Motoneurons and oligodendrocytes are sequentially generated from neural stem cells but do not appear to share common lineage-restricted progenitors in vivo

Olig gene expression is proposed to mark the common progenitors of motoneurons and oligodendrocytes. In an attempt to further dissect the in vivo lineage relationships between motoneurons and oligodendrocytes, we used a conditional cell-ablation approach to kill Olig-expressing cells. Although differentiated motoneurons and oligodendrocytes were eliminated, our ablation study revealed a continuous generation and subsequent death of their precursors. Most remarkably, a normal number of oligodendrocyte precursors are formed at day 12 of mouse development, after all motoneuron precursors have been killed. The data presented herein supports a sequential model in which motoneuron and oligodendrocyte precursors are sequentially generated in vivo from neuroepithelial stem cells, but do not share a common lineage-restricted progenitor.     

Cytotoxic T lymphocyte-induced loss of target cell adhesion and lysis involve common and separate signaling pathways

Recently, we demonstrated that an early event in the CTL-target cell (TC) interaction is loss of TC adherence to substrate. This loss of adhesion is Ag-specific, but distinct from the lytic event because it can ensue in nominally Ca2+-free medium. In this study, we examine further the mechanism of CTL-induced loss of adhesion, concentrating mainly on the signal transduction pathway. Based on the differential sensitivity of CTL to extracellular Ca2+, protein kinase C activation/depletion and inhibition by anti-Lyt-2 (CD8) or anti-CTL receptor (TCR) reagents, we demonstrate that CTL-induced loss of adhesion can be initiated through multiple activation pathways. Although CTL-mediated lysis is restricted to a Ca2+ and protein kinase C-dependent signaling mechanism, CTL-induced loss of adhesion is initiated in the presence or absence of extracellular Ca2+ or functional protein kinase C activity. Furthermore, although under physiologic conditions, anti-CD8 or anti-TCR reagents strongly block both CTL activities, under non-lytic conditions, they fail to inhibit the ability of CTL to promote loss of adhesion. These findings implicate the participation of additional CTL-TC ligand interactions resulting in loss of adhesion, and thus, provide further evidence to support the hypothesis that CTL-induced loss of adhesion can be initiated through multiple triggering pathways.     

Hominins do not share a common postnatal facial ontogenetic shape trajectory

This paper examines the hypothesis raised by recent studies that postnatal trajectories of shape change in the facial skeleton are parallel between, at least, chimpanzees, modern humans and also fossil hominins, specifically australopithecines and possibly Neanderthals. In contrast, other studies point to divergences in postnatal shape trajectories within diverse groups of primates. As such there is some debate regarding the relative contributions of pre and postnatal ontogeny to adult morphological differences. This paper presents a series of geometric morphometric studies of the ontogeny of facial shape in hominins with the specific aim of resolving these issues. The results indicate that many differences in facial shape between hominins are established prenatally, however highly significant divergences of postnatal facial ontogeny are found among living hominins. Our studies point to possible differences between the shape ontogeny of the Australopithecus africanus face and that of African apes on the one hand and humans on the other. However, sampling experiments indicate that the small sample size of available specimens of A. africanus does not permit any conclusions to be drawn regarding comparative shape ontogeny of the face.     

Insulin and hypoxia share common target genes but not the hypoxia-inducible factor-1alpha

Both hypoxia and insulin induce common target genes, including vascular endothelial growth factors and several glycolytic enzymes. However, these two signals eventually trigger quite different metabolic pathways. Hypoxia induces glycolysis, resulting in anaerobic ATP production, while insulin increases glycolysis for energy storage. Hypoxia-induced gene expression is mediated by the hypoxia-inducible factor-1 (HIF-1) that consists of HIF-1alpha and the aromatic hydrocarbon nuclear translocator (Arnt). Hypoxia-induced gene expression is initiated by the stabilization of the HIF-1alpha subunit. Here we investigated whether insulin-induced gene expression also requires stabilization of HIF-1alpha. Our results indicate that hypoxia but not insulin stabilizes HIF-1alpha protein levels, whereas both insulin- and hypoxia-induced gene expression require the presence of the Arnt protein. Insulin treatment fails to inactivate proline hydroxylation of HIF-1alpha, which triggers recruitment of the von Hippel-Lindau protein and oxygen-dependent degradation of HIF-1alpha. Insulin-induced gene expression is inhibited by the presence of the phosphoinositide (PI) 3-kinase inhibitor LY294002 and the dominant negative mutant of the p85 subunit of PI 3-kinase, whereas hypoxia-induced gene expression is not. Pyrrolidine dithiocarbamate, a scavenger of H2O2, reduces insulin-induced gene expression but not hypoxia-induced gene expression. Although both hypoxia and insulin induce the expression of common target genes through a hypoxia-responsive element- and Arnt-dependent mechanism, insulin cannot stabilize the HIF-1alpha protein. We believe that insulin activates other putative partner proteins for Arnt in PI 3-kinase- and H2O2-dependent pathways.     

Hydrogen exchange in BPTI variants that do not share a common disulfide bond.

Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30-51, 5-55, and 14-38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5-55]Ala and [30-51; 14-38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide-bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native-like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI folding intermediates containing either the 5-55 or the 30-51 disulfide bond.     

Bactericidal antibiotics do not appear to cause oxidative stress in Listeria monocytogenes

Oxidative stress can be an important contributor to the lethal effect of bactericidal antibiotics in some bacteria, such as Escherichia coli and Staphylococcus aureus. Thus, despite the different target-specific actions of bactericidal antibiotics, they have a common mechanism leading to bacterial self-destruction by internal production of hydroxyl radicals. The purpose of the present study was to determine if a similar mechanism is involved in antibiotic killing of the infectious human pathogen, Listeria monocytogenes. We treated wild-type L. monocytogenes and oxidative stress mutants (Δsod and Δfri) with three different bactericidal antibiotics and found no difference in killing kinetics. In contrast, wild-type E. coli and an oxidative stress mutant (ΔsodA ΔsodB) differed significantly in their sensitivity to bactericidal antibiotics. We conclude that bactericidal antibiotics did not appear to cause oxidative stress in L. monocytogenes and propose that this is caused by its noncyclic tricarboxylic acid (TCA) pathway. Hence, in this noncyclic metabolism, there is a decoupling between the antibiotic-mediated cellular requirement for NADH and the induction of TCA enzyme activity, which is believed to mediate the oxidative stress reaction.     

Sec-dependent pathway and DeltapH-dependent pathway do not share a common translocation pore in thylakoidal protein transport.

Thylakoidal proteins of plant chloroplasts are transported to thylakoids via several different pathways, including the DeltapH-dependent and the Sec-dependent pathways. In this study, we asked if these two pathways utilize a common translocation pore. A fusion protein consisting of a 23-kDa subunit of the oxygen evolving complex and Escherichia coli biotin carboxyl carrier protein was biotinylated in E. coli cells and purified. When incubated with isolated pea thylakoids in the absence of avidin, the purified fusion protein was imported into the thylakoids via the DeltapH-dependent pathway. However in the presence of avidin, the fusion protein became lodged in the thylakoid membranes, with its N terminus reaching the thylakoidal lumen, while its C-terminal segment complexed with avidin exposed on the thylakoidal surface. The translocation intermediate of the fusion protein inhibited the import of authentic 23-kDa subunit, suggesting that it occupies a putative translocation pore for the DeltapH-dependent pathway. However the intermediate did not block import of the 33-kDa subunit of the oxygen evolving complex, which is a substrate for the Sec-dependent pathway. These results provide evidence against the possibility of a common translocation pore shared by the Sec-dependent pathway and the DeltapH-dependent pathway.     

Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis

Purified cytolytic T lymphocyte (CTL) proteases granzyme (gzm)A and gzmB with sublytic dose of perforin (perf) initiate distinct proapoptotic pathways. Their physiological relevance in CTL-mediated target cell apoptosis is elusive. Using ex vivo virus-immune CD8(+) T cells from mice deficient in perf, gzmA and/or gzmB, and the Fas-resistant EL4.F15 tumor target cell, we show that (a) CTL from gzmA(-/-) or gzmB(-/-) mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, Delta Psi(m) loss, and reactive oxygen radical generation, though with distinct kinetics; (b) CTL from gzmA(-/-) but not from gzmB(-/-) mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA(-/-) or gzmB(-/-) mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzm-induced apoptotic features analyzed depend critically on perf. Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes. The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors.     

Doppel and PrP(C) do not share the same membrane microenvironment

Doppel is a paralog of the normal prion protein, PrP^C. It has been suggested that Doppel can compensate for the absence of PrP^C in PrP^0/0 mice. In this work, we tested whether Doppel and PrP^C share the same cell location, thereby sharing the same neighboring cell components, probably required to share the same cell function. Our results show that, at detergent conditions in which membrane rafts were intact, neither PrP^C and Doppel co-immunoprecipitate with the appropriate antibodies, nor was Doppel retained by a Cu²⁺IMAC resin, as PrP^C does. This indicates that, although Doppel is a raft-associated protein as is PrP^C, both proteins are not present in the same membrane microenvironment, and they probably do not perform the same function.     

Possible mechanism for target cell lysis by cytotoxic T-cells

The mechanism of the lysis of target cells by cytotoxic T-cells (Tc) is still obscure; there is no evidence for transfer of material from the Tc and prior to lysis, despite intimate contact, the plasma membranes of both types of cell appear to remain intact. The effects on the target cell lysosomes of brief contact between anti-viral Tc and targets bearing both the appropriate histocompatibility and viral antigens, have been examined cytochemically. Both the distribution of acid phosphatase activity and the percentage bound lysosomal naphthylamidase activity indicated that, in virus-infected target cells exposed to Tc, the lysosomal membranes became totally labilized. Thus the contact between Tc and targets appears to cause sufficient perturbation of the target plasma membrane as to cause the intracellular release of some agent that activates 'suicide capsule' lysosomes.     

Cutting edge: regulatory T cells do not mediate suppression via programmed cell death pathways

Regulatory T cells (Tregs) play a critical role in the immune system to regulate peripheral tolerance and prevent autoimmunity. However, the relative importance of different mechanisms of Treg function remains obscure. In this article, we reveal a limited role for programmed cell death pathways in mediating Treg suppression of conventional T cells. We show that Tregs are able to suppress the proliferation of conventional T cells that are resistant to apoptosis (Bim(-/-), Bim(-/-)Puma(-/-), Bcl-2 transgenic) or receptor-interacting serine-threonine kinase-dependent necrosis (also referred to as regulated necrosis or necroptosis) (Ripk3(-/-)) in several in vitro and in vivo assays. These data suggest that programmed cell death pathways, such as apoptosis and receptor-interacting serine-threonine kinase-dependent necrosis, are not required for Treg-mediated suppression.     

Arrest of the cell cycle reduces susceptibility of target cells to perforin-mediated lysis

Cytotoxic T lymphocytes secrete a pore-forming cytolysin, perforin, that damages membranes of target cells. They also ligate Fas receptors on target cells and provoke apoptotic death. A20 (B lymphoma) and P815 (mastocytoma) cell lines were examined for their susceptibility to perforin-mediated lysis and to Fas-induced apoptosis after blockade of the cell cycle at the G₁/S interface. Cells were arrested at the G₁/S interface by inhibition of DNA synthesis with thymidine or aphidicolin. Subsequently, the treated cells were incubated either with CTL cytotoxic granules or the Fas-specific monoclonal antibody Jo-2. We show that arrest of the cell cycle at the G₁/S interface markedly reduced the susceptibility of target cells to perforin-mediated lysis. In contrast, growth arrest with thymidine or aphidicolin increased susceptibility of A20 and P815 cells to Fas-mediated apoptosis. Susceptibility to lysis by intact CTLs was not affected significantly by blockade of target cells with aphidicolin or thymidine. When cells surviving exposure to perforin-containing granules were isolated on Ficoll density gradients and cell-cycle profiles were examined by flow cytometry, the ratio of G₁ to G₂cells increased among the survivors exposed to granules in contrast to controls incubated with buffer alone. The data suggest that cells in G₁ phase of the cell cycle are less susceptible to the perforin pathway than cells in G₂and S phases but are more susceptible to the Fas pathway. J. Cell. Biochem. 69:425–435, 1998. © 1998 Wiley-Liss, Inc.     

Infant and adult human B cell responses to rotavirus share common immunodominant variable gene repertoires

Ab repertoires exhibit marked restrictions during fetal life characterized by biases of variable gene usage and lack of junctional diversity. We tested the hypothesis that Ab repertoire restriction contributes to the observed poor quality of specific Ab responses made by infants to viral infections. We analyzed the molecular determinants of B cell responses in humans to two Ags of rotavirus (RV), a common and clinically important infection of human infants. We sequenced Ab H and L chain V region genes (V(H) and V(L)) of clones expanded from single B cells responding to RV virus protein 6 or virus protein 7. We found that adults exhibited a distinct bias in use of gene segments in the V(H)1 and V(H)4 families, for example, V(H)1-46, V(H)4-31, and V(H)4-61. This gene segment bias differed markedly from the V(H)3 dominant bias seen in randomly selected adult B cells. Recombinant Abs incorporating any of those three immunodominant V(H) segments bound to RV-infected cells and also to purified RV particles. The RV-specific B cell repertoires of infants aged 2-11 mo and those of adults were highly related when compared by V(H), D, J(H), V(L), and J(L) segment selection, extent of junctional diversity, and mean H chain complementarity determining region 3 length. These data suggest that residual fetal bias of the B cell repertoire is not a limiting determinant of the quality of Ab responses to viruses of infants beyond the neonatal period.     

Capacity of thymic cells to effect target cell lysis following treatment with concanavalin A

The capacity of lymph node, thymus and bone marrow cells to mediate a cytolytic effect on target monolayers in the presence of concanavalin A was investigated. It was found that freshly prepared rat lymph node cells, but not thymus or bone marrow cells, lyse mouse fibroblasts in cultures treated with concanavalin A. A population of thymic cells did, however, manifest cytolytic capacity in the presence of concanavalin A after having been first sensitized and selected on mouse fibroblasts. Thus, sensitized thymus cells possess a lytic potency which can be activated under proper conditions.     

The human and murine influenza-specific B cell repertoires share a common idiotope

We have dissected the human influenza-specific B cell repertoire by performing Epstein-Barr virus (EBV) limiting dilution analysis of lymphocytes obtained from donors before and after immunization with a commercially available influenza vaccine. In addition to an analysis of precursor frequency and light chain diversity, we studied sera and culture supernatants containing human anti-influenza antibodies with a panel of murine monoclonal antibodies specific for idiotopes identified on murine anti-PR8 and anti-X-31 antibodies. An idiotypic specificity present on the X-31-specific murine monoclonal PY206 has previously been shown to be shared by murine antibodies specific for PR8, X-31, and other influenza viruses. We observed little correlation among the following parameters: anti-viral titer, serum idiotope content, precursor frequency and immune status. More interestingly, there was a striking predominance of human influenza-specific antibodies that utilized lambda light chains. In addition, 12 of 26 human anti-influenza monoclonals strongly inhibited the binding of one of the murine anti-idiotopes to the labeled murine antibody, PY206. This is the same idiotope that is shared among murine antiinfluenza antibodies and all six individuals studied contained clones reactive with this anti-idiotope. Seven of these 12 idiotope-positive human antibodies gave partial cross-reactivity in a second anti-idiotypic system. These observations imply that a significant level of homology exists between the binding sites of human and murine influenza-specific antibodies and suggest that idiotypic manipulation of the human immune response to influenza virus may have important therapeutic implications.     

Lack of target cell participation in cytotoxic T lymphocyte-mediated lysis.

Data on the subject of cell-mediated cytotoxicity suggest that no single mechanism is likely to provide a satisfactory explanation of this process. Lytic pathways have been proposed that involve both the effector cell and the target cell as active participants. In this report we describe a system in which the target cell is rendered unable to participate in its own demise. Using sheep E derivatized with CD3 antibodies, we show that metabolic inhibition of SRBC by depleting intracellular ATP with iodoacetamide, or even conversion of SRBC to "ghosts" by hypotonic lysis and resealing, has no effect on cytolysis. In the presence of EGTA or cholera toxin, both of which inhibit CTL degranulation, there is a strong suppression of both serine esterase release and cytolysis. These data show clearly that in some situations CTL are able to lyse target cells without any active participation by the target cells themselves.     

Dark and photoactivated rhodopsin share common binding modes to transducin

The structure of the photoactivated deprotonated rhodopsin intermediate was compared with two different structures of dark rhodopsin. Structure comparisons relied on the computation of molecular indices and on docking simulations with heterotrimeric transducin (Gt). The results of this study provide the first evidence that dark and photoactivated rhodopsins share a common recognition mode to Gt, characterized by the docking of the Gt alpha C-tail in the proximity to the E/DRY motif of rhodopsin.