Generation of Xenomitochondrial Embryonic Stem Cells for the Production of Live Xenomitochondrial Mice.

The unique features of the mitochondrial genome, such as its high copy number and lack of defined mechanisms of recombination, have hampered efforts to manipulate its sequence to create specific mutations in mouse mtDNA. As such, the generation of in vivo mouse models of mtDNA disease has proved technically challenging. This chapter describes a unique approach to create mitochondrial oxidative phosphorylation (OXPHOS) defects in mouse ES cells by transferring mtDNA from different murid species into Mus musculus domesticus ES cells using cytoplasmic hybrid ("cybrid") fusion. The resulting "xenocybrid" ES cells carry OXPHOS defects of varying severity, and can be utilized to generate live mouse models of mtDNA disease.

The crystal chemistry of ferric oxyhydroxyapatite.

Ferric hydroxyapatites (Fe-HAp) and oxyapatites (Fe-OAp) of nominal composition [Ca(10-x)Fe(x)(3+)][(PO(4))(6)][(OH)(2-x)O(x)] (0 < or = x < or = 0.5) were synthesized from a coprecipitated precursor calcined under flowing nitrogen. The solid solubility of iron was temperature-dependent, varying from x = 0.5 after firing at 600 degrees C to x approximately 0.2 at 1000 degrees C, beyond which Fe-OAp was progressively replaced by tricalcium phosphate (Fe-TCP). Crystal size (13-116 nm) was controlled by iron content and calcination temperature. Ferric iron replaces calcium by two altervalent mechanisms in which carbonate and oxygen are incorporated as counterions. At low iron loadings, carbonate predominantly displaces hydroxyl in the apatite channels (Ca(2+) + OH(-) --> Fe(3+) + CO(3)(2-)), while at higher loadings, "interstitial" oxygen is tenanted in the framework (2Ca(2+) + (vac) --> 2Fe(3+) + O(2+)). Although Fe(3+) is smaller than Ca(2+), the unit cell dilates as iron enters apatite, providing evidence of oxygen injection that converts PO(4) tetrahedra to PO(5) trigonal bipyramids, leading to the crystal chemical formula [Ca(10-x)Fe(x)][(PO(4))(6-x/2)(PO(5))(x/2)][(OH)(2-y)O(2y)] (x < or = 0.5). A discontinuity in unit cell expansion at x approximately 0.2 combined with a substantial reduction of the carbonate FTIR fingerprint shows that oxygen infusion, rather than tunnel hydroxyl displacement, is dominant beyond this loading. This behavior is in contrast to ferrous-fluorapatite where Ca(2+) --> Fe(2+) aliovalent replacement does not require oxygen penetration and the cell volume contracts with iron loading. All of the materials were paramagnetic, but at low iron concentrations, a transition arising from crystallographic modification or a change in spin ordering is observed at 90 K. The excipient behavior of Fe-OAp was superior to that of HAp and may be linked to the crystalline component or mediated by a ubiquitous nondiffracting amorphous phase. Fe-HAp and Fe-OAp are not intrinsically suitable magnetic agents for drug delivery but may be useful in reactive cements that promote osteoblast proliferation.

Cytotoxic T cells specifically induce Fas on target cells, thereby facilitating exocytosis-independent induction of apoptosis.

Cytotoxic T (Tc) cells deficient in perforin lyse Fas-negative targets after lengthy incubation periods. This process is independent of granzymes, and killing occurs via the Fas pathway for the following reasons. Interaction of perforin-deficient Tc cells with Fas-negative targets leads to an up-regulation of Fas that is dependent on Ag recognition, de novo synthesis, and transport of proteins to the target cell surface. Treatment of effectors with brefeldin A, but not with the exocytosis inhibitor concanamycin, inhibited this process. Lysis of targets is inhibited by anti-Fas Abs, soluble mouse Fas-Fc, and the caspase-cascade inhibitor, crm-A. Targets from Fas-mutant lpr mice are refractory to lysis, and Tc cells from mice deficient in Fas- and perforin-mediated lysis do not lyse Fas-negative targets. The possible relevance of this exocytosis-independent cytolytic process in the regulation of T cell activity and control of pathogens is discussed.

Hsp25-induced radioresistance is associated with reduction of death by apoptosis: involvement of Bcl2 and the cell cycle.

We previously demonstrated the protective effect of the small heat-shock protein against oxidative damage induced by tumor necrosis factor alpha. Here we have extended our studies of the possible role of Hsp25 in ionizing radiation-induced damage. For these studies, we transfected murine fibroblast L929 cells with the Hsp25 gene and selected three stably transfected clones. Hsp25 overexpression conferred radioresistance as detected by clonogenic survival and induction of apoptosis. Interestingly, the Hsp25-transfected cells showed an increase in the level of the anti-apoptosis molecule Bcl2. We also observed alterations of cell growth in the Hsp25-transfected cells. The cell cycle time of Hsp25-transfected cells was 3-4 h slower than that of vector-transfected control cells. Flow cytometry analysis of synchronized cells at late G(1) phase by mimosine treatment also showed the growth delay in Hsp25-overexpressing cells. In addition, reduced cyclin D1, cyclin A and Cdc2 levels and increased levels of Cdkn1a (also known as p21(Waf)) were observed in Hsp25-transfected cells, which probably caused the reduction in cell growth. In addition, synchronization by mimosine treatment only partially altered radioresistance in the Hsp25-transfected cells. Taken together, these data suggest that Hsp25-induced radioresistance is associated with growth delay as well as induction of Bcl2.

Inhibition of fibroblast cell adhesion on substrate by coating with 2-methacryloyloxyethyl phosphorylcholine polymers.

Fibroblast adhesion and growth behavior were examined on various polymers coated on a poly(ethylene telephthalate) (PET) substrate. The polymers are poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylatel copolymer (PMB)s with different MPC unit compositions, and poly(2-hydroxyethyl methacrylate). Surface analysis by dynamic contact angle measurement revealed that the mobility of the polymer chain on the PET substrate depended on the MPC unit composition, but there was no significant difference between the PMBs with 3-10 mol% MPC units and poly(HEMA). Fibronectin adsorption on the polymer surface from a cell culture medium was determined by immunoassay. The adsorbed fibronection was evenly distrubuted in every polymer, however, the amount was reduced with an increase in the MPC unit composition in the PMB. This result suggested that the MPC unit could weaken the interaction between the polymer surface and proteins. When fibroblast L-929 cells, were cultured on the polymers, the cells adhered and the number of cells increased on not only the hydrophobic poly(BMA) but also on the hydrophilic poly(HEMA). However, the number of cells that adhered on the PMB surface decreased with an increase in the MPC unit composition. This was a result of the fibronectin adsorption behavior. Thus, it could be concluded that since the PMB could suppress cell adhesion proteins e.g. fibronectin, the PMB showed excellent cell adhesive resistance properties.

Non-fouling properties of polysaccharide-coated surfaces.

Hyaluronic acid and alginic acid, examples of hydrophilic polysaccharides whose biological and technological properties are deeply related to strong interaction with water, have been coupled to substrate surfaces. These coatings can prevent mammalian cells adhesion and greatly reduce bacterial cells adhesion in vitro and in several in vivo applications. The anti-adhesive properties of these surfaces are discussed in terms of the surface fractional coverage by the polysaccharide, as evaluated by XPS analysis and water contact angle. The implications of chemical-molecular considerations to the properties of these coatings are discussed from an analytical and mechanistic point of view.

Ca(2+)-independent cell-adhesion activity of claudins, a family of integral membrane proteins localized at tight junctions.

In multicellular organisms, various compositionally distinct fluid compartments are established by epithelial and endothelial cellular sheets. For these cells to function as barriers, tight junctions (TJs) are considered to create a primary barrier for the diffusion of solutes through the paracellular pathway [1] [2] [3]. In ultrathin sections viewed under electron microscopy, TJs appear as a series of apparent fusions, involving the outer leaflets of plasma membranes of adjacent cells, to form the so-called kissing points of TJs, where the intercellular space is completely obliterated [4]. Claudins are a family of 16 proteins whose members have been identified as major integral membrane proteins localized exclusively at TJs [5] [6] [7] [8]. It remains unclear, however, whether claudins have the cell-adhesion activity that would explain the unusual intercellular adhesion at TJs. Using mouse L-fibroblast transfectants expressing various amounts of claudin-1, -2 or -3, we found that these claudins possess Ca(2+)-independent cell-adhesion activity. Using ultrathin-section electron microscopy, we observed many kissing points of TJs between adjacent transfectants. Furthermore, the cell-adhesion activity of occludin, another integral membrane protein localized at TJs [9] [10] [11], was negligible when compared with that of claudins. Thus, claudins are responsible for TJ-specific obliteration of the intercellular space.

Dehydroepiandrosterone and related steroids alter 3T3-L1 preadipocyte proliferation and differentiation.

The purpose of the present study was to determine if the anti-adipogenic effects of dehydroepiandrosterone (DHEA) are mediated solely by DHEA or by one or more of its downstream metabolites. In Experiment 1, preconfluent proliferating cultures of 3T3-L1 preadipocytes were incubated for either 24 or 72 h with 0, 1, 5 or 25 microM DHEA, DHEA sulfate (DHEAS), testosterone, estrone and 17beta-estradiol. Pregnenolone, a precursor of DHEA(S), was also tested at these concentrations. After 24 h of incubation, DHEAS, 17beta-estradiol and estrone at the 1 microM level stimulated preadipocyte proliferation. In contrast, DHEA and 17beta-estradiol at the 25 microM level attenuated proliferation to a greater extent than all other steroids. After 72 h of incubation, DHEA and 17beta-estradiol at the 25 microM level attenuated proliferation to a greater extent than all other steroids. In Experiment 2, post-confluent cultures of differentiating 3T3-L1 preadipocytes were incubated for 6 days with 0, 5, 30, or 60 microM levels of these steroids. Preadipocyte differentiation, as assessed by lipid content and glycerol-3-phosphate dehydrogenase activity, decreased markedly when treated with 30 and 60 microM DHEA, 17beta-estradiol, estrone and pregnenolone. In contrast, DHEAS had no impact on preadipocyte proliferation or differentiation. These results suggest that the anti-adipogenic actions of DHEA in adipose tissue may be mediated, in part, by one or more of its distal metabolites, including 17beta-estradiol.

M-cadherin-mediated cell adhesion and complex formation with the catenins in myogenic mouse cells.

M-cadherin is a member of the multigene family of calcium-dependent intercellular adhesion molecules, the cadherins, which are involved in morphogenetic processes. Amino acid comparisons between M-cadherin and E-, N-, and P-cadherin suggested that M-cadherin diverged phylogenetically very early from these classical cadherins. It has been shown that M-cadherin is expressed in prenatal and adult skeletal muscle. In the cerebellum, M-cadherin is present in an adherens-type junction which differs in its molecular composition from the E-cadherin-mediated adherens-type junctions. These and other findings raised the question of whether M-cadherin and the classical cadherins share basic biochemical properties, notably the calcium-dependent resistance to proteolysis, mediation of calcium-dependent intercellular adhesion, and the capability to form M-cadherin complexes with the catenins. Here we show that M-cadherin is resistant to trypsin digestion in the presence of calcium ions but at lower trypsin concentrations than E-cadherin. When ectopically expressed in LMTK- cells, M-cadherin mediated calcium-dependent cell aggregation. Finally, M-cadherin was capable of forming two distinct cytoplasmic complexes in myogenic cells, either with alpha-catenin/beta-catenin or with alpha-catenin/plakoglobin, as E-and N-cadherin, for example, have previously been shown to form. The relative amount of these complexes changed during differentiation from C2C12 myoblasts to myotubes, although the molecular composition of each complex was unaffected during differentiation. These results demonstrate that M-cadherin shares important features with the classical cadherins despite its phylogenetic divergence.

The effect of etoposide (VP-16) on mouse L fibroblasts: modulation of stress response, growth and apoptosis genes.

Molecular events were studied in mouse L cells treated with etoposide (VP-16) a drug widely used in cancer therapy. Modulation of the expression of stress response genes belonging to the hsp70 family (hsp70, hsc73, grp78), growth- and cycle-related genes (c-myc, c-fos, c-jun, histone H3) and apoptosis-related genes (p53, TRPM-2, tTG) was monitored at different time points in the cells that remained adherent to the substrate up to 96 hours after exposure to VP-16. The steady state level of mRNA was determined by Northern blot analysis and hybridization with specific probes, and the relative rate of gene transcription was monitored by run on transcription with isolated nuclei. Our results indicate that protracted VP-16 treatment of 1. cells induces, within 24 hours, the arrest of DNA synthesis, repression of growth-related genes and transient induction of the tTG gene. Altogether these molecular events may contribute to the cytotoxic effect of VP-16. However in cells surviving a longer exposure to the drug, the expression of growth-related genes resumes, even if a blockade in DNA replication persists, and expression of the grp78 gene significantly increases. These data suggest that under continuous treatment with VP-16 a fraction of L cells showing increased resistance to the drug may emerge.

[A model for the assessment of fibroblast morphology]. / Model' dlia otsenki morfologii fibroblastov.

A model for comprehensive assessment of morphologic parameters of a cell (area, nucleus and cytoplasm area, degree of spreading), based on the use of a scanning device, has been developed. The model comprises an original method for staining the preparations, which permits assessment of the degree of cell spreading by microdensitometry, and updating the equipment and software. The programs help assess the integral and derivative parameters, such as the area of the interface zone of a cell, the shape factor, spreading, and nucleus to cytoplasm ratio. Using this model, the effect of peripheral blood mononuclears on the morphology of transformed murine fibroblasts L929 upon a contact exposure was assessed. Addition of mononuclears to fibroblast culture led to alteration of their morphology: increase of the mean cellular area, degree of spreading, and of shape factor and reduction of the nucleus/cytoplasm ratio. This was true for both the mean values and the population composition.

Programmed cell death (apoptosis) of mouse fibroblasts is induced by the topoisomerase II inhibitor etoposide.

The mechanism by which etoposide, a topoisomerase II inhibitor, killed replicating mouse L929 fibroblasts was investigated. Etoposide at 10 microM killed 70% of the cells within 4 days, a result that was accompanied by DNA fragmentation. A characteristic "ladder" pattern of DNA fragmentation was confirmed by agarose gel electrophoresis. Simultaneous exposure of the cells to 10 microM etoposide plus 1 microM cycloheximide reduced both the extent of cell killing and the fragmentation of DNA. Delayed addition of cycloheximide protected cells only if cycloheximide was added 1-6 hr after exposure to etoposide. When added 6-24 hr after treatment with etoposide, cycloheximide lost the ability to protect cells. Cell growth was completely inhibited by either etoposide or cycloheximide. Furthermore, DNA synthesis was inhibited by either etoposide or cycloheximide within 6 hr. Protein synthesis, however, was not inhibited by etoposide. Thus, the ability of cycloheximide to protect cells correlated with inhibition of protein synthesis, rather than inhibition of DNA synthesis. A 1-hr exposure to 2.5 mM N-methyl-N-nitrosourea similarly inhibited DNA synthesis within 6 hr. without affecting protein synthesis. However, no loss of viability accompanied N-methyl-N-nitrosourea treatment. Thus, an imbalance between protein synthesis and DNA synthesis cannot explain the cell killing by etoposide. H-7, a protein kinase C inhibitor, prevented the cell killing and DNA fragmentation, whereas aurintricarboxylic acid, an endonuclease inhibitor, reduced the extent of DNA fragmentation but did not have an effect on cell killing. The data document that the killing of replicating mouse fibroblasts by etoposide represents an example of programmed cell death (apoptosis) that depends on protein synthesis. Although protein synthesis is required during the first 24 hr of exposure to etoposide, cell death is delayed until several days later.

[The long-term limitation of the proliferation of cells in culture does not lead to their proliferative aging]. / Dlitel'noe ogranichenie proliferatsii kletok v kul'ture ne privodit k ikh proliferativnomu stareniiu.

A hypothesis was tested about a long-term limitation of cell proliferation as the primary cause of their senescence and, consequently, the senescence of the whole organism. Both half-yearly and yearly maintenance of mouse fibroblast-like cells L-929 in a high density permanent culture with low level of mitotic activity (less than 1 mitosis per 10,000 cells) has not revealed any irreversible changes in their proliferative features and has not confirmed the hypothesis under examination.

Platelet/endothelial cell adhesion molecule-1 (CD31)-mediated cellular aggregation involves cell surface glycosaminoglycans.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kDa integral membrane glycoprotein expressed on endothelial cells, platelets, and leukocytes. Experiments analyzing the aggregation of mouse L-cells stably transfected with full-length PECAM-1 cDNA have demonstrated that PECAM-1 is capable of mediating calcium-dependent heterophilic aggregation. In this report the ligand interactions involved in the aggregation process were studied. This aggregation was inhibited by heparin and chondroitin sulfate, but not by other glycosaminoglycans. Enzymatic removal of cell surface glycosaminoglycans confirmed a PECAM-1-glycosaminoglycan interaction and suggested that this interaction involved glycosaminoglycans on adjacent cells. PECAM-1 contains a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like domain of the molecule's extracellular domain. A comparable region in the related adhesion protein N-CAM has been shown to mediate the adhesive properties of N-CAM. Cells expressing mutant PECAM-1 protein missing the second domain failed to aggregate. Synthetic peptides mimicking the consensus glycosaminoglycan binding sequence, L-K-R-E-K-N, inhibited aggregation. These results demonstrate that PECAM-1-mediated aggregation is dependent on the binding of PECAM-1 to specific glycosaminoglycans on adjacent cells via a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like homology domain.

Eukaryotic cells grown on microcarrier beads offer a cost-efficient way to propagate Chlamydia trachomatis.

The use of microcarrier cell culture as a method for the in vitro propagation of the obligate intracellular bacterial parasite, Chlamydia trachomatis, is described. The microcarrier beads proved to be a more cost-effective means to propagate C. trachomatis than traditional tissue culture flasks or roller bottles without sacrificing yields or infectivity. In addition, microcarrier cell culture was found to be a much simpler technique to study the intracellular development of these bacteria.

[The mitotic cycle and DNA repair in UV-irradiated murine neuroblastoma cells]. / Mitoticheskii tsikl i reparatsiia DNK v UF-obluchennykh kletkakh neiroblastomy myshei.

A correlation has been shown between a reduced rate of movement of UV-irradiated neuroblastoma cells from G1 into S phase, an essential increase of cells in S phase while progressing through the cell cycle, and a defect in free DNA synthesis on a damaged template. These indices may reflect one and the same cell response to the UV light.

The stimulatory effect of actinomycin D on avian reovirus replication in L cells suggests that translational competition dictates the fate of the infection.

Indirect immunostaining of avian reovirus S1133-infected L-cell monolayers showed that most of the cells can support viral replication. However, the number of cells in which the virus was actually replicating depended on the multiplicity of virus infection. The presence of actinomycin D during infection increased viral protein synthesis, viral growth, and the number of actively infected cells at late infection times. The antibiotic elicited these effects by triggering viral replication in cells that already contained unproductive cytoplasmic virus but that would not get productively infected in the absence of the drug. From these results, we propose a model for the interaction between L cells and avian reovirus S1133 in which viral versus host mRNA competition for the translational machinery determines the fate of the virus infection.

The uvomorulin-anchorage protein alpha catenin is a vinculin homologue.

The cytoplasmic region of the Ca(2+)-dependent cell-adhesion molecule (CAM) uvomorulin associates with distinct cytoplasmic proteins with molecular masses of 102, 88, and 80 kDa termed alpha, beta, and gamma catenin, respectively. This complex formation links uvomorulin to the actin filament network, which seems to be of primary importance for its cell-adhesion properties. We show here that antibodies against alpha catenin also immunoprecipitate complexes that contain human N-cadherin, mouse P-cadherin, chicken A-CAM (adherens junction-specific CAM; also called N-cadherin) or Xenopus U-cadherin, demonstrating that alpha catenin is complexed with other cadherins. In immunofluorescence tests, alpha catenin is colocalized with cadherins at the plasma membrane. However, in cadherin-negative Ltk- cells, alpha catenin is found uniformly distributed in the cytoplasm, suggesting some additional biological function(s). Expression of uvomorulin in these cells results in a concentration of alpha catenin at membrane areas of cell contacts. We also have cloned and sequenced murine alpha catenin. The deduced amino acid sequence reveals a significant homology to vinculin. Our results suggest the possibility of a new vinculin-related protein family involved in the cytoplasmic anchorage of cell-cell and cell-substrate adhesion molecules.

The construction of human somatic cell hybrids containing portions of the mouse X chromosome and their use to generate DNA probes via interspersed repetitive sequence polymerase chain reaction.

Interspersed repetitive sequence polymerase chain reaction (IRS-PCR) has become a powerful tool for the rapid generation of DNA probes from human chromosomes present in rodent somatic cell hybrids. We have constructed a somatic cell hybrid containing a major portion of the mouse X chromosome in a human background (clone 8.0). IRS-PCR was developed for the specific amplification of mouse DNA using either of two primers from the rodent-specific portion of the murine B1 repeat. Amplification was subsequently performed with clone 8.0 and a subclone, 8.1/1, which retains a small murine X-chromosomal fragment including Hprt and the Gdx locus. A total of 15-20 discrete PCR products ranging in size from less than 500 to greater than 3000 bp were obtained from clone 8.0 with each primer. In clone 8.1/1, a subset of these bands plus some additional bands were observed. Nine bands amplified from clone 8.1/1 have been excised from gels and used as probes on Southern blots. All of the fragments behaved as single-copy probes and detected domesticus/spretus variation. They have been regionally mapped using an interspecific backcross. The probe locations are compatible with those of markers known to be present in clone 8.1/1. These results demonstrate the feasibility of this method as applied to the mouse genome and the high likelihood of generating useful DNA probes from a targeted region.

Tumor necrosis factor-induced interleukin-6 expression and cytotoxicity follow a common signal transduction pathway in L929 cells.

Interleukin (IL)-6 gene induction was studied in response to treatment with Tumor Necrosis Factor (TNF) in the sensitive murine L929 cell line. Under conditions where TNF-mediated cytotoxicity was either increased or decreased, depending on addition of activators or inhibitors, we found that the TNF-induced IL6 gene expression was likewise enhanced or repressed. We conclude that the signal (or part of the signals) going to the nucleus and responsible for gene activation is conducted along the reaction mechanism leading to cellular toxicity.