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PubMed2005

Intra-operative acoustic hemostasis of liver: production of a homogenate for effective treatment.

OBJECTIVE: We have shown that High-Intensity Focused Ultrasound (HIFU) can effectively control bleeding from injuries to solid organs such as liver, spleen, and lung. Achievement of hemostasis was augmented when a homogenate of tissue and blood was formed. The objective of this study was to investigate quantitatively the effect of homogenate production on HIFU application time for hemostasis. Possible mechanisms involved in homogenate production were also studied. METHODS: Ten anesthetized rabbits had laparotomy and liver exposure. Liver incisions, 15-25 mm long and 3-4 mm deep, were made followed immediately by HIFU application. Two electrical powers of 80 and 100 W corresponding to focal acoustic intensities of 2264 and 2829 W/cm(2), respectively were used. Tissue and homogenate temperatures were measured. Smear and histological tissue sample analysis using light microscopy were performed. RESULTS: In treatments with homogenate formation, hemostasis was achieved in 76+/-1.3 s (Mean+/-Standard Error Mean: SEM) at 80 W. In treatments without homogenate formation (at 80 W), hemostasis was achieved in 106+/-0.87 s. At 100 W, hemostasis was achieved in 46+/-0.3 s. The time required for homogenate formation, at 80 and 100 W were 60+/-2.5 and 23+/-0.3 s, respectively. The homogenate temperature was 83 degrees C (SEM 0.6 degrees C), and the non-homogenate tissue temperature at the treatment site was 60 degrees C (SEM 0.4 degrees C). The smear and histological analysis showed significant blood components and cellular debris in the homogenate, with some intact cells. CONCLUSION: The HIFU-induced homogenate of blood and tissue resulted in a statistically significant shorter HIFU application time for hemostasis. The incisions with homogenate had higher temperatures as compared to incisions without homogenate. Further studies of the correlation between homogenate formation and temperature must be done, as well as studies on the long-term effects of homogenate in achieving hemostasis.

Animals↗
PubMed2000

Apparent viscosity of chicken muscle homogenates. Influence of pH and muscle type.

The effect of pH and type of muscle on apparent viscosity of chicken breast, thigh and combined B/T muscles was investigated. The apparent viscosity of thigh muscle homogenate at pH from 5.8 to 6.6, and combined B/T muscle homogenate at pH from 5.8 to 6.3 was increasing. The apparent viscosity of breast muscle homogenate increased with pH increase, reaching a maximum at pH 6.3 and then decreased. When pH raised from 5.8 to 6.3, breast muscle homogenate apparent viscosity increased 3.5-6.0 times more than apparent viscosity of thigh muscle homogenate. An increase of combined B/T muscle homogenate apparent viscosity under shear rate 0.3333-48.6 (s-1) was approximately an average of increases for its individual muscles. At pH 5.8 and 6.0, apparent viscosity of thigh muscle homogenate was approximately two times higher than that of breast muscle homogenate, and reversibly, at pH 6.3, breast muscle homogenate apparent viscosity was about 20% higher than that of thigh muscle homogenate. The apparent viscosity of combined B/T muscle homogenate at pH 5.8 and 6.0, was greater than apparent viscosity of breast muscle homogenate, and at pH 6.3, was greater than apparent viscosity of thigh muscle homogenate. The present data extend the results reported by other researches that there are remarkable differences not only in functional and rheological properties of myofibrillar proteins (SSP, myosin) but also in those of homogenates from chicken white and red muscles.

Animals↗
PubMedDate not supplied

Experimental reproduction of malabsorption syndrome with different combinations of reovirus, Escherichia coli, and treated homogenates obtained from broilers.

Attempts to reproduce malabsorption syndrome (MAS) by oral inoculation with several different combinations including intestinal homogenate, reovirus, and hemolytic Escherichia coli obtained from MAS-affected chickens and intestinal homogenate from healthy chickens (healthy homogenate) were performed in 1-day-old specific-pathogen-free (SPF) broilers. The MAS homogenate, serving as a positive control, induced weight gain depression and intestinal lesions such as cystic crypts of Lieberkuhn, villus atrophy, and lymphoid and/or granulocytic infiltration. The healthy homogenate, the formalin-treated MAS homogenate, the formalin-treated healthy homogenate, and phosphate-buffered saline caused neither weight gain depression nor intestinal lesions. We were able to reproduce both weight gain depression and intestinal lesions by inoculation of reovirus either combined with the formalin-treated MAS homogenate or combined with healthy homogenate. Surprisingly, when hemolytic E. coli was added to the combination of reovirus with formalin-treated MAS homogenate, this did not cause weight gain depression although this combination caused the described intestinal lesions. Identical results were obtained with the combination of formalin-treated MAS homogenate with hemolytic E coli or the combination of reovirus with hemolytic E. coli. The intestinal lesions were more severe and developed faster by combinations including reovirus and formalin-treated MAS homogenate. This study indicates that a combination of enteropathogenic reovirus with other agents or substances that are present in an intestinal homogenate from MAS-affected and healthy chickens can induce MAS in SPF broilers. Escherichia coli is not essential for induction of weight gain depression but can play a role in development of intestinal lesions. Furthermore, intestinal lesions alone will not always result in weight gain depression.

Animals↗
PubMed1965

THE METABOLISM OF PROGESTERONE BY ANIMAL TISSUES IN VITRO. SEX AND SPECIES DIFFERENCES IN CONJUGATE FORMATION DURING THE METABOLISM OF (4-14C)PROGESTERONE BY LIVER HOMOGENATES.

1. Sex and species differences during the metabolism of [4-(14)C]progesterone by liver homogenates from rat, rabbit, guinea pig and hamster have been investigated. 2. Liver homogenate from male rat formed ;water-soluble' metabolites faster and in significantly larger amounts than did liver homogenate from female rat. About 65-70% of the added progesterone was conjugated as glucuronide by liver homogenate from male rat and about 45-50% by that from female rat. Liver homogenate from male rat also formed glucuronides faster than did liver homogenate from female rat. Sulphate formation was low (8-16%) in liver homogenates from both male and female rats. 3. Hamster-liver homogenate did not show any sex difference in the rate of formation of ;water-soluble' metabolites, but a sex difference was observed in the amount of free steroids recovered at low tissue:steroid ratios. Liver homogenate from female hamster formed glucuronides faster and in significantly larger amounts than did liver homogenate from male hamster, the reverse of what was found in rat liver. 4. Liver homogenates from male and female rabbits and guinea pigs formed ;water-soluble' metabolites that were almost entirely glucuronides. 5. Neither rabbit liver nor guinea-pig liver showed any significant sex difference in the rate or amount of formation of total ;water-soluble' metabolites or glucuronides, but guinea-pig liver was considerably less active than rabbit liver. 6. Glucuronides were quantitatively the major type of conjugate formed by the liver homogenates from both sexes of all species except the male hamster.

Animals↗
PubMed1984

The isolation of large quantities of undamaged cellular organelles and cytosolic enzymes using a low-shear continuous tissue homogenizer.

There is often a need to isolate large quantities of subcellular components such as membrane-coated organelles (e.g., nuclei, lysosomes, and mitochondria), cell membranes, and soluble (cytosolic) proteins. Instruments which can homogenize relatively large masses of tissue, primarily those with rapidly rotating blades and cylinders, are excessively vigorous, often resulting in damaged and/or low yields of the subcellular components. This paper describes procedures for obtaining high yields of undamaged subcellular components using a continuous bulk tissue homogenizer which performs with low shear (the low-shear continuous homogenizer or LSC). This homogenizer is simple in operation, durable and can be used with a variety of tissues. Fibrous tissues are more difficult to homogenize using this instrumentation and require a premincing to small pieces (0.2 to 1.0-cm diam) followed by filtration through 2-4 mesh (two to four apertures per inch). Methods for bulk preparations with enhanced recoveries of undamaged nuclei, and a typical soluble multimeric enzyme, phosphofructokinase, are presented. Electron microscope views of the homogenates show the preserved state of the other subcellular components. The LSC homogenizer requires less physical effort with no "hands on" operation and thus is safer. This homogenizer requires less homogenization time compared to the smaller, hand-held Potter-Elvehjem-type homogenizers. Operations requiring low temperature can be performed at room temperature as long as the continuously passing homogenate solutions are kept chilled.

Animals↗
PubMed1992

Age-related alterations in antioxidant capacity and lipid peroxidation in brain, liver, and lung homogenates of normal and vitamin E-deficient rats.

Age-related alterations in both antioxidant capacity and lipid peroxidation in the cerebrum, lung and liver homogenates of normal and vitamin E-deficient rats were investigated. The antioxidant capacity, which includes superoxide dismutase, catalase and glutathione peroxidase activities and vitamin E (alpha-tocopherol) concentration, was relatively stable throughout the lifespan. It was observed, however, that catalase and glutathione peroxidase activities in livers of old rats decreased and that vitamin E concentration in lung and liver increased with age. In vitamin E-deficient animals, catalase activity in liver increased and glutathione peroxidase activity in liver and lung decreased. Lipid peroxidation was monitored by use of three different indices, i.e. the thiobarbituric acid (TBA) value, oxygen absorption and conjugated-diene formation. In the absence of any initiator, neither oxygen absorption into tissue homogenates nor conjugated-diene formation in lipid extracts from the homogenates occurred. The TBA value of each cerebrum homogenate incubated under air or an oxygen atmosphere was larger than that of the corresponding unincubated cerebrum homogenate. From comparison between the TBA value and oxygen absorption, this increase in the TBA value was suggested to be due to some reactions other than lipid peroxidation. Although tissue homogenates examined contained TBA-reacting materials, no lipid peroxidation seems to arise during incubation of them. No age-related alterations in the TBA value and oxygen absorption in rat tissue homogenates were observed. Vitamin E deficiency had no effect on the TBA values of cerebrum and lung homogenates, while it seemed to increase the TBA values of liver homogenates. Vitamin E deficiency had no effect on oxygen absorption in these tissue homogenates. The induction period of initiator-induced conjugated-diene formation in lipid extracts from liver and lung homogenates from normal and vitamin E-deficient rats tended to be extended with age. Vitamin E deficiency decreased the induction period of initiator-induced conjugated-diene formation. As a result, the length of the induction period was found to be proportional to vitamin E concentration in lipid extracts. The overall antioxidant capacity of rat tissues appears to be maintained without large variation during ageing. Decreases in the capacity of some antioxidant factors may be compensated by increases in the capacity of other factors.

Aging↗
PubMed1980

Phenolic and tyrosyl ring deiodination of iodothyronines in rat brain homogenates.

Conversion of thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)) in rat brain has recently been shown in in vivo studies. This process contributes a substantial fraction of endogenous nuclear T(3) in the rat cerebral cortex and cerebellum. Production of T(4) metabolites besides T(3) in the brain has also been suggested. To determine the nature of these reactions, we studied metabolism of 0.2-1.0 nM [(125)I]T(4) and 0.1-0.3 nM [(131)I]T(3) in whole homogenates and subcellular fractions of rat cerebral cortex and cerebellum. Dithiothreitol (DTT) was required for detectable metabolic reactions: 100 mM DTT was routinely used. Ethanol extracts of incubation mixtures were analyzed by paper chromatography in t-amyl alcohol:hexane:ammonia and in 1-butanol:acetic acid. Rates of production of iodothyronines from T(4) and T(3) were greater at pH 7.5 than at 6.4 or 8.6 and greater at 37 degrees C than at 22 degrees or 4 degrees C. Lowering the pH, reducing the protein or DTT concentrations, and preheating homogenates to 100 degrees C all increased excess I(-) production but reduced iodothyronine production. In cerebral cortical homogenates from normal rats, products of T(4) degradation were as follows (percent added T(4)+/-SEM in nine experiments): T(3), 1.9+/-0.5%; 3,3',5'-triiodothyronine (rT(3)), 34.0+/-2.4%; 3,3'-diiodothyronine (3,3'-T(2)), 5.8+/-1.6%; 3'-iodothyronine (3'-T(1)), </=2.5%; and excess I(-), 4.7+/-1.2%. In the same experiments, products of T(3) degradation were 3,3'-T(2), 63.3+/-5.5%, and 3'-T(1), 12.6+/-1.4%. Cerebral cortical homogenates from hyperthyroid rats and normals were similar in regard to T(4) to T(3) deiodination. In contrast, in cerebral cortical homogenates from hypothyroid rats, phenolic ring deiodination rates were increased and tyrosyl ring deiodination rates were decreased compared with normals.T(4) to T(3) conversion rates in cerebellar homogenates were greater than rates in cerebral cortical homogenates from the same normal rats and less than rates in cerebellar homogenates from hypothyroid rats. T(4) and T(3) tyrosyl ring deiodination rates were greatly diminished in cerebellar homogenates compared with cerebral cortical homogenates in normal and hypothyroid rats. High-speed (1,000-160,000 g) pellets from cerebral cortical homogenates were enriched in phenolic and tyrosyl ring deiodinating activities relative to cytosol. Fractional conversion of T(4) to T(3) was inhibited by T(4), iopanoic acid, and rT(3), but not by T(3). Tyrosyl ring deiodination reactions were inhibited by T(3), T(4), and iopanoic acid, but not by rT(3). These studies demonstrate separate phenolic and tyrosyl ring iodothyronine deiodinase enzymes in rat brain. The brain phenolic ring deiodinase serves in vivo as a T(4) 5'-deiodinase and closely resembles anterior pituitary T(4) 5'-deiodinase in physiological and biochemical characteristics. The physiological significance of the tyrosyl ring iodothyronine deiodinase enzyme is unclear; it shares several properties with rat hepatic T(4) 5-deiodinase.

Animals↗
PubMedDate not supplied

Effect of infrared analyzer homogenization efficiency on repeatability of uncorrected fat A and fat B signals.

Poor repeatability by infrared milk analyzers may be caused by inefficient homogenization as a result of light scattering and the Christiansen effect. The objectives of this study were to identify instruments with good and poor homogenization efficiency and to determine if a difference exists in repeatability performance between instruments with good vs poor homogenization efficiency. Unhomogenized and homogenized portions of the same milk were tested 20 times consecutively on 22 instruments. An instrument was considered to have poor homogenization efficiency if the mean difference in the uncorrected signal between unhomogenized and homogenized portions of the same milk was > or = 1.43% of the fat test (i.e., > or = 0.05% at 3.5% fat). Instruments were evaluated for repeatability by calculating the sample standard deviation and the range of the latter 19 uncorrected readings for unhomogenized and homogenized milks. When repeatability was evaluated as a function of homogenization efficiency, there was a significant (p = 0.001) correlation between poor homogenization efficiency and poor repeatability when testing unhomogenized milk but not when testing homogenized milk. Improved homogenizer performance within infrared milk analyzers is needed to improve the repeatability of raw milk testing.

Animals↗
PubMedDate not supplied

Effect of infrared analyzer homogenization efficiency on linearity of uncorrected fat A and fat B signals.

The objective of the survey was to determine if poor homogenizer performance causes nonlinear behavior of the uncorrected fat A or fat B signal that is not detected when an instrument's residual nonlinearity is determined by using dilutions of homogenized milk instead of unhomogenized milk. Unhomogenized and homogenized (17238 kPa) portions of the same 6.1% fat milk were tested on 20 instruments to determine homogenization efficiency. Instruments with differences of > or = 0.087% fat between homogenized and unhomogenized portions of the same milk had inefficient homogenization, on the basis of criteria established in a previous study. Four and 12 instruments out of 20 demonstrated inefficient homogenization for the fat A and fat B channels, respectively. Uncorrected signal linearity for the fat channels was evaluated quantitatively by using a series of dilutions of homogenized (17238 kPa) and unhomogenized milks. Most instruments passed the linearity evaluation for dilutions of either homogenized or unhomogenized milk, even though many of the same instruments failed the homogenization efficiency evaluation. Thus, using dilutions of homogenized milk is valid for linearity evaluation of instruments being used for testing unhomogenized milk in the range of fat concentrations used for payment testing.

Animals↗
PubMedDate not supplied

Equipment for rapid homogenization of high numbers of plant tissue for electrophoretic analysis of proteins.

A homogenizing system was developed to process multiple samples of various plant tissues. The equipment consists of a motor-driven stainless steel homogenizer that fits perfectly into a hole of a disposable plate. Hard plant material like melon or squash seeds (maximal length of 1.4 cm) could be homogenized within 10 s by forcing the homogenizer towards the bottom of a 24- or 48-well tissue culture plate. Smaller seeds could be homogenized within 10 s in a similar way using a 96-well tissue culture plate. Leaf tissue, or other types of soft plant tissue, could be homogenized with the same equipment. For homogenization of small samples, e.g. pistils of small flowers, a 60-well Terasaki plate was used. For homogenization of several hundreds of leaf tissue samples, an apparatus was developed that contains 12 homogenizers driven simultaneously by an electromotor. A tissue culture plate containing 96 leaf samples could be handled in this way in less than 10 min. The performance of the equipment was evaluated by analyzing homogenates of several types of plant tissue by different electrophoretic techniques.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Drop Coalescence during Emulsion Formation in a High-Pressure Homogenizer for Tetradecane-in-Water Emulsion Stabilized by Sodium Dodecyl Sulfate.

The present study investigates the effects of homogenizer pressure, surfactant concentration, ionic strength, and dispersed phase fraction on the coalescence rate of tetradecane-in-water emulsions during their formation in a high-pressure homogenizer. Experiments were conducted in a recirculating system consisting of a Rannie laboratory-scale single-stage homogenizer and a stirred vessel for tetradecane-in-water emulsions stabilized by sodium dodecyl sulfate (SDS). The initial evolution of the number concentration of droplets in the stirred tank was measured when subjected to a negative stepchange in the homogenizer pressure. The average drop coalescence rate constant in the homogenizer was inferred by fitting the experimental evolution of the number concentration of drops to a simple model accounting for the coalescence in the homogenizer under the assumption of a quasi steady state in the homogenizer. The residence time of the emulsion in the homogenizer was evaluated from the analysis of radial turbulent flow between disks. The step down homogenizer pressure was varied in the range 20.7-48.3 MPa, the drop size in the range 174-209 nm, the dispersed phase fraction in the range 5%-15%, SDS concentration in the range 0.0033-0.25 wt%, and ionic strength in the range 0.01-0.1 M. The coalescence rate constants were found to be in the range from 3.34x10(-17) to 2.43x10(-16) m(3) s(-1). The coalescence rate constant was found to be higher for higher homogenizer pressures, smaller drop sizes, lower dispersed phase fractions, and lower SDS concentrations and was insensitive to variations in ionic strength. Copyright 2001 Academic Press.

Journal Article↗
PubMed1995

Immunoblotting studies on artifactual contamination of enamel homogenates by albumin and other proteins.

The reason for the presence of albumin and other serum, cytoskeletal, cytosolic, and extracellular matrix proteins in enamel fractions was investigated by immunoblotting using homogenates prepared from freeze-dried and freshly dissected rat incisors, and antibodies capable of resolving at least 1 ng of the primary antigen. The data indicated that most of the 16 antibodies examined in this study reacted with antigens present only within "cell" homogenates (enamel organ cells + adhering labial connective tissue and blood vessels). One exception was rat serum albumin which was detected routinely in enamel homogenates prepared from freshly dissected, wiped incisors but rarely within enamel homogenates prepared from freeze-dried incisors. Another exception was calbindin-D 28 kDa which was consistently found within secretory stage enamel homogenates irrespective of preparative technique. A third exception was enamel proteins (amelogenins) which were enriched in secretory and early maturation stage enamel homogenates compared with cell homogenates and distributed as multiple molecular weight, antigenic bands in enamel homogenates (14-30 kDa), but mostly as a single antigenic band in cell homogenates (near 27 kDa). Overall, the results of this study suggest that developing rat incisor enamel naturally contains few exogenous proteins such as albumin. High concentrations of albumin (or other serum proteins) in crude homogenates, or purified fractions, derive mostly from blood and/or tissue fluids soaking into the enamel during sample preparation. This type of artifact can be avoided by using freeze-dried teeth for biochemical analyses.

Amelogenin↗
PubMed1985

Evidence for exocytotic release of dopamine beta-hydroxylase from rabbit heart and of vasopressin from rat neurohypophyses during homogenization and fractionation. Effects of gadolinium ions, cytochalasin B, gallopamil and different temperatures.

Isolated rabbit hearts were perfused according to a modified Langendorff method for 1 h (unstimulated hearts). In different hearts, release of dopamine beta-hydroxylase activity into the transmyocardial fluid draining the interstitium was evoked by electrical field stimulation for six periods of 1 min at 30 min intervals (stimulated hearts). The hearts were then homogenized and fractionated into 100,000 g supernatant and sedimented at 4 degrees C. In homogenates from unstimulated hearts, the soluble dopamine beta-hydroxylase (determined in the supernatant) accounted for 17% of the total dopamine beta-hydroxylase (determined in the homogenate). In stimulated hearts the soluble fraction of dopamine beta-hydroxylase was reduced by 65%. The dopamine beta-hydroxylase released into the transmyocardial fluid by electrical stimulation, expressed as fraction of the total activity, corresponded well to the loss of enzyme from the supernatant demonstrating that the soluble dopamine beta-hydroxylase determined from the supernatant represents the releasable pool. Gadolinium ions (Gd3+) added to the homogenization medium of unstimulated hearts reduced the soluble fraction of dopamine beta-hydroxylase up to 63%, with the maximum effect at 200 microM. Similarly, when neurohypophyses were homogenized and spun at 0-4 degrees C, the fraction of vasopressin in the soluble phase was about 50% of the total. Gd3+ reduced this fraction by maximally 60%, an effect which was accompanied by an increase of vasopressin in the sedimentable fraction. When cytochalasin B (10 microM) was present during the homogenization of the hearts the soluble fraction of dopamine beta-hydroxylase was reduced to the same extent as in the presence of Gd3+. However, cytochalasin B had no effect on the distribution of vasopressin in the soluble and sedimentable fractions of homogenates of neurohypophyses. Gallopamil, when present during the homogenization of the hearts at a maximum effective concentration of 1 microM, reduced the soluble fraction of dopamine beta-hydroxylase by only 40%. However, the electrically evoked noradrenaline release from perfused hearts was completely blocked at 100-300 microM gallopamil. When neurohypophyses were homogenized and fractionated at room temperature only 13% of the total vasopressin was found in the soluble fraction and Gd3+ did not further reduce this fraction. When unstimulated hearts were homogenized and fractionated at room temperature the fraction of soluble dopamine beta-hydroxylase was reduced by 40% compared to the experiments at 0-4 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Regional resistivity variations in lens homogenates.

The response of the lens to changes in the distribution and concentration of electrolytes can be measured by characterizing the changes in resistivity that occur secondary to variations in intracellular composition. Regional intracellular cytoplasmic resistivities of lens fiber cells can be approximated by homogenates isolated from the cortex and nucleus of the lens. The cortical, transitional, and nuclear calf lens homogenate resistivities at physiologic water concentrations were established to be 186.04 +/- 12.79, 297.69 +/- 18.95, and 421.74 +/- 69.64 omega cm respectively. The physiologic water content of the calf lens is 70.25 +/- 0.94% in the cortex and 54.33 +/- 1.28% in the nucleus. These data suggest that the increasing cortico-nuclear resistivity gradient is accompanied by a decreasing electrolyte concentration and a progressive cortico-nuclear dehydration. The corresponding mature bovine cortical and nuclear homogenate resistivities are 204.82 +/- 13.92 and 628.30 +/- 111.81 omega cm. When compared with calf homogenates the mature bovine cortical resistivity is nearly identical; while the elevated nuclear resistivity is attributable to a continual nuclear dehydration that occurs secondary to aging. Resistivity measurements performed on decapsulated lens homogenates reveal a close correspondence between calf and human lenses. Whereas, the elevated resistivity of the mature bovine decapsulated lens homogenate is attributable to a progressively dehydrated nucleus that increases in size with age. Supplementary data gathered from hydration and dehydration experiments performed on cortical and nuclear homogenates were used to characterize anatomical variations in resistivity as a function of total water content. These data indicate that the resistivity of the cortical homogenate is inversely proportional to the electrolyte concentration, whereas the resistivity of the nuclear homogenate is primarily dependent upon the mobilities of the electrolyte species with the highest concentrations. As the water concentration of the nuclear homogenate is increased to non-physiologic levels the resistivity becomes concentration-dependent, and is no longer is no longer dominated by the mobility.

Aging↗
PubMed1984

Peptide-degrading enzymatic activities in GH3 cells and rat anterior pituitary homogenates.

The activities of a number of peptide-degrading enzymes were compared in homogenates of GH3 cells and rat anterior pituitaries. The enzymes studied were prolyl endopeptidase (EC 3.4.21.26), a soluble metalloendopeptidase, pyroglutamyl peptide hydrolase (EC 3.4.11.8), a multicatalytic protease complex, cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), aminopeptidase (EC 3.4.11.2), and a membrane-bound neutral metalloendopeptidase (EC 3.4.24.11). Specific substrates were used to measure the activities, and active-site-directed inhibitors were used to verify the identities of the enzymes studied. Of the two lysosomal enzymes studied, cathepsin B, the enzyme with the highest activity in both preparations, had 5 times the activity in GH3 cell homogenates as in anterior pituitary homogenates. Cathespin D had a somewhat higher activity in the anterior pituitary homogenates than in the GH3 cell homogenates. Soluble metalloendopeptidase and prolyl endopeptidase, both cytoplasmic enzymes, had about twice the activity in GH3 cell homogenates as in anterior pituitary homogenates. Membrane-bound neutral metalloendopeptidase in the GH3 cell homogenates had 25% of the activity of the anterior pituitary homogenates. Of the two TRH-degrading enzymes, the activity of prolyl endopeptidase in GH3 cell homogenates was about 25 times higher than that of pyroglutamyl peptide hydrolase. Since the secretory function of the pituitary is in part controlled by neuropeptides, the knowledge of the enzyme profiles of the GH3 cells and the anterior pituitary should be of value in studying the metabolism of neuropeptides and peptide hormones in these systems.

Animals↗
PubMed2000

Melatonin suppresses autoxidation and hydrogen peroxide-induced lipid peroxidation in monkey brain homogenate.

OBJECTIVES: Melatonin, the major secretory product of the pineal gland, is known as an effective antioxidant and neuroprotector. Its neuroprotective actions and mechanisms have been documented in a variety of rodent brain models. However, little is known of melatonin's antioxidative capacity in the brain of primates. Herein, we investigated whether melatonin would suppress autoxidation and exogenous hydrogen peroxide-induced lipid peroxidation in monkey cerebral cortical homogenates. MATERIALS AND METHODS: The monkey brain was dissected during routine autopsy and immediately frozen at -80 degrees C until the experiment. A sample of cerebral cortex (50 mg) was homogenized in 1 ml ice cold phosphate buffer (20 mM, pH 7.4) at 0-4 degrees C. Four different treatments of cerebral cortical homogenates were performed: 1) homogenates incubated in a water bath at different temperatures (4 degrees C, 25 degrees C or 37 degrees C, respectively) for two hours to induce autoxidation; 2) homogenates co-incubated with different concentrations of melatonin at 37 degrees C for 2 hours; 3) homogenates co-incubated with 1 mM vitamin C and different concentrations of hydrogen peroxide at 37 degrees C for 1 hour to induce membrane lipid peroxidation; 4) homogenates incubated with different concentrations of melatonin and 1 mM H2O2 plus 1 mM vitamin C. After incubation, homogenates were analyzed for products of lipid peroxidation (malondialdehyde and 4-hydroxy-alkenals). RESULTS: The levels of lipid peroxidation products significantly increased in monkey cerebral cortical homogenates as a consequence of autoxidation or after the addition of H2O2 plus vitamin C. Melatonin not only suppressed the increase in lipid peroxidation induced by H2O2 plus vitamin C but also inhibited lipid breakdown resulting from autoxidation. The concentrations of melatonin required to suppress lipid peroxidation resulting from autoxidation or induced by exogenous oxidants in monkey cerebral cortical homogenates were in the same dose range. CONCLUSION: The results show for the first time that melatonin functions as an antioxidant and neuroprotector in primate brain tissue as was observed previously in rodent brain. The data provide information supporting the use of melatonin in the treatment of neurodegenerative disorders that involve oxidative damage to brain lipids.

Journal Article↗
PubMed1979

Intracellular lipase activities in heart and skeletal muscle homogenates. The absence of trierucin cleavage by the heart: a possible biochemical basis for erucic acid lipidosis.

Rat heart and skeletal muscle homogenates were compared for their intracellular lipolytic activity towards a series of saturated and unsaturated triglycerides from trilaurin (C12:0) to trierucin (C22:1). It is shown that for all triglycerides esterified with fatty acids from C12 to C18, lipolytic activity in heart homogenates was higher than in skeletal muscle homogenates. For these triglycerides there was no relationship between the fatty acid chain length and the lipolytic activity. In both homogenates cleavage of unsaturated triglycerides was higher than cleavage of the homologous saturated triglyceride. Lipolysis of tri-delta-11-eicosenoin (C20:1) was similar in both homogenates but much lower than lypolysis of other triglycerides. Although cleavage of trierucin (C22:1) was very low in skeletal muscle homogenates, it was undetectable in heart homogenates, even when enzyme concentration was increased. A mixture of triglycerides did not show preferential hydrolysis of any simple triglyceride. Trierucin was the only triglyceride that did not complete for lipolytic activity and only with heart homogenates, which shows that that lipase(s) do not cleave trierucin. The absence of lipolytic activity towards trierucin in heart homogenates could explain the selective accumulation of erucic acid-rich triglycerides in hearts of animals fed a diet with a high erucic acid content.

Animals↗
PubMed1998

Starvation depresses acylglycerol biosynthesis in bovine subcutaneous but not intramuscular adipose tissue homogenates.

The primary objective of this study was to document the effects of starvation on acylglycerol biosynthesis in homogenates of intramuscular and subcutaneous adipose tissues. Adipose tissue samples were obtained from 8th-13th thoracic rib sections from 12 Angus cattle (six steers plus six heifers). Three steers and three heifers were starved for 72 h prior to slaughter while the remainder were slaughtered 4 h after food was withheld. Fat-free 700 x g centrifugal fractions were used to measure the esterification of radiolabeled sn-glycerol 3-phosphate (G-3-P) into acylglycerols at 1.0 mM palmitic or stearic acid, or 0.2 mM oleic, linoleic, or alpha-linolenic acid. There were significant tissue x fatty acid interactions for rates of incorporation into diacylglycerols and triacylglycerols; in subcutaneous, but not intramuscular homogenates, palmitic > stearic = oleic = linoleic = alpha-linolenic acid. Subcutaneous homogenates incorporated a greater percentage of G-3-P into triacylglycerols, and a lesser percentage into phospholipid, than intramuscular homogenates (P < 0.05). In intramuscular homogenates, the primary product of G-3-P esterification to saturated fatty acids was phospholipids. When unsaturated fatty acid served as substrates, triacylglycerols and phospholipids were produced in equal proportions in intramuscular homogenates, and triacylglycerols were the predominant product in subcutaneous homogenates. Intramuscular adipose tissue homogenates exhibited no response to starvation, whereas triacylglycerol and diacylglycerol synthesis was depressed by approximately 50% in subcutaneous adipose tissue homogenates. Similarly, phosphatidic phosphohydrolase activity, initially greater in subcutaneous than in intramuscular adipose tissue, was decreased by approximately 50% by starvation in subcutaneous adipose tissue, but not in intramuscular adipose tissue. We conclude that differences in rates of diacylglycerol and triacylglycerol biosynthesis, and response to starvation, between intramuscular and subcutaneous adipose tissues were due to dissimilarities in the activity of phosphatidic phosphohydrolase.

Adipose Tissue↗