Density of TEPC-15 plasmacytoma cells as function of size

TEPC-15 plasmacytoma cells were observed under the microscope, as they sedimented at 1 G in Dulbecco's modified Eagle's medium. A mean density of 1.0422 g/cm3 (30 degrees C) was found. The density difference of smaller cells (15 micron diameter) was significantly higher than the density difference of larger cells (25 micron diameter). The ratio in density difference was close to 3.     

Droplet sorting of large particles

The systematics of droplet formation conditions for orifices with diameters up to 200 micron are described. Sorting recovery experiments indicate that particles up to 44 micron in diameter can be recovered by charged droplet deflection of two drops with at least 75% recovery. By reducing the jet velocity, a deflection of greater than 1 cm was obtained for all droplet sizes.     

Calibration of beam deflection produced by cellular forces in the 10(-9)--10(-6) gram range

An experimental procedure and method of analysis are presented for calibration of a thin-beam force transducer. The beam transducer can be produced and calibrated with a minimum coefficient of 10 ng (10(-5) dyne) force per micron (10(-4) cm) deflection, i.e., kB approximately 0.1 dyne/cm. Since beam deflections on the order of 0.1 micron can be detected, forces of a few nanograms can be resolved. Such forces are common in mechanical experiments on microscopic bodies, e.g., biological cells, artificial membrane capsules, droplets, etc.     

Morphometric analysis of Leydig cells in the normal rat testis

Leydig cells are thought to be the source of most, if not all, the testosterone produced by the testis. The goal of this study was to obtain quantitative information about rat Leydig cells and their organelles that might be correlated with pertinent physiological and biochemical data available either now or in the future. Morphometric analysis of Leydig cells in mature normal rats was carried out on tissue fixed by perfusion with buffered glutaraldehyde, and embedded in glycol methacrylate for light microscopy and in Epon for electron microscopy. In a whole testis, 82.4% of the volume was occupied by seminiferous tubules, 15.7% by the interstitial tissue, and 1.9% by the capsule. Leydig cells constituted 2.7% of testicular volume. Each cubic centimeter (contained approximatelyy 1 g) of rat testis contained about 22 million Leydig cells. An average Leydig cell had a volume of 1,210 micron3 and its plasma membrane had a surface area of 1,520 micron2. The smooth endoplasmic reticulum (SER), the most prominent organelle in Leydig cells and a major site of steroidogenic enzymes, had a surface area of approximately 10,500 micron2/cell, which is 6.9 times that of the plasma membrane and is 60% of the total membrane area of the cell. The total surface area of Leydig SER per cubic centimeter of testis tissue is approximately 2,300 cm2 or 0.23 m2. There were 3.0 mg of Leydig mitochondria in 1 g of testis tissue. The average Leydig cell contained approximately 622 mitochondria, measuring on the average 0.35 micron in diameter and 2.40 micron in length. The mitochondrial inner membrane (including cristae), another important site of steroidogenic enzymes, had a surface area of 2,920 micron2/cell, which is 1.9 times that of the plasma membrane. There were 644 cm2 of inner mitochondrial membrane/cm3 of testis tissue. These morphometric results can be correlated with published data on the rate of testosterone secretion to show that an average Leydig cell secretes approximately 0.44 pg of testosterone/d or 10,600 molecules of testosterone/s. The rate of testosterone production by each square centimeter of SER is 4.2 ng/d or 101 million molecules/s: the corresponding rate for each square centimeter of mitochondrial inner membrane is 15 ng testosterone/d or 362 million molecules/s.     

The variation of characteristics of twitch and tetanic contractions with sarcomere length in isolated muscle fibres of the frog.

The relation between sarcomere length, tension and time course of tension development in twitch and tetanic contractions at 20 degrees C was determined for isolated fibres from the semitendinosus muscle of the frog (Rana esculenta). In twenty fibres at about 2.15 micron sarcomere length, the peak twitch tension, the maximum tetanic tension and the twitch/tetanus ratio ranged, respectively, from 0.22 to 1.6 kg/cm2, from 2.13 o 3.96 kg/cm2 an from 0.07 to 0.53. The peak twitch tension was found to be: i) directly correlated with the twitch/tetanus ratio and the time to the peak of the first derivative of the twitch tension, ii) inversely correlated with the time to the peak of the first derivative of tetanic tension. No significant correlation was found between the maximal tetanic tension and the peak twitch tension or the twitch/tetanus ratio. Peak twitch tension and twitch/tetanus ratio were not correlated with the fibre cross-sectional area which ranged from 1.052 to 6,283 micron2. Sarcomere length-tension curves for twitch and tetanic isometric contractions at 20 degrees C were determined in twelve fibres. Increases in sarcomere length from about 2.15 to 2.85 micron produced, depending on the peak twitch tension or the twitch/tetanus ratio at about 2.15 micron, either decrease and no change or increase in peak twitch tension, but constantly enhanced the twitch/tetanus ratio and the degree of this potentiation was inversely correlated with the twitch/tetanus ratio at 2.15 micron. Increase in sarcomere length above 2.15 micron did not alter the course of the early development of twitch and tetanic tensions, reduced considerably the variation in peak twitch tension and twitch/tetanus ratio, without altering that of tetanic tension and swamped the correlation between the peak twitch tension and the time to peak of the differentiated twitch tension. However, the peak twitch tension at about 2.85 micron resulted to be directly correlated with the peak twitch tension at about 2.15 micron and in addition the relative length-dependent change in the time of the peak of the first derivative of the twitch tension resulted to be directly correlated with the relative length-dependent change in the peak twitch tension. It is concluded that both the duration of the active state and the rate factors of activation contribute to the determining of the large variation in peak twitch tension at about 2.15 micron, whereas the length-dependent increase in twitch/tetanus ratio appears to be mainly determined by prolongation of the active state duration.     

Mitochondrial DNA from Podospora anserina. I. Isolation and characterization.

Mitochondrial (Mt) DNA from Podospora anserina was isolated and characterized with respect to density in CsCl, contour length and endonuclease restriction enzymes. The density of Mt DNA for four races examined was 1.694 g/cm3, compared with 1.712 g/cm3 for nuclear DNA. Extraction in the presence of a nuclease inhibitor, aurintricarboxylic acid and isolation in DAPI CsCl gradients allowed us to isolate high molecular weight DNA. Mt DNA isolated by total DNA extraction contained ca. 1% of circular molecules, 31 micron in contour length; Mt DNA isolated from purified mitochondria contained 2--4% of these 31 micron circles. Analysis with Eco RI restriction endonuclease revealed that each of the four races examined, s, A, T and E had a characteristic fragment pattern. Races s and A Mt DNA differed by only one fragment after Eco RI enzymatic digestion; similarly, these two DNA differed by only one or two fragments after Hae III digestion.     

Thin sectioning of slice preparations for immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue. Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle. Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm. We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections. The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed. We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice. This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice. A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT). This is then quickly frozen with crushed dry ice and the resulting wafer can be position easily for cryostat sectioning. Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.     

Identification of normal or neoplastic murine mesenchymal cells in chimeric tissues or heterotransplants

An improved method for identifying murine mesenchymal cells in chimeric tissues or heterotransplants using Hoechst dye 33258 is described. Following fixation in formalin-saline, tissues are embedded in JB-4 plastic. Sections 3 micron thick are then stained in a 10 microgram/ml solution of Hoechst 33258 in Hanks' balanced salt solution for 5-10 min at 4 C. After rising, the sections are coverslipped using a modified polyvinyl alcohol mounting medium. This approach offers several advantages over existing techniques: 1) uniform section thickness is more easily obtained than with paraffin or cryostat microtomy, thereby allowing improved resolution and more reliable identification of mesenchymal cells with small nuclei such as skeletal muscle myocytes or fibroblasts, 2) the preparations are stable over long periods and can be repeatedly viewed or photographed, and 3) calcified tissues can be examined without prior decalcification. An example is shown of species identification using rat chondrosarcoma cells grown in nude mice.     

Multiple recording of electromyograms in a chronic experiment

The authors describe electromyographic electrodes (EMG electrodes) for recording the activity of several muscles in chronic experiments on cats. An EMG electrode is a loop made of a silver wire 200 micron in diameter and 1.5-2 cm long, to which a flexible copper wire insulated with teflon 300 micron in diameter is soldered. The soldering site is insulated with biologically indifferent material. The EMG electrodes turned out fairly good in numerous experiments.     

Decidual cells in the human ovary at term: II. Morphometric analysis of cytoplasmic processes and organelles

Morphometric analysis of human ovarian decidual cells was performed with a Videoplan computer, and mean values were established for the area and perimeter of cellular processes and organelles. Two-hundred forty electron micrographs representing 160 cells were analyzed. The mean decidual cell area was 218.7 microns2, of which 34.5 microns2 was occupied by the nucleus (15.8% of the cytoplasmic area); the nucleus contained 1.74 micron2 of nucleolar material (0.8%). The endoplasmic reticulum occupied 13.63 microns2 (6.2%). Mitochondria occupied 7.3 microns2 (3.3%) and the Golgi network 5.49 microns2 (2.5%). Decidual secretory bodies occupied 0.91 micron2 (0.42%) and cytoplasmic processes 1.89 micron2 (0.94%). The remainder of the cytoplasm, containing inclusions and cytoskeleton, represented 71% of the cell area. Perimeter measurements indicated an average decidual cell was surrounded by 87.8 microns of plasma membrane. The mean nuclear membrane measured 28.3 microns (representing 32.3% of the plasma membrane, pm, or 4.1% of total cellular membranes, cm). Outer mitochondrial membranes measured 156.6 microns (178% pm, 23.5% cm); endoplasmic reticulum membranes measured 350.3 microns (400% pm, 52.6% cm); Golgi membrane measured 30.77 microns (35% pm; 4.5% cm) and membrane surrounding secretory bodies measured 9.8 microns (11.2% pm; 1.4% cm). A mean of 280 secretory bodies per ovarian decidual cell was calculated. The plasma membranes of evaginated cytoplasmic processes represented 22.3% of the total pm (19.6 microns or 2.9% cm). A mean of seven such processes was observed per 87.8 microns of plasma membrane (160/cell). These morphometric data provide a baseline for comparisons of human ovarian decidual cells with uterine decidua, in vivo and in vitro, as well as with decidual cells of other species.     

Interaction energies in lectin-induced erythrocyte aggregation

Two N-acetylgalactosamine-reactive lectins, Helix pomatia (HPA) and Dolichos biflorus (DBA), were used to study the energies involved in cell-cell interactions through the specific binding of these lectins to their membrane receptors on genotype AO human erythrocytes (red blood cells) (RBCs). The energy required to dissociate a unit of aggregated membrane area (gamma d) of two RBCs bridged by lectin molecules was determined from the shear force needed to dissociate two-cell aggregates in a flow channel. When HPA were used as bridging molecules, gamma d (0.4 X 10(-4) to 3.8 X 10(-4) dyn/cm) was proportional to the density (D = 175 to 1,060 molecules/micron 2) of HPA molecules bound on the RBC membrane. A similar gamma d/D ratio was also obtained for DBA. These results indicate that the number of lectin molecules bound on the interface plays an important role in determining the energy required for cell-cell dissociation. The aggregation energy per unit membrane area (gamma a) in lectin-induced aggregates was calculated from the degree of encapsulation of a lectin-bound, heat-sphered human RBC by a normal discoid RBC. A minimum of approximately 1,800 HPA molecules/micron 2 on the spheres was required to form stable aggregates with the RBC. By using spheres having a surface HPA density of 1,830 to 2,540 molecules/micron 2, or 1.1-1.5 X 10(12) combining sites/cm2, the gamma a value for HPA-induced aggregation was found to be 2.2 X 10(-3) dyn/cm. This higher value of gamma a than gamma d has been explained on the basis of several differences in aggregation and disaggregation processes. The gamma a value for DBA-induced aggregation was not obtainable by the sphere encapsulation method because of the relative low D values. A comparison of the present results with the published value of the free energy change of 5 kcal/mol for the interactions of HPA and DBA with their ligands suggests that only a small fraction of the lectin molecules bound to RBC surface participate in the bridging of adjacent cells.     

Air embolism due to limited high grade barometric depression as evidenced in vivo

The Authors supply documentary evidence for the aeroembolism of auricular lobe vessels photographed in transillumination of living albino rabbits exposed to high grade barometric depression (850 g/cm2) limited on the top of the head.     

ESTIMATING NATURAL MORTALITY RATE IN REPRODUCTIVELY ACTIVE FEMALE SOUTHERN RIGHT WHALES, EUBALAENA AUSTRALIS

Cow-calf pairs of southern right whales on the South African coast have been photographed in aerial surveys in October each year since 1979. In this paper 469 resightings of 177 individually identified cows photographed in the first six years of surveys have been analyzed in two ways to produce estimates of natural mortality rate. Both methods assume that all females calve either two, three, or four years after their previous calf. In Method A there is assumed to be no systematic trend with time in the probability of a female being photographed on each calving occasion. Natural logarithms of the numbers photographed 2-4, 5-7, 8-10, etc. yr after being first seen are regressed against time, the slope of which provides an estimate of natural mortality rate of 0.0255 ± 0.0071. The intercept value for this regression provides an estimate of the detection probability, or 0.769 ± 0.011. In Method B it is assumed that if a female has not been photographed for at least nine years then it is dead. Annual mortality estimates are obtained from the proportional reduction in the numbers of females known to be alive at each three-year interval after being first photographed, up to at least 6-8 yr from the present. Method B produces an estimate of natural mortality of 0.0260 ± 0.0190 (corrected to 0.0227 ± 0.0192 using the average detection probability). Both methods may be subject to various biases that tend to inflate estimates of natural mortality.     

Dielectric properties of yeast cells as determined by electrorotation.

Electrorotational spectra of yeast cells, Saccharomyces cerevisiae strain R XII, were measured over a frequency range of nearly 7 decades. The physical properties of distinct cell parts were simultaneously determined for individual cells by comparison with an electrical two-shell model: The conductivity of the cytoplasm, cell wall and cytoplasmic membrane of living cells were found to be 5.5 mS/cm, 0.1 to more than 0.5 mS/cm and less than 0.25 nS/cm to 4.5 microS/cm, respectively. The conductivity of the cytoplasmic membrane was dependent on the conductivity of the medium. Membrane behaviour is interpreted as an opening of membrane channels when the environment becomes more physiological. The specific membrane capacitance was determined to be 1.1 microF/cm2 and the thickness of the cell wall was calculated as 0.11 micron. Heat treated cells showed an increased membrane conductivity of more than 0.1 microS/cm (at 25 microS/cm medium conductivity) and a drop in cytoplasmic conductivity to between 0.1 and 0.8 mS/cm, depending on the length of time the cells were suspended in low conductivity water (25 microS/cm), indicating a perforation of the membrane. A slightly decreased spinning speed scaling factor for dead cells suggests a modification to the cellular surface, while the principal structure of the cell wall appears to be uneffected. It can be demonstrated by these observations, that cellular electrorotation permits the simultaneous investigation of the different cellular compartments of individual cells in vivo under various environmental conditions.     

Cyclic AMP transport in human erythrocyte ghosts.

10(-5) M cyclic AMP has high permeability in human erythrocyte ghosts (p = 0.061-10(-6) cm.s-1). Saturation of influx and efflux occurs. Koizt = 4.43 mM. Voizt = 259.6 micron.min-1-Kiozt = 0.475 micron. Viozt = 28.3 micron.min-1 at 30 degrees C. Equilibrium exchange entry of cyclic AMP has similar kinetics to zero trans influx, though the system does show counterflow. Cytochalasin B is an apparent competitive inhibitor of cyclic AMP exit. (Ki = 3.9.10(-7) M). Control experiments indicated that cyclic AMP remains intact during incubation with red blood cell ghosts and is contained within the intravesicular space during the transport experiments.     

Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance

The relationship of tight junction permeability to junction structure and composition was examined using two strains of Madin-Darby canine kidney (MDCK) cells (I and II) which differ greater than 30-fold in transepithelial resistance. This parameter is largely determined by paracellular, and hence junctional, permeability under most conditions. When these two strains of cells were grown on permeable filter supports, they formed monolayers with equivalent linear amounts of junction/area of monolayer. Ultrastructural analysis of these monolayers by thin section EM revealed no differences in overall cellular morphology or in tight junction organization. Morphometric analysis of freeze-fractured preparations indicated that the tight junctions of these two cell strains were similar in both number and density of junctional fibrils. Prediction of transepithelial resistance for the two strains from this freeze-fracture data and a published structure-function formulation (Claude, P. 1978, J. Memb. Biol. 39:219- 232) yielded values (I = 26.5 omega/cm2, II = 35.7 omega/cm2) that were significantly lower than those observed (I = 2,500-5,000 omega/cm2, II = 50-70 omega/cm2). Consistent with these structural studies, a comparison of the distribution and cellular content of ZO-1, a polypeptide localized exclusively to the tight junction, revealed no significant differences in either the localization of ZO-1 or the amount of ZO-1 per micron of junction (I = 1,415 +/- 101 molecules/micron, II = 1,514 +/- 215 molecules/micron).     

Gel electrophoresis of micron-sized particles: a problem and a solution

The gel electrophoresis of spherical particles with a radius above 0.2 micron has not been reported yet. In the present study, video phase-contrast light microscopy is used to observe the motion of individual latex spheres, 0.52 micron in radius, during electrophoresis in 0.1% agarose gels. At 2 V/cm, the spheres initially migrate in the direction of the electrical field. However, each sphere eventually undergoes a cessation of all motion. Brownian motion is restored when the electrical potential gradient is reduced to zero. Arrest can be prevented by periodically inverting the direction of the electrical field. These observations are explained by electrical field-induced steric trapping of the spheres by gel fibers. Inversion of the electrical field should assist the application of agarose gel electrophoresis to micron-sized cellular organelles and cells.     

Acanthocheilonema sabanicolae n. sp. (Filarioidea: Onchocercidae) from the savanna armadillo (Dasypus sabanicola) in Venezuela, with comments on the genus Acanthocheilonema

A new species of Acanthocheilonema, Acanthocheilonema sabanicolae n. sp., is described from the subcutaneous tissues of the savanna armadillo (Dasypus sabanicola) in Venezuela. The adults (females 25-30.6 mm long by 61-78 micron wide; males 10.9-15.3 mm long by 42-44 micron wide) and the microfilariae (112-131 micron long by 3-5 micron wide) are the smallest within the genus. The microfilaria, a skin dweller, assumes a characteristic C-shaped curve, when fixed in 2% formalin, which serves to distinguish A. sabanicolae from others in the genus. Moreover, the spicules are notably smaller than any others in the Acanthocheilonema. Acanthocheilonema sabanicolae is the first species in this genus to be described from edentates, and, along with A. reconditum of dogs and A. pricei of opossums, is the third species of Acanthocheilonema to be reported from South America. Morphological features, vectors, and vertebrate hosts of the genus Acanthocheilonema are reviewed.     

The static elastic properties of 45 human thoracic and 20 abdominal aortas in vitro and the parameters of a new model

Segments of 45 human thoracic and 20 abdominal aortas, including 13 pairs, aged 30-88 yr at autopsy, were perfused with 37 degrees C Tyrode's solution at in-situ length. Diameter changes due to 20 mmHg pressure steps, between 20 and 180 mmHg, were measured to 1 micron accuracy with balanced transducers. Absolute diameter at 100 mmHg was measured to 50 micron accuracy. At 100 mmHg, cross-sectional area ranged from 2.6 to 7.6 for thoracic and from 1.0 to 3.2 cm2 for abdominal segments. Compliances ranged from 1.9 to 17 for thoracic and from 0.6 to 4.4 mm3/mmHg.cm for abdominal segments. An arctangent model with three free parameters A(p) = Am(1/2 + tan-1 [p-p0)/p1)/pi) explained over 99% of the variance in area with pressure for each aorta. Changes in compliance, characteristic impedance and propagation velocity are equally well described. Abdominal fits on the average appeared down scaled by a factor of 2 and shifted 20 mmHg towards lower pressures from paired thoracic (significant at p = 0.001).