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PubMed2001

Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception.

BACKGROUND: With an increase in the use of assisted reproduction technologies the requirements of the diagnostic semen analysis are constantly changing. METHODS: Spermatozoa from patients undergoing IVF were analysed by examining the conventional semen parameters and DNA/chromatin integrity, using in-situ nick translation (NT) and the Chromomycin A(3) fluorochrome, which indirectly demonstrates a decreased presence of protamine. Samples were examined before and after preparation using discontinuous density gradient centrifugation. RESULTS: Density gradient centrifugation enriched samples by improving the percentage of morphologically normal forms by 138% and sperm nuclear integrity by 450%. Sperm nuclear integrity as assessed by in-situ nick translation (NT) demonstrated a very clear relationship with sperm concentration, motility and morphology. Morphology correlated with fertilization rates of patients undergoing IVF, while NT values of the spermatozoa post-preparation were significantly lower in pregnant patients. CONCLUSIONS: We have demonstrated that along with the classical semen parameters, the assessment of nuclear integrity improves the characterization of the semen sample and may be used as a tool for allocating patients to specific assisted reproduction treatments.

Adult↗
PubMed1992

Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction.

The genomic RNA of hepatitis C virus (HCV) in the plasma of volunteer blood donors was detected by using the polymerase chain reaction in a fraction of density 1.08 g/ml from sucrose density gradient equilibrium centrifugation. When the fraction was treated with the detergent NP40 and recentrifuged in sucrose, the HCV RNA banded at 1.25 g/ml. Assuming that NP40 removed a lipid-rich surface coat from HCV, the 1.08 g/ml and 1.25 g/ml HCV RNA may correspond to intact HCV virions and nucleocapsids, respectively. The extraordinarily low density of the virion is unusual in comparison to the density of classified viruses.

Base Sequence↗
PubMed2004

Reduced senescence and retained nuclear DNA integrity in human spermatozoa prepared by density gradient centrifugation.

PURPOSE: To investigate whether removal of extraneous cells and immotile spermatozoa from a sperm preparation by density gradient centrifugation could help to maintain normal spermatozoa in a viable state and retain their deoxyribonucleic acid integrity. METHODS: Sperm motility was assessed on a daily basis in aliquots of neat semen, extended semen, and spermatozoa prepared on a PureSperm density gradient. At the same time, aliquots of each sperm sample were preserved for TUNEL assay and nick translation. RESULTS: Spermatozoa prepared using density gradient centrifugation survived three times as long as spermatozoa in neat semen or in extended semen. Both deoxyribonucleic acid integrity and sperm motility were retained in the gradient preparations. CONCLUSIONS: Preparing spermatozoa by density gradient centrifugation is advantageous in prolonging sperm survival and maintaining deoxyribonucleic acid integrity, presumably by removing sources of reactive oxygen species. Stored spermatozoa could be used for a second attempt at fertilization if oocyte immaturity was suspected.

Adult↗
PubMed1987

Purification of Cowdria ruminantium by density gradient centrifugation.

The isolation of Cowdria ruminantium by differential and isopycnic density gradient centrifugation is reviewed with special reference to the suitability of Percoll as density gradient medium. Infected sheep brain, Amblyomma hebraeum nymphae and various mouse organs were used as starting material. By these methods, partially purified viable populations of the organism with distinctly different densities were obtained. The conclusions are based upon results of analyses of density fractions by inoculation into sheep or mice, protein determination, electron microscopy and enzyme-linked immunosorbent assay. Morphological differences were observed in the density fractions obtained from infected brain tissue and A. hebraeum.

Animals↗
PubMed1975

Use of iodinated organic compounds for the density gradient centrifugation of viruses.

Use of Urografin and Conray for the equilibrium centrifugation of viruses is described. These pharmaceuticals, which consist of iodinated arylic compounds, reach densities of 1.6 g/cm3 and have low intrinsic viscosities. Poliovirus, Newcastle disease virus, and lymphocytic choriomeningitis virus were centrifuged to equilibrium in gradients made of these substances. Viral infectivities were not measurably affected, which is especially noteworthy in the case of the very labile lymphocytic choriomeningitis virus. Buoyant densities were found to be significantly lower than densities obtained with gradients made of CsCl and sucrose.

Centrifugation, Density Gradient↗
PubMed1969

Bovine splenic nerve: characterization of noradrenaline-containing vesicles and other cell organelles by density gradient centrifugation.

1. Homogenates of bovine splenic nerves were subjected to differential and sucrose density gradient centrifugation. From the low-speed supernatant a high-speed sediment (mitochondria, lysosomes, microsomes and noradrenaline (NA) vesicles) was obtained. By density gradient centrifugation of this sediment it was shown that NA vesicles are slightly less dense than mitochondria, but denser than microsomes.2. In further experiments a mitochondrial and a microsomal sediment were obtained. The mitochondrial sediment was fractionated with a short centrifugation time over a density gradient ranging from 0.6 to 1.2 M sucrose. Mitochondria (fumarase and succinate-dehydrogenase) and lysosomes (acid ribonuclease and deoxyribonuclease) sedimented to the bottom of the tube. The highest concentration of NA vesicles was found in a medium position. There was only a small amount of microsomes (glucose-6-phosphatase) present.3. The microsomal sediment was centrifuged for 150 min over a density gradient ranging from 0.8 to 1.4 M sucrose. The microsomes remained on the top of the gradient. There were also some mitochondria and lysosomes present. The NA vesicles were found in highest concentration in the middle of the gradient (at about 1.2 M sucrose).4. With the use of these two density gradients, the subcellular distribution of dopamine-beta-hydroxylase, monoamine oxidase and ATPase was studied. Dopamine-beta-hydroxylase was found to be localized in the NA vesicles. Monoamine oxidase was mainly recovered in mitochondria; a small part of the enzyme appeared to be microsomal. ATPase was present in microsomal elements.

Adenosine Triphosphatases↗
PubMed1980

Isolation of human NK cells by density gradient centrifugation.

Sedimentation characteristics of human NK cells in discontinuous Percoll density gradients were studied. NK activity against the leukaemic cell line K-562 peaked in a single low density fraction mainly consisting of large granular lymphocytes previously shown to be the principal NK cells in human peripheral blood. Percoll density gradient centrifugation provides a useful tool for analysis of human NK cells and their relationship to other blood mononuclear cells.

Cell Fractionation↗
PubMed1985

Thyrotropin increases the iodine content of rat circulating thyroglobulin as measured by equilibrium density gradient centrifugation.

In previous work we demonstrated that circulating thyroglobulin contains very little or no iodine. We have now characterized circulating thyroglobulin following administration of thyrotropin (TSH) to determine whether its iodine content remains low or increases after stimulation. The iodine content of circulating thyroglobulin was estimated from its density determined by equilibrium density gradient (isopycnic) centrifugation. TSH stimulated thyroglobulin from 182 +/- 28 ng/ml to 571 +/- 83 ng/ml at 8-14 h. Circulating thyroglobulin in the basal state had a density consistent with very little or no iodine. Its density increased following TSH to a maximum at 8-14 h which was nearly the same as the density of thyroglobulin extracted directly from the thyroid. To determine whether selective peripheral metabolism, based on the degree of iodination, could account for the density shift, purified rat thyroid thyroglobulin was injected into thyroidectomized rats. The density of thyroglobulin remained unchanged for 25 h during which time it was metabolized by more than 97%. Therefore, selective metabolism of thyroglobulin based on iodine content did not occur. We conclude that TSH causes a marked increase in the iodine content of circulating thyroglobulin. It is most likely that in the basal state circulating thyroglobulin comes from selective release of poorly iodinated molecules, while after TSH, it comes from release of previously synthesized, iodinated and stored molecules.

Animals↗
PubMed1979

Density gradient centrifugation for separation of different stages of Toxoplasma gondii.

Suitable procedures for separation of various stages of Toxoplasma gondii by density gradient centrifugation have been delineated using peritoneal exudate of infected mouse as a model. Separation from host cells was effected by gradient centrifugation at 450 g for 30 minutes. Using Ficoll, dextran and sucrose, average parasite recoveries by pooling up to and including the peak fraction of 73.65%, 66.18% and 65.68% respectively were obtained. Toxoplasma trophozoites peaked at density of 1.040 g/ml with Ficoll, 1.060 g/ml with dextran and 1.110 g/ml with sucrose. In view of successful separation of exo-enteric stages of Toxoplasma by density gradient centrifugation, possible application of this method to isolation of various endo-enteric stages is discussed.

Animals↗
PubMed1981

Separation of Isospora (Toxoplasma) gondii cysts and cystozoites from mouse brain tissue by continuous density-gradient centrifugation.

A simple, quick and reproducible method consisting of density-gradient centrifugation of homogenized infected mouse brain tissue on Percoll is described for the isolation and purification of cysts of Isospora (Toxoplasma) gondii. A 100% recovery of cysts, with 74.2% in a single fraction with a specific gravity of 1.056, was obtained by overlaying homogenates of infected mouse brains on a pre-formed Percoll gradient and centrifugation at low g forces. With this procedure recovery was independent of the age of the cysts. Titration of purified cystozoites showed there to be no loss of infectivity.

Animals↗
PubMed1972

Separation of HeLa cells by colloidal silica density gradient centrifugation. I. Separation and partial synchrony of mitotic cells.

Using a colloidal silica density gradient, HeLa cells in mitosis were found to have a density of 1.040-1.046 g/cc, lighter than the remaining interphase cells. The mitotic cells could be harvested and cultured after centrifugation, showing growth synchrony by measurement of a peak in mitotic index 21 hr after establishing the culture. By using Colcemid or vinblastine sulfate, HeLa cells were arrested in metaphase and centrifuged on the colloidal silica density gradient. The blocked metaphase cells were lighter in density than the interphase cells but somewhat more dense than untreated cells selected by the density gradient centrifugation. Near-equilibrium conditions were established during the centrifugation of cells so that cell density measurements could be made, and the gradient medium employed was not measurably toxic to those cells tested.

Cell Separation↗