone catabolism by mouse liver homogenate: strain difference in dependence on added NADP.

The C57BL/10ScSn and DBA/2J strains of mice differed in the rate at which corticosterone was reductively catabolised by liver homogenates when the animals had been fed a diet low in fat. The addition of an NADPH generating system to the homogenates increased the catabolism of corticosterone. Its greatest effect was on the homogenates from DBA mice given a fat-free diet and its least effect on those from C57 mice fed a standard diet. The addition of NADP was similar in its effect to the addition of the complete NADPH generating system. Glucose-6-phosphate dehydrogenase stimulated corticosterone breakdown only in homogenates from C57 mice on the standard diet. The addition of glucose-6-phosphate had no effect. NADP was the only effective stimulator of corticosterone reduction in homogenates from CBA/FaCam mice.

Animals↗

PubMed2003

Homogeneity check of agricultural and food industries samples using near infrared spectroscopy.

Samples distributed in proficiency testing schemes (PTS) need to be homogeneous in order to be sure that if a laboratory has a result different from the other laboratories, its error can be attributed to its analysis method and not to its sample. This control must be done according to the ISO 13528 draft standard before sending the samples to the laboratories. It can be done by determining homogeneity targets by sub-contracting to accredited laboratories using reference methods, but this engenders logistic and financial problems. That is why a homogeneity check using Near Infrared Spectroscopy (NIR) has been developed for agricultural and food industries samples prepared for PTS at Bipea (Bureau Interprofessionnel d'Etudes Analytiques). To evaluate the homogeneity among samples, this procedure involves a comparison of NIR spectra, the determination of global homogeneity criteria and the use of control charts. The method of control developed and carried out at Bipea allows the rapid and easy monitoring of the performance of the sample preparation.

Crops, Agricultural↗

PubMed2003

Lineage-specific homogenization of the polyubiquitin gene among human and great apes.

Ubiquitin is a highly conserved protein, and is encoded by a multigene family among eukaryote species. The polyubiquitin genes, UbB and UbC, comprise tandem multiple ubiquitin coding units without a spacer region or intron. We determined nucleotide sequences for the UbB and UbC of human, chimpanzee, gorilla, and orangutan. The ubiquitin repeat number of UbB was constant (3) in human and great apes, while that of UbC varied: 6 to 11 for human, 10 to 12 for chimpanzee, 8 for gorilla, and 10 for orangutan. The heterogeneity of the repeat number within closely related hominoid species suggests that a lineage-specific unequal crossover and/or gene duplication occurred. A marked homogenization of UbC occurred in gorilla with a low level of synonymous difference (p(s)). The homogenization of UbC also occurred in chimpanzee and less strikingly in human. The first and last ubiquitin coding units of UbC were clustered independently between species, and less affected by homogenization during the hominoid evolution. Therefore, the homogenization of ubiquitin coding units is likely due to an unequal crossing-over inside the ubiquitin units. The lineage-specific homogenization of UbC among closely related species suggests that concerted evolution has a key role in the short-term evolution of UbC.

Animals↗

PubMed2000

Comparison of the spatial homogeneity of physico-chemical parameters and bacterial 16S rRNA genes in sediment samples from a dumping site for dredging sludge.

The homogeneity of the microbial community structure of a sediment landfill was examined by a culture-independent method and compared with physico-chemical parameters, i.e. organic matter, CaCO3 content, pH, and texture. Total genomic DNA was extracted from samples derived from different places and depths. After amplification with two different primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints of different sediment samples taken in regular patterns at the same depth were similar, which indicates a spatial homogeneity in the numerically dominant bacterial populations in a landfill over 10,000 m2 in size. In a vertical column of approx. 10 m, only some differences in a few bands of the bacterial community structure were observed between samples taken from different depths. This DNA homogeneity coincided with a similar homogeneity of the physico-chemical parameters in the landfill at this site. Nevertheless, the DGGE technique revealed small differences in less prominent bacteria and was capable of separating the upper and lower samples of one column into two clusters. It therefore seems more sensitive than the physico-chemical approach for characterising the homogeneity of an environmental habitat.

Belgium↗

PubMed2004

Optimal operation of high-pressure homogenization for intracellular product recovery.

An optimal control methodology for the homogenization of bacterial cells to recover intracellular products is presented. A Fluent computational fluid dynamics (CFD) model is used to quantify the hydrodynamic forces present in the homogenizer, and empirical models are used to relate these forces to experimentally obtained cell disruption and product recovery data. The optimal homogenizer operation, in terms of either constant cell breakage or maximum intracellular product recovery, is determined using these empirical models. We illustrate this methodology with an Escherichia coli bacterial system used to produce DNA plasmids. Homogenization is performed using an industrial APV-Gaulin high-pressure homogenizer. The modeling and optimization results for this E. coli-DNA plasmid system show good agreement with the experimental data.

Cell Fractionation↗

PubMed2006

Near-infrared imaging for studying homogeneity of protein-sugar mixtures.

PURPOSE: To investigate the applicability of near-infrared (NIR) imaging for assessing the homogeneity of dried protein-sugar formulations. METHODS: Physical mixtures of lysozyme and trehalose in different ratios were prepared and analyzed by near-infrared (NIR) imaging with a spatial resolution of 10 or 40 microm. To define and select the best imaging strategy, besides visual inspection of the images, several approaches for data processing were tested: single wavelength intensity, peak/height ratio of two specific wavelengths, correlation coefficient with a reference spectrum and principal component analysis (PCA). In order to relate the contrast directly to concentration differences of lysozyme and trehalose, quantitative models were created based on correlation coefficient and partial least squares (PLS) regression. The selected imaging method was applied to compare the homogeneity of a supercritical fluid (SCF) dried and a freeze-dried lysozyme-trehalose mixture. RESULTS: All tested methods confirmed each other and showed spatial heterogeneity in the lysozyme and trehalose contents of the physical mixtures. However, multivariate data processing methods (correlation coefficient and PCA/PLS) resulted in more distinct contrasts than univariate approaches (single wavelength analysis) and allowed a quantitative estimation of the homogeneity. As shown by NIR imaging in combination with the correlation coefficient or the PLS method, the SCF dried lysozyme-trehalose formulation was at least as homogeneous as its lyophilized counterpart, at 10 microm pixel size resolution. CONCLUSIONS: NIR imaging is a useful tool for studying the homogeneity of dried protein-sugar formulations.

Algorithms↗

PubMed1985

A micro-technique for preparing homogenates of biological samples.

A method is described for preparing homogenates of insects, mites, nematodes, or fungal spores for electrophoretic analysis which overcomes the disadvantages of existing methods. It is also applicable to the homogenization of microsamples of animal tissue. The method involves grinding the samples in glass tubes, closed at one end, prepared from melting-point capillaries. A range of plungers is fabricated from the stainless-steel plungers from broken microsyringes, or from stainless-steel wire. The plunger is operated by hand to homogenize a sample in 2-20 microliter of buffer in a tube. The techniques for handling the samples before and after homogenization and for labeling and centrifuging them are described. Compared with existing methods, the procedure minimizes sample loss and risk of cross-contamination and eliminates the possibility of overheating the sample during homogenization.

Animals↗

PubMed1988

Hydrolysis of the four stereoisomers of soman catalyzed by liver homogenate and plasma from rat, guinea pig and marmoset, and by human plasma.

Stereoselective hydrolysis at pH 7.5 and 37 degrees of C(+/-)P(+/-)-soman by liver homogenate and plasma from rat, guinea pig and marmoset, and by human plasma is studied by using the four single stereoisomers. The fast hydrolysis of the C(+/-)P(+)-isomers is monitored titrimetrically, whereas the decay of the much slower reacting C(+/-)P(-)-isomers is followed by gas chromatographic determination of the residual concentration. Values of Km and Vmax are evaluated for the enzymatic hydrolysis of the two relatively nontoxic C(+/-)P(+)-isomers. The plasma enzymes have a high affinity for these isomers (Km: 0.01-0.04 mM); the Km values of the liver enzymes vary between 0.04 and 0.7 mM. Except for rat liver homogenate, only first-order rate constants can be obtained for catalyzed hydrolysis (kc) of the highly toxic C(+/-)P(-)-isomers: most measurements with C(+/-)P(-)-isomer concentrations greater than 0.3 mM are complicated by epimerization to C(+/-)P(+)-isomers, which may conceal enzyme saturation with the C(+/-)P(-)-isomers. The first-order rate constants of catalyzed hydrolysis (Vmax/Km or kc) by all liver homogenates and plasmata decrease in the order: C(+)P(+)- greater than C(-)P(+)- much greater than C(-)P(-)- greater than C(+)P(-)-soman. The highest P(+)-/P(-)-stereoselectivity is found for rat plasma. Rat liver homogenate is more potent than the other liver homogenates in catalyzing the hydrolysis of both the C(+/-)P(+)- and the C(+/-)P(-)-isomers. Rat plasma shows the highest activity for degradation of the C(+/-)P(+)-isomers, but is approximately as active as marmoset and human plasma for degradation of the C(+/-)P(-)-isomers.

Animals↗

PubMed1994

Hemichrome formation, lipid peroxidation, enzyme inactivation and protein degradation as indexes of oxidative damage in homogenates of chicken kidney and liver.

The change in relative hemichrome formation (RHF) was investigated as a potential marker of oxidative damage in kidney and liver homogenates prepared from chicks fed diets deficient or adequate in vitamin E. RHF gave an earlier indication of oxidative damage in tissue homogenates than the formation of thiobarbituric acid reactive substances (TBARS) or decrease in glutathione peroxidase activity (GPXA). RHF correlated significantly with both TBARS and GPXA. The correlations were 0.64 (P < 0.0001) and -0.57 (P = 0.0002) in kidney homogenates and 0.53 (P = 0.0006) and -0.71 (P < 0.0001) in liver homogenates. The correlation between RHF and the sum of TBARS and GPXA was also highly significant in both kidney and liver homogenates.

Animals↗

PubMed1991

Characteristics of hexosaminidase A in homogenates of white blood cells using methylumbelliferyl-N-acetyl-beta-D-glucosaminide-6-sulphate as substrate.

Using methylumbelliferyl-N-acetyl-beta-D-glucosaminide-6-sulphate as a substrate hexosaminidase A activity was measured in leukocyte homogenates with a one-step method without the need of thermoinactivation. According to the buffer system used--acetate or citrate--the enzyme has pH optima of 3.5 and 4, respectively, and is linear during at least 1 h of incubation. The enzyme is resistant to mechanical inactivation by homogenization or to freezing and thawing and is thermostable between 0 and 37 degrees C but it is rapidly inactivated at 50 degrees C. This thermoinactivation is retarded by acidification of the homogenate. The latency of the enzyme is in agreement with its known lysosomal localization. Like most lysosomal enzymes, hexosaminidase A binds to Concanavalin A. After homogenization of leukocytes and repeated freezing and thawing, about 60% of the enzyme is present in the supernatant. Hexosaminidase A activity was stimulated by taurocholate between 0.05 and 0.1 M and inhibited by dimethylsulfoxide, dimethylformamide, N-acetylglucosamine and N-acetylgalactosamine. No pronounced difference was noted in specific activity of a homogenate of mononuclear cells, granulocytes or total leukocytes.

Binding Sites↗

PubMed1987

Seasonal changes of the 5'-monodeiodination activity in kidney and skin homogenates of male Rana ridibunda: relation to plasma thyroxine (T4) and testosterone.

Plasma concentrations of thyroxine (T4) and testosterone were studied together with the in vitro 5'-monodeiodination activity in liver, kidney, and skin homogenates in male Rana ridibunda during 12 consecutive months. Plasma T4 levels increase in February, drop in April, and increase again in May. In July a sharp decrease is seen and the values remain low during the rest of the year. Testosterone plasma levels also exhibit month to month variations. A maximum is found in February and March, and minimal values are measured in August, September, and October. A 5'-monodeiodination activity with a resulting 3,3',5-triiodothyronine (T3) production could be demonstrated in kidney and skin homogenates but not in liver homogenates. Addition of 1,4-dithio-DL-threitol (DTT) and EDTA to the homogenates significantly increases the T3 production. In kidney homogenates incubated with DTT and EDTA a significant annual variation in the T3 production is seen. This T3 production is very high in April, May, and June and is followed by a minimum in July and August. It is concluded that the opposite changes in 5'-monodeiodination activity and plasma T4 concentrations may indicate a peripheral monodeiodination of T4 to T3. During reproduction, which is preceded by a testosterone peak, the increase in the 5'-monodeiodination activity may reflect a greater need for T3.

Animals↗

PubMed1985

Effect of various agents and conditions on palmitate oxidation by homogenates of rat liver and rat and human skeletal muscle.

The effect of various inhibitors of fatty acid transport and of respiratory chain on palmitate oxidation was investigated in homogenates and mitochondria of rat muscle and homogenates of rat liver and human muscle. Inhibition of fatty acid transport by carnitine omission, malonyl-CoA, tetradecylglycidic acid and mersalyl decreased oxidation more with muscle than with rat liver. Antimycin and KCN decreased markedly palmitate oxidation and caused a larger accumulation of peroxisomal oxidation products. Inhibition of mitochondrial long-chain fatty acid transport decreased accumulation of peroxisomal products in comparison to the control. The effect of malonyl-CoA was dependent on the nutritional state, the pH and the palmitate-albumin ratio with liver homogenates, and only on the latter parameter with muscle homogenates. Effects observed were comparable for rat and human muscle homogenates.

Animals↗

PubMed1991

Peroxisomal oxidases and catalase in liver and kidney homogenates of normal and di(ethylhexyl)phthalate-fed rats.

1. Activities of peroxisomal oxidases and catalase were assayed at neutral and alkaline pH in liver and kidney homogenates from male rats fed a diet with or without 2% di(2-ethylhexyl)phthalate (DEHP) for 12 days. 2. All enzyme activities were higher at alkaline than at neutral pH in both groups. 3. The effect of the DEHP-diet on the peroxisomal enzymes was different in kidney and liver. Acyl-CoA oxidase activity was raised three- and sixfold in kidney and liver homogenates, respectively. The activity of D-amino acid oxidase decrease in liver, but increased in kidney homogenates. In liver homogenates, urate oxidase activity was not affected by the DEHP diet. The catalase activity was twofold induced in liver, but not in kidney. 4. The differences suggest that the changes of peroxisomal enzyme activities by DEHP treatment are not directly related to peroxisome proliferation. 5. DEHP treatment caused a marked increase of total and peroxisomal fatty acid oxidation in rat liver homogenates. 6. In the control group the rate of peroxisomal fatty acid oxidation was higher at alkaline pH than at neutral pH. 7. This rate was equal at both pH values in the DEHP-fed group, in contrast to the acyl-CoA oxidase activity. These results indicate that after DEHP treatment other parameters than acyl-CoA oxidase activity become limiting for peroxisomal beta-oxidation.

Animals↗

PubMed1983

Internal radiotherapy for hepatic metastases I: The homogeneity of hepatic arterial blood flow.

Internal radiotherapy, in the form of arterially infused yttrium-90-labeled microspheres, theoretically appears encouraging as a method of treatment for hepatic metastases. Previous investigators have assumed a homogeneous distribution of these microspheres and given dosages of isotope based solely on an estimated liver mass. The purpose of this study has been to establish the homogeneity of isotope distribution in liver substance when 15 micrometers microspheres are arterially injected. This has been done in three mammalian species, with the results expressed as a mean percentage coefficient of variation of 28 +/- 5%. Also demonstrated is the fact that 15 micrometers particles, while not penetrating to the venous circulation, achieve a more homogeneous spread throughout the liver than larger particles. It has been demonstrated that to achieve this maximum homogeneity distribution, 4000 beads/g of liver tissue are required. This equates in the therapeutic situation to a maximum activity of 4 Ci/g of infused microspheres. These results are considered significant in that they indicate criteria necessary to achieve the maximum homogeneity of therapeutic agent within liver substance when it is administered by this method, and will allow confidence limits to be attached to direct in vivo measurement of hepatic irradiation.

Animals↗

PubMed1990

Insulin and proinsulin proteolysis in mucosal homogenates of the albino rabbit: implications in peptide delivery from nonoral routes.

The objective of this study was to determine (a) the rate of hydrolysis of insulin and proinsulin in homogenates of various non-oral absorptive mucosae relative to the ileal mucosa, and (b) the inhibitory effect of various protease inhibitors on the degradation of these peptides. Overall, insulin was somewhat more susceptible to hydrolysis than proinsulin in all mucosal homogenates. Proteolytic activity was highest in the nasal and rectal homogenates, followed by the ileal, vaginal, conjunctival and buccal homogenates in that order. The rate of insulin and proinsulin proteolysis differed by a factor of about 6 to 7 between the least and the most proteolytically active mucosa. A 5-fold maximum reduction in proteolytic rate was seen in the presence of Na glycocholate, aprotinin and p-chloromercuriphenyl-sulfonic acid (PCMPS). The rank order of effectiveness was Na glycocholate (1%) greater than aprotinin greater than PCMPS in all but the conjunctival and vaginal homogenates. Extrapolating the above in vitro findings to in vivo, both insulin and proinsulin are expected to be degraded to varying degrees during absorption across the various mucosae. Absorption may therefore be enhanced in the presence of protease inhibitors such as aprotinin, PCMPS, or Na glycocholate.

4-Chloromercuribenzenesulfonate↗

PubMed1986

Ultrasound speed and attenuation in homogenates of bovine skeletal muscle.

The attenuation and speed of ultrasound were measured in homogenates of post-rigor bovine skeletal muscle, and found to increase in proportion to the concentration of muscle. Extrapolation of the data to tissue concentrations yielded an attenuation of 7.5 dB cm-1 at pH 5.7, 20 degrees C and 7.3 MHz. This was close to that measured in the minced tissue, 8.3 dB cm-1, and between values previously recorded across and along the fibres of intact muscle. Corresponding measurements for the speed of ultrasound in homogenates, extrapolated to the native tissue concentration, were: 1555 +/- 9 m s-1 at 0 degree C, 1592 +/- 10 m s-1 at 20 degrees C and 1616 +/- 9 m s-1 at 37 degrees C. These were not significantly different from measurements of minced muscle at the same temperatures. Measurements of the attenuation of 7.3 MHz ultrasound in suspensions of myofibrils indicated that attenuation by the myofibrils caused at least 64% of the attenuation in muscle homogenates at pH 5.7. Re-analysis of the viscous loss arising from relative movement of the myofibrils in their surrounding fluid, indicated that this mechanism could account for no more than 15% of the attenuation in muscle homogenates. Attenuation due to scattering was calculated to be at least two orders of magnitude smaller than that observed in either homogenates or suspensions of myofibrils. It was concluded that the contribution of scattering to the attenuation was small, and that the attenuation was caused by processes involving an absorption of energy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Determination of polychlorinated biphenyl levels in the serum of residents and in the homogenates of seafood from the New Bedford, Massachusetts, area: a comparison of exposure sources through pattern recognition techniques.

We measured the residues of polychlorinated biphenyls (PCBs) in the serum of 23 residents of the New Bedford, Massachusetts, area and from two homogenates each of bluefish and lobsters from the same area. We used congener-specific and total Aroclor quantitative approaches, both of which involved gas chromatography with electron capture detection. Using gas chromatography mass spectrometry (electron ionization mode), we confirmed the presence of PCBs in the combined serum samples and in the aliquots of bluefish and lobsters. In measuring the PCB levels in serum, we found good agreement between the two electron capture detector approaches (r > or = 0.97) when the serum of specific congeners was compared to total Aroclor. We used univariate and multivariate quality control approaches to monitor these analyses. Analytical results for bluefish showed a better agreement between the two techniques than did those for lobsters; however, the small number of samples precluded any statistical comparison. We also measured levels of chlorinated pesticides in the serum samples of two groups of New Bedford residents, those with low PCB levels (< 15 ng/ml) and those with high PCB levels (> or = 15 ng/ml). We found that residents with high PCB levels also tended to have higher levels of hexachlorobenzene (HCB) and 1,1-dichloro-2,2-di-(p-chlorophenyl) ethylene (p,p'-DDE). The higher concentration of all three analytes appears to be influenced by employment in the capacitor industry, by seafood consumption, or both. Using Jaccard measures of similarity and principal component analysis we compared the gas chromatographic patterns of PCBs found in the serum of New Bedford area residents with high serum PCBs with the patterns found in homogenates of lobsters (inclusive of all edible portions except the roe), in homogenates of bluefish fillets taken from local waters, and in serum from goats fed selected technical Aroclors (e.g. Aroclors 1016, 1242, 1254, or 1260). The patterns found in human serum samples were similar to the patterns found in lobster homogenates. Both of these patterns closely resembled patterns found in the serum samples of the goat fed aroclor 1254, as demonstrated by both pattern recognition techniques. In addition, the chromatographic patterns of human serum and of lobsters and bluefish homogenates all indicated the presence of PCBs more characteristic of Aroclors 1016 or 1242.

Analysis of Variance↗