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Steroid synthesis in ovarian homogenates from immature mice treated with diethylstilboestrol in neonatal life.

Female mice of the NMRI strain were treated with the synthetic oestrogen diethylstilboestrol (DES) for the first 5 days after birth. Pools of ovaries were removed from groups of 6-, 12-, 21-, 28- and 56-day-old females. An homogenate of an ovarian pool was incubated for 1 h in the presence of [3H]pregnenolone. Synthesized steroids were extracted and separated in a two-dimensional thin-layer chromatography system. Homogeneity of tentative steroids was verified with recrystallization to constant specific activity. Synthesis of [3H]progesterone and [3H]testosterone was demonstrated at 6 days, [3H]androstenedione at 12 days, [3H]17 alpha-hydroxyprogesterone at 21 days, and [3H]oestradiol-17 beta at 28 days. Up to 28 days (21 days for progesterone), the synthetic activity was lower in homogenates of DES-exposed ovaries than in control homogenates. After 28 days, values for recovered [3H]progesterone, [3H]androstenedione and [3H]oestradiol-17 beta were higher in DES homogenates than in control homogenates while the reverse was true for [3H]17 alpha-hydroxyprogesterone and [3H]testosterone. The results are compatible with an early and direct DES inhibitory effect on ovarian steroidogenesis and, later in immature life, a DES-induced disruption of the normal FSH-LH stimulation of ovarian development.

17-alpha-Hydroxyprogesterone↗

Production of microparticles by high-pressure homogenization.

A high-pressure homogenization method for the production of aqueous suspensions of poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) was investigated. Depending on the production conditions it was possible to produce micro--as well as nanoparticulate systems without the use of organic solvents. The influence of different homogenization temperatures and different dispersion media on particle size and charge was investigated. Additionally, various polymer/surfactant ratios were investigated. Homogenization in phosphate buffer at temperatures above the glass transition temperature (Tg) of the polymers resulted in wide particle size distributions with a high percentage of particles in the nanometer range. The effect of a second homogenization step at a temperature below Tg was examined at two different homogenization pressures. Additional homogenization cycles at 10 degrees C led to smaller particle size distributions and the average particle sizes were smaller. The stability of the particles was affected by the concentration of surfactant as well as by the zeta potential of the particles. Phosphate buffer kept the pH of the suspension in a range that provided a high surface charge of the particles because of deprotonization of the carboxylic functions of the polymer.

Drug Delivery Systems↗

Enhanced luminescence study of liver homogenate response to oxidative stress.

An enhanced luminescence technique was used to monitor the response of liver homogenates stressed with sodium perborate. Rat liver homogenates were subjected to oxidative stress with sodium perborate, and the light signals, generated by a suitable system, containing luminol and compounds producing enhancement of light emission such as sodium benzoate and indophenol, were detected by a luminometer. The intensity of light emission (E) was found dependent on homogenate concentration (C). When C increased, E at first increased as well and, then, decreased rapidly. The graphic expression of this phenomenon resulted as a curve that can be described by the equation: E = a.C/exp(b.C). It is proposed that the a value represents the capacity of the tissue to catalyze the production of .OH radical species. The b value might be related to the capacity of the tissue to scavenge such radicals, since it increases when homogenates are supplemented with antioxidants and decreases when homogenates are treated with prooxidant. The results obtained by supplementing homogenates with iron containing substances, or using model systems, suggest that cell substances catalyzing the luminescent reaction, such as the hemoproteins, are "scavengers" as well as radical producers. The concentration-emission curve obtained with suitable model system is described by the equation: E = a.C/exp(b.Ck). It is suggested that, using the k value, information can be obtained on the relative capacity of hemoproteins and antioxidant systems to interact with .OH radicals.

Animals↗

Effect of high pressure homogenization on microbial and chemico-physical characteristics of goat cheeses.

The objective of this work was to compare goat cheeses obtained from milk previously subjected to high pressure homogenization (1000 bar) with those produced from untreated milk and milk subjected to sanitization (61 degrees C; 20 min) or to pasteurization (72 degrees C; 15 s). The pressure homogenization treatment had both direct and indirect effects on cheese characteristics and their evolution during ripening. The direct effects were principally linked to the change in water-binding capacity of proteins as shown also by the lower whey separation. The indirect effects involved the microbial growth or activity and, particularly, modifications of the population of the lactic acid bacteria that occurred naturally and their evolution as well as a more precocious yeast and mold growth with a consequent rapid rise in pH. Although the treatment proved to enhance both proteolytic and lipolytic activities according to Fourier transform infrared analysis, which was used to obtain a rapid description of the biochemical modification, the cheeses homogenized under high pressure showed relevant qualitative differences only in the zone corresponding to amide I and amide II signals of proteins. The activation of these enzymatic activities observed in the homogenized cheeses could be either an indirect effect of the shift of the microbial population or a consequence of a different exposure of the macromolecules to the enzymatic activity. Scanning electron microscopy analyses of goat cheeses revealed that cheeses homogenized under high pressure had a more homogeneous microstructure than did the others.

Animals↗

The effects of ultrapasteurization with and without homogenization on the chemical, physical, and functional properties of aseptically packaged liquid whole egg.

The effects of ultrapasteurization with and without homogenization on some chemical, physical, and functional properties of liquid whole egg were observed. Heat treatments of 64, 68, and 72 C each at 30, 60, and 95 s were conducted on liquid whole egg, unhomogenized and homogenized [10.34 mPa (1,500 psi)]. The viscosity of the egg increased with increased processing temperature, and unhomogenized egg was more viscous than homogenized egg. alpha-Amylase was inactivated at 68 C for 30 s. Although cake height was highly correlated to soluble protein content, this relationship did not hold for all time-temperature combinations. Due to homogenization effects on soluble protein results, soluble protein is not a reliable predictor of egg functionality when homogenization is used in the process. Homogenization had no effect on the population reduction of aerobic bacteria in all but three of the nine time-temperature combinations.

Bacteria, Aerobic↗

Methods for measurement of LDL-cholesterol: a critical assessment of direct measurement by homogeneous assays versus calculation.

BACKGROUND: Because LDL-cholesterol (LDL-C) is a modifiable risk for coronary heart disease, its routine measurement is recommended in the evaluation and management of hypercholesterolemia. We critically examine here the new homogeneous assays for direct determination of LDL-C. APPROACH: This review relies on published studies and data of the authors using research and routine methods for LDL-C determination. We review experience with methods from their earlier use in lipid research laboratories through the transition to routine clinical testing and the recent development of homogeneous assays. We focus on comparative evaluations and characterizations and the performance of the assays. CONTENT: Homogeneous assays seem to be able to meet current National Cholesterol Education Program (NCEP) requirements for LDL-C testing for precision (CV <4%) and accuracy (bias <4%), when samples collected from nonfasting individuals are used. In addition, all five currently available assays have been certified by the Cholesterol Reference Methods Laboratory Network. The homogeneous methods also appear to better classify individuals into NCEP cutpoints than the Friedewald calculation. However, the limited evaluations to date raise questions about their reliability and specificity, especially in samples with atypical lipoproteins. CONCLUSIONS: Available evidence supports recommending the homogeneous assays for LDL-C to supplement the Friedewald calculation in those cases where the calculation is known to be unreliable, e.g., triglycerides >4000 mg/L. Before the homogeneous assays can be confidently recommended to replace the calculation in routine practice, more evaluation is needed.

Autoanalysis↗

[Development of homogeneous assay for HDL-C in serum].

Recent development of several homogeneous assays for measuring HDL-cholesterol (HDL-C) in serum without pretreatment has been reviewed. Several problems on the five homogeneous assays based on PEG-modified enzyme/alpha-cyclodextrin sulfate (PEGME), polyanion-polymer/detergent (PPD), immunoinhibition, catalase/detergent and 4-sulfonyl calyx[8]areine are discussed comparing with CDC reference method. The two homogeneous assays (PEGME and PPD) used in many laboratories were precise and correlated well with CDC reference method (mean percentage bias: < 5%). The discrepancies of recoveries for apo E-rich HDL-C were observed among reagents of homogeneous assays in serum from patients with hepatobiliary diseases and cholesterol ester transfer protein (CETP) deficiency. Apo E-rich HDL slightly contained in normal serum was most recovered by homogeneous methods, but not by precipitation methods. The differences between the normal values for HDL-C obtained by homogeneous assay (PEGME method) and precipitation method (dextran sulfate-phosphotungstate-MgCl2) were statistically significant (p < 0.01).

Adult↗

Stability of ketoprofen-dextran ester prodrugs in homogenates of various segments of the pig GI tract.

Initial velocities of ketoprofen formation from ketoprofen-dextran ester prodrugs incubated in homogenates of various segments of the pig GI-tract were determined. Enzyme-mediated drug release was found in caecum and colon homogenates with their contents, whereas release rates in the stomach, duodenum, jejunum and ileum homogenates were comparable to those determined in pure buffer solutions of identical pH. In colon homogenates adjusted to various pH values between 6.0 and 7.9, little variation in release rates was observed. However, the contribution of enzyme-catalyzed drug regeneration to the overall initial velocity of ketoprofen formation increased significantly as a function of decreasing pH. The presence of several antibiotics and betamethasone in colon homogenates did not affect the drug activation process, whereas the addition of various enzyme inhibitors slowed down the ketoprofen release rates. During incubation in colon homogenates the average molecular weight of the dextran esters decreased. The drug release may therefore involve an initial fragmentation of the drug-liganded dextran chains carried out by dextranases secreted from the microflora which reside in the pig's large bowel.

Animals↗

[Enzyme, electron microscopic and polarographic characteristics of isolated rat brain mitochondria. III. Quantitative assessment of their distribution in fractions of the homogenate].

Activities of succinate dehydrogenase, succinate- and NAD-H-cytochrome c--reductases, and cytochrome c--oxidase was compared in 1 g tissue homogenate and homogenate fractions made from 1 g brain tissue using various solutions. Fractionation resulted in the increased activities of NADH- and succinate cytochrome reductases, and in the loss of succinate dehydrogenase activity, cytochrome oxidase was less influenced. These phenomena are regarded as signs of the interrelation between mitochondria and other constituents of brain cell within homogenates. Maximal quantity of mitochondria isolated from homogenates is no more than 20% of all the mitochondrial homogenates (according enzyme data). The electronogram of the brain mitochondrial preparation isolated in the Krebs--Ringer solution without glucose pointed out to a high homogeneity of mitochondria in the residue.

Animals↗

[Thoracic radiography. Comparison of asymmetric screen-film, technic of mechanical homogenization, and conventional technics].

This study was aimed at comparing three different systems, i.e., asymmetric screen-film, mechanical homogenized and conventional techniques, in standing frontal and lateral plain radiographs of the chest. Two hundred consecutive patients with normal posteroanterior (PA) and lateral films were randomly subdivided into 4 groups. Each group was submitted to chest radiographs with a different technique: asymmetric screen-film systems (InSight HC and High Light GCA), mechanical homogenized (Tau-Gil Homogenized) and conventional high-kilovoltage techniques. The exposure values for frontal projections ranged 110 kV (InSight HC) to 141 kV (Tau-Gil), while for lateral projections they ranged 123 kV (conventional technique) to 143 kV (Tau-Gil). Statistically significant differences were observed between the two asymmetric systems as regards exposure values, High Light exhibiting higher mean values in the frontal projection (t-test p < 0.05). Image quality was studied jointly by 3 experienced chest radiologists. The observers were asked to grade, on a 3-point ordinal scale, the conspicuity of mediastinal borders, of pulmonary vessels and of selected areas of lung parenchyma (i.e., retrocardiac, retrosternal and apical regions), as well as overall image quality on the frontal projection. The statistical analysis of paired differences was performed with the Mann-Whitney U-test. The asymmetric and the mechanical homogenized techniques were much better than the conventional technique in depicting tracheobronchial tree, retrocardiac parenchyma, azygos-esophageal recess and thoracic spine (p < 0.05). The mechanical homogenized system provided best overall image quality on frontal films, being superior to both InSight HC and conventional techniques, but not to the High Light GCA system; only the frontal projection obtained with the homogenized technique was compared, no filter being available for the lateral projection. When the two asymmetric systems were compared, the High Light system better depicted vascular structures on frontal films (p < 0.05), while apical areas were better demonstrated with the InSight system, namely with lateral films (p < 0.05).

Female↗

[Homogenized thoracic radiography].

The major obstacle to optimal radiographic depiction of the chest has always been its anatomical features and, in particular, the considerable difference in X-ray beam attenuation between pulmonary and mediastinal structures. At least 40% of this region cannot be visualized in an optimal way. Therefore, much technological work was done to overcome this limitation, which has recently led to the development of electronic (Amber) and mechanical (homogenization) filtration systems. This study was aimed at assessing the capabilities of mechanical filtration, in terms of anatomical representation and lower biologic cost, the latter intended as lower exposure and absorbed doses. Therefore, eight radiologists studied 40 negative radiographs, i.e., a homogenized radiograph and a non-homogenized one per patient, twenty patients in all. The statistical analysis of the radiographs demonstrated the homogenized technique to allow better visualization of all the anatomical structures examined, i.e., trachea, carina, right and left main bronchi, aorta, spine and retrocardiac region. In contrast, the two radiographic techniques yielded much the same results in the depiction of the pulmonary pattern. As for dosimetry, good results were also obtained, because the homogenizer made it possible to reduce both exposure and absorbed doses to the lung and thyroid, while in the mediastinal region the absorbed dose remained similar to that measured with the non-homogenized even though exposure dose was increased mildly.

Humans↗

Carbon metabolic homogenization is linked to microbial competition and antimicrobial resistance in soils under forest-to-cropland conversion.

Global agricultural expansion by converting natural forests into croplands often leads to soil functional homogenization and antimicrobial resistance enhancement, threatening ecosystem services. However, the associations between microbial carbon metabolic homogenization and antimicrobial resistance remain largely unknown. Here, we collected 240 paired forest and cropland soil samples from the most intensively farmed Yangtze River Basin in China, and constructed a novel framework based on microbial functional traits to decipher the role of carbon metabolic homogenization on antimicrobial resistance via microbial competition for metabolites. Using genome-scale metabolic models, we found that carbon metabolic homogenization was associated with a shift in microbial interactions from cooperation toward competition, with a 45.6% increase in competitive interactions that coincided with a 35.6% higher antimicrobial resistance gene (ARG) diversity. This shift was accompanied by smaller genome sizes and higher 16S rRNA copy numbers, indicating fast-growing, resource-acquisitive microbial strategies. Metabolic transfer analyses further revealed less cooperation relationships among microbial communities in cropland soils than in forest soils, indicating an intensified battle for communal metabolites and an attenuated exchange for complementary metabolites. Together, these findings provide a new framework to understand the association between carbon metabolic homogenization and soil antimicrobial resistance risks from the perspective of microbial traits and interactions under land use change.

Soil Microbiology↗

Robustness and power of analysis of covariance applied to data distorted from normality by floor effects: homogeneous regression slopes.

We investigate through computer simulations the robustness and power of two group analysis of covariance test applied to small samples distorted normality by floor effects when the regression slopes are homogeneous. We consider four parametric analysis of covariance tests that vary according to the treatment of the homogeneity of regression slopes and two t-tests on unadjusted means and on difference scores. Under the null hypothesis of no difference in means, we estimated actual significance levels by comparing observed test statistics to appropriate values from the F and t distributions for nominal significance levels of 0.10, 0.05, 0.02 and 0.01. We estimated power by similar comparisons under various alternative hypothesis. The hierarchical approach (that adjusts for non-homogeneous slopes if found significant), the test that assumes homogeneous regression slopes, and the test that estimates separate regression slopes in each treatment were robust. In general, each test produced power at least equal to that expected from normal theory. The textbook approach, which does not test for mean differences when there is significant non-homogeneity, was conservative but also had good power. The t-tests were robust but had poorer power properties than the above procedures.

Analysis of Variance↗

In vitro activation of the corticosteroid ciclesonide in animal nasal mucosal homogenates.

Ciclesonide, a new corticosteroid for allergic rhinitis, is administered as an inactive parent compound that is converted by esterases to the pharmacologically active metabolite, desisobutyryl-ciclesonide (des-CIC). This study investigated the in vitro activation of ciclesonide in nasal mucosa of multiple animal species. Nasal mucosal homogenates from rats, guinea-pigs, rabbits and dogs were incubated with ciclesonide 0.5 micromol/l (0.271 microg/ml) or 5 micromol/l (2.71 microg/ml) for up to 120 min. Concentrations of ciclesonide and des-CIC were measured by high-performance liquid chromatography with tandem mass spectrometry. Ciclesonide was metabolized to des-CIC in nasal mucosal homogenates of each species. The initial velocities of des-CIC formation ranged from 0.0038 to 0.0150 nmol/min/mg protein and 0.0319 to 0.0983 nmol/min/mg protein in nasal mucosal homogenates incubated with ciclesonide 0.5 micromol/l and 5 micromol/l, respectively. Furthermore, the initial velocities of ciclesonide metabolism ranged from 0.0032 to 0.0142 nmol/min/mg protein and 0.0445 to 0.1316 nmol/min/mg protein in nasal mucosal homogenates incubated with ciclesonide 0.5 micromol/l and 5 micromol/l, respectively. This study confirms that ciclesonide is converted to des-CIC in nasal mucosal homogenates without any marked differences among animal species.

Adrenal Cortex Hormones↗

Non-homogeneous Markov processes for biomedical data analysis.

Some time ago, the Markov processes were introduced in biomedical sciences in order to study disease history events. Homogeneous and Non-homogeneous Markov processes are an important field of research into stochastic processes, especially when exact transition times are unknown and interval-censored observations are present in the analysis. Non-homogeneous Markov process should be used when the homogeneous assumption is too strong. However these sorts of models increase the complexity of the analysis and standard software is limited. In this paper, some methods for fitting non-homogeneous Markov models are reviewed and an algorithm is proposed for biomedical data analysis. The method has been applied to analyse breast cancer data. Specific software for this purpose has been implemented.

Algorithms↗

Relaxometry of calf lens homogenates, including cross-relaxation by crystallin NH groups.

We studied the magnetic field dependence of the longitudinal relaxation rates of water protons (1/T1 nuclear magnetic relaxation dispersion (NMRD) profiles) in transparent homogenates of calf lens. The samples included nuclear homogenates with total (heterogeneous) crystallin contents between 34% (v/v) (native) and 14% (diluted) as well as cortical homogenate, 21% (native) and 34% (concentrated). The NMRD profiles had two components: a monotonic dispersive component (analogous to that of both globular protein solutions and diamagnetic tissue) and "14N quadrupolar peaks." 14N peaks have never been reported for protein solutions, only for tissues and dehydrated proteins. These peaks occur between 0.5 and 5 MHz proton Larmor frequency and arise from interactions of solvent water protons with NH moieties of proteins. The 14N peaks in lens cytoplasm are very large and may correlate with the crystallin structure and interactions required to maintain short-range order and lens transparency. The monotonic and 14N quadrupolar components were largest in concentrated samples, but with different concentration dependencies. The dispersive components of samples above approximately 19% protein concentration had a fixed functional form, the amplitude of which varied with protein volume fraction, f, by the multiplicative factor f/(1 - f), suggesting spatial organization and dynamics of the solute proteins that are relatively independent of water content. In contrast, at concentrations less than 19%, the NMRD profiles are concentration dependent, indicating a dependence of the orientational relaxation time of the proteins on protein-protein interactions seen previously in other globular proteins at these concentrations. The 14N peaks are not resolved below approximately 19% protein and increase linearly with incremental volume fraction at protein concentrations above 19%. In addition, the 14N peaks in nuclear homogenates are 50-100% larger than those of cortical homogenates at the same concentrations. Partial substitution of solvent D2O for H2O decreases the peak heights, indicating that an exchangeable proton mediates the interaction between solvent protons and protein 14N nuclei.

Animals↗

What is the correct value for Na, K-ATPase activity in homogenates of cerebral cortex?

Activity of Na, K-ATPase in homogenates of fresh cerebral cortex of rats was compared with that of cortex frozen under different conditions. Activity yields after rapid in situ freezing of the exposed cerebral cortex were twice, higher (26.1 U) than in homogenates of the fresh cortex (13.3 U). Fresh brain kept on ice for 60 and 300 s and subsequently frozen in liquid nitrogen yielded activities comparable to those of the tissue frozen in situ (24.1 U and 24.9 U for 60 s and 300 s periods, respectively). Inhibition of Na, K-ATPase by 10(-7) M vanadate was significantly stronger (38%) in homogenates of the fresh brain then in those of the cortex frozen in situ (28%). High Na, K-ATPase activity (47.6 U) in suspensions of synaptosomal membranes (SM) prepared from fresh cortical homogenates was only slightly inhibited by 10(-7) M vanadate (12%). Various treatments of homogenates or SM suspensions, like increase of piston rotation speed, repeated freezing and thawing procedure or vigorous shaking did not significantly affect the enzyme activity. Mg-ATPase activity and its sensitivity to vanadate was also modified by tissue treatment but the effect was much less pronounced.

Adenosine Triphosphatases↗

Oral administration of lens homogenate suppresses antibody production in mice injected with beta-crystallin emulsified in CFA.

Auto-antibodies (Abs) against lens antigens (Ags) are present in most patients with age-related cataract, and with complement they kill lens epithelial cells (LECs) in vitro. We studied, in an animal model, whether cytotoxic Abs against lens Ags can be suppressed by oral administration of the Ags. Mice were fed calf lens homogenate, 4 mg/mouse, every 4 days for 4-5 weeks, or bovine serum albumin (BSA) before and after immunisation with beta-crystallins emulsified in complete Freund's adjuvant (CFA). Sera from these animals were analysed for Abs to beta-crystallins by enzyme-linked immunosorbent assay (ELISA) and protein blot analysis. In addition, we studied the proliferative response of T-lymphocytes to beta-crystallins. The titer of anti-beta-crystallin Abs in the control animals fed BSA gradually increased to 1.5 x 10(-6) by the 5th week after the first injection. In contrast, the titer of anti-beta-crystallin Abs in animals fed calf lens homogenate was reduced to 30-70% of the control. Feeding lens homogenate prior to or concomitant with beta-crystallins immunization, was more effective than feeding after immunization (65% suppression vs. 30% suppression, respectively). Also the proliferative response of T-lymphocytes to beta-crystallins in mice fed homogenate was suppressed significantly. Thus, oral administration of lens homogenate is a specific and nontoxic method of suppressing anti-beta-crystallin Ab production in mice. We are exploring the therapeutic value of oral administration of lens proteins in age-related cataract.

Animals↗