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At least 1,315 records · Page 73Linked to original sources
PubMed2005

The virulence-associated two-component PhoP-PhoR system controls the biosynthesis of polyketide-derived lipids in Mycobacterium tuberculosis.

Two-component regulatory signal transduction systems are important elements of the adaptative response of prokaryotes to a variety of environmental stimuli. Disruption of PhoP-PhoR in Mycobacterium tuberculosis dramatically attenuates virulence, implying that this system directly and/or indirectly coordinates the expression of important virulence factors whose identity remains to be established. Interestingly, in knockingout the PhoP-PhoR two-component system in M. tuberculosis Mt103, dramatic changes in the colonial morphology, cording properties, and reactivity of the mutant strain to the basic dye neutral red, all intrinsic properties of tubercle bacilli known to correlate with virulence, were noted. Because deficiencies in the ability of the mutant to form serpentine cords and stain with the dye are likely the results of alterations of its cell envelope composition, we undertook to analyze the lipid content of phoP and phoP-phoR mutants constructed in two different strains of M. tuberculosis. Our results indicate that PhoP coordinately and positively regulates the synthesis of methyl-branched fatty acid-containing acyltrehaloses known to be restricted to pathogenic species of the M. tuberculosis complex, namely diacyltrehaloses, polyacyltrehaloses, and sulfolipids. Evidence is also provided that PhoP but not PhoR is required for the production of these lipids. This work represents an important step toward the functional characterization of PhoP-PhoR and the understanding of complex lipid synthesis in M. tuberculosis.

Bacterial Proteins↗
PubMed1997

Modulation of cholestane-3 beta,5 alpha,6 beta-triol toxicity by butylated hydroxytoluene, alpha-tocopherol and beta-carotene in newborn rat kidney cells in vitro.

Cholesterol oxidation products (COP) have been reported to influence vital cellular processes such as cell growth, cell proliferation, membrane function and de novo sterol biosynthesis. The objectives of the present study were: (1) to develop an in vitro model using newborn rat kidney (NRK) cells to investigate the actions of COP; (2) to investigate the effect of COP on cell viability, endogenous antioxidant enzymes activities, i.e. superoxide dismutase (EC 1.15.1.1; SOD) and catalase (EC 1.11.1.6; CAT), and the extent of lipid peroxidation in this model; (3) to determine whether the addition of 100-1000 nM-alpha-tocopherol, beta-carotene or butylated hydroxytoluene (BHT) could protect against COP-induced cytotoxicity. NRK cells were cultured in the presence of various concentrations (5-50 microM) of cholesterol or cholestane-3 beta,5 alpha,6 beta-triol (cholestantriol) for a period of 24 h. Cholesterol over the range 5-50 microM did not induce cytotoxicity as indicated by the neutral-red-uptake assay or the lactate dehydrogenase (EC 1.1.1.27)-release assay. However, cell viability was compromised by the addition of > 10 microM-cholestantriol (P < 0.05). The addition of beta-carotene (100-1000 nM) did not increase cell viability significantly in cholestantriol-supplemented cells. However, the addition of alpha-tocopherol (1000 nM) and BHT (1000 nM) significantly increased percentage cell viability above that of the cholestantriol-supplemented cells but not back to control levels. SOD and CAT activities in NRK cells significantly decreased (P < 0.05) following incubation with cholestantriol. The addition of > 750 nM-alpha-tocopherol, beta-carotene or BHT returned SOD and CAT activities to that of the control. Lipid peroxidation was significantly induced (P < 0.05) in the presence of cholestantriol. Supplementation of the cells with alpha-tocopherol (250, 500 or 1000 nM) or BHT (750 or 1000 nM) resulted in a reduction in the extent of lipid peroxidation (P < 0.05). The addition of beta-carotene over the concentration range of 250-1000 nM did not reduce lipid peroxidation significantly compared with cells exposed to cholestantriol alone. These findings suggest that addition of exogenous antioxidants may be beneficial in the prevention of COP-induced toxicity in vitro.

Animals↗
PubMed2000

Effects of ochratoxin A on cytotoxicity and cell differentiation in cultured rat embryonic cells.

In the present study, the effects of ochratoxin A (OTA) on cytotoxicity, cell differentiation, and other cell functions in the embryonic midbrain cells, which are dopaminergic, were compared to those in the limb bud cells, which are nondopaminergic, to assess the selectivity of OTA central action. Twelve-day rat embryo midbrain and limb bud cells were cultured in Dulbecco's modified Eagle's medium nutrient and Ham's F12 (1:1) mix ture containing 10% Nuserum for 96 h in the presence of various concentrations of OTA. OTA signicfiantly reduced the levels of protein, DNA and glutathione, and [H]thymidine incorporation into DNA in both embryonic midbrain and limb bud cells in a similar concentration-dependent manner. The IC50 values for cytotoxicity measured by neutral red uptake were 1.10 microM in the midbrain cells and 1.05 microM in the limb bud cells. The IC50 values of cell differentiation were 1.10 microM in the midbrain cells and 1.0 microM in the limb bud cells. The addition of exogenous glutathione (32.5 microM) did not change the OTA-induced fall in protein and DNA levels, or the IC50 values of cytotoxicity and differentiation in the midbrain and limb bud cells. Data show that OTA does not appear to exert a selective toxic dopaminergic cell action and that OTA-induced cytotoxicity and inhibition of cell differentiation were not prevented by exogenous glutathione.

Animals↗
PubMed1996

Proteinases in renal cell death.

The role of proteinases in renal proximal tubule (RPT) cellular death was examined using specific inhibitors of proteinases. Rabbit RPT suspensions were incubated with antimycin A for 1 h or tetrafluoroethyl-L-cysteine (TFEC) for 4 h in the absence or presence of the specific cysteine proteinase inhibitor L-trans-epoxysuccinyl-leucylamido (4-guanidino)butane (E-64), the serine proteinase inhibitors N-p-tosyl-L-lysine chloromethyl ketone (TLCK) or 3,4-dichloroisocoumarin (DCS), the serine and cysteine proteinase inhibitors leupeptin or antipain, or the aspartic proteinase inhibitor pepstatin. E-64 and pepstatin decreased lactate dehydrogenase (LDH) release, a marker of cell death, from RPT exposed either to antimycin A or TFEC. TLCK, DCS, leupeptin, or antipain did not decrease antimycin A- or TFEC-induced cell death. Bromohydroquinone- or t-butylhydroperoxide-induced cell death was not decreased by any of the proteinase inhibitors. Loss of lysosomal membrane potential, indicated by neutral red release, occurred prior to the onset of antimycin A-induced cell death. Extensive inhibition of lysosomal cathepsins B and L by E-64 was correlated with cytoprotection. However, E-64 was only protective after some cell death had occurred. These results suggest that lysosomal cysteine and aspartic proteinases, but not serine proteinases, play a role in RPT cell death induced by antimycin A or TFEC. The observation that E-64 was only protective after some cell death had occurred suggests that lysosomal cathepsins are released from dying cells and subsequently attack the remaining viable cells.

Animals↗
PubMed2006

Effect of chitosan-coated alginate microspheres on the permeability of Caco-2 cell monolayers.

Alginate microspheres were prepared by emulsification/internal gelation and coated with chitosan. The ability of chitosan-coated alginate microspheres to increase the paracellular transport across Caco-2 cell monolayers was evaluated in comparison to uncoated microspheres and chitosan solutions. Transport studies were performed by using a permeability marker, Lucifer Yellow (LY), and by measuring the transepithelial electric resistance (TEER) variations. Furthermore, the occurrence of cytotoxic effects was assessed by evaluating neutral red uptake in viable cells and lactate dehydrogenase (LDH) release from damaged cells. A 3-fold increase on LY permeability was obtained for coated microspheres when compared to chitosan solutions. TEER variations were in agreement with permeability results. Chitosan solutions exhibited a dose-dependent toxicity, but coated microspheres did not decrease the viability of cells. Chitosan-coated alginate microspheres have potential to be used as carriers of poorly absorbable hydrophilic drugs to the intestinal epithelia and possibly increase their oral bioavailability.

Alginates↗
PubMed2005

Enhancement of the cell-killing effect of ultraviolet-C radiation by short-term exposure to a pulsed magnetic field.

PURPOSE: To investigate whether low frequency pulsed magnetic field (PMF) exposures produce alterations in the cell killing induced by ultraviolet C (UVC) radiation. MATERIALS AND METHODS: MCF-7 breast cancer cells of exponentially growing cultures were exposed to PMF (25 Hz, 0.75 mT) and UVC (from 6.6 J/m2 to 59.4 J/m2) in two different protocols: (a) cells were exposed to PMF for 30 min and then exposed to UVC at different doses; (b) cells were exposed to PMF for 30 min. After 15 min of the PMF exposure they were exposed simultaneously to PMF+different doses of UVC. After an additional time of 72 h of incubation, viability was measured by the neutral red stain cytotoxicity test. RESULTS: Both exposure protocols produced a significant decrease in the post UVC survival at 13.2 J/m2 and 19.8 J/m2, as compared to controls. The simultaneous exposition of PMF and UVC produced an additional increment in cell killing at 26.4 J/m2, being the greater effects obtained for this second exposure protocol. CONCLUSIONS: Results of the present study show that PMF in combination with UVC have the ability to augment the cell killing effects of UVC radiation. In addition, the effects appear to be greater when PMF and UVC are applied at the same time.

Breast Neoplasms↗
PubMed2005

Infrared microspectroscopy study of gamma-irradiated and H2O2-treated human cells.

PURPOSE: Fourier transform infrared microspectroscopy (FT-IRM), which allows simultaneous detection of biochemical changes in the various cellular compartments, was used as a new analytical tool to study early radiation- and oxidation-induced cellular damage at the molecular level in single human cells. MATERIALS AND METHODS: HaCaT keratinocytes were given a single dose of 6 Gy (137Cs) or 650 microM H2O2, neither of which is cytotoxic (neutral red assay) but both of which result in less than 10% clonogenic survival, and deposited on zinc sulphur (ZnS) windows for infra-red (IR) spectra acquisition, immediately and 2 h after treatment. DNA damage was assessed by comet assays in alkaline conditions. RESULTS: Comet assays showed that the yield of DNA damage was higher after H2O2 treatment than after gamma-irradiation. The comparison between spectra of irradiated and H2O2-treated cells showed common changes, but H2O2 treatment presented a broader spectrum of cellular oxidation than ionizing radiation. The bands characteristic of deoxyribose/ribose in nucleic acids centered at 966 and 997 cm(-1), the bands characteristic of nucleic acid bases centered at 1572, 1599, and 1691 cm(-1), as well as the bands characteristic of ordered secondary structure of DNA centered at 1713-1716 cm(-1), were changed in absorbance, sometimes accompanied by a shift. The bands characteristic of proteins centered at 1515, 1530, 1544 and 1640 cm(-1) were changed in absorbance indicating a decrease in secondary structure of proteins. Moreover, the absorbance of the bands at 1515 and 1630 cm(-1) was correlated the yield of reactive oxygen species. Two hours after both treatments most changes were persistent, suggesting either irreversible or not easily repaired damage or persistent oxidative stress. CONCLUSION: As we previously demonstrated in radiation-induced apoptosis studies, these results show that FT-IRM, in correlation with other cellular biology techniques, might be useful for assessing immediate radiation- and oxidative-induced damage to nucleic acids and proteins in single human cells.

Apoptosis↗
PubMed2002

Oxidative stress in rat cortical neurons and astrocytes induced by paraquat in vitro.

Oxidative stress has been discussed as crucial mechanism of neuronal cell death in the adult brain. However, it was not clear until now whether neurons are more sensitive to oxidative stress than the other cells in the brain, e.g. astrocytes. Therefore both cell types were exposed to oxidative stress provoked by the redox-cycling compound paraquat. Cortical neurons were found to be more sensitive towards paraquat toxicity than astrocytes as shown by MTT and Neutral Red assay, two different cytotoxicity assays. Mitochondrial functions were determined by the mitochondrial membrane potential and intracellular ATP concentrations. Again cortical neurons were more severely impaired (by paraquat than astrocytes). The production of reactive oxygen species after paraquat exposure was much higher in cortical neurons than in astrocytes and correlated with a higher depletion of GSH (intracellular glutathion). Lipid peroxidation could be shown in astrocytes via the breakdown product malondialdehyde (MDA) whereas in cortical neurons 4-hydroxynonenal (4-HNE) was detected as this endpoint. If and how oxidative stress influences the antioxidant defense was determined via changes in the expression of antioxidant enzymes. Paraquat exposure lead to a 2-3 fold increase of catalase, MnSOD and CuZnSOD mRNA expression in astrocytes. In contrast to astrocytes, in cortical neurons catalase and MnSOD mRNA levels were only marginally elevated above 1.5-fold by treatment with paraquat. Expression levels of glutathione peroxidase (GPx) mRNA were the only one that were not changed in both cell types after paraquat exposure. It is concluded that the more marked increase in expression levels of antioxidant enzymes may render astrocytes more resistant to oxidative stress than neuronal cells.

Journal Article↗
PubMed2002

Relationship between tumor cell load and sensitivity to the cytostatic effect of two novel platinum-bile acid complexes, Bamet-D3 and Bamet-UD2.

Based on the organotropic characteristics of bile acids towards the liver and the intestine, two novel compounds of the Bamet family, containing at least one bile acid moiety bound to platinum(II), have been synthesized and their cytostatic effect compared to their ability to become accumulated in tumor cells of hepato-intestinal origin. Bamet-UD2 [cis-diammine-bis-ursodeoxycholic platinum(II)] induced a marked inhibition of cell growth, which was more marked in human hepatoblastoma HepG2 and mouse hepatoma Hepa 1-6 cells than in rat hepatoma McA-RH7777 and human colon adenocarcinoma LS 174T cells. This effect was similar to that observed for cisplatin and stronger than that previously reported for other members of this family, such as Bamet-H2 and Bamet-R2. By contrast, Bamet-D3 [(N'N'' cis-dichloro N(3-3-amminepropylammine)propyl) glycocholamide platinum (II)] was only effective in reducing growth in human hepatoblastoma HepG2 cells. Because the in vitro DNA-reactivity was approximately 5-fold higher for Bamet-D3 than for Bamet-UD2, an additional cause for the difference in their cytostatic abilities was sought, investigating the relationship between cell load and the cytostatic effect of the drugs. Drug uptake by two cell lines, Hepa 1-6 and HepG2, with different sensitivities to these compounds was measured. The cellular uptake of Bamet-D3 and Bamet-UD2 was several-fold higher than that of cisplatin. No significant difference in the amount of both drugs taken up by these cell types was found. A study on sodium-dependency and substrate specificity indicated that Hepa 1-6 cells take up Bamet-D3 and Bamet-UD2 via similar mechanism(s), whereas these compounds do not seem to share the uptake pathways in HepG2 cells. Measurement of cell viability by formazan formation from tetrazolium salts and by neutral red uptake, after short-term (6 h) exposure to the desired drug, indicated that no acute toxic effect occurs in the presence of cisplatin or Bamet-D3 in either HepG2 or Hepa 1-6 cells. By contrast, in both cell lines Bamet-UD2 induced acute cell toxicity in a dose-dependent fashion. In sum, the results indicate that tumor cells efficiently take up these two novel compounds of the Bamet family. Although the exact uptake mechanism remains unknown, it seems to be dependent on the cell type. However, the cell load does not account for the differences in the anti-proliferative properties of the drugs. The strong and promising cytostatic activity of Bamet-UD2 is additionally related to its ability, absent in Bamet-D3, to acutely alter cellular functions other than proliferation.

Animals↗
PubMed1995

Classification of contaminants by mode of action based on in vitro assays.

A concept for toxicity assessments based on in vitro assays of variant levels (i.e., from whole cells to isolated enzymes) is presented. Due to the complexity of organisms of different species, it is evident that no single in vitro test can represent the entire spectrum of toxic potency of chemicals, rather a carefully designed battery of tests has to be employed to account for the various targets attacked in organisms yielding the different modes of action. Cytotoxicity tests like the Neutral-Red Assay predominantly reflect non-specific toxicity, which can be modelled according to a log Pow dependent baseline QSAR. Specific toxicants (e.g., decouplers, acetylcholinesterase inhibitors or photosystem II inhibitors) may be identified based on according in vitro tests and eventually modelled by the respective mode of action related QSARs employing also, e.g., steric or polarizability descriptors to account for specific interactions. The complementation and partial replacement of in vivo (eco)toxicological testing by in vitro assays depends on two criteria: (a) the sensitivity of the tests to reliably detect environmentally relevant concentrations of toxicants and (b) the specificity of the assays to provide an unambiguous classification of toxicants by modes of action: the pattern of interaction with the various targets allows to recognize those compounds of specific effects that frequently occur as outliers in QSAR analyses.

Acetylcholine↗
PubMedDate not supplied

Polyacrylamide-chitosan hydrogels: in vitro biocompatibility and sustained antibiotic release studies.

Controlled drug delivery is gaining importance over the conventional methods of drug administration because of its inherent benefits. Self-regulated release from the delivery vehicle may enhance drug potency with a sustained action. The present study describes a novel hydrogel blend of polyacrylamide with chitosan for controlled delivery of antibiotics. Hydrogel was synthesized by cross-linking acrylamide-chitosan mixture (8:2 v/v) with N,N' methylene bisacrylamide. Hydrogel was characterized for surface morphology, hydrophilicity, pH-dependent swelling properties, cytotoxicity, and control release properties. Scanning electron microscopy (SEM) revealed the macroporous surface morphology of the matrix with average pore size at 104 +/- 7.61 mu. Hydrogel was found to be highly hydrophilic as assessed by octane contact angle (154.5 + 0.572) measurement. Hydrogel showed no cytotoxic effects on NIH3T3 and HeLa cells up to 40% of extract concentrations as determined by MTT and neutral red assay. This showed hydrogel biocompatibility and thus absence of deleterious effects of the hydrogel on cell viability and functionality. Hydrogels did not show any pH-dependent swelling profile, and they swelled considerably to achieve a swelling ratio of approximately 16.0 at the end of 24 hr. Amoxicillin was incorporated in the hydrogel matrix as a candidate antibiotic for release studies. In vitro release studies of amoxicillin revealed the sustained nature of delivery and matrix released 56.47 + 1.12% and 77.096 + 1.72% of amoxicillin at the end of 24 and 75 hr, respectively. Although in vivo studies are awaited, the present study provides enough documentation to consider polyacrylamide-chiotsan hydrogel as a possible candidate for controlled delivery of antibiotics.

3T3 Cells↗
PubMed2004

Cytotoxicity of resorcinol under short- and long-term exposure in vitro.

Cytotoxicity of resorcinol to 3T3 fibroblast in short- (3 hrs) and long-term (72 hrs or 6 weeks) exposure was investigated. The effects of resorcinol on cell viability (neutral red uptake, NRU assay), mitochondrial function (MTT assay) and total cell protein (Kenacid Blue assay) were estimated. As a model for long-term exposure an INTEGRA CL 6-WELL bioreactor was used. The concentrations of resorcinol producing 20, 50 and 80% inhibition of cell growth in the NRU test were lower than in the MTT test after 3 hrs of exposure. The use of an INTEGRA CL 6-WELL bioreactor allows continuous culturing and exposure to test chemical of cells for several weeks, but the strong adhesiveness of fibroblast and forming aggregates make it difficult to remove them from chambers. Resorcinol in concentration of 1 microg/cm(3) did not decrease the viability of cells to 50% of control in long-term exposure in the bioreactor.

Animals↗
PubMed2002

Effects of dibutyl phthalate and monobutyl phthalate on cytotoxicity and differentiation in cultured rat embryonic limb bud cells; protection by antioxidants.

This present study was undertaken to examine the effects of DBP and its metabolite mono-n-butyl phthalate (MBuP) on cytotoxicity and differentiation in cultured rat embryonic limb bud cells. When limb bud cells extracted from rats on gestation d 12.5 were treated with DBP or MBuP for 96 h, induction of cytotoxicity and inhibition of cell differentiation were observed in a concentration-dependent manner. However, MBuP elicited a toxic effect at higher concentrations than DBP. The IC50 values of DBP for cytotoxicity (measured by neutral red uptake) and cell differentiation (measured by alcian blue staining) were 25.54 microg/ml (91.75 microM) and 21.21 microg/ml (76.20 microM), respectively. The IC50 values of MBuP for cytotoxicity and cell differentiation were 307.24 microg/ml (1.38 mM) and 142.61 microg/ml (0.64 mM), respectively. in order to determine whether free radicals are related to induction of cytotoxicity and inhibition of differentiation by DBP in limb bud cells, DBP was coadministered with several antioxidants, including catalase and vitamin E acetate to limb bud cells. Cotreatment with catalase and vitamin E acetate decreased induction of cytotoxicity and inhibition of differentiation by DBP in limb bud cells. However, these compounds did not show any protective effect against MBuP. Results indicate that DBP and MBuP induced developmental toxicity in rat embryonic limb bud cells and suggest that this effect of DBP might be exerted through oxidative stress.

Animals↗
PubMed2005

Comparative cytotoxicity potential of soft contact lens care products.

PURPOSE: To determine the cytotoxicity potential of soft contact lens care products and benzalkonium chloride (BAK) by two colorimetric in vitro assays on an immortalized human corneal epithelial cell line (HCE-T). METHODS: Four commercial soft contact lens care solutions were tested at 1:3 dilution in growth medium. The positive controls for cytotoxicity were BAK in Dulbecco's phosphate buffered saline (DPBS) at 10, 5, 2.5, and 1.25 ppm and 1:3 dilution of DPBS as negative control. Cell viability was assayed using a novel tetrazolium compound and an electron coupling reagent (MTS/PES), and by cell membrane integrity using neutral red dye uptake and release (NRUR). Mean spectrophotometric optical density of the test samples was compared to mean optical density of the controls at 24 and 48 hours of exposure to test product. Significance was measured by ANOVA/Tukey HSD test. RESULTS: Cell viability and cell membrane integrity tests were not significantly different between lens care solutions and the negative controls for CIBA Vision SOLOcare PLUS, Advanced Medical Optics COMPLETE Comfort PLUS, or BAK at 1.25 ppm. Exposure to Bausch and Lomb ReNu MultiPlus, Alcon OPTI-FREE Express with Aldox, BAK 10, 5, and 2.5 ppm yielded significant reduction in cell viability and membrane integrity compared to negative controls. DISCUSSION: Assays of HCE-T cell viability by MTS/PES, and cell membrane integrity by NRUR cells yielded data that were similar to that previously reported with mouse L929 cells in tests based on the USP Elution Test. For the MTS/PES and NRUR assay methods, the solutions in order of increasing cytotoxicity potential were: SOLOCare = COMPLETE Comfort Plus < ReNu < < OPTI-FREE Express with Aldox.

Journal Article↗
PubMed2005

Comparison of several toxicity tests applied to complex wastewaters and mussel biomarkers in receiving waters.

In this study, the complex wastewaters/effluents discharged to coastal regions of Turkey and Greece were sampled, and various toxicity tests were applied. The bioassays used included the assessment of the luminescence inhibition of the bacterium Vibrio fisheri, the photosynthesis inhibition (14C uptake rate) and growth inhibition of the algal species Phaeodactylum tricornutum, and the mortality of the crustacean Artemia franciscana and rotifer Brachionus plicatilis. Additionally, "neutral red retention" and "filtration rate" sublethal biomarker techniques were performed by utilizing the mussel species Mytilus galloproviancialis, inhabiting the points of discharges in Turkey. All discharges tested were found to be acutely toxic to P. tricornutum and slightly toxic to V. fisheri, A. franciscana, and B. plicatilis. Test results showed that the 14C uptake rate toxicity test was the most sensitive one among the other bioassays. The biomarker results showed that the health status of mussels in the coastal areas decreased significantly, indicating the pollution of the receiving waters in Turkey. The conclusions drawn highlight the necessity for an intesive ecotoxicological monitoring scheme that will incorporate the most suitable bioassays and biomarkers to adequately contribute to the upgrading and maintenance of the ecological quality of the coastal waters in Greece and in Turkey.

Aliivibrio fischeri↗
PubMed1957

The Golgi apparatus in neurons and epithelial cells of the common limpet Patella vulgata.

1. In view of widely diverse views held about the identity and structure of the Golgi apparatus in neurons of Mollusca, particularly gastropods, a study has been made on neurons of the common limpet, Patella vulgata, both by light and electron microscopy. A report is given also of observations made on epithelial cells of Patella by electron microscopy. 2. As revealed by Kolatchev's method, the Golgi apparatus in neurons consists basically of black filaments lying to one side of the nucleus. The filaments generally anastomose to form networks of various complexity. Rarely some cells contain only discrete filaments. Associated with some of the filaments is a weakly osmiophilic substance identified as archoplasm. Kolatchev's method also revealed spheroidal bodies (neutral red bodies, "lipochondria," etc.). 3. It has not been possible to demonstrate the Golgi apparatus using either iron-haematoxylin or Sudan black. 4. Examination of Kolatchev's preparations by electron microscopy has revealed that some of the Golgi filaments consist of chromophilic and chromophobic components. The chromophilic component consists of dense lamellae. 5. After fixation in buffered osmium tetroxide solution and examination by electron microscopy, it has been concluded that (a) the chromophilic component of the Golgi apparatus corresponds to a system of paired membranes (which usually enclose an inner dense substance), (b) the chromophobic component corresponds to a substance lying within small dilations of the paired membrane, and (c) the archoplasm corresponds to numerous small vesicles. 6. The paired membranes branch, anastomose, and can often be traced back to a common source. They are interpreted as lamelliform folds, and occasionally tubular processes, of essentially a single Golgi membrane. In cells containing a Golgi network it is suggested that the membrane extends through the whole of the apparatus in such a way that the substance it encloses may be regarded as being in a continuous phase. 7. Epithelial cells of Patella contain a juxtanuclear Golgi apparatus with an ultrastructure similar to that described for neurons.

Cell Count↗
PubMed1968

Studies on lysosomes. XII. Redistribution of acid hydrolases in human lymphocytes stimulated by phytohemagglutinin.

Subcellular fractions were isolated by differential centrifugation from pure suspensions of human blood lymphocytes incubated with and without phytohemagglutinin (PHA). Between 30 and 120 min after addition of PHA to intact cells, redistribution of acid hydrolases (beta glucuronidase, acid phosphatase), from a 20,000 g x 20 min granular fraction into the corresponding supernatant, was observed. No increase in total acid hydrolase activity was found at these times. The mitochondrial marker enzyme, malate dehydrogenase, did not undergo redistribution. Granules derived from PHA-treated cells became more fragile upon subsequent incubation with membrane-disruptive agents in vitro (streptolysin S, filipin). These changes were associated with an increase in the over-all permeability of the stimulated cell to substances in the surrounding medium, such as neutral red. Augmentation of dye entry into lymphocytes required intact metabolism as judged by response to temperature and inhibitors (cyanide, antimycin A, 2,4-dinitrophenol). PHA, however, did not release enzyme activity from hydrolase-rich granules in vitro or render them more susceptible to subsequent challenge with membrane-disruptive agents. These studies suggest that PHA induces early changes in the surface of lymphocytes. The consequent redistribution of acid hydrolases may play a role in remodeling processes of the stimulated cells.

Acid Phosphatase↗
PubMed1974

Protein degradation in cultured cells. II. The uptake of chloroquine by rat fibroblasts and the inhibition of cellular protein degradation and cathepsin B1.

The degradation of cellular proteins in fibroblasts, both those of rapid and those of slow turnover rates, was inhibited by low concentrations of chloroquine or neutral red in the medium. Cells inhibited by chloroquine can be inhibited further by fluoride. Chloroquine was taken up by the fibroblasts and the concentration in the cells reached several hundred times that in the medium. Isopycnic fractionation studies showed that within the cells the chloroquine was concentrated in the lysosomes, and that these chloroquine-containing lysosomes had a lower equilibrium density than the lysosomes of untreated cells. Chloroquine, at concentrations attained inside the lysosomes, inhibited cathepsin B(1) but not cathepsin D. It is concluded that chloroquine impairs the breakdown of cellular proteins after these have entered the lysosome system, probably through inhibition of cathepsin B(1).

Animals↗