Formation Of Cytoplasmic Vacuoles Research Articles

VMP1 Expression Correlates with Acinar Cell Cytoplasmic Vacuolization in Arginine-Induced Acute Pancreatitis

Background: Recently, we described the cloning of VMP1 (vacuole membrane protein 1). In vitro expression of VMP1 promotes formation of cytoplasmic vacuoles followed by cell death. In order to test if VMP1 expression is related to the cytoplasmic vacuolization of the acinar cells during acute pancreatitis, we studied the in vivo expression of the new gene during arginine-induced acute pancreatitis. Methods: Male Wistar rats injected with 500 mg/100 g of L-arginine were time-course sacrificed and pancreas tissue removed. Results: Northern blot analysis showed maximal induction of VMP1 after 24 h remaining high after 48 h of arginine administration. Significant increase in the number of TUNEL-stained cells were found at those periods. After 24 and 48 h of arginine administration, light micrographs from thin plastic toluidine blue sections revealed numerous vacuoles in the cytoplasm of acinar cells. In situ hybridization studies showed high expression of VMP1 in acinar cells with cytoplasmic vacuolization. VMP1 mRNA highly and significantly correlated with vacuole formation. Conclusion: These results suggest that VMP1 expression may be involved in the cytoplasmic vacuolization of acinar cells during the early stage of acute pancreatitis.

Proteasome inhibitor-induced apoptosis of glioma cells involves the processing of multiple caspases and cytochrome c release.

The proteasome is a multiprotein complex that is involved in the intracellular protein degradation in eukaryotes. Here, we show that human malignant glioma cells are susceptible to apoptotic cell death induced by the proteasome inhibitors, MG132 and lactacystin. The execution of the apoptotic death program involves the processing of caspases 2, 3, 7, 8, and 9. Apoptosis is inhibited by ectopic expression of X-linked inhibitor of apoptosis (XIAP) and by coexposure to the broad-spectrum caspase inhibitor, benzoyl-VAD-fluoromethyl ketone (zVAD-fmk), but not by the preferential caspase 8 inhibitor, crm-A. It is interesting that specific morphological alterations induced by proteasome inhibition, such as dilated rough endoplasmic reticulum and the formation of cytoplasmic vacuoles and dense mitochondrial deposits, are unaffected by zVAD-fmk. Apoptosis is also inhibited by ectopic expression of Bcl-2 or by an inhibitor of protein synthesis, cycloheximide. Further, cytochrome c release and disruption of mitochondrial membrane potential are prominent features of apoptosis triggered by proteasome inhibition. Bcl-2 is a stronger inhibitor of cytochrome c release than zVAD-fmk. XIAP and crm-A fail to modulate cytochrome c release. These data place cytochrome c release downstream of Bcl-2 activity but upstream of XIAP- and crm-A-sensitive caspases. The partial inhibition of cytochrome c release by zVAD-fmk indicates a positive feedback loop that may involve cytochrome c release and zVAD-fmk-sensitive caspases. Finally, death ligand/receptor interactions, including the CD95/CD95 ligand system, do not mediate apoptosis induced by proteasome inhibition in human malignant glioma cells.

Venom from the Endoparasitic Wasp Pimpla hypochondriaca Adversely Affects the Morphology, Viability, and Immune Function of Hemocytes from Larvae of the Tomato Moth, Lacanobia oleracea

During oviposition, the endoparasitic wasp Pimpla hypochondriaca injects its pupal hosts with venom. This complex fluid has toxic properties and recently several venom components were characterized. In addition, it was suggested that venom might be involved in host immune suppression. For this to be the case, venom would have to adversely affect hemocytes and this aspect was further addressed in the current study utilizing the larval stage of the tomato moth Lacanobia oleracea as a model system. Using sublethal venom injections we investigated the effects of venom on encapsulation and hemocyte concentration. Additionally, the effects of venom on hemocyte morphology, viability, and phagocytic capability were determined in vitro. Injection of 16 μg of venom protein into sixth instar larvae was sufficient to reduce the ability of hemocytes to encapsulate Sephadex A25 beads by more than 50% in four of five insects examined. Hemocyte concentration in sixth instar larvae 32 h after injection with 16 μg of venom was reduced by 56% compared to that in controls. Damaged hemocytes and cell debris were also observed in hemolymph from venom-treated insects, suggesting that P. hypochondriaca venom has cytotoxic properties. In vitro incubation of washed hemocytes for 20 h with 500 ng/μl venom resulted in disintegration of a high proportion of hemocytes, leaving only parts of the plasma membrane and nucleus intact. Treatment with low concentrations of venom (1.6 ng/μl) resulted in an absence of spread plasmatocytes, which were abundant on control monolayers. High-resolution microscopy of hemocyte cultures exposed to 320 ng/μl venom for 3.5 h on glass slides indicated that venom induced a variety of effects on cellular morphology, including blebbing of the plasma membrane, degranulation, and the formation of cytoplasmic vacuoles. Incubation of hemocytes with 320, 64, or 3.2 ng/μl venom for 3.5 h reduced cell viability to 70, 90, and 92%, respectively, confirming that venom is cytotoxic to hemocytes. Treatment with 320 ng/μl venom reduced the capacity of hemocytes to phagocytose Escherichia coli by 85%. Together, these results demonstrate that at sublethal doses venom has a potent anti-hemocyte action and can impair hemocyte-mediated immune responses.

Early ultrastructural changes of apoptosis induced by fumonisin B1 in rat liver.

A time sequential study was performed to investigate the histological and ultrastructural findings of fumonisin B1-induced apoptosis in the male Sprague-Dawley rat liver. Six hours after administration of FB1, marked morphologic changes of hepatocytes included the appearance of small vacuoles along the margin of cell membrane. Twelve hours after injection of FB1, acidophilic degeneration of cells occurred, but no fragmented nucleus was evident around the centrilobular area, with few apoptotic cells. By electron microscope, the degenerated acidophilic cells revealed following changes: characteristic formation of cytoplasmic vacuoles, condensed cytoplasm, detachment from neighboring cells, and as well as margination of nuclear chromatin and swollen mitochondria with amorphous matrical deposit. The number of apoptotic cells or bodies was further enhanced at 24 hours in the vicinity of dense acidophilic cells, resulting in a marked increase over the values of control rats. Serum analysis revealed the elevation of cholesterol levels from the beginning to the end of this experiment. Morphologic data and serum findings in this study support the theory that FB1-induced alteration of membrane lipid constituents of the hepatocytes are likely to be early key events in explaining the FB1 apoptotic effect.

Early Changes of Gene Expression During Cerulein Supramaximal Stimulation

Administration of supramaximal doses of cerulein results in acute interstitial pancreatitis. To understand the pathogenesis of this disease, it would be of great importance to elucidate the changes during the early phase of the process. We report changes of gene expression in the pancreas during the first 6 h of cerulein supramaximal stimulation. The expression of genes, including the secretory enzyme amylase, the lysosomal enzyme cathepsin B, as well as the housekeeping genes beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPD), was investigated in this study. The most prominent alteration in gene expression is beta-actin messenger RNA (mRNA), which increased continuously after cerulein infusion. Immunostaining for beta-actin was observed along the membrane of large cytoplasmic vacuoles in pancreatic acinar cells. The level of amylase mRNA decreased during the first 30 min of cerulein infusion, recovered to the control level at 1 h and increased twofold at 2 h. An obvious increase in cathepsin B mRNA was observed after 3 h of cerulein infusion and reached sixfold of the control at 6 h. A significant increase of GAPD mRNA level was observed at 6 h of cerulein stimulation. In conclusion, this study provides direct evidence that the changes in gene expression, such as cathepsin B and amylase, after supramaximal cerulein stimulation, are regulated at the transcriptional level. It also suggests that beta-actin is involved in the formation of cytoplasmic vacuoles during supramaximal cerulein administration. Finally, this study indicates that beta-actin and GAPD may not be appropriate as RNA-loading controls for Northern blot analysis of pancreatic tissue.

Development of the Endosperm of Myrsine laetevirens (Myrsinaceae). I. Cellularization and Deposition of Cell‐Wall Storage Carbohydrates

Myrsine laetevirens has a nuclear type of endosperm development. Vacuole formation in peripheral coenocytic cytoplasm precedes cytokinesis and compartmentalizes the cytoplasm. Cellularization starts as a series of anticlinal walls projecting centripetally. Periclinal cell walls are formed from expanding cell plates following nuclear divisions. The developing walls are flanked by endoplasmic reticulum cisternae with dilated and short profiles and few ribosomes. Subsequent anticlinal wall growth and periclinal divisions elongate the endosperm into the central vacuole, resulting, finally, in the convergence of the advancing tissue. After that, the endosperm increases in size by anticlinal, periclinal, and oblique divisions in all cells of the endosperm and involving phragmoplast and cell plate. Carbohydrate deposition in the cell walls begins 40 d after pollination (DAP) with deposition of pectins in the cell layers next to the embryo. At ca. 50 DAP, the cell walls of the bulk endosperm begin to thicken, sho...

Reversal of multidrug resistance by a liposome-MDR1 ribozyme complex.

Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. We examined whether cationic liposome-mediated transfer of a ribozyme could reverse MDR. A ribozyme which cleaved codon 196 of MDR1 mRNA was constructed from synthetic oligonucleotides. The MDR1 ribozyme was mixed with N-(1-(2,3-dileoyloxy)propyl)-N,N,N-trimethylammonium methyl sulfate (DOTAP) to form a liposomal complex. The complex was used to treat two P-glycoprotein-producing MDR cell lines: MCF-7/R human breast cancer cells resistant to doxorubicin and MOLT-3/TMQ800 human ALL cells resistant to trimetrexate (TMQ). In order to investigate the differential sensitivity of these two cell lines to the liposome-ribozyme complex, cellular pharmacological studies including phase-contrast and confocal microscopic studies were performed. Treatment with the liposome-ribozyme complex resulted in reversal of vincristine (VCR) resistance in MCF-7/R cells, but not in MOLT-3/TMQ800 cells. In MCF-7/R cells the treatment resulted in decreases in MDR1 mRNA expression and P-glycoprotein production, whereas no changes in these parameters were seen in MOLT-3/TMQ800 cells. Phase-contrast microscopy revealed that in MCF-7/R cells treatment with DOTAP led to the formation of cytoplasmic vacuoles, and treatment with latex beads resulted in the development of a shiny material in the cytoplasm. In contrast, in MOLT-3/TMQ800 cells hardly any morphological changes occurred. Confocal microscopic imaging showed cytoplasmic fluorescence in MCF-7/R cells after treatment with DOTAP/FITC-dextran or FITC-conjugated latex beads. In MOLT-3/TMQ800 cells no fluorescence was detected. Treatment with cytochalasin B abolished fluorescence in MCF-7/R cells after treatment with DOTAP/FITC-dextran or FITC-conjugated latex beads. These studies show that MCF-7/R cells have high endocytotic activity whereas MOLT-3/TMQ800 cells have little activity. Endocytotic activity was correlated with the success of cationic liposome-mediated transfer of MDR1 ribozyme. Determination of endocytotic activity of target tumor cells may be predictive of efficacy of liposome-mediated gene transfer.

Cell death during luteal regression in the marmoset monkey (Callithrix jacchus).

The mechanism controlling luteal regression in primates is unknown but may involve cell death by apoptosis. Marmoset ovaries containing corpora lutea were studied at different stages of the normal ovarian cycle. Two additional groups of animals underwent induced luteolysis with either the prostaglandin F2 alpha analogue, cloprostenol, or the GnRH antagonist, antarelix, at the mid-luteal phase. Apoptosis in ovarian sections was estimated both by counting the number of cells exhibiting morphological features of apoptosis and by in situ labelling the 3' ends of the DNA fragments with digoxigenin-11-dUTP. Apoptosis was found to be significantly increased in corpora lutea in the early follicular phase (equivalent to the later stage of luteal lifespan) compared with the mid-luteal phase corpora lutea, as judged by either computerized morphometry or 3' end labelling. Apoptosis was also increased by the administration of either cloprostenol or antarelix when using the 3' end labelling end point, but only after cloprostenol when using computerized morphometry. A further form of cell death, characterized by the formation of cytoplasmic vacuoles, was also observed in corpora lutea undergoing both induced and spontaneous regression. These results demonstrate that apoptosis within the primate corpus luteum is increased in both physiological and induced luteal regression. In addition, they show that an alternative form of cell death is involved in both spontaneous and induced luteal regression, although the relative importance of the two mechanisms remains to be determined.

Regulation of Parathyroid Hormone-Related Protein Secretion and mRNA Expression in Normal Human Keratinocytes and a Squamous Carcinoma Cell Line

Parathyroid hormone-related protein (PTHrP) has been identified as a causative factor in the pathogenesis of humoral hypercalcemia of malignancy (HHM). The regulation and mechanisms of PTHrP secretion in most normal and malignant cells are unknown. PTHrP secretion, mRNA expression, and transcription were measured in neoplastic human squamous carcinoma cells (A253) and normal human foreskin keratinocytes (NHFK) by radioimmunoassay, RNase protection assay, and transient transfections of the 5′-flanking region of humanPTHrPin a luciferase expression vector. Mechanisms of PTHrP secretion were investigated using chemicals (monensin, colchicine, cytochalasin B, guanosine 5′-[γ-thio]triphosphate (GTPγS)) that interfere with or facilitate intracellular transport. Monensin inhibited PTHrP secretion in both NHFK and A253 cells. Ultrastructurally, monensin caused dilatation of rough endoplasmic reticulum and the formation of numerous cytoplasmic secretory vacuoles in both cell lines. Colchicine decreased PTHrP production in NHFK cells and stimulated PTHrP production and mRNA levels in A253 cells. Colchicine also stimulated transcription of the PTHrP–luciferase reporter gene. Cytochalasin B stimulated PTHrP secretion and mRNA expression in A253 cells, but had no effect in NHFK cells. GTPγS had no effect on PTHrP secretion in either cell line. It was concluded that PTHrP secretion is dependent on the constitutive movement of secretory vesicles to the cytoplasmic membrane and regulation of PTHrP secretion and mRNA expression are altered in squamous carcinoma cells compared to normal human keratinocytesin vitro.

Repair of naphthalene-injured microdissected airways in vitro.

Nonciliated bronchiolar epithelial (Clara) cells, as both the primary target for metabolically activated pulmonary toxicants and the progenitor cell for repair after bronchiolar injury, are critical for distal airway epithelial function and regeneration. Previously, we described a model system whereby differentiated Clara cells can be maintained in culture using explants of microdissected distal airways. The purpose of this study is to establish whether distal airway explants can be used to study bronchiolar epithelial repair in vitro. Lungs from adult mice treated with naphthalene, a metabolically activated Clara cell cytotoxicant, or vehicle were inflated with agarose and distal airways were microdissected. Distal airway explants were cultured for up to 7 days in serum-free medium. Clara cells in explants from naphthalene-treated mice exhibited the characteristic cytotoxic responses previously reported in vivo when maintained in vitro: cell swelling, formation of cytoplasmic vacuoles, and exfoliation of injured cells into the airway lumen 1 to 2 days after injury (DAI). Epithelial cells squamated to cover the injured area 2 to 4 DAI. At 7 DAI, the epithelium generally consisted of cuboidal cells. Proliferating cells and marker proteins for differentiated Clara cells (Clara cell 10 kD secretory protein, or CC10, and cytochrome P450 monooxygenase isozyme 2B, or CYP2B) were detected immunochemically and their pattern of distribution during the injury and repair response in vitro paralleled the pattern of cell regeneration seen previously in vivo. We conclude that Clara cells in explants from defined regions of murine tracheobronchial airways can be used to study the early phases of the repair response to naphthalene injury, including differentiation and proliferation, in vitro.

The homeobox gene GLABRA2 is required for position-dependent cell differentiation in the root epidermis of Arabidopsis thaliana.

The role of the Arabidopsis homeobox gene, GLABRA 2 (GL2), in the development of the root epidermis has been investigated. The wild-type epidermis is composed of two cell types, root-hair cells and hairless cells, which are located at distinct positions within the root, implying that positional cues control cell-type differentiation. During the development of the root epidermis, the differentiating root-hair cells (trichoblasts) and the differentiating hairless cells (atrichoblasts) can be distinguished by their cytoplasmic density, vacuole formation, and extent of elongation. We have determined that mutations in the GL2 gene specifically alter the differentiation of the hairless epidermal cells, causing them to produce root hairs, which indicates that GL2 affects epidermal cell identity. Detailed analyses of these differentiating cells showed that, despite forming root hairs, they are similar to atrichoblasts of the wild type in their cytoplasmic characteristics, timing of vacuolation, and extent of cell elongation. The results of in situ nucleic acid hybridization and GUS reporter gene fusion studies show that the GL2 gene is preferentially expressed in the differentiating hairless cells of the wild type, during a period in which epidermal cell identity is believed to be established. These results indicate that the GL2 homeodomain protein normally regulates a subset of the processes that occur during the differentiation of hairless epidermal cells of the Arabidopsis root. Specifically, GL2 appears to act in a cell-position-dependent manner to suppress hair formation in differentiating hairless cells.

Effects of contrast media on renal epithelial cells in culture.

Proximal and distal tubular cells in culture have been exposed to various roentgen contrast media (CM) at concentrations of 0 to 100 mg I/ml for 22 hours to study cellular mechanisms that may be involved in CM-induced nephropathy. The effects on cell morphology were assessed by electron microscopy and cell viability was evaluated. Levels of brush border and lysosomal marker enzymes in the culture medium were assayed biochemically. Morphological examination showed that CM induced a concentration-dependent formation of large cytoplasmic vacuoles in both cell lines. Cellular damage was observed more frequently after exposure to low-osmolal rather than the iso-osmolal CM iodixanol; the low-osmolal CM causing more cell death and inhibiting cellular growth to a greater degree than did iodixanol. In cultures of both cell lines the CM produced a concentration-dependent increase in brush border marker enzyme activity. While an increase in lysosomal enzyme activity was seen at low concentrations, a decrease in activity occurred at high concentrations. Earlier investigations have demonstrated that the nonionic CM have less pronounced effects on the cell lines studied than ionic CM. The results presented here indicate that the effects of the iso-osmolal nonionic CM (iodixanol) on both the investigated cell lines are less marked than those of the low-osmolal nonionic CM investigated.

Induction of Membrane Proliferation by Poliovirus Proteins 2C and 2BC

Poliovirus infection leads to the appearance of a number of cytoplasmic vacuoles involved in the replication of virus genomes. To characterize the viral proteins involved in membrane proliferation different poliovirus proteins have been expressed in HeLa cells. Two recombinant vaccinia viruses have been obtained that express poliovirus protein 2C, one under the 5′ untranslated (UTR) sequence of poliovirus and another under the leader region of EMC virus. Expression of 2C was very efficient in both cases, although better results were obtained when poliovirus 2C was expressed under the 5′UTR sequence of EMC virus. Transient expression of poliovirus proteins 2B, 2C or 2BC placed under a T7 promoter was analyzed using a recombinant vaccinia virus that contains the bacteriophage T7 RNA polymerase. The expression of 2C, or 2BC, contrary to 2B, was able to induce the proliferation of vacuoles morphologically similar to those found during poliovirus infection. These findings indicate that poliovirus protein 2C, in addition to its NTPase and RNA binding activities,is also endowed with the capacity to induce the formation of cytoplasmic vacuoles.

The rice culture filtrate of Bacillus cereus isolated from emetic-type food poisoning causes mitochondrial swelling in a HEp-2 cell.

Rice culture filtrates of Bacillus cereus SA-50, an emetic-type strain, produced a toxin which caused cytoplasmic vacuole formation in HEp-2 and HeLa cells. Electron microscopic observation revealed that the apparent vacuoles in HEp-2 seen under a light microscope were actually swollen mitochondria. The oxygen consumption of HEp-2 cells was accelerated by the addition of the rice culture filtrate as was measured with a polarographic oxymeter; a respiratory control ratio was 1.0 for control cells, while 1.4 for ones with the filtrates. The culture filtrates showed a similar effect on the isolated mouse liver mitochondria; respiratory control ratios for the mitochondria with and without the filtrates were 3.6 and 1.0, respectively. The affecting manner of the culture filtrates on the oxygen consumption of mitochondria was similar to that of 2,4-dinitrophenol, suggesting that the culture filtrate contains a toxin acting as an uncoupler of oxidative phosphorylation in mitochondria. It is likely that the culture filtrates containing the emetic toxin of B. cereus causes mitochondrial swelling with a close relationship to the uncoupling of the oxidative phosphorylation of mitochondria.

Resistance of human tracheal epithelial cells to killing by neutrophils, neutrophil elastase, and Pseudomonas elastase.

The airway disease of cystic fibrosis (CF) is characterized by massive polymorphonuclear leukocyte (PMN) infiltration and the presence of variable but substantial quantities of uninhibited elastases derived from both PMNs and the common infecting organism Pseudomonas aeruginosa. In order to determine whether these agents inflict fatal injury on the airway epithelium, we exposed primary cultures of human tracheal epithelial (HTE) cells to activated PMNs, PMN elastase (PMNE), and elastase from P. aeruginosa (PSE) and monitored cytotoxicity by 51Cr release assay. Activated PMNs did not kill HTE cells, and fewer than 2% of the added PMNs adhered to the HTE cell layer. Pretreatment of HTE cells with lipopolysaccharide, incubation of PMNs with cytochalasin B, or increasing the incubation period to 8 h did not increase PMN adherence or target cell killing. However, poor PMN adherence was not by itself responsible for lack of cytotoxicity, since PMNs were not cytotoxic for 9HTEo- cells, a HTE cell line to which PMNs adhere in large numbers. Purified PMNE, but not exogenous H2O2, caused a small but significant increase in cytotoxicity after 6 h of incubation, but only at the highest concentrations tested (10 and 50 micrograms/ml). The PMNE remained fully active throughout the incubation period. Some detachment of the cell layer occurred after 4 h of incubation with 10 micrograms/ml PMNE. PSE at concentrations > 1 micrograms/ml also caused slight cytotoxicity and removal of the cell layer from the culture substratum. Ultrastructural studies showed only minor cytoplasmic vacuole formation. We conclude that cultured HTE cells are resistant to cytolysis by PMNs and elastases.

Detection of rabbit haemorrhagic disease virus particles in the rabbit liver tissues

Liver tissues from rabbits experimentally infected with rabbit haemorrhagic disease virus (RHDV) were studied electron microscopically. The earliest change in hepatocytes of the rabbits infected with RHDV was hydropic degeneration. Rough endoplasmic reticulum was dilated with a mild increase in polysomes and cytoplasmic cisternae in degenerated hepatocytes. Characteristic cytopathological changes of necrotic hepatocytes included shrinkage of the cell body, formation of cytoplasmic vesicles, vacuoles or cisternae and karyolysis. A large number of viral particles resembling a calicivirus in size and morphology was demonstrated in the cytoplasm of many necrotic hepatocytes. The particles had accumulated mainly in the membrane-bound cisternae or scattered around the membrane-bound vacuoles of the necrotic hepatocytes. Western blot analysis demonstrated that RHDV antigen was present in the infected hepatocytes. RHDV particles were also detected by immunoelectron microscopy. Replicating patterns of RHDV particles and subsequent cytopathology resembled those in other calicivirus infections.

Inhibition of Growth, Morphological and Morphometrical Changes Induced by Amido-anthraquinone Derivatives in NCTC 2544 and HeLa Cells

In this work, we have studied cytotoxicity induced in NCTC 2544 and HeLa cell lines by two newly-synthesised potential anticancer drugs, the amido-anthraquinone derivatives 8-diethylaminopropionamido-1,4,5-trihydroxy-9,10-anthracenedione bromide (A888) and 5,8-bisdiethylaminopropionamido-1,4-dihydroxy-9,10-anthracenedione dibromide (A890). The results have been compared to the results obtained with mitoxantrone, a well-known anticancer agent. Using the neutral red uptake assay, A888 showed less cytotoxic action compared to mitoxantrone and A890. According to the results of morphological analysis after exposure for five hours, anthraquinone derivatives induced the formation of cytoplasmic vacuoles and changes in the arrangement of nucleus in both cell lines. On the basis of morphometrical analysis, we have observed an increase in total cellular area with a decrease in the nuclear/cytoplasmic ratio. We have found that the increase in total cellular area was higher in NCTC 2544 cells than in HeLa cells. From these data, we can conclude that NCTC cells are more sensitive to the compounds tested than HeLa cells. However, the growth inhibition assay and morphological analysis did not confirm these results.

Salivary glands of the tsetse Glossina pallidipes Austen infected with Trypanosoma brucei and virus particles: Ultrastructural study

The effects of infection by Trypanosoma brucei and DNA virus on the ultrastructure of the salivary gland cells in Glossina pallidipes Austen were investigated. Cytoplasm of uninfected cells contains a dense ribosomal population and rough endopiasmic reticulum, scattered Golgi areas and mitochondria. In infected ceils of salivary glands the ultrastructural integrity of the cytoplasm is profoundly changed due to cellular proliferation giving rise to a stratified epithelium and gland enlargement; cell degeneration characterized by formation of cytoplasmic vacuoles, chromatin margination, disorganization and elimination of cell organelles and gland hypertrophy. The virus particles are found in the nucleoplasm as well as the cytoplasm of cells. The ultrastructural evidence indicates that the virus are largely assembled in nuclei of cells and virions pass through the nuclear membrane. The trypanosomes are found within the degenerating cytoplasm and lumen of the cell. The implications of the features observed in the infected cells are discussed, and possible suggestions are made regarding alteration of the metabolic functions due to infection.

Intravenous bilirubin infusion causes vacuolization of the cytoplasm of hepatocytes and canalicular cholestasis

To examine whether intravenous bilirubin infusion causes cholestasis and impairs liver metabolism, bile secretion and ethanol clearance were measured in 34 anaesthetized pigs before and after intravenous infusion of 0.5 mumol kg-1 min-1 bilirubin for 4.5 hours. Bilirubin infusion increased plasma bilirubin to 556 +/- 76 mumol l-1 and hepatic tissue bilirubin to 3.5 +/- 1.3 mmol kg tissue weight-1. Bilirubin infusion depressed bilirubin secretion and net hepatic uptake of cholate and taurocholate, and caused a 86 +/- 6% reduction of cholate-induced bile secretion. Bilirubin caused formation of large cytoplasmic vacuoles in hepatocytes and dilatation of bile canaliculi. Ethanol clearance and secretin-dependent ductular bile secretion were unaffected by bilirubin. We conclude that intravenous infusion of unconjugated bilirubin causes accumulation of bilirubin in the liver, vacuolization of the hepatocyte cytoplasm and canalicular but not ductular cholestasis. The canalicular cholestasis is not due to impaired hepatic mitochondrial energy metabolism, but may be due to inhibition of a common pathway for lipid, bilirubin and bile salt secretion from hepatocytes.

The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells

Okadaic acid, a specific and potent inhibitor of protein phosphatases 2A and 1, was tested for its effect on the morphology of a number of cell types: freshly isolated rat hepatocytes in suspension or in primary culture, the human mammary carcinoma cell line MCF-7, the human neuroblastoma cell line SK-N-SH, rat pituitary adenoma GH3 cells, and rat promyelocytic IPC-81 cells. All the cell types reacted within a few hours to okadaic acid in the concentration range 0.1 to 1 μ M with profound morphological alterations. Among the changes noted were: condensation of chromatin, shedding of cell contents via surface bleb formation, redistribution and compacting of cytoplasmic organelles, formation of cytoplasmic vacuoles, and hyperconvolution of the nuclear membrane. In some cells nuclear fragmentation was noted. In addition, cells growing as monolayers rounded up and detached from the substratum. The treated cells had no swollen mitochondria and retained the ability to exclude trypan blue until the final stage of dissolution, supporting the hypothesis that the changes were apoptotic rather than necrotic. In hepatocytes the action of okadaic acid was mimicked by another phosphatase inhibitor, microcystin, and was accompanied by shrinkage of the cell volume, as judged by Coulter counter analysis. The action of phosphatase inhibitor was not abolished by protein synthesis inhibitors, Ca 2+-depleted medium, or phorbol ester. Although hepatocyte DNA replication was very sensitive to inhibition by okadaic acid, DNA fragmentation was less pronounced in response to okadaic acid than other agents inducing the morphological appearance of apoptosis.