Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells

During differentiation in vitro, embryonic stem (ES) cells generate progenitors for most hemato-lymphoid lineages. We studied the developmental potential of two ES cell subpopulations that share the fetal stem cell antigen AA4.1 but differ in expression of the lymphoid marker B220 (CD45R). Upon transfer into lymphoid deficient mice, the B220+ population generated a single transient wave of IgM+ IgD+ B cells but failed to generate T cells. In contrast, transfer of the B220− fraction achieved long-term repopulation of both T and B lymphoid compartments and restored humoral and cell-mediated immune reactions in the recipients. To assess the hemato-lymphopoietic potential of ES cell subsets in comparison to their physiological counterparts, cotransplantation experiments with phenotypically homologous subsets of fetal liver cells were performed, revealing a more potent developmental capacity of the latter. The results suggest that multipotential and lineage-committed lymphoid precursors are generated during in vitro differentiation of ES cells and that both subsets can undergo complete final maturation in vivo.

Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures

Neuronal apoptosis was observed in the rat dentate gyrus in two experimental models of human limbic epilepsy. Five hours after one hippocampal kindling stimulation, a marked increase of in situ terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) of fragmented DNA was observed in nuclei located within and on the hilar border of the granule cell layer and in the polymorphic region. Forty kindling stimulations with 5-min interval produced higher numbers of labeled nuclei compared with one stimulation. The increase of TUNEL-positive nuclei was prevented by the protein synthesis inhibitor cycloheximide but not affected by the N-methyl-d-aspartate receptor antagonist MK-801. Kainic acid-induced seizures lead to a pattern of labeling in the hippocampal formation identical to that evoked by kindling. A large proportion of cells displaying TUNEL-positive nuclei was double-labeled by the neuron-specific antigen NeuN, demonstrating the neuronal identity of apoptotic cells. Either 1 or 40 kindling stimulations also gave rise to a marked increase of the number of cells double-labeled with the mitotic marker bromodeoxyuridine and NeuN in the subgranular zone and on the hilar border of the dentate granule cell layer. The present data show that single and intermittent, brief seizures induce both apoptotic death and proliferation of dentate gyrus neurons. We hypothesize that these processes, occurring early during epileptogenesis, are primary events in the development of hippocampal pathology in animals and possibly also in patients suffering from temporal lobe epilepsy.

Transmembrane β-barrel of staphylococcal α-toxin forms in sensitive but not in resistant cells

Staphylococcal α-toxin is a 293-residue, single-chain polypeptide that spontaneously assembles into a heptameric pore in target cell membranes. To identify the pore-forming domain, substitution mutants have been produced in which single cysteine residues were introduced throughout the toxin molecule. By attaching the environmentally sensitive dye acrylodan to the sulfhydryl groups, the environment of individual amino acid side chains could be probed. In liposomes, a single 23-amino acid sequence (residues 118–140) was found to move from a polar to a nonpolar environment, indicating that this sequence forms the walls of the pore. However, periodicity in side chain environmental polarity could not be detected in the liposomal system. In the present study, the fluorimetric analyses were extended to physiological target cells. With susceptible cells such as rabbit erythrocytes and human lymphocytes, the 23 central amino acids 118–140 were again found to insert into the membrane; in contrast to the previous study with liposomes, the expected periodicity was now detected. Thus, every other residue in the sequence 126–140 entered a nonpolar environment in a striking display of an amphipathic transmembrane β-barrel. In contrast, human granulocytes were found to bind α-toxin to a similar extent as lymphocytes, but the heptamers forming on these cells failed to insert their pore-forming domain into the membrane. As a consequence, nonfunctional heptamers assembled and the cells remained viable. The data resolve the molecular organization of a pore-forming toxin domain in living cells and reveal that resistant cells can prevent insertion of the functional domain into the bilayer.

The selectivity of the hair cell’s mechanoelectrical-transduction channel promotes Ca2+ flux at low Ca2+ concentrations

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca2+ entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel’s open probability, an understanding of adaptation requires estimation of the magnitude of Ca2+ influx. To determine the Ca2+ current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K+, Na+, or Ca2+. For modest concentrations of a single permeant cation, Ca2+ carried much more receptor current than did either K+ or Na+. For higher cation concentrations, however, the flux of Na+ or K+ through the transduction channel exceeded that of Ca2+. For mixtures of Ca2+ and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel’s pore. These results demonstrate not only that the hair cell’s transduction channel is selective for Ca2+ over monovalent cations but also that Ca2+ carries substantial current even at low Ca2+ concentrations. At physiological cation concentrations, Ca2+ flux through transduction channels can change the local Ca2+ concentration in stereocilia in a range relevant for the control of adaptation.

Functional consequences of NR2 subunit composition in single recombinant N-methyl-d-aspartate receptors

Single-channel recordings were obtained from Chinese hamster ovary cells transfected with the N-methyl-d-aspartate (NMDA) receptor subunit NR1 in combination with NR2A, NR2B, NR2C, or NR2A/NR2B. NMDA-activated currents were recorded under control conditions and in the presence of a thiol reductant (DTT), an oxidant (5,5′-dithio-bis[2-nitrobenzoic acid], DTNB), or the noncompetitive antagonist CP101,606 (CP). For all subunit combinations, DTT increased the frequency of channel opening when compared with DTNB. In addition, channels obtained from NR1/NR2A-transfected cells also exhibited a pronounced difference in mean open dwell-time between redox conditions. CP dramatically reduced both the open dwell-time and frequency of channel opening of NR1/NR2B-containing receptors, but only modestly inhibited NR1/NR2A and NR1/NR2C channel activity. A small number of patches obtained from cells transfected with NR1/NR2A/NR2B had channels with properties intermediate to NR1/NR2A and NR1/NR2B receptors, including insensitivity to CP block but redox properties similar to NR1/NR2B, consistent with the coassembly of NR2A with NR2B. Hence, NMDA receptors containing multiple types of NR2 subunits can have functionally distinguishable attributes.

The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid

The γ-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (α1–6, β1–3, γ1–3, δ1, and ɛ1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the β subunit subtype present within the receptor. Receptors containing β2- or β3-, but not β1 subunits, are highly sensitive to the agent. Here, chimeric β1/β2 subunits coexpressed in Xenopus laevis oocytes with human α6 and γ2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the β3 subunit to Ser (the homologous residue in β1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the β1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk−) cells stably expressing the α6β3γ2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.

Quantitative analysis of the immunopotency of genetically transfected dendritic cells

Dendritic cells (DCs) instruct and activate a naive immune system to mount a response toward foreign proteins. Therefore, it has been hypothesized that an ideal vaccine strategy would be to directly introduce genes encoding antigens into DCs. To test this strategy quantitatively, we have compared the immune response elicited by a genetically transfected DC line to that induced by a fibroblast line, or standard genetic immunization. We observe that a single injection of 500–1,000 transfected DCs can produce a response comparable to that of standard genetic immunization, whereas fibroblasts, with up to 50-fold greater transfection efficiency, were less potent. We conclude that transfection of a small number of DCs is sufficient to initiate a wide variety of immune responses. These results indicate that targeting genes to DCs will be important for controlling and augmenting the immunological outcome in genetic immunization.

Recombinant parvovirus-like particles as an antigen carrier: A novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8+ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8+ class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells.

Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from Parp-disrupted mouse embryonic stem cells

The enzyme poly(ADP-ribose) polymerase (Parp) catalyzes poly(ADP-ribosyl)ation reaction and is involved in DNA repair and cell death induction upon DNA damages. Meanwhile, poly(ADP-ribosyl)ation of chromosome-associated proteins is suggested to be implicated in the regulation of gene expression and cellular differentiation, both of which are important in tumorigenesis. To investigate directly the role of Parp deficiency in tumorigenicity and differentiation of embryonic stem (ES) cells during tumor formation, studies were conducted by using wild-type J1 (Parp+/+) ES cells and Parp+/− and Parp−/− ES clones generated by disrupting Parp exon 1. These ES cells, irrespective of the Parp genotype, produced tumors phenotypically similar to teratocarcinoma when injected s.c. into nude mice. Remarkably, all tumors derived from Parp−/− clones contained syncytiotrophoblastic giant cells (STGCs), which possess single or multiple megalo-nuclei. The STGCs were present within large areas of intratumoral hemorrhage. In contrast, neither STGC nor hemorrhage was observed in tumors of both wild-type J1 cells and Parp+/− clones. Electron microscopic examination showed that the STGCs possess microvilli on the cell surface and contained secretory granules in the cytoplasm. Furthermore, the cytoplasms of STGCs were strongly stained with antibody against mouse prolactin, which could similarly stain trophoblasts in placenta. These morphological and histochemical features indicate that the STGCs in teratocarcinoma-like tumors derived from Parp−/− clones belong to the trophoblast cell lineage. Our findings thus suggest that differentiation of ES cells into STGCs was possibly induced by the lack of Parp during the development of teratocarcinoma.

Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells

Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase δ or ɛ. Here we report the reconstitution of the RFC complex from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified baculovirus-produced RFC appears to contain equimolar levels of each subunit and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase δ-catalyzed PCNA-dependent DNA chain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (iii) binding preferentially to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.

Temporal mapping of gene expression levels during the differentiation of individual primary hematopoietic cells

A hierarchical order of gene expression has been proposed to control developmental events in hematopoiesis, but direct demonstration of the temporal relationships between regulatory gene expression and differentiation has been difficult to achieve. We modified a single-cell PCR method to detect 2-fold changes in mRNA copies per cell (dynamic range, 250–250,000 copies/cell) and used it to sequentially quantitate gene expression levels as single primitive (CD34+,CD38−) progenitor cells underwent differentiation to become erythrocytes, granulocytes, or monocyte/macrophages. Markers of differentiation such as CD34 or cytokine receptor mRNAs and transcription factors associated with their regulation were assessed. All transcription factors tested were expressed in multipotent progenitors. During lineage-specific differentiation, however, distinct patterns of expression emerged. SCL, GATA-2, and GATA-1 expression sequentially extinguished during erythroid differentiation. PU.1, AML1B, and C/EBPα expression profiles and their relationship to cytokine receptor expression in maturing granulocytes could be distinguished from similar profiles in monocytic cells. These data characterize the dynamics of gene expression accompanying blood cell development and define a signature gene expression pattern for specific stages of hematopoietic differentiation.

Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1

Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RAD10 gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1− and ERCC1+ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT− cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT− products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1− cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1− cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1− cells are repaired by illegitimate recombination.

Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the “small-conductance” basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.

Mitosis in vertebrate somatic cells with two spindles: Implications for the metaphase/anaphase transition checkpoint and cleavage

During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK1 cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK1 cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 μm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles x̄ = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase x̄ = 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a “start anaphase” reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.

Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells

Cytochrome b-type NAD(P)H oxidoreductases are involved in many physiological processes, including iron uptake in yeast, the respiratory burst, and perhaps oxygen sensing in mammals. We have identified a cytosolic cytochrome b-type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) containing cytochrome b5 (b5) and b5 reductase (b5R) domains. A genetic approach, using blast searches against dbest for FAD-, NAD(P)H-binding sequences followed by reverse transcription–PCR, was used to clone the complete cDNA sequence of human b5+b5R from the hepatoma cell line Hep 3B. Compared with the classical single-domain b5 and b5R proteins localized on endoplasmic reticulum membrane, b5+b5R also has binding motifs for heme, FAD, and NAD(P)H prosthetic groups but no membrane anchor. The human b5+b5R transcript was expressed at similar levels in all tissues and cell lines that were tested. The two functional domains b5* and b5R* are linked by an approximately 100-aa-long hinge bearing no sequence homology to any known proteins. When human b5+b5R was expressed as c-myc adduct in COS-7 cells, confocal microscopy revealed a cytosolic localization at the perinuclear space. The recombinant b5+b5R protein can be reduced by NAD(P)H, generating spectrum typical of reduced cytochrome b with alpha, beta, and Soret peaks at 557, 527, and 425 nm, respectively. Human b5+b5R flavohemoprotein is a NAD(P)H oxidoreductase, demonstrated by superoxide production in the presence of air and excess NAD(P)H and by cytochrome c reduction in vitro. The properties of this protein make it a plausible candidate oxygen sensor.