Cytoplasmic Volume Fraction Research Articles

Морфологические особенности в тканях грудинно-реберного комплекса у детей при воронкообразной деформации грудной клетки

Objective: to evaluate the reactive changes in chondrocytes of the cartilage of the sterno-costal complex with its morphological characteristics of the components of the sterno-costal complex in children with funnel chest deformity. Methods. This article presents the material of morphological studies of the biopsy material of the costal-cartilage part, taken from 66 children (3-18 years old). For histological examination, intraoperatively removed tissue pieces from the anatomically modified cartilaginous elements of the sternum-costal complex of the anterior chest wall during thoracoplasty, were fixed in an aldehyde-osmium solution, and were placed in aralditis. A quantitative study of reactive changes in chondrocyte cartilage was carried out. Results. The quantitative studies of changes in the chondrocytes of the cartilage of the sterno-costal complex indicate pronounced changes in the chondrocytes of the surface zone with a decrease in the nuclear-cytoplasmic index with a more significant increase in the volume fraction of cytoplasm. The obtained results indicate a rather high degree of severity of compensatory reactions of the cartilage tissue of the sterno-costal complex in children. Conclusions. Morphological studies show that properly selected preoperative administration of patients with funnel chest deformity leads to a significant prevention of residual effects of connective tissue dysplasia, which also reduces the likelihood of the formation of gross morphological changes in the structures of the sterno-costal complex.

Myocardial function after polarizing versus depolarizing cardiac arrest with blood cardioplegia in a porcine model of cardiopulmonary bypass.

OBJECTIVESPotassium-based depolarizing St Thomas' Hospital cardioplegic solution No 2 administered as intermittent, oxygenated blood is considered as a gold standard for myocardial protection during cardiac surgery. However, the alternative concept of polarizing arrest may have beneficial protective effects. We hypothesize that polarized arrest with esmolol/adenosine/magnesium (St Thomas' Hospital Polarizing cardioplegic solution) in cold, intermittent oxygenated blood offers comparable myocardial protection in a clinically relevant animal model.METHODSTwenty anaesthetized young pigs, 42 ± 2 (standard deviation) kg on standardized tepid cardiopulmonary bypass (CPB) were randomized (10 per group) to depolarizing or polarizing cardiac arrest for 60 min with cardioplegia administered in the aortic root every 20 min as freshly mixed cold, intermittent, oxygenated blood. Global and local baseline and postoperative cardiac function 60, 120 and 180 min after myocardial reperfusion was evaluated with pressure–conductance catheter and strain by Tissue Doppler Imaging. Regional tissue blood flow, cleaved caspase-3 activity, GRK2 phosphorylation and mitochondrial function and ultrastructure were evaluated in myocardial tissue samples.RESULTSLeft ventricular function and general haemodynamics did not differ between groups before CPB. Cardiac asystole was obtained and maintained during aortic cross-clamping. Compared with baseline, heart rate was increased and left ventricular end-systolic and end-diastolic pressures decreased in both groups after weaning. Cardiac index, systolic pressure and radial peak systolic strain did not differ between groups. Contractility, evaluated as dP/dtmax, gradually increased from 120 to 180 min after declamping in animals with polarizing cardioplegia and was significantly higher, 1871 ± 160 (standard error) mmHg/s, compared with standard potassium-based cardioplegic arrest, 1351 ± 70 mmHg/s, after 180 min of reperfusion (P = 0.008). Radial peak ejection strain rate increased and the load-independent variable preload recruitable stroke work was increased with polarizing cardioplegia after 180 min, 64 ± 3 vs 54 ± 2 mmHg (P = 0.018), indicating better preserved left ventricular contractility with polarizing cardioplegia. Phosphorylation of GRK2 in myocardial tissue did not differ between groups. Fractional cytoplasmic volume in myocytes was reduced in hearts arrested with polarizing cardioplegia, indicating reduction of cytoplasmic oedema.CONCLUSIONSPolarizing oxygenated blood cardioplegia with esmolol/adenosine/magnesium offers comparable myocardial protection and improves contractility compared with the standard potassium-based depolarizing blood cardioplegia.

Protein Diffusion in the E. coli Cytoplasm and Periplasm under Osmotic Stress

The E. coli cytoplasm contains the nucleoid, ribosomes, mRNA and tRNA, and numerous globular proteins. Much of the water is associated with biopolymer surfaces. Facile diffusion of globular proteins within this complex medium is essential to normal cellular biochemical activity and growth. Remarkably, E. coli grows in minimal medium over a wide range of external osmolalities (0.03-1.8 Osm). The mean cytoplasmic biopolymer volume fraction for such adapted cells varies from phi = 0.16 at 0.10 Osm to phi = 0.36 at 1.45 Osm. For cells grown at 0.28 Osm, a similar range of is obtained by plasmolysis (sudden osmotic upshift) using NaCl as the external osmolyte, after which the cellular response is passive loss of cytoplasmic water. Using fluorescence recovery after photobleaching (FRAP), we measure the effective axial diffusion coefficient DGFP of green fluorescent protein in the cytoplasm of live E. coli cells as a function of for both plasmolyzed and adapted cells. For adapted cells the median diffusion coefficient DGFPm decreases by only a factor of 2.1 as phi increases from 0.16 to 0.36. In sharp contrast, for plasmolyzed cells DGFPm decreases by a factor of 70 as phi increases from 0.16 to 0.33. Clearly GFP diffusion is not determined by phi alone. By comparison with quantitative models, we show that the plasmolysis data cannot be explained by simple crowding theory in a homogeneous medium. We will also report on measurements of time-resolved fluorescence anisotropy of GFP in the cytoplasm, diffusion of RNA polymerase in the cytoplasm, and diffusion of GFP in the periplasm of E. coli. Time lapse measurements monitor the recovery of cell volume and GFP diffusion after plasmolysis, which may be a key determinant of the time scale of the recovery of growth.

Cytoplasmic Protein Mobility in Osmotically Stressed Escherichia coli

Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction ((phi)) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar phi range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D(GFP)) in the cytoplasm of E. coli cells as a function of (phi) for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D(GFP) (D(GFP)(m)) decreases by a factor of 70 as (phi) increases from 0.16 to 0.33. In sharp contrast, for adapted cells, D(GFP)(m) decreases only by a factor of 2.1 as (phi) increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by (phi) alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.

Adaptations of the Rat Vagina in Pregnancy to Accommodate Delivery

To characterize ultrastructural changes in the rat vagina in pregnancy, delivery, and postpartum, focusing on collagen architecture and smooth muscle cell morphology. The vagina of four virgin, four midpregnant, four late pregnant, four immediate, and four late post-vaginal-delivery rats were examined by transmission electron microscopy. Images were classified into one of four categories based on collagen fibril area fraction, with group 1 containing the highest number of collagen fibers per unit area and group 4 containing the lowest. Smooth muscle cells were characterized into three cell types ("synthetic," "intermediate," and "contractile") based on the volume fraction of cytoplasm occupied by organelles compared with myofibrils. Quantitative analysis demonstrated that 76% of collagen fibers in virgin rats were categorized as group 1 or 2 compared with 49% in midpregnant, 40% in late pregnant, and 23% in immediate postpartum animals (P=0.006). Late postpartum tissue seemed similar to virgin tissue (77%). Midpregnant (37%), late-pregnant (34%) and immediate postpartum animals (43%) contained a higher proportion of synthetic smooth muscle cells compared with virgins (20%) and late postpartum animals (21%) (P=.02). Contractile smooth muscle cells predominated in virgin (64%) and late postpartum animals (70%) compared with midpregnant (42%), late pregnant (50%) and immediate postpartum (50%, P=.05). In pregnancy, collagen fiber area decreased while smooth muscle cells transformed from a contractile to a synthetic phenotype. The late postpartum period returned to prepregnant levels for both collagen and smooth muscle cell morphologies. It is likely that these changes represent adaptations to minimize trauma to the vagina during passage of the fetus.

Crowding and Confinement Effects on Protein Diffusion In Vivo

The first in vivo measurements of a protein diffusion coefficient versus cytoplasmic biopolymer volume fraction are presented. Fluorescence recovery after photobleaching yields the effective diffusion coefficient on a 1-mum-length scale of green fluorescent protein within the cytoplasm of Escherichia coli grown in rich medium. Resuspension into hyperosmotic buffer lacking K+ and nutrients extracts cytoplasmic water, systematically increasing mean biopolymer volume fraction, <phi>, and thus the severity of possible crowding, binding, and confinement effects. For resuspension in isosmotic buffer (osmotic upshift, or Delta, of 0), the mean diffusion coefficient, <D>, in cytoplasm (6.1 +/- 2.4 microm2 s(-1)) is only 0.07 of the in vitro value (87 microm2 s(-1)); the relative dispersion among cells, sigmaD/<D> (standard deviation, sigma(D), relative to the mean), is 0.39. Both <D> and sigmaD/<D> remain remarkably constant over the range of Delta values of 0 to 0.28 osmolal. For a Delta value of > or =0.28 osmolal, formation of visible plasmolysis spaces (VPSs) coincides with the onset of a rapid decrease in <D> by a factor of 380 over the range of Delta values of 0.28 to 0.70 osmolal and a substantial increase in sigmaD/<D>. Individual values of D vary by a factor of 9 x 10(4) but correlate well with f(VPS), the fractional change in cytoplasmic volume on VPS formation. The analysis reveals two levels of dispersion in D among cells: moderate dispersion at low Delta values for cells lacking a VPS, perhaps related to variation in phi or biopolymer organization during the cell cycle, and stronger dispersion at high Delta values related to variation in f(VPS). Crowding effects alone cannot explain the data, nor do these data alone distinguish crowding from possible binding or confinement effects within a cytoplasmic meshwork.

Morphometric analysis of primary graft non‐function in liver transplantation

Primary graft non-function (PNF) is a life-threatening condition that is thought to be the consequence of microcirculation injury. The aim of the present study was to assess, with a computerized morphometric model, the morphological changes at reperfusion in liver biopsy specimens from patients who developed PNF after liver transplantation. Biopsy specimens were obtained at maximum ischaemia and at the end of reperfusion. Morphology included many stereological parameters, such as volumes of all parenchymal components, surface density, size distribution and mean diameter of hepatocytes. Other variables examined were intensive care unit stay, degree of steatosis, serum liver function tests and ischaemic time. In the postoperative period, the PNF group showed elevated serum levels of alanine transferase, decreased daily rate of bile production and prothrombin activity. Blood lactates were significantly higher in the PNF group than in a control group. When comparing groups, the volumetric parameters related to hepatocytes and sinusoids and the surface densities of the hepatic cells showed an inverse relationship. At the end of reperfusion, in PNF group the volume fraction of hepatocyte cytoplasm was decreased; in contrast, the volume fraction of sinusoidal lumen was markedly increased. The cell profiles showed the same inverse trend: the surface density of the parenchymal border of hepatocytes was decreased in PNF when compared with the control group, while the surface density of the vascular border was increased. In the PNF group, the surface density of the sinusoidal bed was directly correlated with alanine transferase, daily rate of bile production, prothrombin activity and cold ischaemic time. The alterations in hepatic architecture, as demonstrated by morphometric analysis in liver transplant recipients that developed PNF, provide additional information that may represent useful viability markers of the graft to complement conventional histological analysis.

The effects of treatment with melatonin on the ultrastructure of mouse Leydig cells: a quantitative study.

Both the presence of receptors for gonadal steroids in the pineal gland and in vitro observations of direct action of melatonin upon Leydig cells, inhibiting testosterone secretion, indicate a direct connection between pineal gland and gonadal function. In the present study, we used a transmission electron microscope to analyze the morphologic parameters of Leydig cells from adult Swiss outbred white mice treated with daily subcutaneous injections of 100 micrograms of melatonin (N-acetyl, 5-methoxytryptamine), during 22 consecutive days, compared with sham-control animals which had only received the melatonin vehicle. The melatonin group of mice showed a decrease in nuclear volume and fractional nuclear volume; smooth and rough endoplasmic reticulum; mitochondria; and Golgi complex. Our data also showed an increase in cytoplasmic volume, fractional cytoplasmic volume, and lysosomes in these same animals. The results suggest that melatonin, directly or indirectly, alters the ultrastructure of mouse Leydig cells and possibly influences their secretory activity by inhibiting their capacity to secrete steroids.

Influence of X-ray on the autophagic-lysosomal system in rat pancreatic acini

Lysosomes have an important role in radiation injury of cells and tissues. Activation of autophagy is frequently observed in different types of pathological tissue degeneration. In radiation response it increases in some cases, and lysosomes are responsible for regulated degradation of the autophagic vacuoles. Lysosomes are also involved in ionizing radiation induced cell death. In apoptosis lysosomes degrade content of the phagocytotic vacuoles derived from engulfed apoptotic blebs. On the other hand lysosomal enzymes discharged from disintegrated cells have a key role in induction of necrotic changes. In this work we investigate autophagy and lysosomal protein degradation in the relatively radiation insensitive exocrine pancreatic acini in vivo and in vitro. Type of cell death induced by X-ray was also examined in relation to the changes of the lysosomal processes. In 5 h after 16 Gy in vivo whole body irradiation we observed significant increase in the cytoplasmic volume fraction of autophagic vacuoles and in the number of apoptotic cells in vivo. But in the acini isolated from irradiated rats we could not detect a change in the lysosomal degradation of intracellular proteins. Therefore irradiation probably influences the autophagy in an earlier step than lysosomal degradation. Extended necrotic lesions were not observed in vivo as long as 48 h. Isolated pancreatic acini usually contain more autophagic vacuoles than in vivo, but we could not observe additional increase in autophagy after 8 Gy, in vitro irradiation. Lysosomal degradation of intracellular proteins was also unaltered after 8 Gy, in vitro irradiation. Other biochemical functional parameters of the isolated pancreatic acini, like protein synthesis and amylase secretion were not changed either after 8 Gy, in vitro X-ray treatment. These results indicate that pancreatic acinar cells in vitro have a high tolerance to irradiation. The observed in vivo radiation induced changes of the exocrine pancreas are possibly indirectly induced by injuries of more sensitive mechanisms.

Quantitative in vivo measurement of glutathione in Arabidopsis cells.

A new, non-destructive assay is described to quantify cytoplasmic glutathione (GSH) levels in vivo in single cells or populations of cells from Arabidopsis suspension cultures. Cytoplasmic GSH was labelled with monochlorobimane (MCB) in situ to give a fluorescent GSH-bimane (GSB) conjugate. At low (10-100 microM) concentrations of MCB, labelling was mediated by a glutathione S-transferase, which confers specificity for GSH. HPLC analysis of MCB-labelled low molecular-weight thiols showed that the assay measures the total GSH pool, including the oxidized glutathione. The progress curve for the labelling could be described using Michaelis-Menten kinetics with an apparent KM of 40 microM and Vmax of 470 micromol lcyt -1 min-1. There was no evidence for de novo synthesis of GSH during the labelling period of 2 h, suggesting that control of GSH synthesis is not mediated by feedback control of gamma-glutamylcysteine synthetase in this system. The total cellular level of GSH was calculated from the plateau value of the progress curve, after appropriate calibration, as 830-942 nmol g-1 FW. The volume fraction of cytoplasm was measured from serial optical sections of bimane-labelled cells collected by confocal laser scanning microscopy (CLSM) with excitation 442 nm, or two-photon laser scanning microscopy (TPLSM) with excitation 770 nm. A value of 42 +/- 3% cytoplasm was determined by manual segmentation, and a value of 37 +/- 2% using stereological techniques. Using these figures, values for cytoplasmic [GSH] were estimated to be between 2.7 +/- 0.3 and 3.2 +/- 0.3 mM for cell populations. In addition, measurement of GSH levels in individual cells using CLSM and TPLSM gave values of 3.0 +/- 0.5 and 3.5 +/- 0.7 mM, respectively.

Relationship of glycosaminoglycan and matrix changes to vascular smooth muscle cell phenotype modulation in rabbit arteries after acute injury

Purpose: The phenotype of vascular smooth muscle cells (SMCs) is altered in several arterial pathologies, including the neointima formed after acute arterial injury. This study examined the time course of this phenotypic change in relation to changes in the amount and distribution of matrix glycosaminoglycans. Methods: The immunochemical staining of heparan sulphates (HS) and chondroitin sulphates (CS) in the extracellular matrix of the arterial wall was examined at early points after balloon catheter injury of the rabbit carotid artery. SMC phenotype was assessed by means of ultrastructural morphometry of the cytoplasmic volume fraction of myofilaments. The proportions of cell and matrix components in the media were analyzed with similar morphometric techniques. Results: HS and CS were shown in close association with SMCs of the uninjured arterial media as well as being more widespread within the matrix. Within 6 hours after arterial injury, there was loss of the regular pericellular distribution of both HS and CS, which was associated with a significant expansion in the extracellular space. This preceded the change in ultrastructural phenotype of the SMCs. The glycosaminoglycan loss was most exaggerated at 4 days, after which time the HS and CS reappeared around the medial SMCs. SMCs of the recovering media were able to rapidly replace their glycosaminoglycans, whereas SMCs of the developing neointima failed to produce HS as readily as they produced CS. Conclusions: These studies indicate that changes in glycosaminoglycans of the extracellular matrix precede changes in SMC phenotype after acute arterial injury. In the recovering arterial media, SMCs replace their matrix glycosaminoglycans rapidly, whereas the newly established neointima fails to produce similar amounts of heparan sulphates. (J Vasc Surg 2001;33:155-64.)

Autophagy in the epithelial cells of murine seminal vesicle in vitro. Formation of large sheets of nascent isolation membranes, sequestration of the nucleus and inhibition by wortmannin and 3-ethyladenine.

The spontaneous autophagic activity in epithelial cells of isolated tissue slices of murine seminal vesicle is strongly enhanced by short (5 min) pretreatment in a medium containing 0.03% Triton X-100. In addition to the significant increase in the cytoplasmic volume fraction and the mean size of autophagic vacuoles, the appearance of shorter or longer smooth membrane pairs located between cisterns of rough endoplasmic reticulum (RER) and in the vicinity of nucleus is also greatly stimulated. Their morphological features observed after application of various fixation methods, freeze-substitution and freeze-fracture techniques show that they are unclosed nascent isolation membranes, representing a unique class of intracellular membranes. They may grow around the nucleus, leading to its complete autophagic sequestration and degradation, which is observed here for the first time. Treatment with 3-methyladenine or wortmannin inhibits the formation of autophagosomes, leading to their regression with a halving time of 7 min. In contrast, these inhibitors cause extremely fast shrinking of nascent isolating membranes, leading to their complete disappearance within 7 min. We propose that the early events of autophagy involve three main steps: initiation, growth and closure, and suggest that the growth of nascent isolation membranes is reversible i.e. the membranes may be subject to disassembly before their closure is completed. Bending and closure of the isolation membrane and the stability of neighbouring cellular structures appear as important determinants of size regulation. These early steps of autophagy are good candidates for very fast accommodation to changing conditions and subtle regulation by phosphoinositide kinases as indicated by wortmannin sensitivity.

Down-regulation of liver RNA breakdown by turpentine administration in the starved rat: autophagy and relevant factors.

To determine whether the inhibition of RNA breakdown observed in ad libitum fed rats 24 h after turpentine administration still occurs in inflamed rats fasted for 24 h and to examine the mechanism and factors involved. RNA breakdown was measured during cyclic in situ perfusion of livers by the accumulation of [14C] cytidine after in vivo RNA labelling. Autophagic activity was determined by the morphometric analysis of lysosomal structures. The decrease in RNA breakdown (53%) observed in the inflamed rats was accompanied by a 38% drop in the fractional cytoplasmic volume of initial and digestive autophagic vacuoles. Among amino acids, only the portal levels of glutamate were significantly enhanced by 83%. In vivo suppression of glucocorticoid activity using RU 38486 in inflamed rats did not affect the inhibition of RNA breakdown. The results show that turpentine-induced inflammation in fasted rats inhibits RNA degradation as well as autophagy and that glucocorticoids do not seem to be involved.

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

Purpose: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. Methods: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and 3H-thymidine incorporation in SMCs in culture. Results: Arterial HSPGs (680 μg) reduced neointimal formation by 35% at 14 days after injury ( P = .029), whereas 2000 μg of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 μg/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1% ± 13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1% ± 15.1%). In contrast, 100 μg/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9% ± 14.6%, controls 35.9% ± 12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID 50 value of 0.4 μg/mL compared with a value of 14 μg/mL for Enoxaparin ( P < .01). Conclusion: When used periadventitially in the rabbit arterial injury model, natural arterial HSPGs are effective inhibitors of neointimal formation. In vitro, the HSPGs maintain SMCs in a quiescent state by inhibiting phenotypic change and DNA synthesis. This study suggests that HSPGs may be a natural agent for the treatment of clinical restenosis. (J Vasc Surg 1998;28:308-18)

Age-dependent decrease in the activity of succinic dehydrogenase in rat CA1 pyramidal cells: a quantitative cytochemical study

A computer-assisted morphometric study has been carried out on the ultrastructure of perikaryal CA1 pyramidal cell mitochondria positive to the copper ferricyanide cytochemical reaction for succinic dehydrogenase (SDH) in rats of 3, 12 and 23 months of age. The cytoplasmic volume fraction occupied by the positive mitochondria (Volume density: V v), the number of organelles/μm 3 of CA1 pyramidal cell cytoplasm (Numerical density: N v) and the average mitochondrial volume ( V) were automatically calculated by means of computer-assisted morphometry. V v was significantly decreased in 23-month-old animals versus the other age groups. N v was unchanged between 3 and 12 months of age, but was decreased to a significant extent in old animals. V did not undergo significant changes in the three age groups taken into account. In the old animals the percent of organelles smaller than 0.16 μm 3 is above 20%, while in the young and adult groups the same size of mitochondria accounts for 7 and 3%, respectively. Thus, a reduction in the number of medium sized organelles appears to be responsible for the decrease in V v due to age. Since SDH activity is known to support maximum rates of respiration, quantitative estimation of the active mitochondria provides information on the metabolic competence of the cells investigated when energy demand is high. In this context, our present findings document that a significant impairment in the efficiency to match actual energy provisions occurs in old CA1 pyramidal cells.

Glucagon administration in vivo stimulates hepatic RNA and protein breakdown in fed and fasted rats

Liver RNA and protein breakdown rates were measured simultaneously in fed and in 24 h-fasted rats during a short-term cyclic perfusion, 1 h after an intraperitoneal injection of glucagon or of saline. RNA was labelled in vivo by an intraperitoneal injection of [6-14C]orotic acid, 60 h before the start of the perfusion. The accumulation of radioactive cytidine and valine in the perfusion medium for 15 min was used to determine RNA breakdown and proteolysis respectively. The portal glucagon/insulin ratio was significantly higher in the fasted glucagon-treated rats than in their fed counterparts. Although glucagon administration significantly increased RNA and protein degradation rates in the fasted and in the fed groups, the effect was greater after 24 h of starvation. The relationship between these biochemical changes and the alterations of the hepatocyte lysosomal system was investigated by determining the fractional cytoplasmic volume of lysosomal structures (autophagic vacuoles and dense bodies) by morphometry in the fasted glucagon-treated rats and in their controls. Hyperlucagonaemia significantly enhanced the relative volume of autophagic vacuoles without affecting that of dense bodies. The results showed that hyperglucagonaemia induced in vivo stimulated both liver RNA and protein breakdown and that this effect was modulated by the nutritional status of the rats.

Hepatocyte Spheroids: Prolonged Hepatocyte Viability for in Vitro Modeling of Nongenotoxic Carcinogenesis

Hepatocyte Spheroids: Prolonged Hepatocyte Viability for in Vitro Modeling of Nongenotoxic Carcinogenesis. Roberts, R. A., and Soames, A. R. (1993). Fundam. Appl. Toxicol. 21, 149-158. To explore peroxisome proliferator-perturbed hepatocyte growth regulation, robust in vitro models of liver are required. This has always posed a problem since isolated hepatocytes show a rapid loss of viability and differentiation status and cease to be useful after 3-4 days in culture. We now describe a model system in which rat hepatocytes are maintained as three-dimensional spheroids. The maintenance of hepatocyte viability and morphology in these cultures is considerably prolonged over that seen in monolayer culture and is comparable to that obtained by the use of collagen gels or dimethyl sulfoxide. The spheroid system is, however, free of any additives that may lead to artifact and free of excessive exogenous protein that may compromise subsequent analyses. Ultrastructural examination reveals extensive interhepatocyte junctional complexes and interdigitation of adjacent membranes together with the presence of bile cannalicular structures. Furthermore, hepatocytes maintained as spheroids retain expression of liver markers such as albumin and also retain their ability to respond to peroxisome proliferators: even after 12 days in culture, treatment with the peroxisome proliferator nafenopin causes a 4.5-fold increase in cytoplasmic volume fraction of peroxisomes. There is a concomitant induction of peroxisomal bifunctional enzyme and cytochrome P4504A, the enzyme markers associated with peroxisome proliferation. The spheroids also maintain expression of the peroxisome proliferator-activated receptor and preliminary data indicate that they are able to undergo replicative DNA synthesis in response to nafenopin. Hepatocyte spheroids will provide us with a model system for studying the early changes in rodent liver nongenotoxic carcinogenesis.

Hepatocyte Spheroids: Prolonged Hepatocyte Viability for in Vitro Modeling of Nongenotoxic Carcinogenesis

To explore peroxisome proliferator-perturbed hepatocyte growth regulation, robust in vitro models of liver are required. This has always posed a problem since isolated hepatocytes show a rapid loss of viability and differentiation status and cease to be useful after 3–4 days in culture. We now describe a model system in which rat hepatocytes are maintained as three-dimensional spheroids. The maintenance of hepatocyte viability and morphology in these cultures is considerably prolonged over that seen in monolayer culture and is comparable to that obtained by the use of collagen gels or dimethyl sulfoxide. The spheroid system is, however, free of any additives that may lead to artifact and free of excessive exogenous protein that may compromise subsequent analyses. Ultrastructural examination reveals extensive interhepatocyte junctional complexes and in terdigitation of adjacent membranes together with the presence of bile cannalicular structures. Furthermore, hepatocytes maintained as spheroids retain expression of liver markers such as albumin and also retain their ability to respond to peroxisome proliferators: even after 12 days in culture, treatment with the peroxisome proliferator nafenopin causes a 4.5-fold increase in cytoplasmic volume fraction of peroxisomes. There is a concomitant induction of peroxisomal bifunctional enzyme and cytochrome P4504A, the enzyme markers associated with peroxisome proliferation. The spheroids also maintain expression of the peroxisome proliferator-activated receptor and preliminary data indicate that they are able to undergo replicative DNA synthesis in response to nafenopin. Hepatocyte spheroids will provide us with a model system for studying the early changes in rodent liver nongenotoxic carcinogenesis.

An in vitro model of rodent nongenotoxic hepatocarcinogenesis

An in vitro model of liver in which rat hepatocytes are maintained as cocultures with nonparenchymal epithelial cells (NPC) derived from liver has been developed and characterized with respect to maintenance of hepatocyte viability and differentiated function. The system was then evaluated as a model for studying peroxisome proliferator-induced rodent liver nongenotoxic carcinogenesis. Within the coculture model, hepatocyte viability and morphology were maintained for 1 month or more within a system that is both easily accessible for microscopic examination and is free of any additives that may lead to artifacts. Even after 1 month or more, hepatocyte cocultures retained expression of the constitutive liver marker albumin. In addition, they maintained the ability to show induction of the peroxisome proliferator-inducible enzymes peroxisomal bifunctional enzyme (PBE) and cytochrome P450IVA1 in response to the peroxisome proliferator nafenopin. After 4 weeks, NPC cocultures showed a six- and a fourfold induction of PBE and cytochrome P450IVA1 expression, respectively, which compared well with the three-and fivefold induction seen in freshly isolated cells. This was paralleled by an increase in the cytoplasmic volume fraction of peroxisomes averaging eightfold. Interestingly, great heterogeneity was exhibited between adjacent hepatocytes in terms of the degree of peroxisome proliferation, a finding reflected by immunocytochemical staining which indicated heterogeneity in the level of expression of the peroxisome proliferator-inducible enzymes. Other cell lines representing different tissue types, morphologies, and species were also examined for their ability to support hepatocyte survival but were found to be ineffective, with the exception of a bovine corneal endothelial cell line. This line supported hepatocyte survival and maintenance of differentiated function but to a lesser extent than that observed with NPC. Ultrastructural examination of NPC cocultures revealed extensive interhepatocyte junctional complexes and interdigitation of adjacent membranes together with the presence of bile canalicular structures. There were no junctional complexes between the hepatocytes and the supporting feeder cells with any contact being limited to a close association of the hepatocytes with the extracellular matrix presumably produced by the NPC. The data demonstrate that hepatocytes maintained in vitro within an NPC coculture system retain differentiated function and the ability to respond to the peroxisome proliferator class of nongenotoxic carcinogens. Cocultures will provide us with a model system for the study of changes in hepatocyte growth regulation during rodent liver nongenotoxic carcinogenesis.

Cotyledon cell development in Arabidopsis thaliana during reserve deposition

Cotyledon cell development in Arabidopsis thaliana L. during reserve deposition has been analyzed qualitatively and quantitatively. Development has been related to the previously defined time scale for Arabidopsis, hours after flowering. Between 144 and 216 h after flowering the major cell changes in the cotyledon are an increase in the cell volume, a decrease in the volume fraction of cytoplasm and plastids, and an increase in lipid and vacuole volume fractions. The endoplasmic reticulum and dictyosome volume fractions are high during early reserve formation (144 – 168 h after flowering) but decrease significantly thereafter. Evidence as to the origin of the storage lipid is inconclusive, although a dual involvement of plastids and rough endoplasmic reticulum is a likely theory. The 3-nm lipid body membrane, which allows the bodies to retain their individuality during accumulation, is probably a half-unit biological membrane, derived from closely associated rough endoplasmic reticulum cisternae. Much of the evidence obtained in this study indicates that both the endoplasmic reticulum and dictyosomes are involved in protein synthesis and transport to the vacuole. The accumulation of reserves occurs in a well-defined and relatively short period during late embryogenesis (144–216 h after flowering). Key words: Arabidopsis, cotyledons, embryogenesis, reserve deposition, stereology.