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As a possible viable and non-invasive method to identify high producing cells, Confocal Raman Microscopy was shown to be able to differentiate CHO host cell lines and derivative production clones. Cluster analysis of spectra and their derivatives was able to differentiate between different producer cell lines and a host, and also distinguished between an intracellular region of high lipid and protein content that in structure resembles the Endoplasmic Reticulum. This ability to identify the ER may be a major contributor to the identification of high producers. PCA enabled the discrimination even of host cell lines and their subclones with inherently higher production capacity. The method is thus a promising option that may contribute to early, non-invasive identification of high potential candidates during cell line development and possibly could also be used for proof of identity of established production clones., (© 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cellular organelles have been shown to shuttle between cells in co-culture. We hereby show that titanium dioxide (TiO2) nanoparticles (NPs) can be transferred in such a manner, between cells in direct contact, along with endosomes and lysosomes. A co-culture system was employed for this purpose and the NP transfer was observed in mammalian cells including normal rat kidney (NRK) and HeLa cells. We found that the small GTPase Arf6 facilitates the intercellular transfer of smaller NPs and agglomerates. Spherical, anatase nano-TiO2 with sizes of 5 (Ti5) and 40 nm (Ti40) were used in this study. Humans are increasingly exposed to TiO2 NPs from external sources such as constituents of foods, cosmetics, and pharmaceuticals, or from internal sources represented by Ti-based implants, which release NPs upon abrasion. Exposure to 5 mg/l of Ti5 and Ti40 for 24 h did not affect cellular viability but modified their ability to communicate with surrounding cells. Altogether, our results have important implications for the design of nanomedicines, drug delivery and toxicity.

A cell pellet biophantom technique is introduced, and applied to the ultrasonic backscatter coefficient (BSC) estimate using Chinese hamster ovary (CHO) cells. Also introduced is a concentric sphere scattering model because of its geometrical similarities to cells with a nucleus. BSC comparisons were made between the concentric sphere model and other well-understood models for mathematical verification purposes. BSC estimates from CHO cell pellet biophantoms of known number density were performed with 40 and 80 MHz focused transducers (overall bandwidth: 26-105 MHz). These biophantoms were histologically processed and then evaluated for cell viability. Cell pellet BSC estimates were in agreement with the concentric sphere model. Fitting the model to the BSC data yielded quantitative values for the outer sphere and inner sphere. The radius of the cell model was 6.8 ± 0.7 μm; the impedance of the cytoplasm model was 1.63 ± 0.03 Mrayl and the impedance of the nuclear model was 1.55 ± 0.09 Mrayl. The concentric sphere model appears as a new tool for providing quantitative information on cell structures and will tend to have a fundamental role in the classification of biological tissues.

Mutations in MPZ, which encodes myelin protein zero (P(0)), may lead to different subtypes of Charcot-Marie-Tooth disease (CMT). The aim of this study was to characterize the cellular manifestations of various MPZ mutations associated with CMT1, Dejerine-Sottas syndrome (DSS) and CMT2, and to correlate their cellular and clinical phenotypes. Nine P(0) mutants associated with CMT1 (P(0)S63F, R98H, R277S, and S233fs), DSS (P(0) I30T and R98C), and CMT2 (P(0)S44F, D75V, and T124M), were investigated. Wild-type and mutant P(0) fused with fluorescent proteins were expressed in vitro to monitor their intracellular localization. An adhesiveness assay was used to evaluate the adhesiveness of the transfected cells. Protein localization and cell adhesiveness of each mutant protein were compared and correlated with their clinical phenotypes. Three different intracellular localization patterns of the mutant P(0) were observed. Wild-type P(0), P(0)I30T, S44F, S63F, D75V, T124M, and R227S were mostly localized on the cell membrane, P(0)R98H, and R98C were found in the endoplasmic reticulum (ER) or Golgi apparatus, and P(0)S233fs formed aggregates within the ER. Cells expressing mutant P(0), as compared with those expressing wild-type P(0), demonstrated variable degrees of reduction in the cell adhesiveness. The molecular patho-mechanisms of MPZ mutations are likely very complex and the clinical phenotype must be influenced by many genetic or environmental factors. This complexity may contribute to the highly variable clinical manifestations resulting from different MPZ mutations.

Objectives: The potential applications and industrial production of multi-wall carbon nanotubes (MWCNT) have raised serious concerns about their safety for human health and the environment. The present study was designed to examine the in vitro cytotoxicity and genotoxicity of MWCNT and UICC chrysotile A (chrysotile)., Methods: Cytotoxicity using both colony formation and lactate dehydrogenase (LDH) assays and genotoxicity including chromosome aberration, micronucleus induction and hgprt mutagenicity were examined by exposing cultured Chinese hamster lung (CHL/IU) cells to MWCNT or chrysotile at different concentrations., Results: The in vitro cytotoxicity of MWCNT depended on the solvent used for suspension of MWCNT and ultrasonication duration of the MWCNT suspension. A combination of DMSO/culture medium and 3-minute ultrasonication resulted in a well-dispersed medium with dispersion and isolation of agglomerated MWCNT by ultrasonication which manifested the highest cytotoxicity. The cytotoxicity was more potent for chrysotile than MWCNT. The genotoxicity of MWCNT was characterized by the formation of polyploidy without structural chromosome aberration, and an increased number of bi- and multi-nucleated cells without micronucleus induction, as well as negative hgprt mutagenicity. Chrysotile exhibited essentially the same genotoxicity as MWCNT, except for marginal but significant induction of micronuclei. MWCNT and chrysotile were incompletely internalized in the cells and localized in the cytoplasm., Conclusions: MWCNT and chrysotile were cytotoxic and genotoxic in Chinese hamster lung cells, but might interact indirectly with DNA. The results suggest that both test substances interfere physically with biological processes during cytokinesis.

Amyloid beta protein (Abeta), a pathogenic molecule associated with Alzheimer's disease, is produced by gamma-secretase, which cleaves the beta-carboxyl terminal fragment (betaCTF) of beta-amyloid precursor protein in the middle of its transmembrane domain. How the cleavage proceeds within the membrane has long been enigmatic. We hypothesized previously that betaCTF is cleaved first at the membrane-cytoplasm boundary, producing two long Abetas, Abeta(48) and Abeta(49), which are processed further by releasing three residues at each step to produce Abeta(42) and Abeta(40), respectively. To test this hypothesis, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify the specific tripeptides that are postulated to be released. Using CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonate)-reconstituted gamma-secretase system, we confirmed that Abeta(49) is converted to Abeta(43/40) by successively releasing two or three tripeptides and that Abeta(48) is converted to Abeta(42/38) by successively releasing two tripeptides or these plus an additional tetrapeptide. Most unexpectedly, LC-MS/MS quantification revealed an induction period, 3-4 min, in the generation of peptides. When extrapolated, each time line for each tripeptide appears to intercept the same point on the x-axis. According to numerical simulation based on the successive reaction kinetics, the induction period exists. These results strongly suggest that Abeta is generated through the stepwise processing of betaCTF by gamma-secretase.

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine repeat tract in the huntingtin protein. Polyglutamine-expanded huntingtin forms intranuclear as well as perinuclear inclusion bodies. Perinuclear aggregates formed by polyglutamine-expanded proteins are associated with a characteristic indentation of the nuclear envelope. We examined the nuclear envelope in cells containing huntingtin aggregates using immunostaining for lamin B1, a major component of the nuclear lamina. Laser confocal microscopy analysis revealed that huntingtin aggregates in a juxtanuclear position were associated with a clear focal distortion in the nuclear envelope in cells transfected with polyglutamine-expanded huntingtin. Lamin B1 distribution was not altered by aggregates of polyglutamine-expanded ataxin-1, that are exclusively intranuclear. Thus lamin immunocytochemistry demonstrates clearly the depression of the nuclear envelope resulting from the formation of perinuclear aggregates by polyglutamine-expanded huntingtin. Lamin immunocytochemistry would be of value to monitor the state of the nuclear envelope in experimental paradigms aimed at establishing the significance of perinuclear aggregates of pathogenic proteins.

Cryo-electron microscopy of vitrified specimen is the method of choice to explore cellular ultrastructure at high resolution as close as possible to the native state and environment. In this study, we investigated the Golgi apparatus - the main organelle of the secretory pathway. Cultured mammalian cells were fixed by high-pressure freezing, sectioned in vitreous ice and subjected to cryo-electron microscopy and cryo-electron tomography. Although the overall morphology of Golgi stacks was comparable to well prepared and plastic-embedded samples, in detail we reached much higher resolution in terms of distinction between biological structures based on their native density. On cisternal buds and peri-Golgi vesicles--some associated with microtubules--we detected two different subtypes of COPI coats: (1) a homogenous coat and (2) an inhomogeneous spiky coat, providing an 8-9 nm regularity, clearly distinct from clathrin coat. Next, we monitored the secretion of cargo, namely, procollagen I, through the Golgi complex. Temporally correlated with fluorescence microscopy, we performed three-dimensional cryo-electron tomography analysis and detected Golgi cisternae enlarged to saccules, containing cargo and showing inter-cisternal connections. Our work provides a first step towards the high-resolution description of the secretory pathway in native vitrified samples and describes the challenges associated with this attempt.

Since GAD65 undergoes post-translational processing and targeting to subcellular compartments and membranes, it may exhibit different immunochemical properties in the cell context compared with the soluble protein expressed in the cell-free eukaryotic system used in the reference method for GADA assessment (radioligand binding assay (RBA)). In the present work, we detected and characterized GADA in 72 sera from patients with type 1 diabetes mellitus (DM) and 14 sera from adult-onset diabetes patients using analytical systems in which GAD65 is expressed in a cellular context: confocal indirect immunofluorescence (IIF) and electron microscopy after immunogold labeling on monolayers of transfected Chinese hamster ovary (CHO) cells, and immunoprecipitation (IP) of metabolically labeled GAD65. Eighteen serum samples, 16 from type 1 diabetes patients and two from adult-onset diabetes patients, were positive by confocal IIF but scored negative by RBA. All of these 18 sera immunoprecipitated a 65 kDa protein, supporting the existence of the GADA marker in such patients. It may be concluded that GADA negativity by the conventional RBA method using the soluble antigen, as well as negativity for other common markers measured by similar methods, is not enough to rule out the existence of the specific autoimmune component in childhood or adult-onset diabetes. Other analytical methods based in a more physiological presentation of the autoantigen structure, as confocal IIF and IP, bring an extra support to assess the complete repertoire of specific autoantibodies to native-like and membrane-bound, or denatured, beta-cell antigens.

Lipid rafts are small cholesterol- and glycosphingolipid-enriched membrane subdomains. Here we compared the mu opioid receptor (MOR)-lipid rafts relationship in the rat brain, where neurons have non-caveolae rafts, and in CHO cells stably transfected with HA-rat MOR (CHO-HA-rMOR), which are enriched in caveolae. Membranes of rat caudate putamen (CPu) and thalamus or CHO-HA-rMOR cells were homogenized, sonicated in a detergent-free 0.5 M Na(2)CO(3) buffer and fractionated through sucrose density gradients. Western blot and [(3)H]diprenorphine binding showed that approximately 70% of MOR in CHO-HA-rMOR was present in low-density (5-20% sucrose) fractions enriched in cholesterol and/or ganglioside M1 (GM1) (lipid rafts) in plasma membranes, whereas about 70% and 45% of MOR in CPu and thalamus, respectively, were associated with lipid rafts. Incubation with a saturating concentration of etorphine or morphine at 37 degrees C for 30 min failed to change the MOR location in rafts in CHO-HA-rMOR, indicating that the internalized MOR does not move out of rafts, in contrast to the delta opioid receptor. In vivo, rafts association of MOR in CPu and thalamus was not affected significantly in rats implanted with two 75-mg morphine pellets for 72 h. In addition, cholesterol reduction by methyl-beta-cyclodextrin (MCD) disrupted rafts and shifted MOR to higher density fractions in both CHO-HA-rMOR and CPu membranes. However, MCD treatment had opposite impacts on MOR signaling in the two tissues: it attenuated MOR-mediated [(35)S]GTPgammaS binding in CPu but enhanced it in CHO-HA-rMOR.

Several causal missense mutations in protein kinase C gamma (gamma PKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that mutant gamma PKC found in SCA14 is susceptible to two types of aggregation, cytoplasmic dot-like and perinuclear massive aggregation, and causes cell death in Chinese hamster ovary cells. Long-term time-lapse imaging revealed that firstly accumulated dot-like aggregation of mutant gamma PKC-green fluorescent protein (GFP) gradually formed perinuclear massive aggregations, followed by cell death. However, it remains unclear how aggregate formation of mutant gamma PKC causes cell death. In the present study, we examined whether these mutant aggregations affect the ubiquitin-proteasome system (UPS) and endoplasmic reticular (ER) stress. Two mutant gamma PKC-GFPs (S119P and G128D) were strongly ubiquitinated, and dot-like aggregations of these mutants were ubiquitin-positive and colocalized with proteasome 20S. Furthermore, proteasome activity in cells with aggregates, especially massive ones, was significantly decreased. Aggregate formation of mutant gamma PKC-GFP induced phosphorylation of PERK (PKR-like ER kinase) and nuclear expression of CHOP (C/EBP homologous protein), hallmarks of ER stress and subsequently activated caspase-3. These results indicate that aggregate formation of mutant gamma PKC found in SCA14 impairs UPS and induces ER stress, leading to apoptotic cell death.

A glycosyl hydrolase family 38 enzyme, neutral alpha-mannosidase, has been proposed to be involved in hydrolysis of cytosolic free oligosaccharides originating either from ER-misfolded glycoproteins or the N-glycosylation process. Although this enzyme has been isolated from the cytosol, it has also been linked to the ER by subcellular fractionations. We have studied the subcellular localization of neutral alpha-mannosidase by immunofluorescence microscopy and characterized the human recombinant enzyme with natural substrates to elucidate the biological function of this enzyme. Immunofluorescence microscopy showed neutral alpha-mannosidase to be absent from the ER, lysosomes, and autophagosomes, and being granularly distributed in the cytosol. In experiments with fluorescent recovery after photo bleaching, neutral alpha-mannosidase had slower than expected two-phased diffusion in the cytosol. This result together with the granular appearance in immunostaining suggests that portion of the neutral alpha-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man(9)GlcNAc to Man(5)GlcNAc in the presence of Fe(2+), Co(2+), and Mn(2+), and uniquely to neutral alpha-mannosidases from other organisms, the human enzyme was more activated by Fe(2+) than Co(2+). Without activating cations the main reaction product was Man(8)GlcNAc, and Cu(2+) completely inhibited neutral alpha-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral alpha-mannosidase in hydrolysis of soluble cytosolic oligomannosides.

G protein-coupled receptors (GPCRs) constitute important targets for drug discovery against a wide range of ailments including cancer, inflammatory, and cardiovascular diseases. Efforts are underway to screen selective modulators of GPCRs and also to deorphanize GPCRs with unidentified natural ligands. Most GPCR-based cellular screens depend on labeling or recombinant expression of receptor or reporter proteins, which may not capture the true physiology or pharmacology of the GPCRs. In this paper, we describe a noninvasive and label-free assay for GPCRs that can be used with both engineered and nonengineered cell lines. The assay is based on using cell-electrode impedance to measure minute changes in cellular morphology as a result of ligand-dependent GPCR activation. We have used this technology to assay the functional activation of GPCRs coupled to different signaling pathways and have compared it to standard assays. We have used pharmacological modulators of GPCR signaling pathways to demonstrate the specificity of impedance-based measurements. Our data indicate that cell-electrode impedance measurements offer a convenient, sensitive, and quantitative method for assessing GPCR function. Moreover, the noninvasive nature of the readout offers the added advantage of performing multiple treatments in the same well to study events such as desensitization and receptor cross-talk.

Polycystin-1 (PC-1) is the product of the PKD1 gene, which is mutated in autosomal dominant polycystic kidney disease. We show that the Na,K-ATPase alpha-subunit interacts in vitro and in vivo with the final 200 amino acids of the polycystin-1 protein, which constitute its cytoplasmic C-terminal tail. Functional studies suggest that this association may play a role in the regulation of the Na,K-ATPase activity. Chinese hamster ovary cells stably expressing the entire PC-1 protein exhibit a dramatic increase in Na,K-ATPase activity, although the kinetic properties of the enzyme remain unchanged. These data indicate that polycystin-1 may contribute to the regulation of Na,K-ATPase activity in kidneys in situ, thus modulating renal tubular fluid and electrolyte transport.

Research on the subtelomeric region has considerably increased because this chromosome segment (1) keeps the chromosome number constant, (2) intervenes in cancer and cell senescence processes, (3) presents more crossovers than other regions of the genome and, (4) is the site of cryptic chromosome aberrations associated with mental retardation and congenital malformations. Quantitative microphotometrical scanning and computer graphic image analysis enables the detection of differentially distributed Giemsa-stained structures in T-banded subtelomeric segments of human and Chinese hamster ovary (CHO) chromosomes. The presence of high density stain patterns in the subtelomeric region was confirmed using endoreduplicated chromosomes as a model. Besides, prolonging the incubation in the T-buffer, specific holes were induced in subtelomeric segments. Hole specificity was confirmed inducing them in complex CHO chromosome aberrations obtained by AluI. The method was also used to detect minute sister chromatid exchanges in the T-banded subtelomeric area (t-SCEs). The presence of t-SCEs was suspected to reflect, at the microscope level, the high crossover activity prevailing in the region. Due to the fact that the fluorescent signals obtained with subtelomeric probes seem to be colocalized with subtelomeric high density areas, measurements on the position of both structures with respect to the diffraction and chromosome edges were carried out. Data obtained showed comparable values suggesting that the high density segments were located where telomeric probes usually fluoresce. The possible relationship of the high density patterns, the production of specific holes, the localization of fluorescent areas and the detection of minute SCEs in the subtelomeric segment observed in T-banded CHO and human chromosomes is briefly reviewed., (Copyright 2003 S. Karger AG, Basel)

The DNA lesions responsible for the formation of sister chromatid exchanges (SCEs) have been the object of research for a long time. SCEs can be visualized by growing cells for either two rounds of replication in the presence of 5-bromo-2'-deoxyuridine (BrdU) or for one round with BrdU and the next without. If BrdU is added after cells were treated with a DNA-damaging agent, the effect on SCEs can only be analyzed in the second post-treatment mitosis. If one wishes to analyze the first post-treatment mitosis, cells unifilarily labeled with BrdU must be treated. Due to the highly reactive bromine atom, BrdU interacts with such agents like ionizing and UV radiation enhancing the frequency of SCEs. However, its precise role in this process was difficult to assess for a long time, because no alternative technique existed that allowed differential staining of chromatids. We have recently developed a method to differentially label sister chromatids with biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-dUTP) circumventing the disadvantage of BrdU. This technique was applied to study the SCEs induced by ionizing and UV radiation as well as by mitomycin C, DNaseI and AluI. This article is a review of the results and conclusions of our previous studies., (Copyright 2003 S. Karger AG, Basel)

SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed., (Copyright 2003 S. Karger AG, Basel)

Sister chromatid exchanges (SCEs) are symmetrical exchanges between newly replicated chromatids and their sisters. While homologous recombination may be one of the principal mechanisms responsible for SCEs, the full details of their molecular basis and biological significance remain to be elucidated. Following exposure to ultraviolet light B (UVB), mitomycin C (MMC) and cisplatin, we analyzed the location of SCEs on metaphase chromosomes in Chinese hamster CHO cells. The frequency of SCEs increased over the spontaneous level in proportion to the agent's dose. UVB-induced SCEs occurred frequently in telomere regions, as cisplatin-induced SCEs did, differing from MMC-induced ones. The remarkable difference of intrachromosomal distribution among the three mutagens may be attributed to the specificity of induced DNA lesions and structures of different chromosome regions. Telomeric DNA at the end of chromosomes is composed of multiple copies of a repeated motif, 5'-TTAGGG-3' in mammalian cells. Telomeric repeats may be potential targets for UVB and cisplatin, which mainly form pyrimidine dimers and intrastrand d(GpG) cross-links, respectively, resulting in SCE formation. UVB irradiation shortened telomeres and augmented the telomerase activity. The possible implications of the frequent occurrence of SCEs in telomere regions are discussed in connection with the maintenance of telomere integrity., (Copyright 2003 S. Karger AG, Basel)

Radiation-induced chromosome damage can be measured in interphase using the Premature Chromosome Condensation (PCC) technique. With the introduction of a new PCC technique using the potent phosphatase inhibitor calyculin-A, chromosomes can be condensed within five minutes, and it is now possible to examine the early damage induced by radiation. Using this method, it has been shown that high-LET radiation induces a higher frequency of chromatid breaks and a much higher frequency of isochromatid breaks than low-LET radiation. The kinetics of chromatid break rejoining consists of two exponential components representing a rapid and a slow time constant, which appears to be similar for low- and high-LET radiations. However, after high-LET radiation exposures, the rejoining process for isochromatid breaks influences the repair kinetics of chromatid-type breaks, and this plays an important role in the assessment of chromatid break rejoining in the G2 phase of the cell cycle., (Copyright 2003 S. Karger AG, Basel)

In a recent paper we reported the results of an experiment carried out by analysing chromosomal damage in Chinese hamster (CHO) cells exposed to low doses of X-rays. The present investigation was undertaken in order to validate those results using a different approach, the single cell gel electrophoresis assay (comet assay) immediately after irradiation. Cells were cultured during 14 cycles, irradiation treatment was performed once per cycle when the cells were at 90-95% of confluence. Doses of 2.5, 5.0 and 10.0 mSv were used. Sequential irradiation of CHO cells induced a decrease of cells without migration and an increase of cells showing DNA damage with the three doses employed. Significant increases of low-level damaged cells (p < 0.001) were found for the 14 exposures when compared to controls except for the first irradiations with 2.5 and 10 mSv, respectively. No significant increase of the frequency of cells with severe damage was observed in any case. These findings could be explained by assuming a complex interactive process of cell recovery, DNA damage and repair together with the induction of genomic instability, the incidence of bystander effects as well as some kind of radioadaptative response of the cells. If these phenomena are limited to the cell line employed deserves further investigation., (Copyright 2003 S. Karger AG, Basel)

The degradation of misfolded and unassembled proteins by the endoplasmic reticulum (ER)-associated degradation (ERAD) has been shown to occur mainly through the ubiquitin-proteasome pathway after transport of the protein to the cytosol. Recent work has revealed a role for N-linked glycans in targeting aberrant glycoproteins to ERAD. To further characterize the molecular basis of substrate recognition and sorting during ERAD in mammalian cells, we expressed a mutant yeast carboxypeptidase Y (CPY*) in CHO cells. CPY* was retained in the ER in un-aggregated form, and degraded after a 45-min lag period. Degradation was predominantly by a proteasome-independent, non-lysosomal pathway. The inhibitor of ER mannosidase I, kifunensine, blocked the degradation by the alternate pathway but did not affect the proteasomal fraction of degradation. Upon inhibition of glucose trimming, the initial lag period was eliminated and degradation thus accelerated. Our results indicated that, although the proteasome is a major player in ERAD, alternative routes are present in mammalian cells and can play an important role in the disposal of both glycoproteins and non-glycoproteins.

Surfactant protein C (SP-C) is a small lipopeptide of which the main part consists of a typical valyl-rich transmembrane domain. The protein is expressed as a propeptide (proSP-C) which is processed and sorted via the regulated secretory pathway to the lamellar body, where mature SP-C is stored before secretion into the alveolar space. In this study we investigated the identity of the compartment to which proSP-C is sorted in cells that do not have a regulated secretory pathway, such as CHO cells. By electron microscopy we determined that proSP-C was localized in an uncommon membrane compartment with very regular morphology, which was not present in control cells. This membrane compartment is not influenced by the palmitoylation of proSP-C and is probably derived from the endoplasmic reticulum. However, proSP-C chimeras with artificial transmembrane domains induced a membrane compartment with a different morphology. Therefore we propose that the typical amino acid sequence of the transmembrane domain of proSP-C plays a role in membrane formation and morphology, which may be relevant under physiological conditions.

Prion diseases are infectious, sporadic and inherited fatal neurodegenerations that are propagated by an abnormal refolding of the cellular prion protein PrP(C). Which chaperones assist the normal folding of PrP(C) is unknown. The linkage of familial Gerstmann- Sträussler-Scheinker (GSS) syndrome with proline substitutions in PrP raised the prospect that peptidylprolyl cis-trans isomerases (PPIases) may play a role in normal PrP metabolism. Here we used cyclo sporin A (CsA), an immunosuppressant, to inhibit the cyclophilin family of PPIases in cultured cells. CsA-treated cells accumulated proteasome-resistant, 'prion-like' PrP species, which deposited in long-lived aggresomes. PrP aggresomes also formed with disease-linked proline mutants when proteasomes were inhibited. These results suggest mechanisms whereby abnormally folded cytosolic PrP may in some cases participate in the development of spontaneous and inherited prion diseases.

The centrosome usually replicates in a semiconservative fashion, i.e., new centrioles form in association with preexisting "maternal" centrioles. De novo formation of centrioles has been reported for a few highly specialized cell types but it has not been seen in vertebrate somatic cells. We find that when centrosomes are completely destroyed by laser microsurgery in CHO cells arrested in S phase by hydroxyurea, new centrosomes form by de novo assembly. Formation of new centrosomes occurs in two steps: approximately 5-8 h after ablation, clouds of pericentriolar material (PCM) containing gamma-tubulin and pericentrin appear in the cell. By 24 h, centrioles have formed inside of already well-developed PCM clouds. This de novo pathway leads to the formation of a random number of centrioles (2-14 per cell). Although clouds of PCM consistently form even when microtubules are completely disassembled by nocodazole, the centrioles are not assembled under these conditions.

Chinese hamster ovary cells stably expressing a rat dopamine transporter (designated D8 cells) and neuroblastoma SK-N-SH cells were used as two model systems to study dopamine neurotoxicity. Within 24 h, 1-10 mM dopamine induced D8 cells into apoptosis while 20-200 microM dopamine induced SK-N-SH cells into cell death. The viability of both cell types decreased in a dose-dependent manner. However, the dopamine uptake activity of D8 cells at 10 mM was not significantly higher than the uptake at 100 microM, suggesting that it was not the high concentration of intracellular dopamine that induced D8 cells into apoptosis, but rather dopamine found in the extracellular space. Furthermore, cocaine, an inhibitor of dopamine uptake, could not block cell death induced by dopamine. Forskolin, an agonist of protein kinase A (PKA), stimulated dopamine uptake in D8 cells and blocked apoptosis induced by the drug. These results suggest that the dopamine transporter mediates a dopamine-dependant apoptotic signal transduction pathway that is independent of dopamine uptake into the cell.

Annexins are a family of calcium-dependent phospholipid-binding proteins, which have been implicated in a variety of biological processes including membrane trafficking. The annexin 6/lgp120 prelysosomal compartment of NRK cells was loaded with low-density lipoprotein (LDL) and then its transport from this endocytic compartment and its degradation in lysosomes were studied. NRK cells were microinjected with the mutated annexin 6 (anx6(1-175)), to assess the possible involvement of annexin 6 in the transport of LDL from the prelysosomal compartment. The results indicated that microinjection of mutated annexin 6, in NRK cells, showed the accumulation of LDL in larger endocytic structures, denoting retention of LDL in the prelysosomal compartment. To confirm the involvement of annexin 6 in the trafficking and the degradation of LDL we used CHO cells transfected with mutated annexin 6(1-175). Thus, in agreement with NRK cells the results obtained in CHO cells demonstrated a significant inhibition of LDL degradation in CHO cells expressing the mutated form of annexin 6 compared to controls overexpressing wild-type annexin 6. Therefore, we conclude that annexin 6 is involved in the trafficking events leading to LDL degradation., (Copyright 2001 Academic Press.)

Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated how B. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca(2+)- and Mg(2+)-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.

This unit describes the isolation of Golgi membranes on a preparative basis. Methods are provided for rapid isolation of dextran-treated Golgi stacks from rat liver using a sucrose density barrier, and Golgi isolation by floatation from a light mitochondrial fraction, along with an alternate procedure using a self-generated iodixinol gradient. If the Golgi tends to vesiculate during homogenization (commonly the case with cultured cells), a primary requirement is to separate these vesicles from other microsomal compartments, so a procedure is described for a discontinuous gradient of sucrose for cultured cells, and an alternate protocol describes a continuous iodixinol gradient that may provide greater resolution. A self-generated iodixinol gradient is described to prepare Golgi membranes from a microsomal fraction of rat hepatocytes, and a standard Golgi enzyme marker assay is given in a support protocol.

In this article we present the development of a multibeam two-photon laser scanning microscope. A new type of beam splitter to create the multitude of laser beams is described. This type of beam splitter has higher transmission and generates more uniform beams than can be achieved with the microlens approach used by other groups. No crosstalk exists between the different foci due to small temporal delays between the individual beams. The importance of dispersion compensation to obtain maximum efficiency of the microscope is discussed. With optimum compensation the fluorescence signal was raised by a factor of 14. Different modes of detecting the fluorescence signals and their effect on imaging speed and resolution are discussed.

Syndecans are cell-surface heparan sulfate proteoglycans, which perform a variety of functions in the cell. Most important, they are co-receptors for growth factors and mediate cell-cell and cell-matrix interactions. Four syndecans (syndecan 1-4) have been described in different species. The aim of this work was the cloning and characterization of human syndecan-3. The human syndecan-3 sequence has high homology to the rat and mouse sequences, with the exception of the 5'-region. Syndecan-3 mRNA is mostly expressed in the nervous system, the adrenal gland, and the spleen. When different cell lines were transiently transfected with full-length syndecan-3 cDNA, it was localized to the membrane and induced the formation of long filopodia-like structures, microspikes, and varicosities. Consequently, the actin cytoskeleton was re-organized, since actin staining was mostly found in the cellular extensions and at the cell periphery, co-localizing with the syndecan-3 staining. The development of the phenotype depended on the presence of sugar chains, as transfected glycosaminoglycan-deficient Chinese hamster ovary (CHO) 745 cells did not show these structural changes, nor did transfected CHO K1 cells in the presence of heparin. The similarity of the cloned DNA sequence with that of other mammalian species and the high expression in the nervous system led us to the assumption that human syndecan-3 could perform comparable functions to those described for syndecan-3 in rat and mouse. Additionally, transient transfection experiments suggest a role of human syndecan-3 in the organization of cell shape by affecting the actin cytoskeleton, possibly by transferring signals from the cell surface in a sugar-dependent mechanism.

The objective of the present study was to evaluate in vitro, using Chinese hamster ovary (CHO-K1) cells, the genotoxicity of genipin, a naturally occurring crosslinking agent. Glutaraldehyde, the most commonly used crosslinking agent for biologic tissue fixation, was employed as a reference chemical. The selected procedures for this evaluation were the micronucleus (MN) and sister chromatid exchange (SCE) assays with or without the addition of a metabolic activation system (S9 mix). Before starting the genotoxicity assays, the maximum noncytotoxic amounts of glutaraldehyde and genipin were determined using the MTT assay. The results obtained in the MTT assay revealed that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde with or without S9 mix. The frequencies of MN observed in the cases drugged with varying concentrations of glutaraldehyde or genipin were not statistically different from those seen in the negative controls (blank) in the presence or absence of S9 mix. However, it was noted that glutaraldehyde significantly inhibited the cell-cycle progression while the cells drugged with genipin did not result in cell-cycle delay. In the SCE assay, the numbers of SCE per cell observed in the cases drugged with varying concentrations of glutaraldehyde were significantly greater than those found in the negative controls with or without S9 mix. Nevertheless, these numbers were still low compared to the numbers of SCE induced by the strong mutagens used as our positive control substances. This suggests that glutaraldehyde may produce a weakly clastogenic response in CHO-K1 cells. In contrast, the numbers of SCE per cell obtained in the cases drugged with genipin were comparable to those observed in the negative controls in those that were except drugged with the highest dose (50 ppm). This suggests that genipin does not cause clastogenic response in CHO-K1 cells provided its concentration is lower than 50 ppm. In conclusion, as far as cytotoxicity and genotoxicity are concerned, genipin is a promising crosslinking agent for biologic tissue fixation., (Copyright 2000 John Wiley & Sons, Inc.)

Chinese hamster ovary (CHO) cells were exposed to thermal and epithermal neutrons, and the occurrence of mutations at the HPRT locus was investigated. The Kyoto University Research Reactor (KUR), which has been improved for use in neutron capture therapy, was the neutron source. Neutron energy spectra ranging from nearly pure thermal to epithermal can be chosen using the spectrum shifters and thermal neutron filters. To determine mutant frequency and cell survival, cells were irradiated with thermal and epithermal neutrons under three conditions: thermal neutron mode, mixed mode with thermal and epithermal neutrons, and epithermal neutron mode. The mutagenicity was different among the three irradiation modes, with the epithermal neutrons showing a mutation frequency about 5-fold that of the thermal neutrons and about 1.5-fold that of the mixed mode. In the thermal neutron and mixed mode, boron did not significantly increase the frequency of the mutants at the same dose. Therefore, the effect of boron as used in boron neutron capture therapy (BNCT) is quantitatively minimal in terms of mutation induction. Over 300 independent neutron-induced mutant clones were isolated from 12 experiments. The molecular structure of HPRT mutations was determined by analysis of all nine exons by multiplex polymerase chain reaction. In the thermal neutron and mixed modes, total and partial deletions were dominant and the fraction of total deletions was increased in the presence of boron. In the epithermal neutron mode, more than half of the mutations observed were total deletions. Our results suggest that there are clear differences between thermal and epithermal neutron beams in their mutagenicity and in the structural pattern of the mutants that they induce. Mapping of deletion breakpoints of 173 partial-deletion mutants showed that regions of introns 3-4, 7/8-9 and 9-0 are sensitive to the induction of mutants by neutron irradiation.

In this paper we show the organisation of the Drosophila gene encoding a Golgi alpha-mannosidase II. We demonstrate that it encodes a functional homologue of the mouse Golgi alpha-mannosidase II. The Drosophila and mouse cDNA sequences translate into amino acid sequences which show 41% identity and 61% similarity. Expression of the Drosophila GMII sequence in CHOP cells produces an enzyme which has mannosidase activity and is inhibited by swainsonine and by CuSO(4.) In cultured Drosophila cells and in Drosophila embryos, antibodies raised against a C-terminal peptide localise this product mainly to the Golgi apparatus as identified by cryo-immuno electron microscopy studies and by antibodies raised against known mammalian Golgi proteins. We discuss these results in terms of the possible use of dGMII as a Drosophila Golgi marker.

To reexamine the existing predictions about the general membrane topology of the high-affinity Na+/glucose cotransporter (SGLT1) and in particular of the large loop at the C-terminal region, a small 6 x Histidine-tag was introduced at different positions of the SGLT1 sequence by site-directed mutagenesis. Eleven His-SGLT1 mutants were constructed and were transiently transfected into COS-7 cells. As demonstrated by immunofluorescent labeling with antipeptide antibodies against SGLT1, all mutants were expressed and inserted into the plasma membrane. Only mutants with the tag in the N-terminal region and the C-terminal region retained Na+/glucose cotransport activity at 0.1 mM D-glucose. The arrangement of the His-tag in the membrane was analyzed by indirect immunofluorescence, using a monoclonal antihistidine antibody. In nonpermeabilized cells the His-tag could be detected at the N-terminal end (insertion at aa 5) and at the C-terminal end (replacement between aa 584-589 and between aa 622-627), suggesting that these portions of the polypeptide are accessible from the extracellular space. Furthermore, an epitope-specific antibody directed against aa 606-630 reacted strongly with the cell surface. To support this topology intact stably transfected SGLT1 competent CHO cells were partially digested with an immobilized trypsin and subsequently subjected to electrophoresis and Western blot analysis. The size of the digestion product suggests that extravesicular trypsin removed the extracellular loop that contains the amino acid residues 549-664. Thus our results indicate that the last large loop (about aa 541-aa 639) towards the C-terminal end faces the cell exterior where it might be involved in substrate recognition.

The irs1 and irs1SF hamster cell lines are mutated for the XRCC2 and XRCC3 genes, respectively. Both show heightened sensitivity to ionizing radiation and particularly to the DNA cross-linking chemical mitomycin C (MMC). Frequencies of spontaneous chromosomal aberration have previously been reported to be higher in these two cell lines than in parental, wild-type cell lines. Microcell-mediated chromosome transfer was used to introduce complementing or non-complementing human chromosomes into each cell line. irs1 cells received human chromosome 7 (which contains the human XRCC2 gene) or, as a control, human chromosome 4. irs1SF cells received human chromosome 14 (which contains the XRCC3 gene) or human chromosome 7. For each set of hybrid cell lines, clones carrying the complementing human chromosome recovered MMC resistance to near-wild-type levels, while control clones carrying noncomplementing chromosomes remained sensitive to MMC. Fluorescence in situ hybridization with a human-specific probe revealed that the human chromosome in complemented clones remained intact in almost all cells even after extended passage. However, the human chromosome in noncomplemented clones frequently underwent chromosome rearrangements including breaks, deletions, and translocations. Chromosome aberrations accumulated slowly in the noncomplemented clones over subsequent passages, with some particular deletions and unbalanced translocations persistently transmitted throughout individual subclones. Our results indicate that the XRCC2 and XRCC3 genes, which are now considered members of the RAD51 gene family, play essential roles in maintaining chromosome stability during cell division. This may reflect roles in DNA repair, possibly via homologous recombination.

Centrosome duplication is indispensable for the formation of the bipolar mitotic spindle. Surprisingly, even if DNA replication or mitosis is inhibited, centrosome duplication can still occur [1] [2] [3] [4] [5]. Thus, it remains unknown how centrosome duplication is coordinated with the cell cycle. Here, we show that centrosome duplication requires cyclin-dependent kinase 2 (Cdk2) in mammalian cells. We have found that in Chinese hamster ovary (CHO) cells, whereas centrosome duplication is not inhibited by hydroxyurea (HU) treatment, which arrests the cells in S phase, it is inhibited by mimosine treatment, which arrests the cells in late G1 phase. Cdk2 activity was higher in HU-treated cells than in mimosine-treated cells. Remarkably, inhibition of the Cdk2 activity in HU-treated cells with butyrolactone I or roscovitine [6], or by expression of the Cdk inhibitor p21(Waf1/Cip1), blocked the continued centrosome duplication. Moreover, overexpression of Cdk2 reversed the inhibition of centrosome duplication by mimosine treatment. These results indicate a requirement of Cdk2 activity for centrosome duplication and therefore suggest an underlying mechanism for the coordination of centrosome duplication with the cell cycle.

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interference with conformational properties of the receptor critical for ligand binding. This distinction is central when employing the antibodies as tools in the elucidation of the structure-function relationship of the protein in question. We have studied the effect of monoclonal antibodies against the urokinase plasminogen activator receptor (uPAR), a protein located on the surface of various types of malignant and normal cells which is involved in the direction of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (BIA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former antibody efficiently blocked the receptor against subsequent ligand binding but was unable to promote the dissociation of a preformed receptor-ligand complex. The latter antibody was capable of binding the preformed complex, forming a transient trimolecular assembly, and promoting the dissociation of the uPA/uPAR complex. The continuous recording of binding and dissociation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this receptor reveals the presence of a determinant which influences the binding properties of a remote site in the molecular structure and which could be an important target for a putative synthetic antagonist.

CHO-K1 cells were examined for their cellular responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (DbATP), and for the presence of mRNA for P2X receptors. Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1-P2X6 subunits. DbATP (EC50 approximately equal to 100 microM) evoked non-desensitizing inward currents which reversed at approximately equal to 0 mV, suggesting activation of a non-selective cation channel. ATP also evoked inward currents but was less potent than DbATP. DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO-PRO-1, in CHO-KI cells. Both responses were inhibited by NaCl and MgCl2. In 280 mM sucrose buffer, 45Ca2+ accumulation was measurable within 10-20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATPgammaS were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and (alphabeta)methylene ATP were inactive at concentrations up to 100 microM. DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60-90 min. These data suggest that CHO-K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO-PRO-1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.

We have previously demonstrated that vascular endothelial growth factor-165 (VEGF), a tumor-secreted angiogenic factor, can acutely and chronically induce fenestrations in microvascular endothelium (Cancer Res 1997, 57:765-772). Because the morphology and function of microvascular endothelium differs from tissue to tissue, we undertook studies to examine whether the neovasculature in tumors also differed depending upon tumor location. Four tumor types implanted in the brain or subcutis in nude mice were studied: a murine rhabdomyosarcoma (M1S), a murine mammary carcinoma (EMT), and two human glioblastomas (U87 and U251). In addition, we studied Chinese hamster ovary cells stably transfected with human VEGF165. As previously reported, tumors grown in the subcutaneous space had a microvasculature that was fenestrated and had open endothelial gaps. The identical tumors when grown in the brain also had fenestrated endothelium and vessels with open endothelial gaps, but they were drastically reduced in occurrence. Open endothelial gaps were not seen in all tumors implanted in the brain (EMT and M1S), although fenestrated endothelium was always seen. VEGF and VEGF receptors were measured in tumors from both locations by immunoblotting and competitive polymerase chain reaction, respectively. VEGF amount was not significantly different between the tumor locations. Interestingly, total tumor vascular mRNA expression of both Flk-1 and Flt-1 was greater in tumor vessels derived from the brain compared with tumor vessels derived from subcutaneous tissues. These results demonstrate that the host microvascular environment determines the morphology and function of the tumor vasculature and that endothelia from different tissues vary in their ability to express the VEGF receptors given identical stimuli.

Stearyl vasoactive intestinal polypeptide has been reported to be a VIP (vasoactive intestinal polypeptide) receptor agonist of high potency with an original bioavailability and action. We synthesized three fatty acyl derivatives, myristyl-, palmityl- and stearyl-[Nle17]VIP, and tested their capacity to recognize recombinant rat- and human VIP1- and VIP2/PACAP (pituitary adenylate cyclase-activating polypeptide) receptors and to stimulate adenylate cyclase activity. The three lipophilic analogues bound with high affinity (from 0.5 to 20 nM) to both receptor subtypes but did not distinguish between them. In preparations expressing a high density of human VIP1/PACAP receptors, the three lipophilic analogues had the same efficacy as VIP and [Nle17]VIP. In preparations expressing the rat receptors, stearyl-[Nle17]VIP had a lower efficacy than the other peptides tested. In preparations expressing a low level of VIP1/PACAP receptors and in those expressing VIP2/PACAP receptors, all analogues behaved like partial agonists. The lowest efficacy was observed for stearyl-[Nle17]VIP on the VIP2/PACAP receptor subclass. Based on our results, a complex pattern of in vivo biological effects of the lipophilic VIP derivatives should be expected: these compounds might behave as full agonists, partial agonists, or antagonists of the VIP response, depending on the number and the subtype of receptor expressed.

Sucrose acetate isobutyrate (SAIB) was tested for potential genotoxic activity in four different in vitro assay systems. Two independent trials of a Salmonella reverse mutation assay (using strains TA98, TA100, TA1535, TA1537 and TA1538) showed no increases in revertant frequencies at doses up to 10,000 microg/plate which was non-toxic but exceeded the solubility limit. Similarly, no mutagenic response was observed at doses up to 1000 microg/ml at the HGPRT locus in cultured CHO cells; SAIB was toxic and its solubility limit was exceeded at 50 microg/ml. No clastogenic activity was detected in cultured CHO cells at concentrations up to 2000 microg/ml. All three preceding in vitro tests were conducted both in the presence and absence of Aroclor 1254-induced rat liver S-9 metabolic activation systems. An unscheduled DNA synthesis assay also was performed using rat primary hepatocyte cultures with doses up to 1000 microg/ml, and no DNA repair was detectable. Thus, SAIB was stringently tested at doses exceeding the solubility limit in culture medium and causing toxicity to CHO cells without obtaining any evidence for genotoxic activity as a mutagen, clastogen, or DNA-damaging agent.

We show in this study that cadherin ligands, either soluble or immobilized on different surfaces, can bind to cells carrying a compatible cadherin and induce long-range signals which affect cell adhesion and dynamics. Addition of recombinant N-cadherin extracellular domain (NEC) to CHO cells expressing N-cadherin (FL4) greatly enhanced the calcium-dependent aggregation of the cells and blocked their migration into an "in vitro wound". Monoclonal antibody which blocks cadherin interactions inhibited the aggregation of suspended FL4 cells and facilitated the "wound closure". As previously shown (Levenberg et al., 1998) synthetic beads coupled to NEC interacted specifically with the surface of FL4 cells and significantly enhanced the formation of adherens junctions. This effect was obtained also with the parental CHO cells, which contain low levels of N-cadherin, and in additional N-cadherin expressing cells such as cultured myoblasts. We further show here that stimulation of adhesion is not affected by the geometry of the NEC-bound surface and that cells plated on flat NEC-coated substratum also develop enhanced adherens junctions. Interaction of cells expressing low levels of endogenous N-cadherin, such as CHO cells with surface-immobilized N-cadherin ligands had a prominent effect also on the total level of N-cadherin and beta-catenin in the cells, probably due to stabilization of the cadherin-catenin complex by the interaction with the external surface.

A new series of arylpiperazide derivatives of 1-naphthylpiperazine of general formula 4 has been prepared and evaluated as 5-HT1B antagonists. Binding experiments at cloned human 5-HT1A, 5-HT1B, and 5-HT1D receptors show that these derivatives are potent and selective ligands for 5-HT1B/1D subtypes with increased binding selectivity versus the 5-HT1A receptor when compared to 1-naphthylpiperazine (1-NP). Studies of inhibition of the forskolin-stimulated cAMP formation mediated by the human 5-HT1B receptor demonstrate that the nature of the arylpiperazide substituent modulates the intrinsic activity of these 1-NP derivatives. Among them, 2-[[8-(4-methylpiperazin-1-yl)naphthalen-2-yl]oxy] -1-(4-o-tolylpiperazin-1-yl)ethanone (4a) was identified as a potent neutral 5-HT1B antagonist able to antagonize the inhibition of 5-HT release induced by 5-CT (5-carbamoyltryptamine) in guinea pig hypothalamus slices. Moreover, 4a was found to potently antagonize the hypothermia induced by a selective 5-HT1B/1D agonist in vivo in the guinea pig following oral administration (ED50 = 0.13 mg/kg).

To improve our knowledge of the bioactive conformation of CCK-B antagonists, we have developed a new series of constrained dipeptoids whose synthesis and biochemical properties are reported here. These compounds, of general structure N alpha-[(2-adamantyloxy)carbonyl]-alpha-methyltryptophanyl-(4 -X)-proline, were designed by introducing a cyclization in the structure of the previously described CCK-B/peptoid antagonist RB 210, N-[N-[(2-adamantyloxy)carbonyl]-DL-alpha-methyltryptophanyl] -N-(2-phenylethyl)glycine (Blommaert et al. J. Med. Chem. 1993, 36, 2868-2877), by means of a five-membered ring. Structure-affinity relationship studies showed that an R configuration of Trp-C alpha and a cis configuration of the pyrrolidine substituents were favorable for receptor recognition. The most potent compounds of this new series had similar affinities for the CCK-B receptor as RB 210 and proved to be far more efficient in inhibiting inositol phosphate production in CHO cells stably transfected with rat brain CCK-B receptor, with IC50 values approaching those of the commonly used antagonists L-365,260 and PD-134,308. Moreover, binding studies performed using transfected CHO cells showed that two affinity states of the CCK-B receptor can be discriminated by some of these compounds which also have different biological profiles and are therefore highly interesting tools for the biochemical and pharmacological characterization of CCK-B receptor heterogeneity.

Previous studies have indicated that neuro-endocrine cells store monoamines and acetylcholine (ACh) in different secretory vesicles, suggesting that the transport proteins responsible for packaging these neurotransmitters sort to distinct vesicular compartments. Molecular cloning has recently demonstrated that the vesicular transporters for monoamines and ACh show strong sequence similarity, and studies of the vesicular monoamine transporters (VMATs) indicate preferential localization to large dense core vesicles (LDCVs) rather than synaptic-like microvesicles (SLMVs) in rat pheochromocytoma PC12 cells. We now report the localization of the closely related vesicular ACh transporter (VAChT). In PC12 cells, VAChT differs from the VMATs by immunofluorescence and fractionates almost exclusively to SLMVs and endosomes by equilibrium sedimentation. Immunoisolation further demonstrates colocalization with synaptophysin on SLMVs as well as other compartments. However, small amounts of VAChT also occur on LDCVs. Thus, VAChT differs in localization from the VMATs, which sort predominantly to LDCVs. In addition, we demonstrate ACh transport activity in stable PC12 transformants overexpressing VAChT. Since previous work has suggested that VAChT expression confers little if any transport activity in non-neural cells, we also determined its localization in transfected CHO fibroblasts. In CHO cells, VAChT localizes to the same endosomal compartment as the VMATs by immunofluorescence, density gradient fractionation, and immunoisolation with an antibody to the transferrin receptor. We have also detected ACh transport activity in the transfected CHO cells, indicating that localization to SLMVs is not required for function. In summary, VAChT differs in localization from the VMATs in PC12 cells but not CHO cells.

Certain hexavalent chromium compounds are documented human carcinogens. Exposure of cells to particulate forms of chromium results in cell-enhanced dissolution of particles in the extracellular microenvironment and chronic production of chromium oxyanions, which are taken up by the cell through an anion transport system and are genotoxic and clastogenic. It was previously shown that apoptosis is the mode of cell death of nearly all of the Chinese hamster ovary cells (CHO-AA8 cell line), which die after high-dose, short-term treatments with soluble sodium chromate. In this report the mode of cell killing by particulate lead chromate and of low-dose continuous treaments of soluble sodium chromate designed to mimic conditions of ionic chromate uptake after lead chromate exposure was examined. CHO-AA8 cells were treated for 24 hr with doses of sodium chromate or lead chromate which cause a 50% decrease in survival in colony-forming effeciency assays. Longer treatments (up to 72 hr) at the same doses did not decrease survival further than the 24-hr exposure. Morphological changes indicative of apoptosis, as well as internucleosomal DNA fragmentation, were detectable by 24 hr after treatment with lead chromate or soluble sodium chromate. All of the cells killed by treatments with lead chromate particles underwent apoptosis as the mode of cell death and this was accurately modeled in cell culture by continuous treatments with low-dose soluble sodium chromate. Exposure of cells to hexavalent chromium compounds causes a spectrum of DNA damage which can be selectively altered by pretreatment of cells with antioxidant vitamins prior to chromium exposure. Here we show that ascorbate and alpha-tocopherol markedly inhibited the chromosomal aberrations induced by both particulate and soluble chromate compounds, even though chromium adduct levels were not decreased by either vitamin pretreatment. Cell survival assays showed that ascorbate, but not alpha-tocopherol, protected cells from apoptosis induced by sodium chromate. The results differentiate chromium-induced apoptosis from both chromosomal damage and adduct levels and suggest that other lesions sensitive to ascorbate but not tocopherol are the proximal inducing signal for chromium-induced apoptosis., (Copyright 1997 Academic Press.)

A novel carbon filter has been developed which primarily reduces the amount of certain vapor phase constituents of tobacco smoke with greater efficiency than the charcoal filters of cigarettes currently in the market. In vitro indicators of genotoxic and cytotoxic potential were used to compare the cigarette smoke condensate (particulate phase) or whole cigarette smoke (vapor phase and particulate phase) from cigarettes containing the novel carbon filter with smoke condensate or whole smoke from commercial or prototype cigarettes not containing the novel carbon filter. Ames bacterial mutagenicity, sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, and neutral red cytotoxicity assays in CHO cells were utilized to assess the genotoxic and cytotoxic potential of the cigarette smoke condensates. SCE and neutral red cytotoxicity assays were utilized to assess the genotoxic and cytotoxic potential of the whole smoke. As expected, the novel carbon filter did not significantly affect the genotoxic or cytotoxic activity of the smoke condensate, although we did observe that the use of low-nitrogen tobacco reduced the mutagenicity of the condensate in Salmonella typhimurium strain TA98. However, the whole smoke from cigarettes containing the novel carbon filter demonstrated significant reductions in genotoxic and cytotoxic potential compared to cigarettes without the novel carbon filter. The toxicity of the smoke was correlated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCE induction) to the aggregate mass of several vapor phase components (acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene, ammonia, NOx, HCN, benzene, isoprene, and formaldehyde) in the smoke of the cigarettes utilized in this study. In conclusion, this novel carbon filter, which significantly reduced the amount of carbonyls and other volatiles in mainstream cigarette smoke, resulted in significant reductions in the genotoxic and cytotoxic activity of the smoke as measured by these assays., (Copyright 1997 Society of Toxicology.)

Tyrosine phosphorylation is a key signaling event in transmembrane and cytoplasmic signal transduction. The m5 muscarinic receptor (m5AChR) responds to ligand stimulation with calcium influx and protein phosphorylation. In contrast, neither of these responses has been associated with m4AChR signaling. We hypothesized that activation of the m5AChR would alter tyrosine phosphorylation patterns spatially within the cell and in a calcium influx-sensitive manner. CHO cells stably transfected with m4- or m5AChRs were assessed for spatial localization and quantity of phosphotyrosylated proteins in response to receptor activation. Results were confirmed by immunoblot of whole cell lysates and cytosol and membrane fractions. m5AChR activation increased tyrosine phosphorylation in all subcellular compartments; coincubation with CAI, a calcium influx inhibitor, reduced phosphorylation below basal levels. Western blot confirmed the change of phosphotyrosylated proteins of M(r) 70, 85, 120, and 180 kDa in whole and fractionated cells. PLC-gamma, used as a marker of m5AChR activity, was increased in quantity and degree of phosphorylation in CHOm5 cell membranes and microvilli in response to receptor activation. Both the quantitative increase and tyrosine phosphorylation of PLC-gamma in membrane fractions was inhibited by CAI. In contrast, CC treatment of CHOm4 cells reduced tyrosine phosphorylation throughout the cell. CC-stimulation of m5AChR cells caused a calcium influx-sensitive increase in phosphotyrosylated proteins throughout the cell, though predominantly in the membrane and microvilli. Activation of the m5AChR induces tyrosine phosphorylation, whereas activation of the m4AChR inhibited tyrosine phosphorylation below baseline, further demonstrating the dichotomy between signaling of these two AChRs.

This study aimed to assess the biocompatibility of two cordierite ceramics (DF and Cord 1014), with similar chemical composition and different porosity, as a potential support for cell growth in a continuous-flow, solid-bed reactor. The Chinese hamster ovary (CHO) cell line transfected with HBV-DHFR recombinant plasmid was seeded on cordierite or polystyrene dishes and evaluated for cell growth and production of recombinant hepatitis B surface antigen. Proliferation of the CHO cells, in terms of cell number, was generally similar in polystyrene and Cord 1014 and always lower in DF. Flow cytometric analysis showed no difference in cell cycle distribution for cells grown on different supports, and showed a two-fold increase in percentage of debris for cells grown on DF than for those grown on Cord 1014 and polystyrene culture dishes. Moreover, the morphology of cells grown on Cord 1014 did not change during the experiment, and cells were well spread and organized. Finally, total recombinant hepatitis B surface antigen production was higher on Cord 1014 than on polystyrene and DF samples. Such evidence suggests that Cord 1014 could be a promising support for growing cells in a continuous-flow, solid-bed reactor.