Characterization of a second highly conserved B-type lamin present in cells previously thought to contain only a single B-type lamin

Previous analyses of the nuclear lamina of mammalian cells have revealed three major protein components (lamins A, B and C) that have been identified by protein sequence homology as members of the intermediate filament (IF) protein family. It has been claimed that mammalian cells contain either all three lamins or lamin B alone. Using monoclonal antibodies specific for B-type lamins and cDNA cloning we identified a second major mammalian B-type lamin (murine lamin B₂), thus showing that lamin composition in mammals is more complex than previously thought. Lamin B₂ is coexpressed with lamin B₁ (formerly termed lamin B) in all somatic cells and mammalian species that we analysed, including a variety of cells currently believed to contain only a single lamin. This suggests that two B-type lamins are necessary to form a functional lamina in mammalian somatic cells. By cDNA cloning we found thatXenopus laevis lamin L_II is the amphibian homolog of mammalian lamin B₂. Lamin expression during embryogenesis of amphibians and mammals shows striking similarities. The first lamins expressed in the early embryo are the two B-type lamins, while A-type lamins are only detected much later in development. These findings indicate that the genomic differentiation into two B-type lamins occurred early in vertebrate evolution and has been maintained in both their primary structure and pattern of expression.     

Loss of Glycosaminoglycan Receptor Binding after Mosquito Cell Passage Reduces Chikungunya Virus Infectivity

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that can cause fever and chronic arthritis in humans. CHIKV that is generated in mosquito or mammalian cells differs in glycosylation patterns of viral proteins, which may affect its replication and virulence. Herein, we compare replication, pathogenicity, and receptor binding of CHIKV generated in Vero cells (mammal) or C6/36 cells (mosquito) through a single passage. We demonstrate that mosquito cell-derived CHIKV (CHIKV_mos) has slower replication than mammalian cell-derived CHIKV (CHIKV_vero), when tested in both human and murine cell lines. Consistent with this, CHIKV_mos infection in both cell lines produce less cytopathic effects and reduced antiviral responses. In addition, infection in mice show that CHIKV_mos produces a lower level of viremia and less severe footpad swelling when compared with CHIKV_vero. Interestingly, CHIKV_mos has impaired ability to bind to glycosaminoglycan (GAG) receptors on mammalian cells. However, sequencing analysis shows that this impairment is not due to a mutation in the CHIKV E2 gene, which encodes for the viral receptor binding protein. Moreover, CHIKV_mos progenies can regain GAG receptor binding capability and can replicate similarly to CHIKV_vero after a single passage in mammalian cells. Furthermore, CHIKV_vero and CHIKV_mos no longer differ in replication when N-glycosylation of viral proteins was inhibited by growing these viruses in the presence of tunicamycin. Collectively, these results suggest that N-glycosylation of viral proteins within mosquito cells can result in loss of GAG receptor binding capability of CHIKV and reduction of its infectivity in mammalian cells.     

Dependence of intrachromosomal recombination in mammalian cells on uninterrupted homology.

Recombination between a 360-base-pair (bp) segment of a wild-type thymidine kinase gene (tk) from each of three different strains (F, MP, and 101) of herpes simplex virus type one and a complete herpes simplex virus type 1 (strain F) tk gene containing an 8-bp insertion mutation was studied. The pairs of tk sequences resided as closely linked repeats within the genome of mouse LTK- cells. The frequency of recombination between sequences exhibiting 232 bp of uninterrupted homology and containing no mismatches other than the insertion mutation was comparable to the frequency of recombination between two sequences exhibiting four additional nucleotide mismatches distributed in such a way to preserve the 232-bp stretch of contiguous homology. In contrast, the placement of only two single-nucleotide mismatches (in addition to the insertion mutation) in such a manner to reduce the longest uninterrupted homology to 134 bp resulted in a 20-fold reduction in recombination. We conclude that the rate of intrachromosomal recombination in mammalian cells is determined by the amount of uninterrupted homology available and not by the total number of mismatches within a given interval of DNA. Furthermore, efficient recombination appears to require between 134 and 232 bp of uninterrupted homology; single-nucleotide heterologies are most likely sufficient to disrupt the minimal efficient recombination target. We also observed that if recombination was allowed to initiate within sequences exhibiting perfect homology, the event could propagate through and terminate within adjacent sequences exhibiting 19% base pair mismatch. We interpret this to mean that heterology exerts most of its impact on early rather than late steps of intrachromosomal recombination in mammalian cells.     

Yeast-based protein delivery to mammalian phagocytic cells is increased by coexpression of bacterial listeriolysin

Yeast-mediated protein delivery to mammalian antigen-presenting cells is a powerful approach for inducing cell-mediated immune responses. We show that coexpression of the pore-forming protein listeriolysin O from Listeria monocytogenes leads to improved translocation of a proteinaceous antigen and subsequent activation of specific T lymphocytes. As the resulting yeast carrier is self-attenuated and killed after antigen delivery without exhibiting any toxic effect on antigen-presenting cells, this novel carrier system suggests itself as promising approach for the development of yeast-based live vaccines.     

An antibody Fc engineered for conditional antibody-dependent cellular cytotoxicity at the low tumor microenvironment pH

Despite the exquisite specificity and high affinity of antibody-based cancer therapies, treatment side effects can occur since the tumor-associated antigens targeted are also present on healthy cells. However, the low pH of the tumor microenvironment provides an opportunity to develop conditionally active antibodies with enhanced tumor specificity. Here, we engineered the human IgG1 Fc domain to enhance pH-selective binding to the receptor FcγRIIIa and subsequent antibody-dependent cellular cytotoxicity (ADCC). We displayed the Fc domain on the surface of mammalian cells and generated a site-directed library by altering Fc residues at the Fc–FcγRIIIa interface to support interactions with positively charged histidine residues. We then used a competitive staining and flow cytometric selection strategy to isolate Fc variants exhibiting reduced FcγRIIIa affinities at neutral pH, but physiological affinities at the tumor-typical pH 6.5. We demonstrate that antibodies composed of Fab arms binding the breast cell epithelial marker Her2 and the lead Fc variant, termed acid-Fc, exhibited an ∼2-fold pH-selectivity for FcγRIIIa binding based on the ratio of equilibrium dissociation constants K_d,7.4/K_d,6.5, due to a faster dissociation rate at pH 7.4. Finally, in vitro ADCC assays with human FcγRIIIa-positive natural killer and Her2-positive target cells demonstrated similar activities for anti-Her2 antibodies bearing the wild-type or acid-Fc at pH 6.5, but nearly 20-fold reduced ADCC for acid-Fc at pH 7.4, based on EC₅₀ ratios. This work shows the promise of mammalian cell display for Fc engineering and the feasibility of pH-selective Fc activation to provide a second dimension of selective tumor cell targeting.     

Synthesis,micellisation and interaction of novel quaternary ammonium compounds derived from l-Phenylalanine with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine as model membrane in relation to their antibacterial activity,and their selectivity over human red blood cells

A series of quaternary ammonium compounds (QUATS) derived from l-Phenylalanine have been synthesized and their antibacterial efficiencies were determined against various strains of Gram-positive and Gram-negative bacteria. The antibacterial activity increased with increasing chain length, exhibiting a cut-off effect at C₁₄ for Gram-positive and C₁₂ for Gram-negative bacteria. The l-Phenylalanine QUATS displayed enhanced antibacterial properties with a higher cut-off point compared to their corresponding l-Phenylalanine ester hydrochlorides. The CMC was correlated with the MIC, inferring that micellar activity contributes to the cut-off effect in antibacterial activity. The hemolytic activities (HC₅₀) of the QUATS against human red blood cells were also determined to illustrate the selectivity of these QUATS for bacterial over mammalian cells. In general, the MIC was lower than the HC₅₀, and assessment of the micellar contribution to the antibacterial and hemolytic evaluation in TBS as a common medium confirmed that these QUATS can act as antibacterial, yet non-toxic molecules at their monomer concentrations. The interaction of the QUATS with the phospholipid vesicles (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) in the presence of 1-anilino-8-naphthalene sulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescence probes showed that the presence of the quaternary ammonium moiety causes an increase in hydrophobic interactions, thus causing an increase in antibacterial activity.     

Intracellular oxygen-sensitive phosphorescent probes based on cell-penetrating peptides

Probing of molecular oxygen in mammalian cells is important for the analysis of mitochondrial function, metabolic responses, and energetic status of the cells. We describe a new panel of intracellular O₂-sensitive probes based on phosphorescent porphyrin dyes conjugated to cell-penetrating peptides. The probes comprising the uncharged derivatives of Pt(II)-coproporphyrin I covalently linked to positively charged TAT-derived peptides are shown to effectively load live mammalian cells without any transfection reagents. The probes work well with all cell types tested, show similar subcellular localization, and produce characteristic responses to cell stimulation with mitochondrial uncouplers and inhibitors. They provide a simple and versatile tool for O₂ monitoring in live cells and in tissue, and an alternative to the existing O₂ probes which require facilitated transport into the cell.     

Effect of dimerization of a β-turn antimicrobial peptide, PST13-RK, on antimicrobial activity and mammalian cell toxicity

PST13-RK (KKKFPWWWPFKKK-NH₂) is an improved derivative of tritrpticin adopting a β-turn structure. In order to investigate the effect of dimerization of PST13-RK on antimicrobial activity and mammalian cell toxicity, we designed and synthesized its Cys- and Lys-linked dimers. The dimerization of PST13-RK resulted in a 2–4 fold decreased antimicrobial activity against Gram-positive and Gram-negative bacteria. However, the dimers showed a large increase in mammalian cell toxicity against mouse NIH-3T3, human MDA-MB-361, and human A549 cells. These results suggested that PST13-RK is active as a monomer to bacterial cells but as an oligomer to mammalian cells. Since the dimeric PST13-RK is much more effective against the cancer cells than the monomer, it might be an attractive candidate for anticancer chemotherapeutic drugs.     

Induction of Apoptosis Due to Lowering the Level of Eukaryotic Initiation Factor 2-Associated Protein, p67, from Mammalian Cells by Antisense Approach

p67, a cellular glycoprotein, protects eIF2α from phosphorylation by inhibitory kinases such as double-stranded RNA dependent eIF2 kinase, PKR, and heme-controlled repressor and thus promotes protein synthesis in mammalian cells. To investigate whether p67 is essential for the survival of mammalian cells, the basal level of p67 was lowered from rat tumor hepatoma cells using antisense approach. The antisense p67 RNA specifically lowered the levels of p67 message and the protein from these cells. As a result, the level of eIF2α phosphorylation increased significantly, the overall rate of protein synthesis decreased, and the rate of DNA synthesis also decreased in mammalian cells with low levels of p67 as compared to that seen in control cells. In addition, the majority of the cells with low levels of p67 are arrested at the G₁phase of the cell cycle and die with apoptosis. Taken together, these results suggest that appropriate levels of p67 is required for normal growth of mammalian cells.     

Chromatin fractionation of normal and virus-transformed cells in culture

A simple technique for the obtaining of purified chromatin fractions from mammalian cells in culture is described. The procedure involves the isolation of clean nuclei in 0.30 M sucrose, 1.5 mM MgCl₂, 0.2 mM CaCl₂, 0.01 M Tris HCl pH 7.2, followed by sonication and differential centrifugation to collect specific chromatin fractions. Heterochromatin of SV-40 and Rous sarcoma virus transformed 3T3 cells showed a 6- to 7-fold enrichment in satellite DNA while a 2- to 3-fold enrichment in repetitive DNA was obtained in established and RSV transformed cells of Microtus agrestis. This method will facilitate the search for the site of integration of oncogenic viruses in the chromatin of mammalian cells.     

Cell cycle kinetics of insect cell cultures compared to mammalian cell cultures

Cell cycle kinetics of lepidopteran cell lines Sf9 (Spodoptera frugiperda) and IZDMb0503 (Mamestra brassicae) were investigated and compared to mammalian cell cycle distributions. The resting phase (G₀) of mammalian cells is characterized by a 2c-DNA content whereas G₀-phase of insect cell lines is characterized by a 4c-DNA content. Flow cytometric data in combination with growth curves of partially synchronized and asynchronously growing cells proved the existence of this phenomenon. Kinetics of cells labeled by the thymidine analog on 5-bromo-2′-deoxyuridine supported these results, which now render the possibility of applying cell cycle analysis in fermentation technology of insect cells.     

Determination of viable mammalian cells by luminol chemiluminescence using microperoxidase

Chemiluminescent assay for menadione-catalyzed H₂O₂ production by mammalian cells was modified by luminol chemiluminescence with microperoxidase instead of peroxyoxalate chemiluminescence with carcinogenic fluorescent materials. Luminol can be used as a common chemiluminescent reagent for the determination of viable mammalian cells and bacteria.     

A tetracycline repressor-based mammalian two-hybrid system to detect protein-protein interactions in vivo

A mammalian two-hybrid system (termed as trM2H) was developed to detect protein-protein interactions in vivo, based on the reconstitution of the functions the of tetracycline repressor (TetR). The system is sensitive enough to detect protein-protein interactions with K_d up to 55 μM in mammalian cells, and the system can be regulated by small molecules. This system can be used as an efficient genetic selection system to map protein-protein interactions in mammalian cells.     

Regulation of the mitochondrial ATPasein situ in cardiac muscle: Role of the inhibitor subunit

The mitochondrial F₁-ATPase inhibitor protein, IF₁, binds to subunits of the F₁-ATPase bothin vitro andin situ under nonenergizing conditions, i.e., under conditions that allow a net hydrolysis of ATP by the mitochondrial ATPase to take place. This reversible IF₁ binding occurs in a wide variety of cell types including (anaerobic) baker's yeast cells and (ischemic) mammalian cardiomyocytes under conditions that limit oxidative phosphorylation. The binding of inhibitor results in a marked slowing of ATP hydrolysis by the undriven mitochondrial ATP synthase. An apparent main function of this reversible IF₁ binding, at least in cells that undergo aerobic-anaerobic switching, is the mitigation of a wasteful hydrolysis of ATP produced by glycolysis during anoxic or ischemic intervals, by the mitochondrial ATPase. While this apparent main function is probably of considerable importance in cells that normally either can or must undergo aerobic-anaerobic switching such as baker's yeast cells and skeletal myocytes, one wonders why a full complement of IF₁ has been retained in certain cells that normally do not undergo such aerobic-anaerobic switching, cells such as adult mammalian cardiomyocytes of many species. While some mammalian species have, indeed, not retained a functional complement of IF₁ in their cardiomyocytes, those that have can benefit significantly from its presence during intervals of myocardial ischemia.This mini-review is dedicated to the memory of Professor Efraim Racker.     

Asymmetrically heteropycnotic X chromosomes in the grasshopper Melanoplus femur-rubrum

In short-horned grasshoppers the X chromosome is negatively heteropycnotic in at least some of the spermatogonia but is positively heteropycnotic (heterochromatic) during prophase I of spermatogenesis. In tetraploid (4n) spermatocytes in prophase I the two Xs present have so far been reported always to be heterochromatic and unpaired. In several males of the grasshopper Melanoplus femur-rubrum (Acrididae), however, some of the 4n primary spermatocytes contained one heterochromatic X (X_h) and one euchromatic X (X_e). This asymmetry of heteropycnosis (AH) was first observed in grasshoppers by M.J.D. White who observed it, however, only in 4n spermatogonia in which one X was negatively heteropycnotic and the other was isopycnotic (euchromatic). In M. femur-rubrum the AH involved both positive and negative heteropycnosis. In some of the 4n cells both Xs were heterochromatic and these cells were usually present in small groups but sometimes comprised whole cysts. The 4n cells with X_e+X_h always comprised several whole cysts in a follicle or whole follicles. The origin of the two cell types may be explained by assuming that heteropycnosis originated prior to the origin of the cysts, that when, as a result of polyploidization, two Xs were present in a 4n cell only one became heteropycnotic, and that the state of the X (X_h vs. X_e) usually persisted into meiosis. The 4n primary spermatocytes exhibiting AH divided regularly during the first meiotic division but following telophase I they usually failed to undergo cytokinesis and to enter the second meiotic division. The available evidence suggests that the arrest of these cells is the result of the genetic activity of the X_e in those stages in which the X is usually heterochromatic and genetically inactive. The relationship between AH and facultative heterochromatinization is discussed and it is concluded that the present observations put into question the validity of previous models attempting to explain facultative heterochromatinization (including that of the X in the mammalian female).     

Turnover of histone acetyl groups in cultured mammalian cells

The relative metobolic turnover rates of labeled histone fractions and of their labeled acetyl groups were determined in random cultures of exponentially-growing mammalian cells. Both the parent histones and their acetyl groups were conserved, neither exhibiting detectable metabolic turnover. The significance of these observations is discussed.     

Sugar transport in the LLC-PK1 renal epithelial cell line: Similarity to mammalian kidney and the influence of cell density

Cells of confluent cultures of the established pig renal epithelial line, LLC-PK₁, accumulate α-methyl-D-glucoside against a concentration gradient. This transport system is strongly inhibited by phlorizin and 6-deoxy-D-glucose, moderately inhibited by phloretin, and only weakly inhibited by 3-0-methyl-D-glucose, paralleling the situation in mammalian kidney. The time course for the uptake of α-methyl-D-glucoside and for the carrier-mediated but passive uptake of 3-0-methyl-D-glucose are identical to those seen in mammalian kidney. Subconfluent cultures of LLC-PK₁ cells are unable to accumulate α-methyl-D-glucoside, and their transport of this glucose analog is less sensitive to phlorizin inhibition than is the transport system in confluent cultures. Transmission electron micrographs show that cells from subconfluent cultures lack the microvillous surface seen in cells from confluent cultures. Cell density is thus a factor in the occurrence of structural and functional differentiated properties related to transport in these cells.