Morphometric analysis of several intracellular events occurring during the vegetative life cycle of the unicellular algaPolytoma papillatum

Summary Quantitative electron microscopy of serial sections was used to study thePolytoma papillatum cell and some of its constituents (nucleus, chondriome, leucoplast) during its vegetative life cycle.The volumes of cells just entering into or passing through mitosis varied considerably and seemed to determine the number of subsequent division processes.Whereas a volumetric balance existed between the cell (100%) and the chondriome (8–9%) during the whole life cycle, there was a correlation between cell and nuclear volumes (8–10%) only during interphase growth and the onset of mitosis. At telophase the nucleus-to-cell-volume ratio was reduced to 2%, but gradually increased during cytokinesis (4.6% at early cytokinesis; 6.5% at late cytokinesis) until it reached the initial value again in newly formed daughter cells. The leucoplast-to-cell-volume ratio (10–26%) varied considerably without any recognizable dependence upon cell cycling.The mean short axis of mitochondrial profiles was proportional to the mean diameter (=thickness) of the mitochondria; the specific surface (outer membrane area per 100 m³ mitochondrial volume), and the surface-to-volume ratio changed rhythmically. Changes in mitochondrial surface-to-volume ratio (Sc/Vc) were apparently correlated with changes in mitochondrial diameter (Dc). This relationship can be approximately described by the function Sc/Vc=4/Dc.Deviations of the surface-to-volume ratios of the nuclei from the surface-to-volume ratios of idealized spheres of equal size, indicating profound changes in nuclear shape, were found mainly during mitosis.Results were compared with those obtained from other morphometric investigations and discussed with regard to their functional meaning.     

Histometric volume determination in aging human thyroid glands and possible errors of this method]

We made histometric volume analyses of hematoxylin and eosin stained paraffin sections of 43 human thyroid lobes by means of the electric integrating apparatus Eltinor 4 A (Optische Werke Rathenow). We investigated ageing changes of the relative amounts of epithel, colloid and connective tissue in the total volume. The volume density of colloid rises up to the 3rd decade, thereafter it shows a small decrease. On the contrary, the amount of epithelial cells decreases until the 3rd decade. The data concerning connective tissue exhibit an increasing trend up to senility. During such measurements errors appear, which are called, among others, total error, systematic and statistic error. We estimated these errors and demonstrated possibilities of their diminution.     

Creatine biosynthesis enzymes in the postnatal development of rats: the role of cyclo-3',5'-AMP and glucagon in the postnatal induction of liver guanidine acetate-N-methyltransferase]

The activity of enzymes of creatin biosynthesis in the rat liver and kidneys has been studied during the postnatal development. The activity of transamidinase of kidneys (E.C. 2.1.4.1.) increases gradually and linearly up to the 20th day after birth, then decreases on the 12th--25th days and increases again up to the level characteristic of the adult organism. The activity of guanidine acetate-N-methyl transferase (E.C. 2.1.1.2.) is rather high during the first days of postnatal development, then decreases and from the 15th day on increases again attaining the maximal level by the 23rd--25th day. The second period of the increase in the enzyme activity begins on the 29th--30th day of postnatal development. The results obtained suggest that the sharp increase of activity of guanidine acetate-N-methyl transferase of the rat liver during the early postnatal development is realized with the participation of cyclic 3',5'-AMP which appears to mediate the glucagon action.     

Soft X-ray microscopy analysis of cell volume and hemoglobin content in erythrocytes infected with asexual and sexual stages of Plasmodium falciparum

Plasmodium falciparum, the most virulent agent of human malaria, undergoes both asexual cycling and sexual differentiation inside erythrocytes. As the intraerythrocytic parasite develops it increases in size and alters the permeability of the host cell plasma membrane. An intriguing question is: how is the integrity of the host erythrocyte maintained during the intraerythrocytic cycle? We have used water window cryo X-ray tomography to determine cell morphology and hemoglobin content at different stages of asexual and sexual differentiation. The cryo stabilization preserves native structure permitting accurate analyses of parasite and host cell volumes. Absorption of soft X-rays by protein adheres to Beer–Lambert’s law permitting quantitation of the concentration of hemoglobin in the host cell compartment. During asexual development the volume of the parasite reaches about 50% of the uninfected erythrocyte volume but the infected erythrocyte volume remains relatively constant. The total hemoglobin content gradually decreases during the 48 h cycle but its concentration remains constant until early trophozoite stage, decreases by 25%, then remains constant again until just prior to rupture. During early sexual development the gametocyte has a similar morphology to a trophozoite but then undergoes a dramatic shape change. Our cryo X-ray tomography analysis reveals that about 70% of the host cell hemoglobin is taken up and digested during gametocyte development and the parasite eventually occupies about 50% of the uninfected erythrocyte volume. The total volume of the infected erythrocyte remains constant, apart from some reversible shrinkage at stage IV, while the concentration of hemoglobin decreases to about 70% of that in an uninfected erythrocyte.     

Ultrastructural qualitative and quantitative data on the sporogenesis of the protozoan Abelspora portucalensis (Microspora, Abelsporidae): a different approach to the study of microsporidia.

The sporogenesis of the microsporidium Abelspora portucalensis was studied with electron microscopy. In qualitative terms, new aspects of the cytoplasmic ultrastructure of the schizont, sporont, and sporoblast are described: the presence of microtubules, of aggregates of small opaque vesicles, and of dispersed larger vesicles with clear matrix. The hypothesis that the opaque vesicles may represent the Golgi apparatus and the clear vesicles may correspond to the smooth endoplasmic reticulum is discussed. The use of standard stereological and statistical techniques gives us a new perspective on the development of this microsporidium. The most relevant quantitative data display that the amount of rough endoplasmic reticulum (either in relative or absolute terms) presents significant differences among the three stages, with the sporont showing the highest values; that the absolute (but not the relative) volume of the large vesicles significantly changes during sporogenesis with the highest values presented by the sporont; that the surface-to-volume ratio of the schizont and sporont cells is similar and significantly greater than that of the sporoblast cell; that the surface density of the nucleus in relation to soma remains constant in the three stages (on the contrary, the surface-to-volume ratio of the nucleus increases and its volumetric density diminishes); and finally, that the nucleolus decreases its relative and absolute volumes. The functional significance of these results is analyzed and the application of similar methodology in quantifying the effects of drugs upon microsporidia is suggested.     

Hyaluronic acid synthesis and secretion by rat liver fat storing cells (perisinusoidal lipocytes) in culture

The ability of rat liver fat storing cells to synthesize and to secrete hyaluronic acid was examined in monolayer cultures. The cells produce [3H] glucosamine-labeled hyaluronic acid, of which about 80% are secreted into the medium. The synthesis rate per cell (mg DNA) of labeled total glycosaminoglycans and hyaluronic acid in the medium increases significantly with culture time, but hyaluronic acid expressed as fraction of total glycosaminoglycans declines from about 0.70 in early cultures (up to the 4th day) down to 0.20 in advanced cultures. Cycloheximide increases and beta-D-xylopyranoside decreases significantly the fraction of hyaluronic acid in the medium, colchicine up to 5 microM was without effect. The synthesis of hyaluronic acid is a newly recognized function of this special type of sinusoidal liver cells. The results suggest that fat storing cells are likely to be a major source of hyaluronic acid in normal and probably also in injured liver.     

Effect of host packed cell volume on the bloodmeal size of male tsetse flies, Glossina pallidipes

aematin contents of engorged, male tsetse flies, Glossina pallidipes Austen, were compared with the packed cell volumes of oxen on which they had fed. Haematin contents icnreased with packed cell volume up to packed cell volumes of approximately 30%. Haematin contents appeared to level off or decline with further increase in packed cell volume. These results support a model of blood-feeding in tsetse flies in which the rate of blood consumption decreases as packed cell volume increases, because of increase in blood viscosity, and tsetse are unable to compensate for the decrease in consumption rate by feeding for a longer time. After allowing for the effects of packed cell volume, bloodmeal sizes of tsetse increased with ox body temperature.     

Changes in activity of the organon vasculosum laminae terminalis in the annual cycle in Rana temporaria.

In 70 sexually mature male and femal Rana temporaria frogs captured in natural habitat, mean nuclear volumes for the cells of the pars ependymalis and pars parenchymalis of the organon vasculosum laminae terminalis (OVLT) were determined in seven characteristic stages in life. The mean nuclear volume for the cells of the pars ependymalis and pars parenchymalis of the OVLT showed distinct annual fluctuation. Maximum nuclear volume of the cells in both investigated parts of the OVLT were observed during the breeding period (Ist decade of April), and minimum volume of the nuclei of the pars ependymalis at the beginning of hibernation (IIIrd decade of October), and in the pars parenchymalis near the end of active life (Ist decade of September).     

A seasonal change in blood cell volume of the Rottnest Island Quokka, Setonix brachyurus

Comparison of the red cell and plasma volumes (T-1824) of wild Quokkas captured in spring (October) with volumes of animals captured in autumn (May) showed that no seasonal change in average plasma volume occurred but a 42% average decrease in red cell volume did occur in autumn. Control animals kept in compounds showed no seasonal change in either average plasma or red cell volume. The available evidence suggests that the seasonal decline in red cell volume results from the semi-starvation suffered by the wild population during the late summer and autumn of each year. The average relative red cell, plasma and whole blood volumes of well-fed Quokkas captured in October were 24.6±3.1; 37.0±3.0; and 61.7±4.5 ml/kg, and equivalent measures of domesticated Quokkas were 28.0±3.2; 40.5±2.8; and 68.5±5.3 ml/kg. These values lie within the ranges of comparaable blood compartment volumes of eutherian mammal species.     

Assessment and consequences of the constant-volume attribute of the four-chambered heart

The constant-volume hypothesis regarding the four-chambered heart states that total pericardial volume remains invariant throughout the cardiac cycle. Previous canine studies have indicated that the pericardial volume remains constant within 5%; however, this hypothesis has not been validated in humans using state-of-the-art technology. The constant-volume hypothesis has several predictable functional consequences, including a relationship between atrial ejection fraction and chamber equilibrium volumes. Using cardiac magnetic resonance (MR) imaging (MRI), we measured the extent to which the constant-volume attribute of the heart is valid, and we tested the accuracy of the predicted relationship between atrial ejection fraction and chamber equilibrium volumes. Eleven normal volunteers and one volunteer with congenital absence of the pericardium were imaged using a 1.5-T MR scanner. A short-axis cine-loop stack covering the entire heart was acquired. The cardiac cycle was divided into 20 intervals. For each slice and interval, pericardial volumes were measured. The slices were stacked and summed, and total pericardial volume as a function of time was determined for each subject. In the normal subjects, chamber volumes at ventricular end diastole, end systole, and diastasis were measured. Pericardial volume remained invariant within 5 +/- 1% in normal subjects; maximum variation occurred near end systole. In the subject with congenital absence of the pericardium, total heart volume, defined by the epicardial surface, varied by 12%. The predictions of the relationship between atrial ejection fraction and chamber equilibrium volumes were well fit by MRI data. In normal subjects, the four-chambered heart is a constant-volume pump within 5 +/- 1%, and constant-volume-based modeling accurately predicts previously unreported physiological relationships.     

Optimization of the performance of the polymerase chain reaction in silicon-based microstructures.

We have demonstrated the ability to perform real-time homogeneous, sequence specific detection of PCR products in silicon microstructures. Optimal design/ processing result in equivalent performance (yield and specificity) for high surface-to-volume silicon structures as compared to larger volume reactions in polypropylene tubes. Amplifications in volumes as small as 0.5 microl and thermal cycling times reduced as much as 5-fold from that of conventional systems have been demonstrated for the microstructures.     

The hepatocyte ultrastructure of the Sevan trout (Salmo ischchan gegarkuni Kessel) in ontogeny]

Hepatocytes of Lake Sevan Salmo were examined at several stages in their life cycles which are different from the point of view of the manner of feeding. Salmo were reared at the fish farm, they were fed with the yolk of the chick eggs. It was revealed that hepatocytes of larva, which was sampled immediately after hatching (endogenous feeding) intensively synthesized the proteins; accumulated and secreted the bile product stored glycogen and lipids. The ultrastructure of larva hepatocytes changed on the 5th and 10th day after larva began to accept food (mixed feeding--endogenous and exogenous). Golgi complex became bigger, glycogen disappeared, lipid droplets became smaller (on the 5th day) and disappeared completely (on the 10th day). Morphological differentiation finishes during the fingerling period (exogenous feeding). Cisternae of granular endoplasmic reticulum (GER) and mitochondria are arranged around nucleus, near bile canaliculus and sinusoids. Big areas of glycogen lie between the organelles. Relative volumes of GER, mitochondria, glycogen increased, but the relative volume of Golgi complex diminished.     

Upper limb muscle volumes in adult subjects

Muscle force-generating properties are often derived from cadaveric studies of muscle architecture. While the relative sizes of muscles at a single upper limb joint have been established in cadaveric specimens, the relative sizes of muscles across upper limb joints in living subjects remain unclear. We used magnetic resonance imaging to measure the volumes of the 32 upper limb muscles crossing the glenohumeral joint, elbow, forearm, and wrist in 10 young, healthy subjects, ranging from a 20th percentile female to a 97th percentile male, based on height. We measured the volume and volume fraction of these muscles. Muscles crossing the shoulder, elbow, and wrist comprised 52.5, 31.4, and 16.0% of the total muscle volume, respectively. The deltoid had the largest volume fraction (15.2%+/-1%) and the extensor indicis propius had the smallest (0.2%+/-0.05%). We determined that the distribution of muscle volume in the upper limb is highly conserved across these subjects with a three-fold variation in total muscle volumes (1427-4426cm(3)). When we predicted the volume of an individual muscle from the mean volume fraction, on average 85% of the variation among subjects was accounted for (average p=0.0008). This study provides normative data that forms the basis for investigating muscle volumes in other populations, and for scaling computer models to more accurately represent the muscle volume of a specific individual.     

Osmotic properties of ehrlich ascites tumor cells during the cell cycle

Ehrlich ascites tumor cells were grown and maintained in continuous spinner culture. The population of dividing cells was synchronized by a double thymidine block technique. Cell cycle phases were determined graphically by plotting mitotic index, cell number, and DNA synthesis against time. Changes in the osmotic properties of Ehrlich ascites tumor cells during the cell cycle are described. Permeability to water is highest at the initiation of S and progressively decreases to its lowest value just after mitosis. Heats of activation for water permeability vary during the cell cycle, ranging from 9–14 kcal/mole. Results may imply changes in the state of water in the membrane during the cycle. The volume of osmotically active cell water is highest during S and early G2 and decreases during the mitotic phase, as cells undergo division. Total water content remains stable at 82% (w/w) during the cycle. Total concentration of the three major ions (Na, K, Cl), expressed as mEq/liter total cell volume, does not change. The fraction of total cell water which is osmotically active (Ponder's R) decreased gradually from 0.75 at S to about 0.56 following mitosis. Findings suggest that a fraction of the total water within the cell exists in a “bound” form and is, therefore, incapable of being shifted under the driving force of osmotic pressure. This fraction of bound water increases during the cell cycle. Possible alterations in membrane fluidity and the state of water in the cell are discussed.     

A comprehensive study of the relationship between size and protein composition in natural bovine casein micelles

Casein micelles of bovine skimmed milk were fractionated by permeation chromatography on porous glass (CPG-10, 50 nm followed by CPG-10, 300 nm) at 30 degrees C. Micelles were pooled in eight eluant fractions and their size distribution was determined by electron microscopy. The composition of casein in the eight fractions was determined by quantitative hydroxyapatite chromatography. Micelle size decreased progressively with increasing elution volume, and volume-to-surface average diameter ranged from 154 nm in fraction 1 to 62 nm in fraction 8. Concurrently there was a decrease in relative proportions of alpha s- and beta-caseins and a large enrichment of kappa-casein, which changed from 4.1% total casein in fraction 1 to 12.1% total casein in fraction 8. At least half the decrease in alpha s-casein proportions was attributed to the alpha s1-casein component, but the data also suggested a decline in proportions of alpha s2-casein in the smallest micelle fractions. A plot of kappa-casein fractional content versus micelle surface-to-volume ratio gave a straight line (correlation coefficient from linear regression 0.98) from which an average kappa-casein surface coverage of 1.5 m2/mg or 47.3 nm2/molecule was obtained. If a constant surface coverage for kappa-casein is assumed, the parameters of the linear equation predict that micelle voluminosity is inversely related to micelle diameter, being approximately 30% larger in fraction 8 compared to fraction 1.     

The relationship between cell injury and osmotic volume reduction: III. Freezing injury and frost resistance in winter wheat

In order to distinguish between several possible mechanisms of frost hardening in winter wheat (Triticum aestivum L.) cells from two hardy and two tender cultivars were plasmolyzed in CaCl₂ solution at room temperature and cell volumes estimated by microscopic examination. Analyses of Boyle-van't Hoff plots of these data reveal that all cells from cultivars progressively increase their intracellular solute concentration up to 20 days hardening. This increase, which we had predicted from published calorimetric data to be the sole mechanism of hardening explained less than half of the increase in hardening seen in the most hardy cultivar, Kharkov. Hardening also increased the osmotically inactive volume.At CaCl₂ concentrations greater than 5%, plasmolyzed protoplasts departed further from the Boyle-van't Hoff prediction, remaining larger than expected until some higher concentration of CaCl₂, where protoplast volume again sharply decreased. In all cultivars except hardened Kharkov, the concentration of CaCl₂ producing this abrupt volume decrease had a freezing point corresponding to the killing temperature. If this concentration was exceeded during plasmolysis, then the protoplasts burst during deplasmolysis at some volume less than their original volume.We interpret these data to mean that, in addition to the often described hardening mechanism of increased cell solute and water binding, winter wheat shows a third mechanism, a mechanical resistance to protoplast shrinkage which produces volumes larger than those predicted during osmotic stress. The resisting element appears to be the plasma membrane itself. Shrinkage brings the membrane under compressive stress, developing tangential pressure within it. Cell injury occurs when the cell membrane area has been reduced to the point at which irreversible loss of membrane material is inevitable. Cell death occurs during deplasmolysis when the protoplast bursts because its membrane contains insufficient material to subtend the area of the cell wall.Of the cultivars tested, hardened Kharkov was unique in avoiding injury. Hardened Kharkov was injured only after the volume inflection had been greatly exceeded. Refractile droplets of lipid appeared in the cytoplasm of hardened Kharkov protoplasts during plasmolysis but disappeared during deplasmolysis suggesting that hardy Kharkov was able reversibly to store membrane lipids in cytoplasmic vesicles and return them to the membrane on deplasmolysis.     

Osmotic loading of in situ chondrocytes in their native environment

Changes in the osmotic environment cause changes in volume of isolated cells and cells in tissue explants, and the osmotic environment becomes hypotonic in cartilage diseases such as osteoarthritis (OA). However, it is not known how cells respond to a hypotonic osmotic challenge when situated in the fully intact articular cartilage. A confocal laser scanning microscope was used to image chondrocytes of intact rabbit patellae in an isotonic (300 mOsm) and hypotonic (172 mOsm) immersion medium. Cell volumes were calculated before and 5, 15, 60, 120 and 240 minutes after the change in saline concentration. Local tissue strains and swelling of the entire tissue were estimated from the relative movements of cells and displacements of single cells, respectively. Cell volumes increased rapidly (< or = 5 minutes, p<0.05) by approximately 22%, after which they remained constant for an hour (p>0.05). However, two and four hours post the hypotonic challenge, cell volumes were statistically greater (p<0.05) than those at all earlier time points, and swelling of the entire tissue continued throughout the four hour loading period. The results of our study suggest that osmotic loading induced volume changes of in situ chondrocytes in their native environment occur quickly and continue for hours. Understanding the behaviour of cells in their native environment provides novel insigth into the cell mechanics in ostearthritic joints and so may help understand the onset and progression of this disease.     

Change of some quantitative indices of the proximal and the distal growth cartilage of the human tibia during the period of the prenatal osteogenesis

The zones of the tibial growth cartilage of human fetuses (6th lunar month), premature newborns (7th-8th lunar month) and mature newborns were micrometrically studied. The zone thickness, the chondrocyte number in a certain area and in the cell columns as well, and the volume density of the chondrocytes were determined. The obtained quantitative data show that the three indices undergo certain changes during the three age periods of the prenatal osteogenesis studied. They differ in the proximal and in the distal cartilages as well. The fetal tibia is found to be growing more intensively at its distal end. The quantitative processes within the growth cartilages of the tibia are more active during the earlier stages of the intrauterine period while the qualitative processes are found to be more active at the end of the intrauterine period.     

Osmotic significance of glycerol accumulation in exponentially growing yeasts.

Natural-abundance 13C-nuclear magnetic resonance spectroscopy has shown glycerol to be the major osmotically significant low-molecular-weight solute in exponentially growing, salt-stressed cells of the yeasts Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Debaromyces hansenii. Measurement of the intracellular nonosmotic volume (i.e., the fraction of the cell that is osmotically unresponsive) by using the Boyle-van't Hoff relationship (for nonturgid cells, the osmotic volume is directly proportional to the reciprocal of the external osmotic pressure) showed that the nonosmotic volume represented up to 53% of the total cell volume; the highest values were recorded in media with maximum added NaCl. Determinations of intracellular glycerol levels with respect to cell osmotic volumes showed that increases in intracellular glycerol may counterbalance up to 95% of the external osmotic pressure due to added NaCl. The lack of other organic osmotica in 13C-nuclear magnetic resonance spectra indicates that inorganic ions may constitute the remaining component of intracellular osmotic pressure.     

Osmotic significance of glycerol accumulation in exponentially growing yeasts

Natural-abundance 13C-nuclear magnetic resonance spectroscopy has shown glycerol to be the major osmotically significant low-molecular-weight solute in exponentially growing, salt-stressed cells of the yeasts Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Debaromyces hansenii. Measurement of the intracellular nonosmotic volume (i.e., the fraction of the cell that is osmotically unresponsive) by using the Boyle-van't Hoff relationship (for nonturgid cells, the osmotic volume is directly proportional to the reciprocal of the external osmotic pressure) showed that the nonosmotic volume represented up to 53% of the total cell volume; the highest values were recorded in media with maximum added NaCl. Determinations of intracellular glycerol levels with respect to cell osmotic volumes showed that increases in intracellular glycerol may counterbalance up to 95% of the external osmotic pressure due to added NaCl. The lack of other organic osmotica in 13C-nuclear magnetic resonance spectra indicates that inorganic ions may constitute the remaining component of intracellular osmotic pressure.