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PubMed1974

Isopycnic centrifugation of sheared L-cell chromatin in metrizamide gradients.

Isopycnic gradient centrifugation of L-929 cell chromatin in a 38% (W/V) metrizamide solution yields two distinct fractions. The fraction banding at a density of 1.24 gm/cm(2) (H chromatin) contains about 10% of the DNA present in the fraction banding at a density of 1.18 gm/cm(2) (L chromatin). Both fractions contain the same proportions of satellite to main band DNA's. Some differences can be seen in the DNA: protein ratios and types of proteins present in the H and L chromatin fractions.

Animals↗
PubMed1976

Distribution of histones in alkali-denatured chromatin studied by isopycnic centrifugation in alkaline metrizamide density gradients.

Three types of density gradients - neutral metrizamide, alkaline NaOH-metrizamide and alkaline triethanolamine-metrizamide - were used for studying the distribution of histones between the two DNA strands in alkali-denatured chromatin. It was found possible to avoid both protein redistribution and dissociation by using triethanolamine-metrizamide density gradients at pH 10.5. Under these conditions an alkali-denatured mixture of DNA and chromatin was well separated into the original DNA and DNP. When native or sonicated chromatin was denatured at pH 12.2 and centrifuged in a triethanolamine-metrizamide density gradient at pH 10.5 no peak of free DNA appeared. These results show that both DNA strands remain associated with histone molecules upon alkaline denaturation of chromatin.

Animals↗
PubMed1978

Complex of DNA with chromatin proteins investigated by isopycnic centrifugation in metrizamide.

Complexes of mouse main band DNA with a fraction of non-histone proteins (NHP), having a high affinity for DNA, in the absence or presence of histones have been investigated by gradient centrifugation in metrizamide. Two types of complexes were formed at an input ratio of NHP to DNA between 1 and 2.5. In metrizamide gradients a majority of DNA was found in the light complex (at the density of 1.14-1.16 g/cm3) even at the very high NHP to DNA ratio. When histones were present in the reaction mixture, most of the DNA was found in the heavy complex (1.19-1.21 g/cm3). The electrophoretic profiles of the proteins recovered from the heavy and light complexes were different; some fractions of nonhistone proteins were present only in the heavy component.

Animals↗
PubMed1995

Human sperm morphology evaluation pre- and post-Percoll gradient centrifugation.

Previous experiments have established a relationship between the morphological characteristics of human spermatozoa and their fertilizing potential in vitro. To assess further the efficiency of Percoll gradient centrifugation (PGC) as a method of sperm selection, we have examined morphological characteristics of spermatozoa from 86 teratozoospermic patients attending Nottingham University Research and Treatment Unit in Reproduction (NURTURE). Patients were divided into groups according to percentage normal morphology in the fresh sample: group A (n = 14), < 5% normal morphology; group B (n = 41), 5-14% normal morphology; and group C (n = 31), > 14% normal morphology. Morphology slides were prepared using Diff Quik staining techniques and evaluated by Kruger strict criteria, under oil immersion, at a magnification of x1000; specific defects, viz. head, neck, cytoplasmic droplets, tail, immature cells, were assessed individually. Following PGC, a sperm sample with enhanced morphology was recovered for group B (P < 0.01) and C (P < 0.005); however, for group A (very severe teratozoospermia) PGC did not select a sample with significantly improved morphological quality. Specific sperm defects affected by PGC were head, neck and immature cells. No significant difference was found for tail abnormalities or cytoplasmic fragments.

Cellular Senescence↗
PubMed1991

Sperm morphology and IVF pregnancy rate: comparison between Percoll gradient centrifugation and swim-up procedures.

Many groups currently use two methods for the separation of motile spermatozoa, swim-up (S-up) and centrifugation on discontinuous Percoll gradient (PGC), and comparison of results indicates that PGC is superior. In this study we have attempted to identify the factors explaining this difference. This laboratory has long-standing expertise in seminology, thus the parameters of sperm morphology were the obvious first choice for detailed study. First, the respective effects of S-up and PGC on sperm morphology were analysed in different types of ejaculates: 62 semen samples with normal parameters and 41 with poor parameters. Both separation techniques resulted in improved morphology in the final preparation but only the increase of morphologically normal spermatozoa in the final Percoll suspension was significant. Second, application of these techniques in our in-vitro fertilization (IVF) programme revealed that, together with the improvement of sperm morphology, a higher pregnancy rate was obtained after PGC. The ongoing pregnancy rates per oocyte retrieval were 21.1% for the S-up technique and 33.3% for the PGC technique. These data show that spermatozoa selected by PGC present an improved morphology which we believe to be linked to improvement of the quality of the in-vitro fertilized embryos and ultimately the percentage of successful IVF results.

Adult↗
PubMed1993

An auto-controlled study in in-vitro fertilization reveals the benefit of Percoll centrifugation to swim-up in the preparation of poor-quality semen.

The efficiency of spermatozoa prepared by swim-up or by Percoll centrifugation was assessed in an in-vitro fertilization programme on 71 semen samples of a well-defined quality [total number of type A (WHO criteria) motile spermatozoa]: category I (n = 21) with > 100 x 10(6), II (n = 31) with 15-100 x 10(6), III (n = 11) with 5-15 x 10(6) and IV (n = 8) with < 5 x 10(6) type A motile spermatozoa. Oocytes were inseminated 4 h after oocyte retrieval, alternately with spermatozoa derived from swim-up and Percoll preparation. Both selection procedures resulted in a significantly higher (P < 0.001) percentage motility as compared to fresh semen. For low-quality samples (III and IV), however, swim-up was more effective in selecting highly motile (P = 0.004) and morphologically normal spermatozoa (P < 0.05). For high-quality samples, this difference might have been masked by introducing a swim-up step to remove Percoll particles. Regardless of the initial sperm quality, the mean fertilization rate was significantly higher (P = 0.003) when Percoll-treated spermatozoa were used for insemination (51.3 versus 37.8%). For semen of groups I and II, no difference in fertilization capacity was observed according to the sperm preparation method. Despite the lower percentage motility and normal morphology for the Percoll compared to the swim-up treatment in groups III and IV, fertilizing capacity was significantly (P < 0.001) in favour of this selection method (65.3 versus 26.5% in group III, 47.6 versus 11.6% in group IV).(ABSTRACT TRUNCATED AT 250 WORDS)

Centrifugation↗
PubMed1976

Proteoglycan complexes from bovine heart valve. Fractionation by density-gradient centrifugation and gel filtration under dissociative conditions.

Proteoglycan complexes from collagenase [EC 3.4.24.3]-indigestible materials of bovine heart valves were extracted with 4 M guanidinium chloride, purified by ion-exchange column chromatography in a urea-containing solution, then fractionated by density-gradient centrifugation under dissociative conditions. Electrophoretic characteristics and enzymic susceptibility of the density-gradient fractions revealed that the glycosaminoglycans constituting the proteoglycan complexes in this indigestible materials were mainly dermatan sulfate in the top three fractions, and dermatan sulfate and chondroitin sulfates in the bottom fraction; a minor constituent which was common to all the fractions was hyaluronic acid. A gel-like substance (Fr. Ig) at the top of the gradient, amounting to about 25% of the loaded dry sample, contained only a trace of hydroxyproline (less than 1%) and was composed of proteodermatan sulfate, glycoprotein, and a small amount of hyaluronic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of Fr. Ig with 2-mercaptoethanol showed that the major part of the proteins in this gel-like substance was cross-linked by disulfide bridges. Chromatography of Fr. Ig on Sepharose 4B in buffered 4 M guanidinium chloride containing 2-mercaptoethanol, together with the electrophoretic patterns of the resulting fractions, suggested that proteodermatan sulfate was not associated with hyaluronic acid through covalent bonds. The amino acid composition of Fr. Ig was very similar to that reported in the literature for "dermatan sulfate-protein complex", and "structural glycoprotein" or "acidic structural protein".

Amino Acids↗
PubMed1981

Isolation of a 5S RNA-protein L5 complex from 60S subunits of rat liver ribosomes by cesium sulfate density-gradient equilibrium centrifugation.

Upon CS2SO4 density-gradient equilibrium centrifugation, 60S subunits of rat liver ribosomes were dissociated to form three bands at the densities of 1.55, 1.40, and 1.30 g/ml. The bands at 1.55 and 1.30 g/ml were shown to contain exclusively RNA or proteins, respectively, whereas the band at 1.40 g/ml contained both RNA and a protein. The RNA in the 1.40 g/ml band was identified as 5S RNA and the protein in this band was protein L5. An empirical calculation suggested that the 1.40 g/ml band contained an equimolar 5S RNA-protein L5 ribonucleoprotein complex. This finding may indicate that protein L5 is located in very close proximity to 5S RNA in large ribosomal subunits.

Animals↗
PubMedDate not supplied

Modified fabrication techniques lead to improved centrifugal blood pump performance.

The authors are developing an implantable centrifugal blood pump for short- and medium-term (1-6 months) left ventricular assist. They hypothesized that the application of result dependent modifications to this pump would lead to overall improved performance in long-term implantation studies. Essential requirements for pump operation, such as durability and resistance to clot formation, have been achieved through specialized fabrication techniques. The antithrombogenic character of the pump has been improved through coating at the cannula-housing interfaces and the baffle seal, and through changing the impeller blade material from polysulfone to pyrolytic carbon. The electronic components of the pump have been sealed for implantable use through specialized processes of dipping and potting, and the surfaces of the internal pump components have been treated to increase durability. The device has demonstrated efficacy in five chronic sheep implantation studies of 14, 10, 28, 35, and 154 day duration. Post mortem findings from the 14 day experiment showed stable fibrin entangled around the impeller shaft and blades. After pump modification, autopsy findings of the 10 day study showed no evidence of clot. Additionally, the results of the 28 day experiment showed only a small (2.0 mm) ring of fibrin at the shaft-seal interface. In the 35 and 154 day experiments, redesign of the stators have resulted in improved motor corrosion resistance. The 35 day study showed a small, 0.5 mm wide fibrin deposit at the lip seal, but no motor failure. In the 154 day experiment, the motor failed because of stator fluid corrosion, while the explanted pump was devoid of thrombus. Based on these findings, the authors believe that these pump refinements have contributed significantly to improvements in durability and resistance to clot formation.

Animals↗
PubMedDate not supplied

Improvement in antithrombogenicity in a centrifugal pump with self wash-out structure for long-term use.

Antithrombogenicity in the initial (NCVC-1) centrifugal pump (CP) developed at our institute is provided by a central balancing hole in an impeller that promotes self wash-out blood flow (Sf) around the impeller. The current CP (NCVC-2) was ameliorated to obtain better antithrombogenicity, where the balancing hole diameter was widened to improve Sf velocity (Vsf), and the edge of a thrust bearing (TB) was rounded to minimize flow separation. Effects of the modifications were assessed in in vitro and in vivo studies. The Vsf of the NCVC-1 and the NCVC-2 evaluated by Doppler velocimeter were 12.8 and 22.1 cm/sec, respectively. Flow around the TB visualized by a light cutting method confirmed less flow stagnation in the NCVC-2. In vivo antithrombogenicity of the CPs was investigated in three goats. A pulsatile VAD (P-VAD) was installed paracorporeally between the left atrium and the aorta. After 3 weeks, the P-VAD was exchanged for the NCVC-2 and the NCVC-1 in sequence. Each CP was driven for 1 week and disassembled. No anticoagulation was used, except for heparin injection at pump exchange. Thrombus at the TB was found in the two NCVC-1s, and two little thrombi were on the impeller of another NCVC-1, whereas a thrombus smaller than 1 mm3 at the TB was noted in one NCVC-2. These results indicate that the NCVC-2 has better antithrombogenicity and Sf around the impeller and the TB.

Animals↗
PubMedDate not supplied

Simulation study of a selective cerebral perfusion system with a single centrifugal pump.

We previously successfully developed a simple nonroller extracorporeal circulation system (NRECC). In aortic arch surgery, more than two pumps are generally used for systemic perfusion and selective cerebral perfusion (SCP); we developed a new pressure-dependent perfusion system for SCP based on our NRECC and operated by a single centrifugal pump. The cerebral perfusion line was branched from the main perfusion line, and one 15 French and two 12 French cannulae were used for SCP. The perfusion pressure was regulated with a tube occluder. Afterload was changed from 30 to 80 mm Hg, the pressure of the SCP line was increased from 80 to 200 mm Hg, and flow volume was measured. When the afterload was set at 50 mm Hg, according to the increase of perfusion from 80 to 200 mm Hg, the flow volume of the 15 French cannula increased from 280 to 950 ml/min. Under the same conditions, flow volume of the 12 French cannula increased from 160 to 560 ml/min. Sufficient flow volume of the SCP lines was obtained when the SCP line pressure was over 80 mm Hg. As a result of the increased perfusion pressure, the flow volume showed a direct increase. These findings suggest that aortic arch surgery is possible using this SCP system.

Aorta, Thoracic↗
PubMedDate not supplied

A pressure controller of selective cerebral perfusion with single centrifugal pump.

We are developing an original nonroller extracorporeal circulation system (NRECC). However, this NRECC could not perform selective cerebral perfusion (SCP). Therefore, we added cerebral perfusion lines and an automatic pressure controller to the system. The purpose of this study was to evaluate the stability and response of the pressure controller for the SCP in simulated clinical circulation. The NRECC consists of a centrifugal pump, four isolated vacuum suction lines and a conventional module. The SCP line branches from the main perfusion tubing and divides into three lines. Cerebral perfusion is regulated by a pressure controller, which is composed of an actuator, a pressure transducer, and a personal computer. The mock circuit was primed with normal saline, and the actual SCP pressure and flow were measured when the target pressure settled at 60, 80, and 100 mm Hg. The main perfusion flow was maintained at 4 L/min and the main perfusion pressure was altered from 120 to 300 mm Hg. The pressure and flow data were recorded. The SCP pressure was maintained within +/-1.47 mm Hg when the set pressure was 80 mm Hg. Fluctuation of flow in the SCP line was within the range of +/-2.8%. The time needed to reach the steady state pressure was 8+/-1 seconds when the initial setting of the roller occluder was full-open, and it took only 3+/-1 seconds to reach the next initial set pressure. We have developed the NRECC-SCP system. The SCP pressure is stable and quickly reaches steady state via the pressure controller. This system is useful for extracorporeal circulation during aortic arch operation.

Centrifugation↗
PubMed1981

Adaptation of EMIT procedures for maximum cost effectiveness to two different centrifugal analyzer systems.

We have adapted commercial immunoassay (EMIT) procedures for dilantin, phenobarbital, theophylline, carbamazepine, tobramycin, disopyramide, quinidine, procainamide, N-acetyl procainamide, and mysoline to two different centrifugal analyzers (Centrifichem and Multistat) using maximum dilutions of reagents to reduce reagent costs to approximately one-tenth of that incurred using the manufacturer's protocol. This savings due to the dilutions of reagents was primarily because of the ability to prolong the reaction time to achieve acceptably large absorbance changes over the absorbance measurement period. Correlation coefficients between values for drugs analyzed by these modified EMIT protocols and values obtained by other methods ranged from 0.927 to 0.994. Within-run precision and recovery values for these drugs were also entirely acceptable. We found that premature mixing of reagents in the rotors of the Multistat system and variable delivery of reagent in the Centrifichem 400 system were both caused by the surfactant in the EMIT buffer. The former problem is being resolved by the manufacturer; the latter problem was resolved by siliconizing the reagent pipet tips.

Centrifugation↗
PubMed1982

An improved theophylline method by EMIT on the multistat III micro centrifugal analyzer.

We describe several modifications of the Syva EMIT assay for theophylline on the Instrumentation Laboratories Multistat III Micro Centrifugal Analyzer (MCA) designed to improve precision. These modifications include increased sample volume, increased time interval for obtaining the delta optical density, the use of buffer with no detergent to prevent the possibility of premix of reagents, and reversal of the order for loading the reagents. Furthermore, the loading of sample and reagents has been programmed to match the concentrations and sample volume/reagent volume ratios recommended by Syva. Precision analysis of the method yielded coefficient of variation values of less than 10% for controls (n = 52) and standards (n = 69). A cross-correlation study comparing the MCA method and the recommended method on the Gilford 3500 yielded a slope of 0.98, a y intercept of 0.07, and a correlation coefficient of 0.992. The method is precise, fast, and inexpensive, costing ¿63 per patient sample for a full rotor.

Centrifugation↗
PubMed1985

Evaluation of the Du Pont Theophylline Assay adapted to a centrifugal analyzer.

We evaluated the Du Pont Theophylline Assay, a particle-enhanced turbidimetric inhibition immunoassay (PETINIA) for the measurement of theophylline in human serum. The procedure was applied to the Cobas-Bio centrifugal analyzer, and was compared to an enzyme immunoassay (EMIT) method and a high performance liquid chromatographic (HPLC) procedure. Day-to-day precision was 3.7% (1 SD coefficient of variation) at 7.1 mg/L, 3.3% at 17.7 mg/L, and 3.9% at 27.8 mg/L. The assay was linear up to 40 mg/L, and the correlation between the PETINIA, EMIT, and HPLC methods was good [PETINIA/EMIT: y = 0.94x + 0.63, r = 0.98, Syx = 1.13; PETINIA/HPLC: y = 1.00x - 0.89, r = 0.99, Syx = 0.66, where Syx is the standard deviation of the residual error of regression], when evaluated using specimens from 135 patients receiving theophylline. No interference from hemolysis, lipemia, icterus, or other methylxanthines was observed. Of the major metabolites of theophylline, only 1,3-dimethyluric acid showed any significant cross-reactivity (2.1 mg/L apparent theophylline at 20 mg/L 1,3-dimethyluric acid). The method is reliable and cost-effective for the measurement of theophylline in serum.

Centrifugation↗
PubMed1987

Homogeneous enzyme immunoassay (EMIT) protocol for monitoring tricyclic antidepressants on the COBAS-BIO centrifugal analyzer.

EMIT tests are available for quantitative determination of the tricyclic antidepressants amitriptyline (AMI), nortriptyline (NORT), imipramine (IMI), and desipramine (DMI). An extraction step before analysis eliminates cross-reacting polar metabolites. Excellent correlation between EMIT and high-performance liquid chromatography (HPLC) has been previously established. Most published protocols for using EMIT reagents on COBAS-BIO centrifugal analyzers were designed for analytes present at 1-50 mg/L in serum. AMI, NORT, IMI, and DMI are usually present at far less than 1 mg/L. We describe a COBAS-BIO EMIT protocol for assaying these analytes. Patient sample correlation between COBAS-BIO EMIT and EMIT-AutoLab were greater than 0.99. Between-run precision for single-point determinations was comparable to SYVA AutoLab performance [less than or equal to 11% at 40 micrograms/L AMI, NORT, IMI (80 micrograms/L DMI), and less than or equal to 4% at 200 micrograms/L AMI, NORT, IMI (400 micrograms/L DMI)]. With stored-curve updating, working reagents were usable for at least 14 days (AMI) or 23 days (DMI).

Antidepressive Agents, Tricyclic↗
PubMed1986

Development of a simplified counterflow centrifugation elutriation procedure for depletion of lymphocytes from human bone marrow.

Graft-versus-host disease (GVHD) remains a major complication of allogeneic bone marrow (BM) transplantation. Techniques that effectively purge BM of mature T lymphocytes should reduce the incidence of GVHD and improve survival. We have developed a simplified, two-flow rate, fixed rotor speed counterflow centrifugation-elutriation (CCE) procedure that reproducibly depletes 99% of lymphocytes from Ficoll-Hypaque(F/H)-separated BM or BM buffy-coat. Two predetermined flow rates (24 and 28 ml/min) were used to purge small and intermediate-to-large lymphocytes, respectively, whereas faster sedimenting cells were recovered at the termination of the run. Lymphocyte depletion was substantiated by pan-T monoclonal antibody analysis as well as by complete loss of responsiveness to alloantigens and mitogens. Despite the lack of mature T cells, the depleted marrow fraction retained lymphoid colony-forming ability. Lymphocyte-purged marrow was obtained in high yield (72%), and retained high viability (greater than 97%) and hematopoietic colony-forming ability (greater than 99%). The ratio of total myeloid/erythroid colony-forming cells to T lymphocytes was 73-fold higher in the lymphocyte-depleted fraction than in unseparated BM. We concluded that a two-step CCE procedure can be used to rapidly deplete lymphocytes from both F/H-separated and buffy-coat BM inocula without altering hematopoietic capacity as measured by the in vitro clonogenic assays. It may be possible to adapt this procedure to the separation of the large number of marrow cells required for human BM transplantation.

Antibodies, Monoclonal↗
PubMedDate not supplied

Evaluation of floating impeller phenomena in a gyro centrifugal pump.

The Gyro centrifugal pump, developed as a totally implantable artificial heart, was designed with a free impeller in which the rotational shaft (male bearing) of the impeller was completely separated from the female bearing. For this type of pump, it is very important to keep the proper magnet balance (impeller-magnet and actuator-magnet balance) to prevent thrombus formation or bearing wear. When the magnet balance is not proper, the impeller is jerked down into the bottom bearing. On the other hand, if magnet balance is proper, the impeller is lifted off the bottom of the pump housing within a certain range of pumping conditions. In this study, this floating phenomenon was investigated in detail. The floating phenomenon was proven by observation of the impeller behavior by means of a transparent acrylic pump. The impeller floating phenomenon was mapped on a pump performance curve. The impeller floating phenomenon is affected by the magnet-magnet coupling distance and the rotational speed of the impeller. To keep the proper magnet balance and to maintain the impeller floating phenomenon at the driving conditions of right and left pumps, the magnet-magnet coupling distance was altered by a spacer that was installed between the pump and actuator. It became clear that the same pump could handle different conditions (right and left ventricular assist) by changing the thickness of the spacer. When magnet balance is proper, the floating impeller phenomenon occurs automatically in response to the impeller revolution. This is called "the dynamic revolutions per minute suspension."

Centrifugation↗