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At least 19 recordsLinked to original sources
PubMed2025

Absolute Quantification of Cellular and Cell-Free Mitochondrial DNA Copy Number from Human Blood and Urinary Samples Using Real Time Quantitative PCR.

Mitochondrial DNA copy number (mtDNA-CN) in human body fluids is widely used as a biomarker of mitochondrial dysfunction in common metabolic diseases. Here we describe protocols to measure cellular and/or cell free (cf)-mtDNA-CN in human peripheral blood and urine. Cellular mtDNA is located inside the mitochondria where it encodes key subunits of the respiratory complexes in mitochondria and is usually normalized with reference to the nuclear genome as the mitochondrial genome to nuclear genome ratio (Mt/N) in either whole blood, peripheral blood mononuclear cells (PBMCs), or whole urine. Cf -mtDNA is usually found outside of the mitochondria, often released following mitochondrial damage, can trigger inflammatory pathways, and is usually measured as mtDNA-CN per volume of the starting material. Here we describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions and whole urine into urinary supernatant and pellet, (2) prepare DNA from each of these fractions, (3) prepare reference standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Humans↗
PubMed1979

[Advantage of phase contrast for the identification of the cellular constituents of urinary sediment].

Phase contrast microscopic observation of the urinary sediment has to be performed on freshly collected urine. This technique, which permits an excellent definition of the cells and the maintenance of intra cellular motility, gives therefore a great accuracy in differential diagnosis, especially for little round cells. Among these, polynuclear granulocytes are easily recognized, because of their lobulated nuclei and of their numerous intra cytoplasmic motile granulations. Lymphocytes and lymphoblasts have round, regular, homogeneous nuclei. Renal tubular cells exhibit eccentric nuclei, with smudged chromatine, and round, refringent cytoplasmic vacuoles. Rapid and easy, this technic should be used for routine microscopic study or urinary sediments.

Adult↗
PubMed1978

[Phase contrast microscopy studies of the urine of newborn infants].

Phase contrast microscopy allows a much better identification of the urinary sediment constituents than conventional bright field microscopy. The urinary sediment of 100 neonates admitted in a care unit has been examined by phase contrast microscopy. In all cases, squamous cells are far more numerous in newborn girls than in boys. An important rate of abnormal sediments has been observed: in 40 patients cytologic symptoms of renal ischaemia have been observed (hyaline or granulous casts and renal tubular cells, but red blood cells are uncommon). Numerous uric acid cristals were persent in 22 newborns, in correlation with a prolonged perinatal hypoxia. A considerable bacteriuria was seen in 21 neonates, without concomitant leucocyturia. In conclusion, only 20% of the investigated neonates had a normal urinary sediment.

Bacteriuria↗
PubMedDate not supplied

Long-term renal handling of sodium and calcium in spontaneously hypertensive rats.

The renal handling of sodium and calcium in spontaneously hypertensive rats (SHR) was investigated over an extended period (10-75 weeks of age) and compared with age-matched normotensive Wistar-Kyoto controls. The animals were fed a standard rat chow except during screening periods when liquid diet that matched the pellet chow was substituted. Sodium balance, urinary excretion of sodium and calcium, fractional excretion of sodium (FENa), and renal cortical dopamine receptors (DA1 and DA2) were measured at 10, 30, 60 and 75 weeks of age. The results showed no difference between the two strains except for FENa, which was significantly higher in the SHR at 75 weeks coincident with decreased glomerular filtration rate. We conclude that a defect in renal handling of sodium and/or calcium is not a major factor in the maintenance of hypertension in the SHR.

Animals↗
PubMed1978

The development of a test system for inhibition of urinary oxalate lithiasis.

The production of calcium oxalate deposits on zinc pellets in the bladder of rats was induced by the addition of 0.25 to 1.0 per cent ethylene glycol to their drinking water. Sprague Dawley rats requied more than 0.50 per cent ethylene glycol to produce calcium oxalate exclusively. The quantity of deposits varied widely between test animals, but intermediate operation to check on the amount of deposit showed that each rat at the 0.75 per cent ethylene glycol level maintained its individual rate of deposition within an acceptable standard deviation relative to the mean. At 1 per cent ethylene glycol deposition in the kidneys interfered with deposition on the zinc pellet.

Animals↗
PubMed1977

Dissolution of urinary stones by calcium-chelating agents: A study using a model system.

Pellets made by dispersing microcrystals of calcium oxalate monohydrate throughout an organic matrix have served as models for kidney stones in studies of factors governing their dissolution by calcium-chelating agents. These factors include pH, ionic strength, concentration of chelating agent, and addition of other acids and bases. The method shows good reproducibility. Results have been applied to improving a clinical procedure for kidney stone dissolution.

Calcium↗
PubMed1978

[The use of gentamicin-polymethylmethacrylate chains for the treatment of soft-tissue wounds in the region of the urinary tract (author's transl)].

Bactericidal concentrations of gentamicin can be attained by implanting at the site of the infection pellet chains of gentamicin-polymethylmethacrylate (PMMA) that permit the protracted release of the antibiotic for several weeks. Satisfactory healing of the wound was achieved in two cases of extensively infected soft-tissue wounds in the region of the urinary tract after temporary implantation of gentamicin-PMMA chains.

Adolescent↗
PubMed1976

Long-term transplantability and morphological stability of three experimentally induced urinary bladder carcinomas in rats.

Three transitional cell carcinomas induced in Fischer 344 rats by a methylcholanthrene pellet or a foreign body inserted locally into the bladder have been serially transplanted in the syngeneic strain for up to 6.5 years. There have been no changes in the individual morphological characteristics of the tumors during this time. Cells cultured in vitro for varying numbers of passages reproduce regularly the morphology of each tumor when they are injected back into the animals and results from a microcytotoxicity assay for cellular immunity indicate that they retain a common, bladder tumor-specific antigen. These tumors are useful for research in turmo biology and are offered to other scientists seeking transplantable carcinomas for experimentation.

Animals↗
PubMed1979

The utilization of chopped and pelleted lucerne (Medicago sativa) by growing lambs.

1. The efficiency of utilization of the dietary energy and nitrogen contained in a dried lucerne (Medicago sativa cv. Chartainvilliers) given either chopped (CL) or ground (1.96 mm screen) and pelleted (PL), was measured in a comparative slaughter experiment. Growing lambs were given equal amounts of digestible energy in the chopped or pelleted form at each of the three planes of nutrition for a period of 100 d. 2. The initial energy, fat and protein content of both the carcass and the total body of the test lambs was estimated from regression equations between fasted (18 h) live weight and these components, derived from a group of twenty-three comparable lambs. The final energy, fat and protein content of the test lambs was determined directly by chemical analyses. 3. The metabolizable energy (ME) content of the diets was derived at each plane of nutrition from measured faecal and urinary losses and estimated methane losses. The depression in ME content with grinding and pelleting the dried lucerne was small (CL 8.69 MJ/kg dry matter (DM), PL 8.42 MG/kg DM). 4. The efficiency of utilization of the ME of the dried lucerne for growth and fattening was higher (P less than 0.01) when given in the ground pelleted form (0.533), than in the chopped form (0.284). The net energy value of the PL (3.5 MJ/kg DM) was higher than that of CL (2.2 MJ/kg DM). 5. Thus lambs fed on PL grew faster and had a higher caracass weight gain, carcass protein and fat retention than lambs fed on CL. The composition of the carcass was not altered by the physical processing treatment. 6. Digestion studies with these same CL and PL diets had shown that grinding and pelleting depressed digestion in the forestomachs and increased digestion in the small intestine compared with the chopped form. The increased efficiency of utilization of the gross energy and ME and the higher net energy value of PL was attributed primarily to a change in the site of digestion within the alimentary tract. Associated with this change was a higher value for absorbed amino acids : absorbed energy and an increased apparent absorption of methionine for lambs fed on PL. The difference in the energy costs of eating and ruminating the CL and PL was small.

Animal Nutritional Physiological Phenomena↗
PubMed1979

Renal adaptation to a low phosphate diet in rats.

The major renal adaptive changes in response to selective dietary phosphate restriction are a marked reduction in urinary excretion of phosphate and an increased urinary excretion of calcium; at the cellular level, there is selective increase in renal cortical brush border membrane phosphate uptake and increase in specific activity of alkaline phosphatase. In the present study we examined whether these functional and biochemical adaptive changes could be blocked by drugs known to inhibit protein synthesis. Administration of actinomycin D or cycloheximide to rats switched from a diet with normal phosphate content (0.7%) to a diet with low (0.07%) phosphate content either completely (actinomycin D) or partially (cycloheximide) prevented the expected decrease in urinary excretion of phosphate and increase in the urinary excretion of calcium. The specific activity of alkaline phosphatase measured in crude membrane fraction (washed 100,000 g pellet) from renal cortical homogenate in animals fed a low phosphate diet and treated with actinomycin D or with cycloheximide was significantly lower than in control animals also on a low phosphate diet receiving placebo; but there were no differences between treated and untreated animals in the activities of two other brush border enzymes, gamma-glutamyltransferase and leucine aminopeptidase. Actinomycin D administered to rats maintained on a normal phosphate diet throughout the course of the experiment caused an increase in the urinary excretion of phosphate on the last (6th) day of the experiment but did not change urinary excretion of calcium. In acute clearance experiments, infusion of actinomycin D to rats adapted to a low phosphate diet did not increase fractional excretion of phosphate. In separate experiments, using the same dietary protocol as above, brush border membrane fraction (vesicles) was prepared from renal cortex of rats sacrificed at the end of the experiment. In this preparation Na(+)-dependent (32)Pi and d-[(3)H]glucose uptake and activities of brush border enzymes membrane were determined. Brush border membrane vesicles prepared from rats fed a low phosphate diet showed significantly higher Na(+)-dependent (32)Pi uptake compared with rats fed a normal phosphate diet. This increase in (32)Pi uptake was completely prevented when rats on a low phosphate diet were simultaneously treated with actinomycin D. These differences were specific for (32)Pi transport as no differences were observed in d-[(3)H]glucose uptake among the three groups. There was a positive correlation (r = 0.82, P < 0.01) between (32)Pi uptake and specific activity of alkaline phosphatase measured in aliquots of the same brush border membranes, whereas no such correlation was observed with two other brush border membrane enzymes gamma-glutamyltransferase and leucine aminopeptidase. These observations show that actinomycin D prevents both the functional and cellular renal adaptive changes induced by a low phosphate diet. Taken together, these observations suggest that renal adaptation to a low phosphate diet could be prevented by inhibition of de novo protein synthesis.

Animals↗
PubMed1977

Mechanism of resistance to the phosphaturic effect of the parathyroid hormone in the hamster.

The effect of parathyroid hormone and calcitonin on the renal excretion of phosphate, calcium, and cyclic AMP was evaluated in the thyroparathyroidectomized hamster, a mammal apparently reisstant to the phosphaturic effect of parathyroid hormone. Parathyroid hormone did not increase phosphate excretion, although it decreased excretion of calcium and increased urinary excretion of cyclic AMP. This lack of a phosphaturic response to parathyroid hormone was not reversed by administration of 25-OH vitamin D or infusions of calcium or phosphate. Calcitonin, another potentially phosphaturic hormone, also vailed to increase phosphate excretion but markedly elevated urinary excretion of cyclic AMP. In hamsters pretreated with infusion of urinary ammonium chloride, which decreased plasma and urinary pH, both parathyroid hormone and calcitonin increased excretion of phosphate as well as that of cyclic AMP. Acetazolamide had no phosphaturic effect in ammonium chloride-loaded hamsters, and it decreased cyclic AMP and calcium excretion. Alkalinization of urine by acetazolamide did not prevent the phosphaturic effect of parathyroid hormone in ammonium chloride-loaded hamsters, but it blocked the increase in urinary cyclic AMP excretion. Parathyroid hormone and calcitonin both stimulated adenylate cyclase in a cell-free system (600-g pellet) from hamster renal cortex, elevated tissue cyclic AMP levels, and activated protein kinase in tissue slices from hamster renal cortex. In acid medium, the increase in cyclic AMP and activation of protein kinase in response to parathyroid hormone was diminished, but addition of acetazolamide restored responsiveness of both parameters to control values. Acetazolamide, on the other hand, did not influence adenylate cyclase or its response to parathyroid hormone or cyclic AMP phosphodiesterase activity. We conclude that the lack of a phosphaturic effect of parathyroid hormone and calcitonin in the hamster depends on steps in the cellular action of these hormones, steps that are sensitive to pH subsequent to cyclic AMP generation and protein kinase activation. In addition, acetazolamide may potentiate the phosphaturic effect of parathyroid hormone by promoting accumulation of cyclic AMP in tissue. Thus, the hamster is a particularly useful model for studies of syndromes in which there is renal resistance to phosphaturic hormones.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1977

[Collagenase activity in an animal and a human carcinoma (author's transl)].

High amounts of collagenase were found in chemically-induced carcinomas of the mouse skin and in carcinomas of the human urinary bladder. Part of the total enzyme activity was detected in the supernatant after sedimentation of the tumour homogenate at 6000 x g and dialysis for 48 hours. The remainder was extracted from the pellet by the use of 5 M urea. Though the localization of enzyme production and regulation of enzyme activity is still unclear, the collagenases may be implicated in the breakdown of collagen structures during tumour invasion.

Animals↗
PubMed1975

[Changes in renal function in the hours following an intravenous injection of acebutolol].

A study was carried out, of the changes of renal functional value and of urinary electrolytes excretion after rapid intravenous injection of acebutolol in twelve subjects with various glomerular filtration. Renal functional value was estimated by measuring inuline. P.A.H., urea and creatinin clearances during a control period of thirty minutes and during the four hours following administration of acebutolol. The variations in the excretion of electrolytes: chloride, sodium, potassium, calcium and phosphorus were measured during the same periods. The following results were obtained: in subjects with normal renal functional value, administration of acetubolol brings about: a variation which is under 20 p.cent of glomerular filtration and P.A.H. clearance; a 50 p.cent average decrease of natriuresis. in cases of renal insufficiency: clearances of inuline and P.A.H. are unchanged. There is a 20 p.cent average decrease of natriuresis. There is a decrease of urinary output (35-45 p.cent) and of urea clearance (20-50 p.cent).

Acebutolol↗
PubMed1978

[General pharmacological studies on tramadol, a potent analgetic agent (author's transl)].

The general pharmacological properties of 1-(m-methoxyphenyl)-2-(dimethylaminomethyl)-cyclohexan-1-ol (tramadol; Tramal) are described and compared with those of other strong narcotic analgetics. In behavioral studies tramadol in high doses had a primarily stimulating effect in mice and rats and a sedative effect in rabbits and dogs. The Straub tail phenomenon, a reaction typical for mice administered morphine, was observed only after subtoxic doses of tramadol. In i.v. doses tramadol generally caused a weak central inhibition of non-stimulated and electrically stimulated brain activity in unanesthetized rabbits. Muscle tone and motor coordination in rats and mice were only slightly affected by the drug, in contrast to the effect of morphine. Unlike other strong analgesics tramadol in doses of 5--20 mg/kg i.v. did not cause respiratory depression and even clearly increased respiratory volume and rate in conscious rabbits and anesthetized dogs. In cats and dogs i.v. doses of tramadol up to 10 mg/kg were well tolerated in the cardiovascular system. Tramadol has a slight, papaverine-like spasmolytic effect and no effect on gastrointestinal motility or urinary and electrolyte excretion. The drug showed no antipyretic properties in rabbits. It inhibited edema in rats and guinea pigs but had no antiproliferative effect in the cotton pellet test in rats. Tramadol did not inhibit monoamine oxidase activity or cause enzyme induction in the rat liver.

Analgesics↗
PubMed1977

Immunocytochemical localization of lysozymes in respiratory and other tissues.

Immunostaining paraffin sections of appropriately fixed tissues with an antiserum to human urinary lysozyme as the primary step in an immunoglobulin-peroxidase bridge method has localized lysozyme in previously recognized sites such as Paneth cells, renal tubules, and lymph node macrophages in several species. In addition, lysozyme was demonstrated in the ciliary layer of the trachea, and type II pneumocytes, as well as cells of presumed mucoid nature in laryngotracheal glands. Large stellate cells in follicle centers in the lymph nodes and spleen and in the medulla of the thymus evidenced strong lysozyme reactivity. Granular pneumocytes disclosed immunoreactivity for lysozyme also at the ultrastructural level. Lysoplate assay demonstrated lysozyme in abundance in both the cellular pellet and acellular supernatant of rat alveolar wash fluid and in rat lung after repeated washing of alveoli. Hamster lung differed from the others in failing to immunostain for lysozyme and affording no evidence for content of lysozyme as determined by lysoplate assay. Sites stained with antiserum to human urinary lysozyme failed to stain with antiserum to egg white lysozyme. However, the pyloric glands, Golgi elements in intestinal epithelium, the surface of the colon, and the proximal straight renal tubule of the mouse stained exclusively with the antiserum to hen egg white lysozyme. Many sites staining with antiserum to urinary lysozyme in respiratory, renal, and lymphoid tissue lacked reactivity in control sections exposed to this antiserum after it was absorbed with purified urinary lysozyme. However, mucous acini in submandibular glands, although failing to stain with other control procedures, retained towared the absorbed antiserum, possibly through reacting with an antibody other than that for human urinary lysozyme. A number of cell types containing proteinaceous cytoplasmic granules stained in control sections exposed to normal serum in place of antilysozyme serum in the immunoglobulin-peroxidase bridge procedure and, thus, possessed selective, but nonimmunospecific affinity for immunoglobulin. Cell types that stained with antiserum to hen egg white lysozyme lost affinity for the antiserum after its absorption with egg white lysozyme but retained the affinity after absorption with urinary lysozyme.

Animals↗