Relative Centrifugal Force Research Articles

Early adhesion of Blumeria graminis to plant and artificial surfaces demonstrated by centrifugation

The relative strength of adhesion by Blumeria graminis conidia and germlings to barley leaves and to clean (hydrophilic) or silanized (hydrophobic) glass, was assessed using centrifugation. Ungerminated conidia were incubated for 10 or 30 min before centrifugation, while extended incubation, for up to 12 h, allowed germlings to reach different developmental stages before treatment. After incubation, mounts were subjected to relative centrifugal force (RCF) ranging from 0.13 × 10 3 to 26.00 × 10 3. Two key features indicating adhesion strength were the minimum RCF required for displacement of any fungal units, and the percentage of these displaced by the maximum RCF. By these criteria, adhesion was far stronger after germination, presumably because germ tubes adhered to substrata. Ultimately, germlings adhered more strongly to leaves, where appressoria differentiated, than to clean glass where only multiple short germ tubes were formed. Ungerminated conidia adhered relatively weakly to host leaves and most strongly to silanized glass. Increasing incubation time did not increase their adhesion to leaves but conidia incubated for 30 min adhered more strongly to clean and silanized glass than those incubated for only 10 min. Thus, in general, the strength of adhesion by ungerminated conidia correlated to known differences in the speed of conidial extracellular material (ECM) release and to the quantity of ECM released on the different substrata. However, even on leaves where ungerminated conidia showed the weakest adhesion, about 80% of conidia were not displaced by RCF of 0.13 × 10 3. Under these circumstances conidia had been subject to about 4.2 × 10 −9 N force acting to displace them. This is far greater than the forces likely to be generated by wind acting on conidia deposited on the leaves of a barley crop. Thus, adhesion by ungerminated conidia is sufficient to hold the majority of spores in place even under very windy field conditions.

Basic Residues within the Kruppel Zinc Finger DNA Binding Domains Are the Critical Nuclear Localization Determinants of EKLF/KLF-1

EKLF/KLF-1 is an erythroid-restricted transcription factor essential for expression of the adult beta-globin gene. EKLF/KLF-1 is a 358-amino acid nuclear protein with an amino-terminal proline-rich domain and a carboxyl-terminal DNA binding domain. The nuclear localization signal (NLS) of EKLF/KLF-1 has not been empirically determined. We generated a series of epitope-tagged deletion and point mutants and assessed their subcellular localization. Our results delimit the NLS to the 83-amino acid (amino acids 276-358) DNA binding domain that consists of three Kruppel zinc fingers. All three zinc fingers are necessary for efficient nuclear localization; deletion of any individual finger results in cytoplasmic accumulation. Fusion of the three zinc fingers to green fluorescent protein (GFP) targeted GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for nuclear localization. EKLF/KLF-1 containing histidine to alanine mutations that disrupt the structure of all three fingers retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization. We demonstrate that basic residues within the fingers are the critical determinants for nuclear localization; mutations of these basic residues to alanine resulted in cytoplasmic mislocalization. The basic residues of all mammalian Kruppel zinc fingers are highly conserved; therefore we propose that these basic residues are a common NLS shared by all Kruppel family members.

Plantar Pressures during Ski Jumping Take-Off

The Paromed Datalogger® with two insole pressure transducers (16 sensors each, 200 Hz) was applied to study the feasibility of the system for measurement of plantar pressure distribution in ski jumping. The specific aim was to test the sensitivity of the Paromed system to the changes in plantar pressure distribution in ski jumping. Three international level ski jumpers served as subjects during the testing of the system. The Datalogger was fixed to the jumpers’ lower back under the jumping suit. A separate pulse was transmitted to the Datalogger and tape recorder in order to synchronize the logger information with photocell signals indicating the location of the jumper on the inrun. Test procedure showed that this system could be used in ski jumping with only minor disturbance to the jumper. The measured relative pressure increase during the inrun curve matched well the calculated relative centrifugal force (mv2 · r‒1), which thus serves a rough estimation of the system validity. Strong increase in pressure under the big toes compared to the heels (225% and 91%, respectively) with large interindividual differences characterized the take-off. These differences may reflect an unstable anteroposterior balance of a jumper while he tries to create a proper forward rotation for a good flight position.

Centrifuges and Rotors

AbstractThis appendix lists rmax values (maximum value of the rotating radius between the particle being centrifuged and the axis of rotation) for commonly used rotors manufactured by Sorvall, Beckman Coulter, Fisher, and IEC. In addition, nomograms are provided for the determination of the relative centrifugal force (RCF) where speed and rmax are known, or the speed where RCF and rmax are known. This is done by aligning a ruler across the two known values and reading the unknown value at the point where the ruler crosses the remaining column. Also discussed are the characteristics of the various materials used to construct centrifuge tubes, including optical properties, appropriate methods for sterilization, and chemical resistances (tolerance to various media, organic solvents, and alcohols).

Effects of sample pretreatment and sequential fractionation by centrifuge drainage on concentrations of minerals in a calcareous soil solution

Soil solution is the direct source of mineral nutrients to vascular plants. Soil solutions of sieved samples at water-holding capacity of a seminatural calcareous soil, never cultivated or treated by fertilizers, were collected by sequential centrifugation at 24–18,900× g relative centrifugal force (RCF), followed by ultrafiltration (0.2 μm), and analysed for Al, Ca, Fe, K, Mg, Mn, Mo, Na, P, S, Si and pH. Soil solution held by weak forces (low RCF applied) had higher concentrations of base cations, Fe, and Si than soil solutions obtained at higher RCF. Opposite relationships were measured for P-TOT, molybdate-reactive P and Mo, whereas concentrations of Mn and S did not vary much according to RCF level. A comparison between centrifugation of soil cores at undisturbed stratification and mixed samples uncovered no great differences in soil solution pH or concentrations of major constituents, though concentrations of Ca, Si and S were 10–20% lower in solution from undisturbed cores. Possible mechanisms accounting for the observations are discussed.

Factors affecting yield and survival of cells when suspensions are subjected to centrifugation. Influence of centrifugal acceleration, time of centrifugation, and length of the suspension column in quasi-homogeneous centrifugal fields.

The goals of the centrifugation of cell suspensions are to obtain the maximum yield of cells with minimum adverse effects of centrifugation. In the case of mechanically sensitive cells such as mouse sperm, the two goals are somewhat contradictory in that g-forces sufficient to achieve high yields are damaging, and g-forces that yield high viability produce low yields. This paper mathematically analyzes the factors contributing to each goal. The total yield of pelleted cells is determined by the sedimentation rate governed by Stokes' Law, and depends on the relative centrifugal force, centrifugation time, size and shape of the cells, density of the cells and medium, viscosity of the medium, and the length of the column of suspension. Because in the situation analyzed the column is short relative to the rotor radius, the analysis considers the centrifugal field to be quasi-homogeneous. The assumption is that cells are not damaged during sedimentation, but that they become injured at an exponential rate once they are pelleted, a rate that will depend on the specific cell type. The behavior is modeled by the solution of coupled differential equations. The predictions of the analysis are in good agreement with experimental data on the centrifugation of mouse sperm.

The optimum relative centrifugal force and centrifugation time for improved sensitivity of smear and culture for detection of Mycobacterium tuberculosis from sputum

Direct microscopy is the only available method for diagnosis of tuberculosis in most centres in developing countries. Methods to improve the sensitivity of direct smear are an urgent requirement. Sputum specimens artificially seeded with known concentrations of Mycobacterium tuberculosis were liquefied and decontaminated with sodium hydroxide-sodium citrate- N-acetyl- l-cysteine solutions. They were subjected to different centrifugation forces and centrifugation times after which the centrifuged deposits were examined by smear and culture. Statistical analysis of results was carried out using EpiInfo version 6.0. The optimum relative centrifugal force (RCF) and centrifugation time combination was 4000 g for 15 min. The sensitivity of detection at an RCF of 4000 g for 15 min was 5000 organisms/mL and 500 organisms/mL for smear and culture, respectively. When results of 163 clinical samples were analysed after centrifugation at 4000 g for 15 min sensitivity of the direct smear improved from 63% to 92% ( P < 0·05) and negative predictive value from 30·5% to 45% ( P < 0·05) when culture was considered the ‘gold standard’. With the concentrated smear there was a reduction in specificity from 82% to 60% ( P > 0·05). As most laboratories are equipped with a simple centrifuge, smear sensitivity can be improved with this simple modification. The other advantage is that the same centrifuged deposit can be cultured, in contrast to when sodium hypochlorite is used for liquefaction.

Fluorometric Determination of Acid Phosphatase in Cooked, Boneless, Nonbreaded Broiler Breast and Thigh Meat

This method is applicable for determining activity of acid phosphatase (ACP), a heat-labile enzyme, in cooked, boneless, nonbreaded broiler marinated (83.65% meat) and nonmarinated (100% meat) breast and thigh and in a 50:50 blend of breast and thigh meat. The assay uses a self-indicating substrate that, when acted upon by ACP, loses a phosphate radical and becomes a highly fluorescent compound. Cooked meat is added to deionized distilled water in a 1:3 ratio, blended with a hand-held homogenizer, and then centrifuged at 2500 relative centrifugal force for 5 min. ACP activity in the filtrate is measured after shaking on a Vortex mixer 75 microL of the extract with a pH 5.00 acetate buffer containing a nonfluorescent aromatic monophosphoric ester substrate. The rate of fluorophore formation is monitored during a 3 min incubation period (38 degrees C) in a fluorometer, and ACP enzyme activity (mU/kg sample) is calculated. Three laboratories analyzed 6 cooked poultry products (marinated and nonmarinated breast, thigh, and 50:50 breast/thigh blend). Five cooking temperatures were used to generate different ACP activity levels, which were replicated twice with duplicate samples and duplicate sample tests representing 720 data points. Log10 ACP activity (mU/kg sample) performance repeatability and reproducibility standard deviations (sr and sR) and relative standard deviations (RSDr and RSDR) over 5 cooking treatments for 6 products were as follows: marinated breast: sr = 0.02, sR = 0.08, RSDr = 0.60%, RSDR = 2.12%; nonmarinated breast: sr = 0.02, sR = 0.04, RSDr = 0.66%, RSDR = 1.29%; marinated thigh: sr = 0.01, = 0.01, RSDr = 0.37%, RSDR = 0.37%; nonmarinated thigh: sr = 0.02, sR = 0.05, RSDr = 0.53%, RSDR = 1.43%; marinated 50:50 breast/thigh blend: sr = 0.01, sR = 0.05, RSDr = 0.36%, RSDR = 1.31%; nonmarinated 50:50 breast/thigh blend: sr = 0.01, sR = 0.04, RSDr = 0.32%, RSDR = 1.12%.

Influence of centrifugation regimes on motility, yield, and cell associations of mouse spermatozoa.

Mouse sperm are exceptionally sensitive to mechanical forces associated with pipetting and mixing. This characteristic raised the question of the sensitivity of mouse sperm to centrifugation, a step necessary in the removal of cryoprotectants and a common component in the general manipulation of sperm suspensions for experimental purpose. Epididymal spermatozoa from ICR mice were isolated and manipulated to minimize pipetting and mixing damage. The centrifugal accelerations studied were 200, 400, 600, and 800 x g (measured with a stroboscope) for 5, 10, or 15 minutes of centrifugation time. The number of cells and the number of motile cells were counted. The percent motility and longevity, total yield, and motile yield were calculated. Centrifugation at 200 and 400 x g for short times (5 minutes) caused only a small loss in either immediate or 2.5-hour motility, but centrifugation at 600 and 800 x g for 15 minutes produced up to a fivefold loss. Low speed/short time centrifugation pelleted only about half of the cells; the others were lost when the supernatant was removed. The maximum number of motile sperm (motile yield) was obtained at intermediate centrifugal forces (approximately 400 x g for 10-12 minutes), and it is the total number of motile sperm (and not the percent motility) that is important in the use of cryopreserved sperm to regenerate cryopreserved mutant lines. Relative centrifugal force and centrifugation time exhibit reciprocity (e.g., 200 x g for 10 minutes produces similar results to 400 x g for 5 minutes). The spermatozoa must be centrifuged under carefully defined conditions to minimize the damage and to maximize the recovery of viable cells.

Rate Zonal Sedimentation of Proteins in One Hour or Less

Rate zonal sedimentation gives information about the shape and size of proteins, and is useful for investigating protein–protein interactions. However, rate zonal sedimentation experiments typically last approximately 1 day. In contrast, this report describes a rate zonal sedimentation method requiring 1 h or less. This was accomplished by centrifuging small density gradients (200 μl) prepared with sucrose or OptiPrep in a fixed-angle rotor at high relative centrifugal force. By using small gradient volumes, the sample dilution that occurs with larger gradients and with many chromatographic techniques was also avoided. For a variety of proteins, plots ofS20,wversus distance sedimented during centrifugation in a TLA 120.2 rotor were linear. As a practical application, sedimentation of the heterotrimeric stimulatory G protein and its dissociated α-subunit were determined. The results were similar to those obtained with 17- to 22-h centrifugations in an SW 50.1 rotor and agreed with previously published values. Long periods of centrifugation might preclude the study of some unstable proteins or the investigation of protein–protein interactions whose affinities are to low to survive the lengthy centrifugations required to carry out traditional rate zonal sedimentation experiments. A rate zonal sedimentation technique that rivals many chromatographic methods in celerity will help to circumvent these problems.

Rhizosphere soil-water collection by immiscible displacement-centrifugation technique

Progress in determining nutrient availability in the rhizosphere is restricted by a lack of reliable and convenient methods for rhizosphere soil-water collection. A modified centrifugation method with a fluorocarbon (Fluorinert™,FC-70) as an immiscible displacement liquid was developed. Our objectives were to: i) obtain an adequate soil-water volume from a small rhizosphere sample within a reasonable time; ii) collect rhizosphere soil-water at container capacity (≈ 90% of field capacity) to determine soluble soil ions; and iii) evaluate FC-70 as an extractant. The soil used was a Beadle clay loam (fine, montmorillonitic mesic Typic Argiustoll) with low and high levels of CaCO3 (5 and 204g kg-1). Soil samples from the rhizosphere of 30-days-old sordan (sorghum (Sorghum bicolor L.), sudangrass (Sorghum sudanese L.) hybrid seedlings were thin-sectioned at 1-, 2- and 3-mm from the root surface. The extraction parameters (sample size, volume of extractant, relative centrifugal force and centrifugation time) were varied to determine optimal values. We obtained adequate amounts of aqueous solutions from moist soil (≈ 6 g) when mixed with 2 mL of FC-70, packed into a filter unit, and centrifuged for 1 hour at 14,500 × g. The displaced soil-water was analyzed by inductively coupled plasma spectrometry. The modified centrifugation technique with FC-70 offers a reliable, rapid, safe, and contamination-free method for obtaining unaltered soil-water from the rhizosphere, at a moisture content normally found in soil.

Centrifuges and Rotors

This appendix provides rmax (the distance between the axis of rotation and the bottom of the centrifuge tube as it sits in the well or bucket of the rotor) values for commonly used rotors manufactured by Du Pont (Sorvall), Beckman, Fisher, and IEC. Nomograms are also given to determine the relative centrifugal force (RCF) where speed and rmax are known, or the speed where RCF and rmax are known.

Centrifuges and Rotors

This appendix provides r(max) values (the maximum value of the rotating radius between the particle being centrifuged and the axis of rotation) for commonly used rotors manufactured by Du Pont (Sorvall), Beckman, Fisher, and IEC. Additionally, nomograms are provided for determining the relative centrifugal force (RCF) where speed and r(max) are known, or the speed where RCF and r(max) are known.

The effect of chemical, physical and enzymatic treatments on the dewatering of tar sands tailings

Fine tailings (with solids < 106 μm) from hot-water extraction of the Athabasca tar sands were subjected to thermal, physical, chemical and enzymatic treatments in an attempt to modify the dewatering characteristics of the solid particles. A low-speed centrifuge at circa 1500 relative centrifugal force for 15 h was used to accelerate the sedimentation of the solids, and allowed comparison of the ultimate concentration of solids after the various treatments. Fine tailings were subjected to thermal treatment, sonication and modification of the water chemistry. Although the rate of sedimentation was affected by these treatments, the ultimate volume fraction of solids after centrifuging was about 0.3 (50–55 wt% solids). Extraction of the tailings to remove bitumen, humins and other organic components also failed to change the ultimate solids concentration. Oxidation of the organic material in the tailings by hydrogen peroxide in Fenton's reagent, and enzymatic oxidation with lignin peroxidase, manganese peroxidase, cytochrome c, horseradish peroxidase and cowpea peroxidase gave no significant effect on the volume fraction of solids in the sediment after centrifugation. The observation that all of these treated samples gave an ultimate volume fraction of solids of about 0.3 indicated that bitumen, adsorbed organic matter and salts had little effect on the ultimate dewatering of tailings.

Centrifugal enhancement of retroviral mediated gene transfer

Centrifugation has been used for many years to enhance infection of cultured cells with a variety of different types of viruses, but it has only recently been demonstrated to be effective for retroviruses (Ho et al. (1993) J. Leukocyte Biol. 53, 208-212; Kotani et al. (1994) Hum. Gene Ther. 5, 19-28). Centrifugation was investigated as a means of increasing the transduction of a retroviral vector for gene transfer into cells with the potential for transplantation and engraftment in human patients suffering from genetic disease, i.e., gene therapy. It was found that centrifugation significantly increased the rate of transduction into adherent murine fibroblasts and into non-adherent human hematopoietic cells, including primary CD34+ enriched cells. The latter samples include cells capable of reconstitution of hematopoiesis in myeloablated patients. As a step toward optimization of this method, it was shown that effective transduction is: (1) achieved at room temperature; (2) directly related to time of centrifugation and to relative centrifugal force up to 10,000 g; (3) independent of volume of supernatant for volumes > or = 0.5 ml using non-adherent cell targets in test tubes, but dependent upon volume for coverage of adherent cell targets in flat bottom plates; and (4) inversely related to cell numbers per tube using non-adherent cells. The results support the proposal that centrifugation increases the reversible binding of virus to the cells, and together with results reported by Hodgkin et al. (Hodgkin et al. (1988) J. Virol. Methods 22, 215-230), these data support a model in which the centrifugal field counteracts forces of diffusion which lead to dissociation during the reversible phase of binding.

CONTROL of ENZYMATIC BROWNING IN RAW FRUIT JUICE BY FILTRATION and CENTRIFUGATION

Filtration and centrifugation were investigated as means of preventing enzymatic browning in minimally processed fruit juices. the capacity of raw apple, grape and pear juices to undergo browning was associated with particulate fractions that could be removed by filtration with Bentonite and/or diatomaceous earth, microfiltration or ultrafiltration, depending on the commodity and cultivar. Centrifugation prevented browning in pear juice and in Granny Smith, Golden Delicious, and Red Delicious but not McIntosh apple juice, provided that foam was excluded and the relative centrifugal force was sufficient. A method for the preparation of nonbrowning, cloudy juices by recombination of juice supernatant or ultrafiltration permeate with particulates, heat-treated to inactivate polyphenol oxidase, was investigated.

Effects of Extraction Methods and Sample Storage on Properties of Solutions Obtained from Forested Spodosols

AbstractSoil solutions obtained by three methods from Spodosols at a high‐elevation, forested study site were compared. Solutions were extracted by miscible displacement, by centrifugation, and by compressing a soil‐packed syringe. Analyses of pH, inorganic anions, total and reactive Al, and organic C showed few differences between miscible displacement and the syringe‐pressure methods. However, centrifugation at a relative centrifugal force (RCF) of 9700 m s−2 consistently produced solutions with significantly higher pH and F− content than did the other methods. Differences after centrifugation also were found in Cl−, NO−3, SO2−4, but Al, but less frequently. Effect of soil storage time on solutions obtained using the syringe‐pressure technique was studied. Both Oa and Bhs horizon samples showed large increases in solution NO−3 after 24 h of storage at 3°C. During 36 d of storage, NO−3 levels increased by as much as 10‐fold and were accompanied by pH decreases. Measurements in solutions obtained from samples that had been frozen for 36 d were higher than in the original solutions, except for NO−3 and pH. Changes with freezing appeared to be related to large increases in soluble organic C. Our results suggest that soil solution samples should be obtained very quickly after sampling of soils and that high‐speed centrifuge techniques may give erroneous results. The syringe‐pressure method is relatively rapid, simple, and easily performed in the field. However, it may not be practical under dry conditions.

'Spin, cool, and filter' red cells prepared in a regional blood center.

Red cells depleted of leukocytes by the "spin, cool, and filter" (SCF) method are effective in preventing most febrile, nonhemolytic transfusion reactions (FNHTR). To determine whether red cell concentrates may be centrifuged in a blood center and filtered subsequently at an outlying facility, the authors examined how leukocyte removal was affected by the transport and storage of centrifuged red cells before microaggregate filtration (MAF). One hundred fourteen red cell units were each divided into two aliquots. After centrifugation, one aliquot from each unit was retained in the blood center, and the other was transported on a truck for 2 to 12 hours. Aliquots were stored for variable periods, after which the residual leukocytes were counted. Neither transportation nor storage significantly affected leukocyte removal by MAF. However, an unacceptable proportion of all SCF units failed to meet American Red Cross standards for leukocyte-poor blood and studies of factors influencing leukocyte depletion were undertaken. A relative centrifugal force of 6700 X g was needed to produce consistently acceptable units when blood was 8 to 15 days old. These results show that making SCF red cells is a practical, inexpensive way for blood centers to provide hospitals with products that prevent most FNHTR; however, each facility that prepares these products must perform quality control carefully.

Rapid isolation of animal mitochondrial DNA in a small fixed-angle rotor at ultrahigh speed.

In recent years, mitochondrial DNA (mtDNA) sequence analysis has become a powerful tool for the investigation of the evolutionary and population biology of various animal species (Wilson et al., 1985). Most such studies have relied on physical purification of mtDNA, typically by ultracentrifugation in cesium chloride gradients. Thorough descriptions of such methods are given by Lansman et al. (1981) and Wright et al. (1983). Practical concerns over the expensive instrumentation and extensive time required by such methods have been expressed by several workers (e.g., Grill et al., 1983; Powell and Zufiiga, 1983). Several of our colleagues have expressed doubt that purified mtDNA samples can be generated in a reasonable time in numbers sufficient to perform large-scale population studies. We describe here an ultracentrifugal procedure that yields homogeneous mitochondrial DNA from as many as i0 samples in as little as 10 hr. The procedure uses a relatively inexpensive tabletop ultracentrifuge and relies on a self-forming cesium chloride gradient generated in a small fixed-angle rotor that operates at a speed of 100,000 rpm and a relative centrifugal force of 436,000g.

A Rapid Centrifugation Method for Obtaining Soil Solution

AbstractA modified centrifugation technique for obtaining soil solutions was developed and compared to immiscible displacement technique using different immiscible liquids. Coefficients of variation and standard deviations for ionic composition of soil solutions obtained by the modified centrifugation method were consistently lower than those of immiscible liquids. Differences in the estimated concentrations and activities for Ca, Mg, K, and Al among the methods employed to obtain soil solutions were noticed. The largest disagreement between activity values occurred for Al species. Such discrepancies necessitate the use of the modified centrifugation technique to obtain soil solution especially when Al determination and speciation are critical. The modified centrifugation technique offers a reliable, rapid, contamination‐free, and inexpensive method for obtaining soil solution for chemical characterization. Practical aspects of the technique (relative centrifugal force, centrifugation time, and filtering materials) are discussed.