Hamster Fibroblasts Research Articles

Generation and characterization of a novel anti-rat CD40L antibody with inhibitory activities in vitro and in vivo

The CD40–CD40L interaction plays a critical role in cell-mediated immune responses. Blocking this interaction has been shown to be beneficial in the treatment of various diseases studied in murine models. Although rats are widely used to test therapeutic strategies in several disease models, a monoclonal antibody (mAb) to block the CD40–CD40L interaction in rats is not broadly available. In the present study we generated Armenian hamster fibroblasts expressing rat CD40L and used these to generate a novel anti-rat CD40L mAb (AS1). In vitro studies showed that AS1 was able to block CD40L-induced DC maturation and B cell proliferation. Most importantly, in vivo, AS1 inhibited B cell responses in a dose-dependent fashion, as measured by the production of OVA specific antibodies after subcutaneous immunization with OVA. AS1 was shown to be a powerful tool to modulate Ag presentation in vitro and in vivo. Elucidating the effect of AS1 in various rat models for human diseases will provide more insight into blocking the CD40–CD40L interaction as a therapeutic strategy to prevent human diseases.

Regulation of mTORC1 Signaling by Src Kinase Activity Is Akt1-Independent in RSV-Transformed Cells

Increased activity of the Src tyrosine protein kinase that has been observed in a large number of human malignancies appears to be a promising target for drug therapy. In the present study, a critical role of the Src activity in the deregulation of mTOR signaling pathway in Rous sarcoma virus (RSV)-transformed hamster fibroblasts, H19 cells, was shown using these cells treated with the Src-specific inhibitor, SU6656, and clones of fibroblasts expressing either the active Src or the dominant-negative Src kinase-dead mutant. Disruption of the Src kinase activity results in substantial reduction of the phosphorylation and activity of the Akt/protein kinase B (PKB), phosphorylation of tuberin (TSC2), mammalian target of rapamycin (mTOR), S6K1, ribosomal protein S6, and eukaryotic initiation factor 4E-binding protein 4E-BP1. The ectopic, active Akt1 that was expressed in Src-deficient cells significantly enhanced phosphorylation of TSC2 in these cells, but it failed to activate the inhibited components of the mTOR pathway that are downstream of TSC2. The data indicate that the Src kinase activity is essential for the activity of mTOR-dependent signaling pathway and suggest that mTOR targets may be controlled by Src independently of Akt1/TSC2 cascade in cells expressing hyperactive Src protein. These observations might have an implication in drug resistance to mTOR inhibitor-based cancer therapy in certain cell types.

The 'Cosmic silence' experiment: on the potential adaptive role of environmental background radiation

In low dose radiobiology, there is a strong interest in phenomena that could imply a deviation from the linear-no-threshold (LNT) model, which is currently used to estimate the excess relative cancer risk at low doses of ionizing radiation (IR) via a linear extrapolation from high- and intermediate-dose epidemiology risk data. Such phenomena include the adaptive response as well as bystander effects , occurring in cells which do not experience direct radiation exposure. These phenomena suggest that biological response to harmful agents such as ionising radiation may be more complex than that assumed by the LNT model. In particular, environmental background radiation represents a source of chronic low dose-rate exposure which may condition the response of living systems to acute exposure to genotoxic agents, also including IR itself, via an adaptive response. To clarify this aspect is essential not only to better evaluate the risk of chronic occupational exposure, but also to understand the role of environmental background radiation on biological evolution. Reduction of background environmental IR dose rate exposure is achieved at the National Laboratories at Gran Sasso (LNGS) of the Italian Institute for Nuclear Physics (INFN). Located underneath the Gran Sasso mountain range in central Italy, these laboratories offer an excellent opportunity to continuously maintain cell cultures at extremely reduced levels of environmental background radiation. The Cosmic Silence experiment represents the natural extension, to a human cell culture model (TK6 lymphoblastoid cells), of earlier experimental studies on S. Cerevisiae (L. Satta et. al. Mut Res 1995) and V79 Chinese hamster fibroblasts (L. Satta et. al. Radiat Environ Biophys 2002), which indicated a progressive disappearance of an adaptive response in cells kept under reduced environmental background IR conditions. For the Cosmic Silence experiment, TK6 parallel cultures were set up in the underground LNGS laboratories and in Rome and planned to be maintained and assayed for 12 months. Starting at 6 months of continuous, parallel cultures, measurements of DNA damage (micronuclei) or oxidative metabolism (antioxidant enzymatic activities of SOD, GPx, and Catalase) indicate that significant deprivation of environmental background IR renders cells less tolerant to an acute X-ray exposure of 1-2 Gy. These results are compatible with the concept that environmental background IR is behaving as an adaptive agent.

Exploration of the β2-adrenergic receptor regulatory regions: the next step in the holy grail of asthma pharmacogenetics research

exploration of the β2-adrenergic receptor (ADRB2) has been the holy grail of asthma pharmacogenetics research since the gene was cloned two decades ago. The evolution in our understanding of genetic variants in the ADRB2 reflects both the progress and the limitations in exploring the contributions

Microfluorimetric analysis of dynamics of genomic changes in Chinese hamster fibroblasts CHL V-79 RJK with multiple drug resistance

Results of karyological analysis of cells CHL V-79 RJK selected for resistance to ethidium bromide (EB) causing multidrug resistance (MDR) (line Vebr-5) were compared with the data of microfluorimetric determination of DNA content in individual chromosomes of the karyotype. The analysis was performed at the 11th and 88th passages. Karyotyping of Vebr-5 has shown the presence of an additional genetic material (ADM) in the form of homogenously or differentially stained regions (HSRs and DSRs, respectively) in two chromosomes (Z1 and Z6, loci 1 p29-31 and 1q26, respectively). HSRs in Z6, in the region of localization of the wild type of gene mdr, had unstable length and structure characteristic of morphological markers of amplification of genes of the family mdr. During long cultivation of Vebr-5 in the presence of EB (88 passages), the instability of HSRs in Z6 increased. Results of microfluorimetric analysis of Vebr-5 at the 11th passage have shown an increase in the DNA content not only in chromosomes Z1 and Z6 marked by HSRs, but also in three chromosomes (Z5, Z12 and Z13) that have no visual morphological changes. The corresponding analysis at the 88th passage has also revealed non-random changes in the DNA content in four more chromosomes: an increase in Z14, while a decrease in chromosomes 8, Z7, and Z9. A decrease of the DNA content in chromosomes is considered to be a result of a partial loss of genetic material, while its increase is a result of its translocation and (or) amplification. Coefficient of variation of the DNA content changes for large chromosomes amounted to about 9%. while for small chromosomes it is about 26%, which indicates that small chromosomes have greater potential for instability than the large ones. The data obtained not only confirm, but also enlarge the concept of directions and character of destabilization of the cell genetic apparatus in the process of neoplastic transformation due to the MDR acquisition by cells.

Genetic control of telomerase and replicative senescence in human and rodent cells.

The ribonucleoprotein telomerase is detectable in most human cancer cells and immortalized cells but is absent or inactive in the vast majority of normal counterparts. Repression of telomerase activity in human somatic cells, which leads to telomere shortening and replicative senescence, may have evolved as a protective mechanism against immortalization, unfettered clonal evolution and cancer. Rodent cells in culture are far more susceptible to immortalization and malignant progression than human cells. This can be explained by our observation that normal diploid rodent (hamster) fibroblasts possess active telomerase throughout their proliferative life span, and therefore they do not require a telomerase activation step during immortalization. Monochromosome transfer techniques have enabled us to identify powerful telomerase repressive activity specifically associated with the introduction of a single copy of human chromosome 3 into human carcinoma cells. Fine-structure deletion analysis of non-repressed hybrids has permitted us to map the position of the candidate telomerase repressor gene to 3p21.1-3p21.3. A strategy for isolating the gene has been developed involving a combination of fine-structure deletion mapping and functional gene transfer approaches. The availability of cloned telomerase repressor genes will advance our understanding of telomerase regulation in the human soma and its disruption during human cancer development.

Genotoxicity and subchronic toxicity studies of DHA-rich oil in rats

Polyunsaturated fatty acids, including docosahexaenoic acid (DHA), are natural constituents of the human diet. DHA-algal oil is produced through the use of the non-toxigenic and non-pathogenic marine protist, Ulkenia sp. The safety of DHA-algal oil was assessed in a subchronic toxicity study and in genotoxicity studies. In a 90-day study, rats were orally administered water or DHA-algal oil at concentrations of 0, 500, 1000, and 2000 mg/kg in combination with 2000, 1500, 1000 or 0 mg/kg DHA-containing fish oil, respectively. Additional animals were administered water, 2000 mg/kg DHA-algal oil, or 2000 mg/kg fish oil for 90 days, followed by a 4-week recovery phase. No treatment-related effects were observed in clinical observations, food and water consumption, mortality, gross pathology, and histopathology. Increased body weights and liver weights in oil-treated groups were attributed to the large lipid load and were not regarded as toxicologically significant. Furthermore, no treatment-related differences in the measured parameters between the DHA-algal oil and fish oil groups were detected. In genotoxicity experiments, DHA-algal oil exerted no mutagenic activity in various bacterial strains, nor did it induce chromosomal aberrations in Chinese hamster fibroblast cells. These results support the safety of DHA-algal oil as a dietary source of DHA.

Changes in antioxidant defense systems induced by thiram in V79 Chinese hamster fibroblasts

The role of antioxidant defence systems in protection against oxidative damage of lipids and proteins induced by fungicide thiram during in vitro exposure was investigated in cultured Chinese hamster V79 cells with normal, depleted, and elevated glutathione (GSH) levels. We analyzed the catalytic activities of superoxide dismutases (SOD1 and SOD2), Se-dependent and Se-independent glutathione peroxidases (GSH-Px), glutathione reductase (GR), and catalase (CAT), as well as total glutathione/glutathione disulfide ratio (GSH total/GSSG). Thiram treatment resulted in an increase in activities of SOD1, Se-dependent GSH-Px, and GR at the highest tested dose (150 μM). On the contrary, inhibition of CAT and Se-independent GSH-Px activities, and no significant changes in the level of SOD2 activity was observed at any tested doses (100–150 μM). GSH total/GSSG ratio in the 100 μM thiram treated cells was not significantly changed comparing to the control, despite significant decrease of GSH total (50%). In 150 μM thiram treated cells the ratio falls to 43% of control value. Pretreatment with l-buthionine sulfoximine ( l-BSO), an inhibitor of GSH synthesis, significantly enhanced decrease in CAT and Se-independent GSH-Px activities, as well as GSH total/GSSG ratio, and reduced Se-dependent GSH-Px activity, following exposure to thiram. Simultaneously, l-BSO pretreatment enhanced increase in SOD1 activity, and had no effect on SOD2, following thiram exposure. Pretreatment with N-acetyl cysteine (NAC), a GSH precursor, prevented enzymatic changes in CAT, Se-dependent GSH-Px, GR, SOD1 activities, and significantly decreased SOD2 activity following exposure to thiram. GSH total/GSSG ratio was restored to the control value. This study suggests that following the changes in antioxidant defense systems thiram can act through the production of free radicals.

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 μM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 μM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 μM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity.

Copper-Transporting P-Type ATPase, ATP7A, Confers Multidrug Resistance and Its Expression Is Related to Resistance to SN-38 in Clinical Colon Cancer

We and others have shown that the copper transporters ATP7A and ATP7B play a role in cellular resistance to cis-diaminedichloroplatinum (II) (CDDP). In this study, we found that ATP7A transfection of Chinese hamster ovary cells (CHO-K1) and fibroblasts isolated from Menkes disease patients enhanced resistance not only to CDDP but also to various anticancer drugs, such as vincristine, paclitaxel, 7-ethyl-10-hydroxy-camptothecin (SN-38), etoposide, doxorubicin, mitoxantron, and 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11). ATP7A preferentially localized doxorubicin fluorescence to the Golgi apparatus in contrast to the more intense nuclear staining of doxorubicin in the parental cells. Brefeldin A partially and monensin completely altered the distribution of doxorubicin to the nuclei in the ATP7A-expressing cells. ATP7A expression also enhanced the efflux rates of doxorubicin and SN-38 from cells and increased the uptake of SN-38 in membrane vesicles. These findings strongly suggested that ATP7A confers multidrug resistance to the cells by compartmentalizing drugs in the Golgi apparatus and by enhancing efflux of these drugs, and the trans-Golgi network has an important role of ATP7A-related drug resistance. ATP7A was expressed in 8 of 34 (23.5%) clinical colon cancer specimens but not in the adjacent normal epithelium. Using the histoculture drug response assay that is useful for the prediction of drug sensitivity of clinical cancers, ATP7A-expressing colon cancer cells were significantly more resistant to SN-38 than ATP7A-negative cells. Thus, ATP7A confers resistance to various anticancer agents on cancer cells and might be a good index of drug resistance in clinical colon cancers.

Haem oxygenase-1--a culprit in vascular and renal damage?

The haem oxygenase (HO) system is responsible for the catabolism of free haem, a potent pro-oxidant, released during the normal and pathophysiological breakdown of haem-containing proteins. HO degrades haem releasing biliverdin, iron and carbon monoxide (CO) [1]. Biliverdin is converted to bilirubin by biliverdin reductase. There are two well characterized isoforms of active HO: an inducible enzyme, HO-1, and a constitutive isoform, HO-2. They are products of different genes with dissimilar regulation and tissue distribution (reviewed in [2]). HO-1 is a 32 kDa protein that is highly inducible in mammalian tissues by a wide variety of stimuli including haem, heavy metals, growth factors, nitric oxide (NO), peroxynitrite, modified lipids, hypoxia, hyperoxia, cytokines as well as others. HO-2 is a 36 kDa protein that is constitutively expressed in distinct locations including in the brain, endothelium, testis and distal nephron segments (reviewed in [2]). The products of HO-mediated haem degradation (biliverdin, bilirubin, carbon monoxide, ferrous iron) regulate important biological processes, including oxidative stress, inflammation, apoptosis, cell proliferation, angiogenesis and fibrosis, through one or more of these products. The HO field has attracted numerous investigators and there has been an exponential increase in the number of publications on this enzyme ( 5–10 in 1990, to 500 in 2006). Several recent reviews and editorials have highlighted the biological effects of the reaction product(s) and the importance of HO-1 as a potent cytoprotective enzyme in diverse conditions [2–5]. HO activity, however, may not be protective in all instances [6–7]. Each of the products of the HO reaction has potential detrimental effects. Bilirubin can be toxic to neural and non-neural cells at high concentrations, and hyperbilirubinemia is responsible for diseases such as neonatal jaundice, kernicterus, and bilirubin encephalopathy [8]. CO can stimulate mitochondrial generation of free radicals and poison haem proteins [9] and ferrous iron can catalyse free radical reactions [10]. Suttner and Dennery [7], using a tetracycline regulatable system, demonstrated that low levels ( 15-fold) of overexpression actually worsen cell injury caused by hyperoxia in hamster fibroblasts. Thus, optimal levels of HO-1 induction may be required for cytoprotective benefits of HO-1. Whether similar effects related to the level of HO-1 enzyme activity occur in the kidney or vasculature is not known. HO activity may have different effects depending on the cell type and/or environment. It has been demonstrated that HO-1 inhibits the growth of renal tubular epithelial cells, increases endothelial cell cycle progression and formation of capillary-like structures in a 2D Matrigel assay, while inhibiting cell cycle progression and inducing apoptosis of smooth muscle cells [11–14]. Recent in vivo studies demonstrate that HO-1 expression and activity promotes vascular endothelial growth factor (VEGF)-mediated endothelial activation and ensuing angiogenesis, but in contrast, it inhibits lipopolysaccharide-mediated leucocyte invasion and prevents subsequent inflammatory angiogenesis (reviewed in [15]). There is also emerging evidence that HO activity may play a role in the development and/or exacerbation of some tissue pathologies. For example, increased HO activity accelerates tumour angiogenesis [16] and renders tumour cells relatively resistant to anticancer treatment [17,18]. There are several diseases associated with increased HO-1 expression including atherosclerosis, hypertension, transplant rejection, acute renal failure, glomerulonephritis and many others (reviewed in [19]). This review will highlight the specific role of HO-1 in vascular and renal injury. Correspondence and offprint requests to: Anupam Agarwal, MD, Division of Nephrology, ZRB 614, University of Alabama at Birmingham, 703 19th street south, Birmingham, AL 35294, USA. Email: agarwal@uab.edu Nephrol Dial Transplant (2007) 22: 1495–1499

α/β ratio: A dose range dependence study

To investigate the dependence of the alpha/beta ratio determined from in vitro survival curves on the dose ranges. Detailed clonogenic cell survival experiments were used to determine the least squares estimators for the linear quadratic model for different dose ranges. The cell lines used were CHO AA8, a Chinese hamster fibroblast cell line; U-373 MG, a human glioblastoma cell line; and CP3 and DU-145, two human prostate carcinoma cell lines. The alpha, beta, and alpha/beta ratio behaviors, combined with a goodness-of-fit analysis and Monte Carlo simulation of the experiments, were assessed within different dose regions. Including data from the low-dose region has a significant influence on the determination of the alpha, beta, and alpha/beta ratio from in vitro survival curve data. In this region, the values are poorly determined and have significant variability. The mid-dose region is characterized by more precise and stable values and is in agreement with the linear quadratic model. The high-dose region shows relatively small statistical error in the fitted parameters but the goodness-of-fit and Monte Carlo analyses showed poor quality fits. The dependence of the fitted alpha and beta on the dose range has an impact on the alpha/beta ratio determined from the survival data. The low-dose region had a significant influence that could be a result of a strong linear, rather than quadratic, component, hypersensitivity, and adaptive responses. This dose dependence should be interpreted as a caution against using inadequate in vitro cell survival data for alpha/beta ratio determination.

Does the cellular labile iron pool participate in the oxidation of 2′,7′-dichlorodihydrofluorescein?

The fluorogenic probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) is widely used for the estimation of oxidative stress in cells. It is known that 2′,7′-dichlorodihydrofluorescein (H2DCF), product of intracellular hydrolysis of H2DCF-DA, is oxidized to the fluorescent compound, DCF, mainly by hydrogen peroxide (H2O2) in the presence of catalysts. The present study was aimed at answering the question whether the labile iron pool (LIP) may contribute to the oxidation of H2DCF in cellular systems. The membrane-permeable lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) was found to inhibit oxidation of the probe by H2O2 dependent on ferrous ions but not by peroxidase or superoxide dismutase in defined in vitro systems. When applied to cells, the probe inhibited considerably oxidation of H2DCF in V79 Chinese hamster fibroblasts and two murine lymphoma L5178Y(LY) sublines (LY-R, LY-S) differing in LIP level, the extent of inhibition being greater in the LY-R line of higher LIP level. These results demonstrate that LIP is a significant factor determining the rate of intracellular H2DCF oxidation.

Toxicological and Analytical Investigations of Noni (Morinda citrifolia) Fruit Juice

Morinda citrifolia (noni) is known to contain genotoxic anthraquinones in the roots. Because of the widespread use of noni juice, the possible genotoxic risk was examined through a battery of short-term tests. Noni juice was also chemically analyzed for the possible presence of anthraquinones. Noni juice extract in the Salmonella microsome assay showed a slight mutagenic effect in strain TA1537, due to the presence of flavonoids. No mutagenicity was observed in the mammalian mutagenicity test with V79 Chinese hamster fibroblasts. Rats treated with a noni juice concentrate did not show DNA repair synthesis (UDS) in primary rat hepatocytes, nor could DNA adducts or DNA strand breaks be observed. HPLC analysis of noni juice for anthraquinones was negative, with a sensitivity of <1 ppm. In summary, chemical analysis and genotoxicity tests reveal that noni juice does not have a genotoxic potential and that genotoxic anthraquinones do not exist in noni juice.

Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family.

Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with beta-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.

Regulation of c-Src activity by the expression of wild-type v-Src and its kinase-dead double Y416F-K295N mutant

Active, wild-type v-Src and its kinase-dead double Y416F–K295N mutant were expressed in hamster fibroblasts. Expression of the active v-Src induced activation of endogenous c-Src and increased general protein-tyrosine phosphorylation in the infected cells. Expression of the kinase-dead mutant induced hypophosphorylation of Tyr416 of the endogenous c-Src. The inactivation of c-Src was reversible, as confirmed by in vitro kinase activity of c-Src immunoprecipitated from the kinase-dead v-Src-expressing cells. Both activation and inactivation of c-Src may be explained by direct interaction of the v-Src and c-Src that may either facilitate transphosphorylation of the regulatory Tyr416 in the activation loop, or prevent it by formation of transient dead-end complexes of the Y416F–K295N mutant with c-Src. The interaction was also indicated by co-localization of v- and c-Src proteins in immunofluorescent images of the infected cells. These results suggest that dimerization of Src plays an important role in the regulation of Src tyrosine kinase activity.

Receptor dependent cellular uptake of synthetic low density lipoprotein by mammalian cells in serum-free tissue culture

Low density lipoprotein (LDL) is a normal plasma component, which is of interest in a number of research areas such as hypercholesterolaemia, drug targeting in cancer chemotherapy and as a lipid supplement in tissue culture systems. Currently, however, it can only be obtained by extraction from fresh plasma samples, which yields only small quantities. Synthetic LDL (sLDL) has been prepared using readily available lipid components coupled with a synthetic amphiphatic peptide molecule containing the apoprotein B receptor sequence. sLDL was capable of supporting the growth of Chinese Hamster Ovary (CHO) and fibroblast cells in serum-free culture media in a cholesterol-dependent manner that was related to the presence of the receptor peptide molecule. sLDL could be fluorescently labelled with 3,3'-dioctadecyloxalocarbocyanine perchlorate (DiO), and once labelled was assimilated by CHO and fibroblast cells in a time- and temperature-dependent manner that was dependent upon the presence of the receptor peptide. In addition, assimilation was reduced by an excess of unlabelled native LDL. The results indicated that the interaction of sLDL with CHO and fibroblast cells occurred via a receptor dependent system, most likely the LDL cellular receptor. sLDL is therefore a useful, easily obtained substitute for native LDL with potential applications in the areas of drug targeting to cells and serum-free tissue culture systems.

Cytotoxicity and antimicrobial activity of six multipurpose soft contact lens disinfecting solutions1

To assess and compare the cytotoxicity and antimicrobial activity of six multipurpose disinfecting contact lens solutions (MPDS). Six MPDS were assessed and compared: MeniCare Soft, Complete Moisture Plus, Solo-Care Aqua, Opti-Free Express, ReNu MultiPlus and ReNuMoistureLoc. Cytotoxicity was evaluated by means of the colony-forming assay (ISO 10993-5) using one cell line. One hundred Chinese hamster's fibroblast V79 cells in 2 mL of medium were inoculated into 6-well plates and cultured for 24 h. After incubation, the medium was removed and 2 mL of each MPDS was diluted with physiological saline and medium and added individually into wells at 1.25, 2.5, 5 and 10% concentrations. After an additional 6 days of incubation, cells were fixed and stained with Giemsa solution. Subsequently, the number of colonies was counted. Cytotoxicity was expressed in terms of the concentration of the MPDS that suppresses colony formation. Antimicrobial activity was evaluated by means of the stand-alone contact lens disinfection test (ISO 14729). Cytotoxicity--MeniCare Soft and Complete Moisture Plus were not cytotoxic at any of the concentrations tested. The cytotoxicity ranking of the other MPDS was as follows: Solo-Care Aqua approximately equal to Opti-Free Express < ReNu MultiPlus < ReNu MoistureLoc. Antimicrobial activity--All MPDS met the primary criteria of the stand-alone test for bacterial species. ReNu MoistureLoc showed borderline values for Serratia marcescens and Staphylococcus aureus. Complete Moisture Plus failed to meet the stand-alone test criteria for Fusarium solani. Large differences in cytotoxicity and antimicrobial activity were found among the MPDS under investigation. Multipurpose disinfecting contact lens solutions with identical concentrations of polyhexamethylene biguanide can behave differently depending on solution formulation. The new MeniCare Soft showed effective antimicrobial activity and minimal cytotoxicity.

Mutation of Succinate Dehydrogenase Subunit C Results in Increased O2·−, Oxidative Stress, and Genomic Instability

Mutations in genes coding for succinate dehydrogenase (SDH) subunits are believed to contribute to cancer and aging, but the mechanism for this is unclear. Hamster fibroblasts expressing a mutation in SDH subunit C (SDHC; B9) showed 3-fold increases in dihydroethidine and dichlorodihydrofluorescein (CDCFH(2)) oxidation indicative of increased steady-state levels of O2(.-) and H2O2, increases in glutathione/glutathione disulfide (indicative of oxidative stress), as well as increases in superoxide dismutase activity, relative to parental B1 cells. B9 cells also showed characteristics associated with cancer cells, including aneuploidy, increases in glucose consumption, and sensitivity to glucose deprivation-induced cytotoxicity. Expression of wild-type (WT) human SDHC in B9 cells caused prooxidant production, glucose consumption, sensitivity to glucose deprivation-induced cytotoxicity, and aneuploidy to revert to the WT phenotype. These data show that SDHC mutations cause increased O2(.-) production, metabolic oxidative stress, and genomic instability and that mutations in genes coding for mitochondrial electron transport chain proteins can contribute to phenotypic changes associated with cancer cells. These results also allow for the speculation that DNA damage to genes coding for electron transport chain proteins could result in a "mutator phenotype" by increasing steady-state levels of O2(.-) and H2O2.

Redox Activation of Aldose Reductase in the Ischemic Heart

Aldose reductase (AR) reduces cytotoxic aldehydes and glutathione conjugates of aldehydes derived from lipid peroxidation. Its inhibition has been shown to increase oxidative injury and abolish the late phase of ischemic preconditioning. However, the mechanisms by which ischemia regulates AR activity remain unclear. Herein, we report that rat hearts subjected to ischemia, in situ or ex vivo, display a 2-4-fold increase in AR activity. The AR activity was not further enhanced by reperfusion. Activation increased Vmax of the enzyme without affecting the Km and decreased the sensitivity of the enzyme to inhibition by sorbinil. Enzyme activation could be prevented by pretreating the hearts with the radical scavenging thiol, N-(2-mercaptoproprionyl)glycine or the superoxide dismutase mimetic, Tiron, or by treating homogenates with dithiothreitol. In vitro, the recombinant enzyme was activated upon treatment with H2O2 and the activated, but not the native enzyme, formed a covalent adduct with the sulfenic acid-specific reagent dimedone. The enzyme activity in the ischemic, but not the nonischemic heart homogenates was inhibited by dimedone. Separation of proteins from hearts subjected to coronary occlusion by two-dimensional electrophoresis and subsequent matrix-assisted laser desorption ionization time-of-flight/mass spectrometry analysis revealed the formation of sulfenic acids at Cys-298 and Cys-303. These data indicate that reactive oxygen species formed in the ischemic heart activate AR by modifying its cysteine residues to sulfenic acids.