A morphometric study on growth of bile canaliculi after partial hepatectomy

19-33 h after partial hepatectomy there is a time-dependent increase of the bile canalicular luminal volume, of the total length of bile canaliculi, and of their non-microvillous, smooth surface, when measured per unit volume of lobular parenchyma. On average the luminal volume fraction is increased by a factor of 2.8, the length per volume by a factor of 1.4, and the density of smooth surface by a factor of 2.0, when compared with sham-operated controls. On the other hand, the volume and surface density of bile canalicular microvilli are only slightly increased after partial hepatectomy. These findings are interpreted as indicating disproportional growth of bile canaliculi which is due predominantly to the formation of new, at first non-microvillous, membrane.     

Changes in cell surface primary cilia and microvilli concurrent with measurements of fluid flow across the rabbit corneal endothelium ex vivo

Primary cilia and microvilli have been reported on the mammalian rabbit corneal endothelium but their relationship to cell function is undefined. Six corneas from healthy 2 kg female albino rabbits were glutaraldehyde-fixed post mortem (15:00 h) or twelve corneal stroma-endothelial preparations incubated at 37 degrees C under an applied hydrostatic pressure of 20 cm H2O for 4 h prior to fixation. The corneal endothelium was assessed by quantitative scanning electron microscopy. Cells fixed immediately post mortem were decorated with small stubby microvilli (average 21 +/- 13/100 micron 2), and only 25% of the cells were decorated with primary cilia having an average length of 2.44 +/- 1.56 microns. Following 4 h ex vivo incubation with a phosphate-buffered Ringer solution, conspicuous microvilli developed to an average density of 40 +/- 19/100 micron 2 and primary cilia were found on 12% of the cells, having on average length of 2.27 +/- 1.38 microns. Following 4 h incubation in a bicarbonate-buffered Ringer solution, small stubby microvilli developed to a density of 49 +/- 18/100 micron 2, and 40% of the cells showed primary cilia with an average length of 4.31 +/- 1.93 microns; the net trans-endothelial fluid flow in the latter set was 60% greater. These studies indicate that the primary cilia on corneal endothelial cells might be responsive to fluid flow, but that mild mechanical and/or chemical stress could also be the cause of the change since the elaboration of primary cilia can be accompanied by microvilli as well.     

Hepatic organic anion transport kinetics and bile flow during short-term total parenteral nutrition in the rabbit

Plasma disappearance of sulfobromophthalein (BSP) after an intravenous bolus (5 mg/kg) was determined in six lab chow-fed (LCF) rabbits and in six rabbits maintained on total parenteral nutrition (TPN) for 5 days. A common bile duct cannula enabled measurements of bile flow and biliary BSP excretion. Compartmental analysis of the biexponential plasma disappearance curve yielded three fractional transfer rates, plasma to liver (hepatic uptake), liver to plasma (reflux), and liver to bile (canalicular excretion). The transfer rates for hepatic uptake were 0.253 +/- 0.061/min for LCF and 0.147 +/- 0.040/min for TPN (P less than 0.01) and for the canalicular excretion of BSP were 0.038 +/- 0.019/min for LCF and 0.019 +/- 0.002/min for TPN (P less than 0.05). Model-computed rates for BSP excretion in bile over 60 min were lower with TPN (61%) than with LCF (80%); the measured excretory rates were 53% for TPN rabbits and 75% of injected dose for LCF animals. Basal biliary flow was reduced by 50% in the TPN group. With a two-compartmental model, assuming two pools and three transfer rates, we have demonstrated for the first time significant decreases in hepatic uptake and canalicular excretion of the organic anion BSP during TPN. A decrease in hepatic blood flow due to the enteral fast of TPN could have contributed in part to the decreased hepatic uptake. But, because the second exponent of the biexponential curve is independent of hepatic blood flow, the decrease in liver to bile transfer rate is a true approximation of a diminished canalicular excretory capacity during TPN. It is concluded that the movement of organic anions along the hepatic BSP/bilirubin transport system is impaired early during TPN.     

Cellular composition of the cyclic corpus luteum of the cow

The cellular composition of CL from 6 cows on approximately Day 12 of the oestrous cycle, after synchronization with cloprostenol, was studied by ultrastructural morphometry. Point-count measurements of volume density (mean +/- s.d.) showed that large luteal cells occupied 40.2 +/- 7.0% of the luteal tissue, and small luteal cells 27.7 +/- 6.3%. Of the total of 393.4 +/- 52.0 x 10(3) cells per mm3 of luteal tissue, large luteal cells made up only 3.5% and small luteal cells 26.7%, a ratio of 1:7.6. Endothelial cells/pericytes, at 52.3%, were the most numerous cell type. The mean volume per large luteal cell was 29.6 +/- 6.3 x 10(3) microns 3, while that of small luteal cells was 2.7 +/- 0.4 x 10(3) microns 3. In spherical form, these volumes would represent mean diameters of 38.4 microns and 17.2 microns respectively, and are consistent with published measurements on dispersed luteal cells. However, the values for cell numbers are much higher than published values based on luteal tissue dispersion, suggesting that dispersion may result in substantial and possibly selective losses of luteal cells.     

An investigation of the presence of ultramicrocells in natural mineral water

The presence of 'ultramicrocells' in natural mineral water, capable of passing through a 0.2 micron filter, has been demonstrated. Filters allowing the greatest proportion of viable (culturable) cells to pass ranked in the order, 0.4 micron polycarbonate (5.02%) > 0.2 micron polycarbonate (0.02%) > or = 0.45 micron cellulose nitrate (0.02%) > 0.2 micron cellulose acetate (< 0.002%). Following incubation for 4 d at 22 degrees C, viable counts in filtered mineral water increased from < 2-8.7 x 10(2) cfu ml-1(-2).8 x 10(4)-1.9 x 10(6) cfu ml-1. Successive filtration/incubation cycles of mineral water increased the proportion of cells passing through a 0.2 micron cellulose acetate filter from < 0.003% to 0.11% and 0.69%, suggesting selection for 'ultramicrocells'. Cells isolated from this process and grown on liquid R2A medium were thin, Gram-negative rods, of 0.15-0.40 micron wide and 0.50-6.20 microns long. Membrane filtration techniques used for pathogen detection in mineral waters will not retain all the cells present. If pathogens are able to form ultramicrocells, these may go undetected.     

Stereologic estimates of glomerular volume in unilaterally nephrectomized rats

OBJECTIVE: To quantitatively evaluate the changes in glomeruli in unilaterally nephrectomized adult rats by unbiased stereologic techniques. STUDY DESIGN: Adult rats received unilateral nephrectomy (UN) or a sham operation. Four weeks later the remnant kidneys were fixed and sampled by the fractionator method. Consecutive sections were prepared and stained. Morphometric analyses were performed by a light microscope equipped with computer-assisted digitizer. RESULTS: The total glomerular volume increased by 51% (P < .01). The volume density of glomeruli did not change (5.58 +/- 0.22 x 10(-2) vs. 5.51 +/- 0.44 x 10(-2), P > .50). In contrast, the numerical densities of glomeruli decreased by 36% (P < .05). CONCLUSION: Glomerular enlargement, but not the increase in glomerular number contributed heavily to compensatory glomerular growth after UN in rats.     

Responses of enterocyte microvilli in experimental diabetes to insulin and an aldose reductase inhibitor (ponalrestat).

The small intestine of 12-week-old streptozotocin-diabetic rats was examined by light and transmission electron microscopy in order to study the effects of alternative treatments on microvillous morphology. Four groups were examined: untreated diabetic rats, insulin-treated diabetics and rats treated with an aldose reductase inhibitor (ponalrestat) given with and without insulin. Numbers and dimensions of microvilli at the apex of columnar absorptive epithelial cells (enterocytes) were estimated using stereological principles. Values were obtained for the organ as a whole as well as for different sites along its length. In the untreated diabetic intestine, the mean (standard error of mean) number of microvilli was 4.5 (0.8) x 10(12) with a total surface area of 1.9 (0.50) m2. On average, the microvilli were 1.1 (0.08) microns long, 104 (3.8) nm in diameter and packed on the villous surface at a density of 3400 (50) per 100 microns 2. Their length at least varied with intestinal location. Significant effects of insulin therapy were detected. In contrast, the study failed to find any significant effect of aldose reductase inhibition on any variable except microvillous packing density.     

Capillary and fiber geometry in rat diaphragm perfusion fixed in situ at different sarcomere lengths.

To determine the potential range of diaphragm sarcomere lengths in situ and the effect of changes in sarcomere length on capillary and fiber geometry, rat diaphragms were perfusion fixed in situ with glutaraldehyde at different airway pressures and during electrical stimulation. The lengths of thick (1.517 +/- 0.007 microns) and thin (1.194 +/- 0.048 microns) filaments were not different from those established for rat limb muscle. Morphometric techniques were used to determine fiber cross-sectional area, sarcomere length, capillary orientation, and capillary length and surface area per fiber volume. All measurements were referenced to sarcomere length, which averaged 2.88 +/- 0.08 microns at -20 to -25 cmH2O airway pressure (residual volume) and 2.32 +/- 0.05 microns at +20 to +26 cmH2O airway pressure (total lung capacity). The contribution of capillary tortuosity and branching to total capillary length was dependent on sarcomere length and varied from 5 to 22%, consistent with that shown previously for mammalian limb muscles over this range of sarcomere lengths. Capillary length per fiber volume [Jv(c,f)] was significantly greater at residual volume (3,761 +/- 193 mm-2) than at total lung capacity (3,142 +/- 118 mm-2) and correlated with sarcomere length [l; r = 0.628, Jv(c,f) = 876l + 1,156, P less than 0.01; n = 18]. We conclude that the diaphragm is unusual in that the apparent in situ minimal sarcomere length is greater than 2.0 microns.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.     

Coccidia of Brazilian mammals: Eimeria corticulata n. sp. (Apicomplexa: Eimeriidae) from the anteater Tamandua tetradactyla (Xenarthra: Myrmecophagidae) and Eimeria zygodontomyis n. sp. from the cane mouse Zygodontomys lasiurus (Rodentia: Cricetidae)

Feces from a specimen of Tamandua tetradactyla (Linn.) from Portel, Pará State, north Brazil, contained two different coccidial oocysts; one identified as Eimeria tamanduae Lainson 1968, and the other as a new species, described here as Eimeria corticulata n. sp. Oocysts of E. corticulata are ellipsoidal, 37.4 x 30.4 (31.2-43.7 x 23.7-35.0) microns, shape index (length/width) 1.2 (1.0-1.5). Oocyst wall 2.5-3.7 microns thick and composed of two layers; an outer thick, brown-yellow one with radial striations, and a thin inner smooth one: no visible micropyle. Oocyst residuum a large globule of about 10.7 x 10.3 microns, usually accompanied by a number of smaller attached globules. Sporocysts ellipsoidal, 21.0 x 11.0 (20.0-22.5 x 10.0-12.5) microns, with a conspicuous Stieda body; shape index 1.9 (1.6-2.2). Sporocyst residuum a small number of scattered granules: sporozoites 18.7 x 5.0 microns, with a large posterior refractile body. Eimeria zygodontomyis n. sp. is described in feces from Zygodontomys lasiurus (Lund) from the Serra dos Carajás, Pará. Oocysts ellipsoidal to cylindrical, 16.5 x 12.0 (13.7-18.7 x 11.2-12.3) microns, shape index 1.4 (1.2-1.5). Wall colorless, smooth, single-layered and about 0.6 micron thick: no micropyle. No oocyst residuum, but a polar granule of about 1.8 x 1.0 microns is sometimes present. Sporocysts ellipsoidal, 8.4 x 5.5 (7.5-8.7 x 5.0-6.2) microns, shape index 1.5 (1.4-1.7), with a thin colorless wall and a delicate Stieda body. Sporozoites enclose a compact residuum of about 2.5 x 3.7 microns.     

Three-dimensional reconstruction of alveoli in the rat lung for pressure-volume relationships

To determine alveolar pressure-volume relationships, alveolar three-dimensional reconstructions were prepared from lungs fixed by vascular perfusion at various points on the pressure-volume curve. Lungs from male Sprague-Dawley rats were fixed by perfusion through the pulmonary artery following a pressure-volume maneuver to the desired pressure point on either the inflation or deflation curve. Tissue samples from lungs were serially sectioned for determination of the volume fraction of alveoli and alveolar ducts and reconstruction of alveoli. Alveoli from lungs fixed at 5 cmH2O on the deflation curve (approximating functional residual volume) had a volume of 173 X 10(3) microns3, a surface area of 11,529 microns2, a mouth opening diameter of 72.7 microns, and a mean caliper diameter of 91.8 micron (SE). Alveolar shape changes during deflation from total lung capacity to residual volume was first (30 to 10 cmH2O) associated with little change in the diameter of the alveoli (102.7 +/- 2.4 to 100.3 +/- 3.3 microns). In the range overlapping normal breathing (10 to 0 cmH2O) there was a substantial decrease in diameter (100.3 +/- 3.3 to 43.3 +/- 2.3 microns). These measurements and others made on the relative changes in the dimensions of the alveolus suggest that the elastic network, particularly around the alveolar ducts, are predominant in determining lung behavior near the volume expansion limits of the lung while the elastic and surface tension properties of the alveoli are predominant in the volume range around functional residual capacity.     

Stereologic estimation of mean nuclear volume in well-differentiated adenocarcinoma and carcinoma in situ of the gallbladder

OBJECTIVE: To investigate mean nuclear volume of cells in well-differentiated adenocarcinomas (20 cases) and carcinoma in situ (20 cases) of the gallbladder by the principle of estimation of the volume of particles with arbitrary shapes. STUDY DESIGN: Hematoxylin and eosin-stained, 4-micron-thick, vertical sections from formalin-fixed, paraffin-embedded tissue blocks were analyzed by using a projection microscope with a 100:1 oil immersion objective (NA 1.3); the final magnification was 2,500:1. The measurements were carried out in 10 microscopic fields for each slide. Mean nuclear volume was obtained by the stereologic method of point-sampled intercepts for vertical sections. RESULTS: Mean nuclear volume in well-differentiated adenocarcinomas (127.67 +/- 46.95 micron 3) was significantly larger than in carcinoma in situ (69.17 +/- 15.74 micron 3) (P < .000001). CONCLUSION: Stereologic estimation of mean nuclear volume may be helpful in the discrimination of malignant and borderline lesions of the gallbladder.     

Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway

Polymeric IgA (pIgA) is transported by liver parenchymal cells (hepatocytes) from blood to bile via a receptor-mediated process. We have studied the intracellular pathway taken by a TEPC15 mouse myeloma pIgA. When from 1 microgram to 1 mg 125I-pIgA was injected into the saphenous vein of a rat, 36% was transported as intact protein into the bile over a 3-h period. The concentration of transported 125I-pIgA was maximal in bile 30-60 min after injection, and approximately 80% of the total 125I-pIgA ultimately transported had been secreted into bile by 90 min. A horseradish peroxidase-pIgA conjugate (125I-pIgA-HRP) was transported to a similar extent and with kinetics similar to that of unconjugated 125I-pIgA and was therefore used to visualize the transport pathway. Peroxidase cytochemistry of livers fixed in situ 2.5 to 10 min after 125I-pIgA-HRP injection demonstrated a progressive redistribution of labeled structures from the sinusoidal area to intermediate and bile canalicular regions of the hepatocyte cytoplasm. Although conjugate-containing structures began accumulating in the bile canalicular region at these early times, no conjugate was present in bile until 20 min. From 7.5 to 45 min after injection approximately 30% of the labeled structures were in regions that contained Golgi complexes and lysosomes; however, we found no evidence that either organelle contained 125I-pIgA-HRP. At least 85% of all positive structures in the hepatocyte were vesicles of 110-160-nm median diameters, with the remaining structures accounted for by tubules and multivesicular bodies. Vesicles in the bile canalicular region tended to be larger than those in the sinusoidal region. Serial sectioning showed that the 125I-pIgA-HRP-containing structures were relatively simple (predominantly vesicular) and that extensive interconnections did not exist between structures in the sinusoidal and bile canalicular regions.     

Maintenance of the 2 microns circle plasmid in populations of Saccharomyces cerevisiae.

The 2 microns circle plasmid is maintained at high frequencies in populations of yeast cells. To find out how the plasmid is maintained, three forces were measured: the selective advantage or disadvantage conferred by 2 microns circles, the rate of generation of [Cir0] cells, and the rate of illegitimate transfer of 2 microns circles from cell to cell. It was found that under the conditions used, 2 microns circles confer a selective disadvantage of about 1%, that [Cir0] cells are generated at the rate of 7.6 x 10(-5) per [Cir+] cell per generation, and that illegitimate transfer of 2 microns circles occurs at a rate less than 10(-7) per recipient cell per generation. The most likely explanation of 2 microns circle maintenance is that the plasmid is sexually transmitted at such a rate that it spreads through populations despite selection against it.     

5′-nucleotidase

Summary The distribution of 5-nucleotidase activity in rat liver shows a sexual dependence. In male liver the activity in the bile canalicular wall is most pronounced, whereas the activity at the sinusoidal border of the liver parenchymal cell is slightly more in the female rat. Castration and treatment with sex hormones change the distribution pattern. The greatest variations in enzyme activity are seen at the bile canalicular site of the liver cell. These changes are probably an expression of the altered functional state of the liver cell.     

Arrangement and density of junctional feet in crayfish muscle fibres

Junctional feet in tubulo-reticular junctions of crayfish muscle fibres are arranged tetragonally with a centre-to-centre spacing of 30-34 nm. The resulting density of 860-1110 feet per 1 micron 2 of the junctional membrane is similar to that reported for other animal species. Using data of a previous stereological study, there are 150-190 feet per 1 micron 2 of the total T-tubule surface and 6000-7800 feet per 100 microns 3 of the fibre volume.     

Electron microscopic cytochemical characterization of bile canaliculi and bile ducts in vitro.

Electron microscopic cytochemical localization of Mg++-activated adenosine triphosphatase (Mg++-ATPase) and 5-nucleotidase (AMPase) was investigated in bile canaliculus-rich and bile duct-containing fractions isolated from rat liver. Comparative cyochemical studies between prefixed and non-prefixed fractions revealed that the activity of both enzymes could be detected in the fractions under appropriate experimental conditions. However, the cytochemical activity of AMPase was much more sensitive to glutaraldehyde than that of Mg++-ATPase. Mg++-ATPase and AMPase reaction products were localized primarily on bile canalicular microvilli, that is, along the outer (luminal) surface of canalicular plasma membranes, but they were never observed on bile ductal microvilli. AMPase was also detectable on lateral hepatic plasma membranes. Mg++-ATPase demonstrated by the cytochemical technique described is a reliable enzyme marker for isolated bile canalicular membranes. At high magnification, Mg++-ATPase reaction product was also observed on the microfilaments surrounding isolated bile canaliculi. The possibility that the reaction product on the pericanalicular microfilaments may result from the hydrolysis of ATP byan actomyosin ATPase-like enzyme associated with these filaments is briefly discussed.     

Quantitative ultrastructure of isolated Kupffer cells of rat liver

The average volume of isolated Kupffer cells of rat liver is 821 +/- 64 microns 3, the average surface being 423 +/- 24 microns 2 (599 microns 2, with cell processes included). The surface structure (pseudopodia, lamellipodia, filopodia, microvilli) of isolated cells is much less developed than that of Kupffer cells in situ. By morphometric characterization volume densities are 0.1264 +/- 0.0077 (SE) for mitochondria and 0.3591 +/- 0.0169 for lysosomal structures. The volume of mitochondria amount to 0.79 +/- 0.04 microns 3.     

Ethinylestradiol administration selectively alters liver sinusoidal membrane lipid fluidity and protein composition

Administration of high-dose ethinylestradiol to rats decreases bile flow, Na,K-ATPase specific activity, and liver plasma membrane fluidity. By use of highly purified sinusoidal and bile canalicular membrane fractions, the effect of ethinylestradiol administration on the protein and lipid composition and fluidity of plasma membrane fractions was examined. In sinusoidal fractions, ethinylestradiol (EE) administration decreased Na,K-ATPase activity (32%) and increased activities of alkaline phosphatase (254%), Mg2+-ATPase (155%), and a 160-kDa polypeptide (10-fold). Steady-state and dynamic fluorescence polarization was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH) was significantly higher in canalicular compared to sinusoidal membrane fractions. Ethinylestradiol (5 mg/kg per day for 5 days) selectively increased sinusoidal polarization values. Similar changes were demonstrated with the probes 2- and 12-anthroyloxystearate. Time-resolved fluorescence polarization measurements indicated that EE administration for 5 days did not change DPH lifetime but increased the order component (r infinity) and decreased the rotation rate (R). However, 1 and 3 days after EE administration and with low doses (10-100 micrograms/kg per day for 5 days) the Na,K-ATPase, bile flow, and order component were altered, but the rotation rate was unchanged. Vesicles prepared from total sinusoidal membrane lipids of EE-treated rats, as well as phospholipid vesicles, demonstrated increased DPH polarization, as did intact plasma membrane fractions. Liver plasma membrane fractions showed no change in free cholesterol or cholesterol/phospholipid molar ratio, while esterified cholesterol content was increased with high-dose but not low-dose ethinylestradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Knockdown of hepatocyte aquaporin-8 by RNA interference induces defective bile canalicular water transport

Aquaporin-8 (AQP8) water channels, which are expressed in rat hepatocyte bile canalicular membranes, are involved in water transport during bile formation. Nevertheless, there is no conclusive evidence that AQP8 mediates water secretion into the bile canaliculus. In this study, we directly evaluated whether AQP8 gene silencing by RNA interference inhibits canalicular water secretion in the human hepatocyte-derived cell line, HepG2. By RT-PCR and immunoblotting we found that HepG2 cells express AQP8 and by confocal immunofluorescence microscopy that it is localized intracellularly and on the canalicular membrane, as described in rat hepatocytes. We also verified the expression of AQP8 in normal human liver. Forty-eight hours after transfection of HepG2 cells with RNA duplexes targeting two different regions of human AQP8 molecule, the levels of AQP8 protein specifically decreased by 60-70%. We found that AQP8 knockdown cells showed a significant decline in the canalicular volume of approximately 70% (P < 0.01), suggesting an impairment in the basal (nonstimulated) canalicular water movement. We also found that the decreased AQP8 expression inhibited the canalicular water transport in response either to an inward osmotic gradient (-65%, P < 0.05) or to the bile secretory agonist dibutyryl cAMP (-80%, P < 0.05). Our data suggest that AQP8 plays a major role in water transport across canalicular membrane of HepG2 cells and support the notion that defective expression of AQP8 causes bile secretory dysfunction in human hepatocytes.