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PubMed1984

Pseudoreplica electron microscopy for the detection of rotavirus: comparison with high-speed centrifugation electron microscopy and ELISA.

Three-hundred and sixty-three stool specimens from patients with diarrhoea were examined for rotaviruses to compare the sensitivity of the pseudoreplica technique (PSD-EM) to that of high-speed centrifugation EM (HSC-EM) in relation to a commercially available (Rotazyme, Abbott) enzyme-linked immunosorbent assay (ELISA). In ELISA-positive cases, both methods were of equal sensitivity. However, in borderline (+/-) and ELISA-negative specimens, PSD-EM detected 31 of 48 (64.6%) and 18 of 229 (8%) positive specimens respectively, compared to only 22 of 48 (45.8%) and one of 229 (0.4%) positives detected by HSC-EM. PSD-EM detected a significantly higher number of positives compared to HSC-EM (p less than 0.05). In view of its simplicity, sensitivity and the fact that a relatively large number of specimens could be processed compared to HSC-EM, we consider that PSD-EM is a much better procedure for routine screening and diagnosis of viral gastroenteritis than HSC-EM.

Centrifugation↗
PubMed1985

Purification of biologically active Epstein-Barr virus by affinity chromatography and non-ionic density gradient centrifugation.

Epstein-Barr virus was purified by affinity chromatography on ricin agglutinin Sepharose followed by non-ionic density gradient centrifugation on Nycodenz. The purified virus was highly active in three biological assays: stimulation of immunoglobulin synthesis by B lymphocytes, transformation of B lymphocytes, and superinfection of Raji cells, as well as in an indirect immunofluorescent binding assay. Electron microscopy revealed intact viral particles free of contaminating membranous structures. CsCl density gradient analysis of nick-translated DNA showed material only at the expected viral density with no detectable material at the density of cellular DNA. SDS gel electrophoresis of radioiodinated virus revealed the characteristic viral polypeptides. This method produces highly purified, biologically active virions with an overall recovery of about 30%.

Agglutination Tests↗
PubMed1987

Detection of cytomegalovirus by 24-well plate centrifugation assay using a monoclonal antibody to an early nuclear antigen and by conventional cell culture.

During a 12-month period, two methods for detection of cytomegalovirus (CMV) in 1624 clinical specimens were compared: (1) centrifugal inoculation of MRC-5 cells on coverslips in 24-well plates and staining with a monoclonal antibody to CMV early nuclear antigen after incubation for 40 h (EA assay), and (2) conventional tube cell culture. CMV was identified in 183 (11.3%) specimens from 113 different patients. The EA assay was positive for CMV in 144/183 specimens (79%), and CMV was detected by recognition of specific cytopathic effect (CPE) in conventional cell culture in 143/183 (78%). Both methods yielded CMV in 56% of the specimens (104/183). CMV was detected by EA assay alone in 22% (40/183) and only by CPE in 21% (39/183) of the positive specimens. When all specimen types were considered, there was no significant difference in the detection of CMV between the two methods. However, bronchoalveolar lavage (BAL) fluids yielded CMV more frequently by EA assay than by CPE (58 compared to 48 of 574, p = 0.0178), and CMV was detected in blood specimens more often by CPE than by EA assay (20 compared to one of 149, p less than 0.0001). In addition to CMV, other viruses were recovered by conventional tube cell culture, including herpes simplex virus (HSV) type 1 from 17 BAL fluids (two of which were positive for CMV by EA assay) and one liver biopsy and adenovirus serotype 4 from four separate urine specimens and three gastrointestinal tract biopsies from one patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1981

A rapid method for the isolation of intact glycosomes from Trypanosoma brucei by Percoll -gradient centrifugation in a vertical rotor.

A rapid method for the isolation of intact glycosomes from trypanosoma brucei is described. A post large-granule extract is subjected to density-gradient centrifugation on isotonic Percoll in a vertical rotor. This results in a good separation of the glycosomes, which equilibrated at 1.09 g/cm3, from other cellular components. Glycosomes isolated from such a gradient are 9-fold purified relative to the starting homogenate with a yield of 25%. Their glycolytic enzymes exhibit a high latency indicative of an intact glycosomal membrane.

Animals↗
PubMed1985

Cell-cycle differences of HL-60 leukemia cells fractionated by centrifugal elutriation.

HL-60 leukemia cells were fractionated into G1 and S/G2 populations using a rapid centrifugal elutriation technique, and studied for differences between the cell-cycle phases. The G1 fraction was found to contain smaller cells with a sedimentation velocity of 7 mm/h. The S/G2 fraction consisted of larger cells with a sedimentation velocity of 125 mm/h. The latter fraction was found to have a peak level of the enzyme (2'-5')An-binding protein, as compared to the G1 fraction, indicating a possible role for (2'-5')An-binding protein and its products in the growth regulation of these leukemic cells. In addition, cytofluorometric analysis of fractionated HL-60 cells indicates that elutriation is an effective fractionation method, rapidly yielding large numbers of cells for study, without the use of chemical treatments.

Carrier Proteins↗
PubMed1996

The slow rotating centrifuge microscope NIZEMI--a versatile instrument for terrestrial hypergravity and space microgravity research in biology and materials science.

NIZEMI (slow rotating centrifuge microscope) is a tool for optical investigations of small biological and non-biological specimens during variable accelerations. Two laboratory models for ground research designed for accelerations from 1 to 5 x g and 10 x g respectively are used for terrestrial research, especially in gravitational biology. A space facility was developed and built for the Spacelab mission IML-2 during which eight experiments were performed successfully. The specifications and topic design features of the NIZEMI models are presented in this paper.

Biological Science Disciplines↗
PubMed1987

Computer-assisted centrifugal elutriation. I. Detection system and data acquisition equipment.

To optimize cell separations by centrifugal elutriation, we constructed an on-line computer-controlled multiparametric light-scatter system. A bypass sample flow, at the outlet of an elutriation rotor, is hydrofocussed and three scatter parameters of each cell are determined up to a maximum of 15,000 cells/second. The 18-bit representation of the parameter values are cumulatively stored by means of a direct memory access interface. The histogram memory is continuously displayed to provide information on the number and type of cells that are elutriated. A special purpose operating system, implemented on a stand-alone computer configuration, allows a high data-acquisition rate and ample data processing capacity. In addition, a local network driver was constructed to facilitate off-line detailed analysis of the data. The equipment is well suited to monitor the centrigural elutriation process. The flexibility of the system allows an extension of the monitor to computer-controlled elutriation.

Animals↗
PubMed1986

The use of the Reuter centrifugal air sampler for the estimation of bacterial air counts in different hospital locations.

The convenience and portability of the Reuter centrifugal air sampler (RCS) encouraged us to determine a range of 'expected values' for bacterial air counts in nine hospitals at 13 defined locations. Results were recorded as the number of cfu per strip per 4 min. Bacterial air counts were comparable from hospital to hospital; in operating theatres the overall median RCS counts for air-inlets and in empty operating rooms were 13 and 9.8 cfu per strip per 4 min respectively. For surgical wards the median value was 141 and 180 cfu per strip per 4 min with, and without, air conditioning. Increased counts were readily demonstrated during surgical operations, and during bedmaking in wards. Conversion of RCS counts to cfu m-3 of air usually yielded values higher than those established by other methods. Our findings, however, demonstrate that this instrument may replace some of the applications of the slit sampler and facilitate examination of epidemiological problems.

Air Microbiology↗
PubMed1994

Acquisition of viable-like surface properties of sperm cells by adsorption of seminal plasma proteins revealed by centrifugal countercurrent distribution.

By using centrifugal countercurrent distribution in dextran/polyethylene glycol two-phase system, we show that the acquisition of adsorbed seminal plasma proteins by the sperm cell surface modifies the partition behaviour of bull spermatozoa reproducing that of alive cells. Thus, addition of increasing concentrations of proteins from the seminal plasma promoted a saturable enhanced affinity of the cells to the polyethylene glycol-rich phase in which alive cells preferentially partition. On the contrary, treatment with Tween provoked the opposite effect.

Adsorption↗
PubMed1989

Human diploid cell rabies vaccine purified by zonal centrifugation: a controlled study of antibody response and side effects following primary and booster pre-exposure immunizations.

Systemic allergic reactions following booster immunizations have complicated rabies pre-exposure prophylaxis with the human diploid cell rabies vaccine licensed in the US (conventional HDCV). We conducted two studies comparing an HDCV purified by zonal centrifugation to conventional HDCV. In a study of primary pre-exposure immunization, volunteers received one of four regimens: three 1.0-ml intramuscular (i.m.) or 0.1-ml intradermal (i.d.) doses of conventional or purified HDCV over 28 days. Although volunteers vaccinated i.m. had significantly greater rabies neutralizing antibody titres (VNA) 49 days, 91 days and 26 months after immunization began than volunteers vaccinated i.d. (p less than 0.005-p less than 0.05), there were no significant differences between vaccines. In a study of booster immunizations, 77 volunteers immunized with conventional HDCV 2 years earlier received a 0.1-ml i.d. booster with either conventional or purified HDCV. VNA was significantly greater with the conventional HDCV on days 7 and 28 after booster, but not on day 365. A moderate or severe reaction was reported by 5 (13%) of the 40 persons who received boosters with conventional HDCV, versus none of 37 who received the purified HDCV (p = 0.03). Purified HDCV appears to be preferable to conventional HDCV for booster vaccination.

Antibodies, Viral↗
PubMed1989

Influence of gravitoinertial force on vestibular nystagmus in man observed in a centrifuge.

The influence of gravity load on the vestibular system in man was investigated in a centrifuge operating on the free swing principle. The vertical vestibular nystagmus induced by acceleration to 3G was analyzed and compared with reference measurements during 1G. Our data indicate that the effects of increased gravity load include a prolonged decay time constant of upbeat nystagmus and a subject-dependent persisting upbeat nystagmus. In an attempt to explain these findings, an extension of the velocity storage model is proposed, with gravity as a second stimulus function in addition to angular acceleration.

Acceleration↗
PubMed1992

Swimming behavior of Paramecium--first results with the low-speed centrifuge microscope (NIZEMI).

The low-speed centrifuge microscope NIZEMI (= Nieder-Geschwindigkeits-Zentrifugen-Mikroskop) is an excellent tool with which to investigate the effects of slightly increased gravity in the fields of biology and material sciences. We investigated the swimming behavior of Paramecium in the NIZEMI, by aid of a computer-controlled image analysis system. In the range of acceleration (1 g to 5 g), cells retained their swimming capability, did not sediment, and even increased the precision of their negative gravitaxis but reduced their mean swimming velocity.

Animals↗
PubMed1976

Separation of tumor cells by density gradient centrifugation: recent work with human tumors and a discussion of the kind of quantitation needed in cell separation experiments.

The application of centrifugation to the purification of individual kinds of cells from tumors has been reviewed. Recent applications of a previously described isokinetic gradient to the purification of subpopulations of cells from human tumors are presented. Individual kinds of cells have been separated from Hodgkin's disease by a method described previously, and the cells from Hodgkin's disease which contain the tumor associated antigen described by Order and his colleagues have been identified for the first time. The importance of the thorough documentation of cell separation procedures is discussed, and we have specifically listed several kinds of data which we believe are important in the documentation of cell separation techniques.

Adenocarcinoma↗
PubMed1976

Simultation of gradient and band propagation in the centrifuge.

A technique is developed for simulating the behavior of both the gradient-forming solute and macromolecular bands in a centrifuge. The change with time of the density gradient due to diffusion and sedimentation of the gradient-forming solute is calculated by a finite difference method, making use of the results of earlier work on the theory of the equilibrium density gradient. Using a perturbation technique, the concentration profiles of dilute bands of macromolecules are then calculated as they sediment and diffuse through the varying supporting gradient. Results of the stimulaion techniques are compared with experiment.

Centrifugation, Density Gradient↗
PubMed1980

Isolation of rat somatotroph and mammotroph secretory granules by equilibrium density centrifugation in a linear metrizamide gradient.

A procedure has been developed for separating rat somatotroph and mammotroph secretory granules from other membranous organelles. The approach devised is a 6-step procedure involving filtration and equilibrium and rate density-gradient centrifugation in Metrizamide gradients of low ionic strength. The use of Metrizamide minimizes disruption and solubilization of the contents of these granules. The majority of the somatotroph and mammotroph granules can be isolated in a gradient fraction virtually free of any mitochondria, microsomes or other secretory granules.

Animals↗
PubMed1985

Iodine content and density of thyroglobulin investigated by isopycnic centrifugation.

The density of in vitro iodinated thyroglobulin (Tgb) molecules, measured by isopycnic centrifugation, increases linearly with their iodine content. In contrast, the iodine to density (I:D) regression for differently iodinated native rat Tgbs is not constant but varies, depending on experimental conditions: For LID-fed rats the slope of the individual I:D regression line of each Tgb decreases linearly with the decreasing mean degree of iodination of the sample (r = 0.9104, P less than 0.01). On the other hand, in Tgb samples synthesized by propylthiouracil-blocked glands, the I:D regression is no longer linear. Rather, a bimodal pattern is obtained revealing a preexisting highly iodinated Tgb pool together with newly synthesized, non-iodinated protein. The results indicate: (a) the mean degree of iodination of a native Tgb cannot be estimated accurately if only its mean density is known, and (b) the degree of heterogeneity of iodination is identical for any in vivo produced Tgb sample with the same iodine content.

Animals↗
PubMed1991

Effects of activin A on anterior pituitary cells fractionated by centrifugal elutriation.

We have shown previously that activin A increases the number of immunoreactive follicle-stimulating hormone (FSH) cells. To further investigate the action of activin A, we examined its effects on anterior pituitary cells fractionated by centrifugal elutriation. Before activin A treatment, FSH cells were widely distributed among various fractions; a higher proportion of FSH cells was found in larger cell fractions (fractions 5-9), and a lower proportion in smaller cell fractions (fractions 2-4). After culture of the cells in each fraction with activin A (10 ng/ml) for 72 h, the number of FSH cells in fraction 4 only was significantly (P less than 0.05) higher by 225% than that in cells cultured without activin A. The amount of FSH secreted into the medium was minimal or undetectable in fractions 1-4. However, FSH secretion tended to be, or was significantly (P less than 0.01 in fraction 9), stimulated by activin A in fractions 5-9, in which the numbers of FSH cells were not significantly affected. These results suggest a dual mode of action of activin A on FSH: activin A increases the number of FSH cells in a specific type(s) of middle-sized cell fraction, and stimulates FSH secretion at least from larger cells without affecting the number of FSH cells.

Activins↗