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PubMed1980

Development of [3H]muscimol binding to subcellular particles of a culture of mouse brain.

"Specific" binding of [3H]muscimol (11.5 nM) related to GABA receptors, occurred in subcellular particles prepared from 4-, 8-, and 12-day cultures of embryonic (14- to 15-day-old) mouse cerebrum, but not to particles prepared from 20- to 45-day cultures. Phase-contrast microscopy revealed that the presence of neuronal elements (neuroblasts, neurites) paralleled the binding of [3H]muscimol. Further evidence is thus provided for using high-affinity [3H]muscimol binding as a "neuronal marker."

Animals↗
PubMed1983

Morphological and electrophysiological aspects of dissociated cultures of rat CNS.

Rat central nervous system has been cultured up to 6 weeks after complete dissociation. Maturation of different cell types has been followed in the quasi monolayer by phase contrast microscopy. Dorsal root ganglion (DRG) neurones usually differed from central nervous system (CNS) neurones by their spherical shape accompanied by only one or two processes, exact identification of cell types, however, was usually only possible by combining morphology with electrophysiology. Scanning electron-microscopy revealed a more extensive arborization of neurites and a higher number of presumed synaptic structures in cultures after 2 weeks of culturing. Layers of ependymal cells were also found. The different cell types were further identified by determining their membrane properties. Glial cells had higher resting membrane potentials (-56 +/- 9.7 mV) than CNS neurones (-49 +/- 10.2 mV), while the membrane potential of DRG neurones lay in-between the two (-53 +/- 1.7 mV). The sequence for input resistance was: DRG neurones (30 +/- 9.3 M omega) greater than CNS neurones (18 +/- 10.5 M omega) greater than glial cells (9.3 +/- 5.2 M omega). In CNS neurones the input resistance is correlated with the membrane potential, which is not the case for glial cells. Action potentials of DRG neurones exhibited delayed repolarisation increasing the spike duration to three times that of CNS neurones.

Action Potentials↗
PubMed1988

Cytoprotective effect of acetaminophen against taurocholate-induced damage to rat gastric monolayer cultures.

Acetaminophen has recently been reported to protect against drug damage to gastric mucosa in vivo. The present study tested acetaminophen protection in cultured rat gastric mucous cells against sodium taurocholate-induced damage and assessed the role of endogenous prostaglandins. Cell damage was assessed by phase-contrast microscopy and quantitated by Chromium-51 release assay which positively correlated with the trypan blue dye exclusion test (r = 0.98). The effect of acetaminophen on the production of PGE2 and 6-keto-prostaglandin F1a (6KF) was also studied. Sodium taurocholate caused cell death in a dose-dependent manner as indicated by increased 51Cr release. Preincubation with 5 mM acetaminophen significantly reduced 51Cr release caused by 5 mM sodium taurocholate, producing a 40% increase in cell survival. This cytoprotection was not blocked by indomethacin. PGE2 and 6KF of the media did not change after preincubation with nondamaging concentrations of acetaminophen or taurocholate. These results indicate that: (1) acetaminophen exerts a direct protective effect on gastric mucous cells cultured in vitro independent of indirect factors such as blood flow and (2) this protection is not associated with increased prostaglandin production.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

Distinction between vascular smooth muscle cells and myoepithelial cells in primary monolayer cultures of human breast tissue.

We report on the discrimination of vascular smooth muscle cells and myoepithelial cells in primary cultures of human breast tissue. Breast tissue was disaggregated enzymatically and the resulting organoids seeded in monolayer culture on collagen-coated plastic in serum-free medium CDM3a. Two main types of organoids were present after enzymatic digestion. One resembled small blood vessels and the other interlobular ducts or acini of the breast gland epithelium. Within 3 to 8 d after plating the organoids migrated into typical monolayer islets. These monolayer islets were evaluated using phase contrast microscopy and further tagged with monoclonal antibodies for immunocytochemical demonstration of Factor VIII-related antigen, muscle iso-forms of actin, type IV collagen, vimentin, desmin, and keratins. It is concluded that vascular smooth muscle cells resembled myoepithelial cells by expressing vimentin filaments, depositing type IV collagen, and showing immunoreactivity to muscle iso-forms of actin. However, whereas vascular smooth muscle cells were associated with endothelial cells and sometimes expressed desmin, myoepithelial cells appeared together with luminal epithelial cells and expressed cytokeratins.

Actins↗
PubMed1981

Effects of high hydrostatic pressure on normal and neoplastic rat cells in culture.

Four types of rat cells in culture were exposed to hydrostatic pressures in the range 1-1,500 bar. Each applied pressure was constant for half an hour. The morphological effects of pressure application were studied by phase contrast microscopy, and mortality was measured by total cell counts and the trypan blue exclusion test. Morphological changes characterized by cell rounding were observed in secondary fetal brain cells and fibroblasts at about 700 bar. In two permanent neoplastic neurogenic cell lines similar changes occurred at 1,000 to 1,100 bar. When approximately 50% of the cells were rounded, mortality began to increase, as compared with controls. This was gradual in secondary cells and comparatively abrupt in the permanent neoplastic lines. Malignant cells in culture may therefore be more resistant to hydrostatic pressure than their normal counterparts.

Animals↗
PubMed1999

Conditional lethality of cell shape mutations of Salmonella typhimurium: rodA and mre mutants are lethal on solid but not in liquid medium.

Round-cell (rodA, mre, divD) derivatives of a conditional alaS mutant of Salmonella typhimurium were studied under conditions allowing expression of tolerance to lethal cell shape mutations (41 degrees C), and under nontolerant conditions (30 degrees C). The rodA22::Tn10d(Kan) derivative grew normally (OD650 nm) in LB-broth at 30 degrees C; however, doubling of total cell count took much longer (130 min) than at 41 degrees C (57 min). Although the cells were able to divide in LB-broth at 30 degrees C, viable count on LB-agar at 30 degrees C was 10(3)-fold lower than on LB-agar at 41 degrees C. Phase-contrast microscopy of rodA cells incubated under different conditions showed that their size increased on LB-soft agar at 30 degrees C, but they failed to divide and finally lysed. In contrast, division occurred in LB-broth at 30 degrees C and also in LB-broth and LB-soft agar at 41 degrees C. The mre-17::Tn10d(Kan) derivative acted like the rodA strain whereas the divD135::Tn10d(Kan) mutant behaved normally both at 30 degrees C and 41 degrees C. It is concluded that rodA and mre mutations delay cell division, but are lethal only on solid medium. Mutations conferring tolerance to "lethal" rodA and mre mutations improve division performance both in liquid and solid media.

Amdinocillin↗
PubMed2000

Epinephrine, but not dexamethasone, induces apoptosis in retinal pigment epithelium cells in vitro: possible implications on the pathogenesis of central serous chorioretinopathy.

BACKGROUND: The pathogenesis of central serous chorioretinopathy is poorly understood. It is believed to be due to dysfunction of the retinal pigment epithelium and/or choroid and has been associated with elevated levels of epinephrine and administration of corticosteroids. Epinephrine and corticosteroids have previously been shown to induce apoptosis (programmed cell death) in various types of cells. The objective of this study was to investigate whether these agents can induce apoptosis in cultured retinal pigment epithelium cells. This may help elucidate the pathogenesis of central serous chorioretinopathy. METHODS: Third-passage porcine retinal pigment epithelium cells were grown to confluence and incubated for 1-7 days in culture medium containing epinephrine (10(2)-10(9) pg/ml) or a corticosteroid, dexamethasone (4-4x10(4) ng/ml). The cultures were evaluated for apoptosis by phase-contrast microscopy and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. RESULTS: Epinephrine (7x10(7)-10(9) pg/ml) induced apoptosis in a dose- and time-dependent manner. Exposure to lower concentrations of epinephrine (10(2)-6x10(7) pg/ml) and all tested levels of dexamethasone did not result in apoptosis. CONCLUSION: Retinal pigment epithelium cells may undergo apoptosis following exposure to elevated levels of epinephrine. These findings suggest a possible pathophysiologic mechanism for the development of central serous chorioretinopathy.

Animals↗
PubMed2002

Biochemical effects and growth inhibition in MCF-7 cells caused by novel sulphonamido oxa-polyamine derivatives.

The novel polyamine derivatives sulphonamido oxa-spermine (oxa-Spm) and sulphonamido oxa-spermidine (oxa-Spd) exhibited rapid cytotoxic action towards MCF-7 human breast cancer cells with IC50 values of 4.35 and 6.47 pM, respectively, after 24-h drug exposure. Neither compound is a substrate of serum amine oxidase. Both oxa-Spm and oxa-Spd caused cell shrinkage, as determined by phase-contrast microscopy. After incubation with 10 microM of either compound for 8 h, the cells underwent chromatin condensation and nuclear fragmentation. However, no clear DNA ladder was obtained by electrophoresis. The sulphonamido oxa-polyamine derivatives and especially oxa-Spd enhanced the activity of polyamine oxidase (PAO), an enzyme capable of oxidising N1-acetylated spermine and spermidine to spermidine and putrescine, respectively, generating cytotoxic H2O2 and 3-acetamidopropanal as by-products. The intracellular polyamine content was only marginally reduced in response to drug treatment. In conclusion, our data show that these novel sulphonamido oxa-polyamine derivatives possess high cytotoxic activity against MCF-7 cells and indicate that induction of PAO may mediate their cytotoxicity via apoptosis.

Amine Oxidase (Copper-Containing)↗
PubMed2006

Chinese herbal medicines and their efficacy in treating renal stones.

In herbal treatment of kidney stones, antilithics are used to "dissolve" the stones or aid their passing to guard against further retention. Diuretic action is also needed to increase the amount of fluid going through the kidneys and flush out the deposits. Previous clinical studies have shown that herbal medicines and their concoctions could be used to inhibit calcium oxalate crystallization. However, the pharmacodynamics and in-vitro effects of such medicines have not been established. Five Chinese herbal medicines were selected based on their usefulness in treating stone disease. A 96-well plate oxalate-induced turbidity in artificial urine was used to evaluate the efficacies of the different herbal medicines on calcium oxalate crystallization. The metastable limit was determined and the nucleation rate was derived from 12-min time-course measurement of turbidity at 405 nm. Phase-contrast microscopy was used to visualize the crystals. The results showed that with increasing concentrations of herbal extracts, smaller calcium oxalate crystal sizes were observed. Overall, the five herbal medicinal extracts tested were able to promote nucleation of calcium oxalate crystals while at the same time decreasing the size. This in-vitro crystallization confirms that prophylaxis of renal stones could be achieved by reducing overall supersaturation through promotion of small crystal nucleates and concomitant pharmacological diuretic action of herbal medicines. Clinical studies will provide more definitive conclusions.

Calcium Oxalate↗
PubMed1998

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

PURPOSE: Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. We examined whether cationic liposome-mediated transfer of a ribozyme could reverse MDR. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

A function of polar flagellum and anisotropic growth in Vibrio alginolyticus early-phase colonies.

We continuously observed growth of Vibrio alginolyticus early-phase colonies on agar plates by phase-contrast microscopy. Two mutants defective in motility on solid surfaces were used in this study: one (YM4) can swim in liquid environments using its polar flagellum, and the other (NMB198) cannot swim because it lacks any flagella. We found that isolated colonies of YM4 were generally more circular than those of NMB198. This observation suggests that YM4 cells moved slightly within a colony by the function of their polar flagella. For clustered colonies, where the distance between the colonies was short (<50 microm), the colonies of YM4 grew rapidly along the line between them, but they grew slowly in the lateral directions. Some colonies of NMB198 grew toward neighboring colonies. These observations indicate colony-to-colony interaction.

Adaptation, Physiological↗
PubMed1998

Retroviral vector-mediated gene transfer into keratocytes: in vitro effects of polybrene and protamine sulfate.

BACKGROUND: To determine the potential of somatic gene transfer as a novel technique for modulating corneal wound healing on a cellular level, the successful transduction of human keratocytes should be ascertained in vitro. In addition, the ability of different polycations to increase the transduction efficiency and their antiproliferative and cytotoxic effects should be assessed. METHODS: To test transduction efficiency (X-Gal staining), cultured human keratocytes were incubated for 2 h with a retroviral vector bearing the beta-galactosidase gene, with and without the addition of polybrene or protamine sulfate. To test the antiproliferative and cytotoxic effects, cultured human keratocytes were incubated with various concentrations of polybrene and protamine sulfate (0.08 to 800 micrograms/ml) for 2, 24 and 72 h, and evaluations were performed by means of an XTT-based colorimetric assay and phase-contrast microscopy. RESULTS: Human keratocytes in vitro were transduced successfully with the beta-galactosidase gene (3.5 +/- 1.0%). Transduction efficiency was significantly (P < or = 0.01) improved by addition of a polycation (from 12.3 +/- 1.7% to 18.6 +/- 2.3%), but there was no significant difference between the effects of polybrene and those of protamine sulfate. Both drugs induced a highly significant dose-dependent inhibition of proliferation (P < 0.001). ID50 ranged from 11 to 22 micrograms/ml with polybrene and from 15 to 244 micrograms/ml with protamine sulfate. Only with doses of 80 and 800 micrograms/ml did protamine sulfate produce less antiproliferative effects than polybrene (P < or = 0.04). The lowest concentrations induced no morphological signs of cytotoxicity, whereas these signs were mild at 8 micrograms/ml and moderate to severe at the highest concentrations. CONCLUSIONS: Both polybrene and protamine sulfate can significantly improve the in vitro efficiency of successful retroviral vector-mediated gene transfer into keratocytes. Mild cytotoxic and moderate antiproliferative effects are to be expected in cultured keratocytes with a standard transduction procedure (8 micrograms/ml for 2 h).

Cell Division↗
PubMed1999

Growth of human fetal retinal pigment epithelium as microspheres.

BACKGROUND: The aim was to develop a three-dimensional cell culture system for human fetal retinal pigment epithelial (HFRPE) cells for in vitro cellular studies and for possible application in subretinal transplantation. METHODS: Pieces of freshly isolated HFRPE monolayer tissue were grown on crosslinked fibrinogen (CLF) films. The growth pattern and morphologic characteristics of the implanted tissue were studied using phase-contrast microscopy, photography, and light and electron microscopy. The cells were screened immunohistochemically for HLA-ABC, HLA-DR, ICAM-1, B7, and Cytokeratin. Cell proliferation was studied using 5-bromo-2-deoxyuridine incorporation. RESULTS: After attachment to CLF, HFRPE monolayer tissue formed small tumor-like formations, i.e. microspheres. HFRPE microspheres survived and proliferated in a floating state for at least 4 months. After attachment of the microspheres to the culture dish floor, formation of a confluent HFRPE cell monolayer with high proliferative activity was noted around the microspheres. HFRPE cells stained positive for HLA-ABC, ICAM-1, and cytokeratin and negative for B7 and HLA-DR. The microspheres could be easily detached from the dish and they were able to initiate similar growth after reattachment. CONCLUSION: HFRPE grown on CLF resemble a three-dimensional culture system with high yield of pure cells that can be useful for a wide variety of in vitro studies. Because of their adjustable size, spherical shape, and ability to initiate growth of cells with a high proliferative potential, HFRPE microspheres may be successfully utilized as a source of donor cells for subretinal transplantation.

B7-1 Antigen↗
PubMed1996

Axenic culture of reptilian Blastocystis isolates in monophasic medium and speciation by karyotypic typing.

The growth of axenic reptilian isolates of Blastocystis in Iscove's modified Dulbecco's medium (IMDM) was studied and the morphology of the parasite was examined by phase-contrast microscopy. The chromosomal patterns of these reptilian isolates of Blastocystis were examined by pulsed-field gel electrophoresis (PFGE) and compared with those of B. hominis and B. lapemi, a sea snake Blastocystis. IMDM with 10% horse serum supported excellent growth of the reptilian Blastocystis isolates. The parasites from all the isolates were predominantly vacuolar, but multivacuolar and amoeboid forms were also seen. Amoeboid forms with rather elongate pseudopodia were also observed. There were some differences in size, morphology, and growth characteristics in the different reptilian isolates. The karyotypic patterns of the Blastocystis isolates from tortoise, iguana, and python were distinctly different from one another and from those obtained with B. hominis and B. lapemi. On the basis of the above-mentioned differences in chromosomal patterns, the tortoise, iguana, and python isolates are described as new species, viz., B. geocheloni sp. nov. from Geochelone carbonaria (red-footed tortoise), B. cycluri sp. nov. from Cyclura cornuta (rhino iguana), and B. pythoni sp. nov. from Python reticulatus (reticulated python).

Animals↗
PubMed2004

Passive stiffness of Drosophila IFM myofibrils: a novel, high accuracy measurement method.

As the smallest muscle-cell substructure that retains the intact contractile apparatus, the single myofibril is considered the optimal specimen for muscle mechanics, although its small size also poses some technical difficulties. Myofibrils from Drosophila indirect flight muscle (IFM) are particularly difficult to study because their high passive stiffness makes them hard to handle, and too resistant to stretch to produce enough elongation for the accurate measurement of sarcomere length change. In this study, we devised a novel method for accurate stiffness measurement of single relaxed myofibrils using microfabricated cantilevers and phase contrast microscopy. A special experimental protocol was developed to minimize errors, and some data analysis strategies were used to identify and exclude spurious data. Remarkably consistent results were obtained from Drosophila IFM myofibrils. This novel, high accuracy method is potentially an effective tool for detecting small passive stiffness change in muscle mutants.

Animals↗
PubMed1997

Three-dimensional endothelial-tumor epithelial cell interactions in human cervical cancers.

The purpose of this study is to understand the multicellular interaction between tumor epithelial (TEC) and human umbilical vein endothelial cells (HUVEC). The development of in vitro systems in which to coculture these cells as multicellular aggregates is very critical. Cell lines were established from cervical tumor cells (n = 6) and two from HUVEC (n = 2) and they were cultured as three-dimensional (3-D) multicellular-cultures using Cytodex-3 microcarrier beads in the rotating wall vessel (RWV). After a 240-h incubation, TEC and HUVEC proliferated exponentially to 4.2 x 10(7) and 2.2 x 10(7) cells/ml, respectively, without requiring a feeder layer; in contrast to the two-dimensional (2-D) cultures that average about 8 x 10(5) cells/ml. Phase contrast microscopy indicated formation of 3-D aggregates that varied in size from 0.5 to 5 mm. The size of the aggregates (1-5 mm, 6-14 microcarriers) increased over time; however, the number of aggregates (0.5-1 mm, 2-5 microcarriers) decreased over a long-term incubation (240 h) because the cells merged to form large clumps. Maximum aggregation was observed with TEC at 120 h and HUVEC at 96 h. The culture of TEC in the absence of HUVEC produced minimal differentiation in contrast to cocultures. The TEC and HUVEC as cocultures in RWV proliferated at an accelerated rate (1.3 x 10(7) cells/ml, 96 h). The TEC-HUVEC coculture presented tubular structures penetrating the tumor cell masses, forming aggregates larger in size than the monocultures and typically with greater cell mass and number. The cells were viable (trypan blue exclusion) and metabolically active (glucose utilization) until 240 h. These data suggest that RWV provides a new model that allows us to investigate the regulatory factors that govern tumor angiogenesis.

Adenocarcinoma↗
PubMed1996

Non-phospholipid fusogenic liposomes.

We have demonstrated the capacity of non-phospholipid liposomes composed primarily of dioxyethylene acyl ethers and cholesterol to fuse with membranes composed primarily of phospholipid. Phase-contrast microscopy, freeze-fracture electron microscopy and a macromolecular probe indicate that these non-phospholipid liposomes can fuse with the plasma membranes of erythrocytes and fibroblasts. Furthermore, fluorescence probe experiments have demonstrated fusion between phosphatidylcholine liposomes and non-phospholipid liposomes. Mixing of internal contents was shown by a terbium/dipicolinate assay. Mixing of membrane lipid components was demonstrated by measuring (i) fluorescence resonance energy transfer between N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine, after phosphatidylcholine liposomes were mixed with non-phospholipid liposomes, and (ii) reduced concentration quenching of rhodaminephosphatidylethanolamine and octadecylrhodamine incorporated into phosphatidylcholine liposomes after mixing with the non-phospholipid liposomes. The degree of apparent fusion reported by the different probe techniques ranged from 25% to 64%.

3T3 Cells↗
PubMed1988

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture.

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.

Animals↗