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[The influence of mode and intensity of homogenization on the absolute value and stability of oxygen consumption of guinea pig liver homogenates (author's transl)].

The influence of five different methods of homogenisation (1. The method according to Potter and Elvehjem, 2. A modification of this method called Potter S, 3. The method of Dounce, 4. Homogenisation by hypersonic waves and 5. Coarce-grained homogenisation with the "Mikrofleischwolf") on the absolute value and stability of oxygen uptake of guinea pig liver homogenates has been investigated in simultaneous measurements. All homogenates showed a characteristic fall of oxygen uptake during measuring time (3 hours). The modified method according to Potter and Elvehjem called Potter S showed reproducible results without any influence by homogenisation intensity.

Animals↗

New immunoseparation-based homogeneous assay for HDL-cholesterol compared with three homogeneous and two heterogeneous methods for HDL-cholesterol.

We evaluated four new commercial methods for HDL-cholesterol determination. The three completely homogeneous assays were an immunoseparation-based (IS) method from Wako, a polyethylene glycol-modified enzyme (PEG) method from Boehringer Mannheim, and a synthetic polymer-based (SP) method from Genzyme. The fourth method was a new heterogeneous method in which lipoproteins are removed using dextran sulfate-coated magnetic beads and Mg2+ (MB, Reference Diagnostics). We compared these methods with the conventional phosphotungstic acid/MgCl2 precipitation (PTA) procedure. The homogeneous assays had good intraassay imprecision with total CVs <2.3%, whereas the CVs of the MB assay were <5.9%. Adding HDL to serum to achieve HDL-cholesterol (HDL-C) concentrations up to 1000 mg/L revealed nearly complete recoveries in the IS, PEG, and MB assays, whereas the SP assay showed a lower recovery (approximately 70%). The SP HDL-C apparently increased at increasing LDL-cholesterol and VLDL-triglyceride concentrations, whereas the IS, PEG, and MB methods were not influenced by LDL-cholesterol up to 6000 mg/L (MB, 5000 mg/L) and VLDL-triglycerides up to 9000 mg/L. Free fatty acids above approximately 2 mmol/L produced falsely high HDL-C in the IS and SP assays, the error amounting to as much as 50% in some samples. An intermethod comparison in 291 fresh serum samples yielded correlation coefficients of at least r = 0.95 for all assays, when compared with the PTA procedure. The slopes and intercepts of the regression lines were 1.05 and 57 (IS), 1.12 and 9.9 (PEG), 1.00 and 39 (SP), and 1.0 and 38 mg/L (MB), respectively. The new assays are precise and simplify the determination of HDL-C, but in part they lack specificity or are susceptible to interferences, resulting in discrepancies when compared with the established PTA procedure.

Antibodies↗

Deoxyribonuclease II purified from the isolated lysosomes of porcine spleen and from porcine liver homogenates. Comparison with deoxyribonuclease II purified from porcine spleen homogenates.

Porcine spleen DNase II (EC 3.1.22.1), one of the best-characterized DNases II, is subcellularly located in lysosomes because the enzyme is co-sedimented with two of the lysosomal marker enzymes, cathepsin D and acid phosphatase. The physicochemical properties, including the subunit structure, sensitivity to iodoacetate inactivation, native molecular weight and chromatographic behavior, of the DNase II purified from the isolated lysosomes of porcine spleen are indistinguishable from those of the same enzyme purified from the whole porcine spleen homogenate. DNase II can also be extracted from porcine liver with 0.05 M H2SO4 or 0.1 M NaCl and purified from either extract by a series of column chromatographies. The purified liver DNase II from either extract has the same subunit structure (alpha-chain, Mr 35,000 and beta-chain, Mr 10,000) as the purified DNase II of porcine spleen. The two liver extracts as well as the extracts of spleen and gastric mucosa contain DNase II with very similar properties on Sephadex G-100 gel filtration, on acid polyacrylamide gel electrophoresis under non-denaturing conditions, and on isoelectric focusing. The data strongly suggest that, for the same species of animal, the DNase II activities in various tissues are associated with protein molecules of identical structure.

Animals↗

Homogeneous and non-homogeneous distribution of inhibitory and excitatory adrenoceptors in the longitudinal muscle of the guinea-pig ileum.

1 The effects of adrenoceptor agonists and antagonists on spontaneous and evoked membrane activities of longitudinal muscle cells from different parts of the guinea-pig ileum were observed, using microelectrode methods.2 Isoprenaline inhibited the generation of spikes in cells in the terminal (0-3 cm from the ileocaecal valve) and proximal (more than 50 cm from the ileocaecal valve) regions of the ileum, with no change on the membrane potential and ionic conductance of the membrane. These actions of isoprenaline were abolished by propranolol.3 Noradrenaline and phenylephrine depolarized the membrane and increased both the spike frequency and ionic conductance of cell membranes of the terminal ileum, whereas noradrenaline and clonidine hyperpolarized the membrane, increased the ionic conductance of the membrane and inhibited the spontaneously generated spikes from cells of the proximal ileum. The excitatory effect of phenylephrine in the cells of the proximal ileum and the inhibitory effect of clonidine on cells of the terminal ileum were less pronounced.4 The excitatory actions of noradrenaline or phenylephrine were antagonized by prazosin and phentolamine, but not by yohimbine, whereas the inhibitory actions of noradrenaline or clonidine were antagonized by yohimbine and phentolamine but not by prazosin.5 The cholinergic e.j.ps evoked by field stimulation to the tissue were not affected by isoprenaline or phenyleprine but were inhibited by noradrenaline and clonidine, in both the terminal and proximal regions of the ileum. These actions of noradrenaline and clonidine were antagonized by yohimbine but not by prazosin.6 The results indicate that in the myenteric plexus and longitudinal muscle tissues of the guinea-pig ileum there are prejunctional inhibitory (alpha(2)), postjunctional inhibitory (alpha(2) and beta) and postjunctional excitatory (alpha(1)) adrenoceptors. The homogeneous distributions of prejunctional alpha(2)- and postjunctional beta-adrenoceptors in the ileum are responsible for inhibitions of cholinergic excitatory junction potentials (e.j.ps) and spontaneous spike activities, respectively. The density of distribution of the postjunctional alpha(1)-adrenoceptors is higher in the terminal than in the proximal regions, and these distributions are reversed in the case of the postjunctional alpha(2)-adrenoceptors. The postjunctional alpha(1)-adrenoceptors are probably responsible for the membrane depolarization and alpha(2)-adrenoceptors for the hyperpolarization induced by catecholamines.

Animals↗

Subcellular fractionation by centrifugation of homogenates of the neural lobe of the bovine pituitary gland: identification of different pools of hormone in the homogenate and isolation of neurosecretosomes.

1. Homogenates of bovine pituitary neural lobe tissue were subjected to differential centrifugation. Six subcellular fractions (I-VI) were obtained and the distribution among them of various cell organelles was studied by means of markers. Fraction II (800-3000 gav), the nerve-ending fraction (neurosecretosomes), contained sedimentable lactate dehydrogenase, hormone and a large proportion of the total Mg2 + +Na+ +K+-ATPase. 2. Lysis of the neurosecretosomes in hypotonic sucrose solutions led to loss of lactate dehydrogenase and vasopressin. 3. Centrifugation of the granule fraction (IV) on a sucrose gradient (1.3-2.0 M sucrose) gave a bimodal distribution of vasopressin. Purified neurosecretory granules were recovered from the denser band. Centrifugation on modified gradients (0.8-2.0 or 0.4-2.0 M sucrose) increased the yield of hormone in the denser band but the purity of the granules was decreased. 4. Considerable purification of the neurosecretosomes (fraction II) was achieved by "washing". Centrifugation of washed neurosecretosomes on sucrose density gradients led to the accumulation of all activities at a region between 1.4 and 1.5 M sucrose. 5. The distribution of Mg2+ +Na+ +K+-ATPase in centrifugal fractions indicated that the neurosecretosomes had been isolated in relatively high yield.

Adenosine Triphosphatases↗

Novel mutation processes in the evolution of a haploid minisatellite, MSY1: array homogenization without homogenization.

The Y-specific locus MSY1 is the only known haploid minisatellite, and displays an extremely high degree of structural diversity which can be assayed by minisatellite variant repeat PCR (MVR-PCR). One group of alleles, in an African-specific class of Y chromosomes (haplogroup 8), behaves unusually in the conventional MVR-PCR assay, and sequencing demonstrates that this is because repeat units in these alleles contain an additional base substitution. We have designed a new MVR-PCR system to detect these novel variants, and show firstly that they are confined to the haplogroup 8 chromosomes, and secondly that the base substitution has spread through these arrays without the elimination of existing repeat variants. The sharing of a particular base substitution between otherwise distinct repeat types in these alleles represents evidence of a remarkable mutation process in their evolutionary history, in which the variant base must have been spread by a biased repair mechanism operating in very small patches within heteroduplexes.

Genetic Variation↗