Chromosome segregation from cell hybrids. II. Do differences in parental cell growth rates and phase times determine direction of loss?

The hypothesis that the direction of chromosome segregation in cell hybrids is determined by the interaction of parent cell cycles, or S-phase times, predicts that the segregant parent will always be the one with the longer cycle, or the longer S phase, and that late replicating chromosomes will be more frequently lost. We have tested this hypothesis by studying cell cycle parameters of mouse, Chinese hamster, and platypus parent cells and by observing chromosome loss and replication patterns in hybrids between them. Two types of hybrids have been studied: mouse-hamster hybrids showed gradual segregation, in one or other direction, of 10-60% chromosomes, while rodent-platypus hybrids (which could be selected under conditions optimal for either parent cell) showed rapid and extreme segregation of platypus chromosomes. We found no correlation between the direction of segregation and the relative lengths of parental cycle times, or phase times, nor between sequence of replication and frequency with which segregant chromosomes are lost. We therefore conclude that the direction and extent of segregation is not directly determined by the interaction of parental cycle or phase times.     

Engineering cell–cell signaling

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Hydrogen peroxide. Ubiquitous in cell culture and in vivo?

Hydrogen peroxide (H2O2) is widely regarded as a cytotoxic agent whose levels must be minimized by the action of antioxidant defence enzymes. In fact, H2O2 is poorly reactive in the absence of transition metal ions. Exposure of certain human tissues to H2O2 may be greater than is commonly supposed; levels of H2O2 in the human body may be controlled not only by catabolism but also by excretion, and H2O2 could play a role in the regulation of renal function and as an antibacterial agent in the urine. Cell culture is a widely used method for the investigation of "physiological" processes such as signal transduction and regulation of gene expression, but chemical reactions involving cell culture media are rarely considered. Addition of reducing agents to commonly used cell-culture media can lead to generation of substantial amounts of H2O2. Some or all of the reported effects of ascorbic acid and polyphenolic compounds (e.g., quercetin, catechin, epigallocatechin, epigallocatechin gallate) on cells in culture may be due to H2O2 generation by interaction of these compounds with cell culture media.     

Antibody specific for the peptide.major histocompatibility complex. Is it T cell receptor-like?

Antibodies recognizing peptide bound to a major histocompatibility complex (MHC) protein usually have a higher affinity for the composite peptide.MHC (pMHC) ligand than T cell receptors (TCR) with the same specificity. Because the solvent-accessible peptide area constitutes only a small portion of the contacting pMHC surface, we hypothesized that the contribution of the MHC moiety to the TCR-pMHC complex stability is limited, ensuring a small increment of the binding energy delivered by the peptide to be distinguishable by the TCR or the peptide-specific antibody. This suggests that the gain in affinity of the antibody-pMHC interaction can be achieved through an increase in the on-rate without a significant change in the off-rate of the interaction. To test the hypothesis, we have analyzed the binding of an ovalbumin peptide (pOV8) and its variants associated with soluble H-2Kb protein to the 25-D1.16 monoclonal antibody and compared it with the binding of the same pMHC complexes to the OT-1 TCR. This comparison revealed a substantially higher on-rate of the antibody-pMHC interaction compared with the TCR-pMHC interaction. In contrast, both the antibody and the TCR-pMHC complexes exhibited comparably fast off-rates. Sequencing of the 25-D1.16 VH and VL genes showed that they have very few somatic mutations and those occur mainly in framework regions. We propose that the above features constitute a signature of the recognition of MHC-bound peptide antigens by TCR and TCR-like antibodies, which could explain why the latter are rarely produced in vivo.     

5′-nucleotidase activity in cultured cell lines. Effect of different assay conditions and correlation with cell proliferation

Summary The 5′-AMPase activity of the ectoenzyme 5′-nucleotidase has been measured in a variety of cell lines, using intact cells. Human cell types showed two orders of magnitude higher enzyme activity than mouse cell lines. The ectoenzyme is inhibited by adenosine 5′-(α,β-methylene) diphosphate and Concanavalin A. A different extent of 5′-nucleotidase lectin inhibition was observed in the studied cell lines, suggesting that the corresponding ectoenzymes are glycoproteins with a different type or degree, or both, of glycosylation. The 5′-nucleotidase activity increased during subculture and decreased after cell transformation. Generally, the 5′-nucleotidase activity was two-to five-fold higher in monolayer than in suspension cell culture. A relation between cell growth and 5′-AMPase activity was also observed. Enzyme activity increased at the end of the lag phase (glioblastoma cells) or during the exponential phase (the other two cell lines). After confluence, the activity decreased to the initial or even lower range of activity. Observed activity variations with cell proliferation correlate with modifications of 5′-AMPase activity during subculture. This work was supported by grant no. PR84-0359 from the Comisión Asesora de Investigación Científica y Técnica (Spain).     

Genomic instability in mammalian cell hybrids. IV. Is mutation frequency elevated in hybrid cells?

In this study, we set out to determine whether the mutation frequency in cell hybrids is increased over the frequencies in the two parental lines, and whether this increase is related to the evolutionary divergence of the cell parents. Two test loci were chosen: forward mutation at the HPRT locus and mutation to resistance to the drug emetine. We conclude that while some cell combinations do seem to produce hybrids with higher mutation frequencies, this is not consistently so, and, indeed, mutation rates in hybrids may be higher, lower or very similar to rates in the parental lines. Further, evolutionary divergence between the parental lines does not appear to correlate to mutation frequency in the hybrids.     

A new anaerobic bacterium,forming up to five endospores per cell —Anaerobacter polyendosporus gen. et spec. nov.

An obligately anaerobic sporeforming bacterium assigned to a new genus and species Anaerobacter polyendosporus gen. et spec. nov. is described. Characteristic features distinguishing the bacterium from known anaerobic sporeformers were variable cell shape, including spherical, the ability to form up to five endospores per cell, diffusive distribution of reserve polysaccharide throughout the cytoplasm, independence from growth factors. The eubacterial nature of the organism was revealed by its sensitivity to 1 mg/l of streptomycin, rifampicin, penicillin and to lysozyme. It belonged to Firmicutes by the type of cell wall structure. The cell wall consisted of one layer; the outer membrane was absent. The cells were not motile. The spores were spherical or oval, heat-resistant, contained dipicolinic acid and had typical endospore structure. Cortex, coats, spore coare, and in most cases exosporium could be distinguished. The bacterium fermented carbohydrates, but not amino acids. The products of fermentation included ethanol, acetate, lactate, butyrate, butanol, H₂ and CO₂. Sulfate or nitrate could not be used as electron acceptors, but nitrite was reduced to NH ₄ ⁺ in a dissimilatory process. The bacterium was capable of fixing N₂. The G + C content of the DNA was 29 mol %. The bacterium was isolated from meadow-gley soil.     

Malignant and nonmalignant T cell lines from human T cell lymphotropic virus type I-negative patients with Sézary syndrome.

The establishment of an in vitro model for cutaneous T cell lymphomas and Sézary syndrome has been difficult since T cells from individuals with these diseases do not proliferate in response to T cell mitogens. We found that conditioned media, collected from mitogen-activated PBMC from Sézary patients, contain an IL-2 receptor inducing factor. Despite their ostensible proliferative disorder, using a combination Sézary cell-conditioned media and rIL-2, we established IL-2 responsive, human T cell lymphotropic virus type I negative T cell lines from 23 patients, nine of which contain cells with the structural and/or genetic characteristics of neoplastic Sézary T cells.     

Molecular biology of cell nonpermissiveness to retroviruses. Has the time come?

The problem of host cell nonpermissiveness to retrovirus infection is characterized and illustrated on several retroviral models, including the role of viral receptors, cell fusion, and endogenous retroviral genomes as modifiers of the outcome of retroviral infection. Special attention is paid to different barriers against the infection of mammalian cells with avian leukosis/sarcoma viruses (ALV/ASV). Even when avian retroviruses become integrated in mammalian cells, several blocks at the level of provirus expression, processing of viral RNAs, and posttranslational modification prevent virus production in such virogenic cells. The significance of these blocks and new strategies making it possible to overcome some of them are discussed in relation to the development of ALV/ASV-based vectors suitable for gene therapy in mammals.     

Giant cell formation: the way to cell death or cell survival?

The study of giant cells in populations of different tumor cells and evaluation of their role in cancer development is an expanding field. The formation of giant cells has been shown to be followed by mitotic catastrophe, apoptosis, necrosis, and other types of cell elimination. Reports also demonstrate that giant cells can escape cell death and give rise to new cancer cells. However, it is not known if the programmed cell death is involved in this type of cell cycle disorders. Here we describe principal events that are observed during giant cell formation. We also consider the role of giant cells in cancer development, taking into account both published work and our own recent data in this field.     

One cell–one immunoglobin. Origin of limited heterogeneity of myeloma proteins

Plasma-cell tumour 5563 forms a single molecular species of immunoglobulin IgG(2)a, i.e. one variant of heavy chain and one variant of light chain. The molecules formed are labile and undergo alterations in charge properties, which rapidly lead to heterogeneity of the myeloma protein after synthesis. The single immunoglobulin species originally formed is found only after the shortest time-intervals tested, i.e. 10min incubation. Two types of changes in charge properties take place: (1) The originally formed protein (component o) is converted via an intermediate o' into the most basic form of 5563 myeloma protein found in serum (component a). Charge differences between these components are suppressed at pH8.9, but can be studied by chromatography at pH6.5 or by analysis of isoelectric points by isoelectric focusing in polyacrylamide gel. The conversion of components o and o' into component a apparently commences soon after assembly of the molecules and proceeds to completion extracellularly. (2) The second type of charge difference that distinguishes components a, b, c and d is exhibited over the pH range 6.0-8.9, but not at acid pH, and has been studied by electrophoresis at pH8.9, by chromatography and by isoelectric focusing. Conversion of component a into components b, c, d and e is only partial so that all five components can be found at decreasing concentrations in serum. Both types of charge alteration can be effected in vitro in the presence of serum, with optimum pH8.0. None of the charge differences could be attributed to the secretion process, since a component with the same isoelectric point as component o was found in secreted myeloma protein (1h). We have found no evidence to support the idea that the first type of change from component o to component a is due to ring formation of N-terminal [(14)C]glutamine into pyrrolid-2-one-5-carboxylic acid; however, our findings do not exclude this process happening very rapidly to a precursor of component o, possibly the polypeptide chain during or immediately after synthesis. In studying this point we noted that not only the heavy chains but also the kappa-type light chain of mouse 5563 myeloma protein have a blocked N-terminus.     

The cell–cell junctions of mammalian testes: I. The adhering junctions of the seminiferous epithelium represent special differentiation structures

The seminiferous tubules and the excurrent ducts of the mammalian testis are physiologically separated from the mesenchymal tissues and the blood and lymph system by a special structural barrier to paracellular translocations of molecules and particles: the “blood–testis barrier”, formed by junctions connecting Sertoli cells with each other and with spermatogonial cells. In combined biochemical as well as light and electron microscopical studies we systematically determine the molecules located in the adhering junctions of adult mammalian (human, bovine, porcine, murine, i.e., rat and mouse) testis. We show that the seminiferous epithelium does not contain desmosomes, or “desmosome-like” junctions, nor any of the desmosome-specific marker molecules and that the adhering junctions of tubules and ductules are fundamentally different. While the ductules contain classical epithelial cell layers with E-cadherin-based adherens junctions (AJs) and typical desmosomes, the Sertoli cells of the tubules lack desmosomes and “desmosome-like” junctions but are connected by morphologically different forms of AJs. These junctions are based on N-cadherin anchored in cytoplasmic plaques, which in some subforms appear thick and dense but in other subforms contain only scarce and loosely arranged plaque structures formed by α- and β-catenin, proteins p120, p0071 and plakoglobin, together with a member of the striatin family and also, in rodents, the proteins ZO-1 and myozap. These N-cadherin-based AJs also include two novel types of junctions: the “areae adhaerentes”, i.e., variously-sized, often very large cell-cell contacts and small sieve-plate-like AJs perforated by cytoplasm-to-cytoplasm channels of 5–7 nm internal diameter (“cribelliform junctions”). We emphasize the unique character of this epithelium that totally lacks major epithelial marker molecules and structures such as keratin filaments and desmosomal elements as well as EpCAM- and PERP-containing junctions. We also discuss the nature, development and possible functions of these junctions.