Amino terminal glutamate residues confer spermine sensitivity and affect voltage gating and channel conductance of rat connexin40 gap junctions

Connexin40 (Cx40) contains a specific binding site for spermine (affinity ∼100 μ m ) whereas connexin43 (Cx43) is unaffected by identical concentrations of intracellular spermine. Replacement of two unique glutamate residues, E9 and E13, from the cytoplasmic amino terminal domain of Cx40 with the corresponding lysine residues from Cx43 eliminated the block by 2 m m spermine, reduced the transjunctional voltage ( V _j ) gating sensitivity, and reduced the unitary conductance of this Cx40E9,13K gap junction channel protein. The single point mutations, Cx40E9K and Cx40E13K, predominantly affected the residual conductance state ( G _min) and V _j gating properties, respectively. Heterotypic pairing of Cx40E9,13K with wild-type Cx40 in murine neuro2A (N2A) cells produced a strongly rectifying gap junction reminiscent of the inward rectification properties of the Kir (e.g. Kir2.x) family of potassium channels. The reciprocal Cx43K9,13E mutant protein exhibited reduced V _j sensitivity, but displayed much less rectification in heterotypic pairings with wtCx43, negligible changes in the unitary channel conductance, and remained insensitive to spermine block. These data indicate that the connexin40 amino terminus may form a critical cytoplasmic pore-forming domain that serves as the receptor for V _j -dependent closure and block by intracellular polyamines. Functional reciprocity between Cx40 and Cx43 gap junctions involves other amino acid residues in addition to the E or K 9 and 13 loci located on the amino terminal domain of these two connexins.     

A Francisella tularensis locus required for spermine responsiveness is necessary for virulence

Tularemia is a debilitating febrile illness caused by the category A biodefense agent Francisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by which F. tularensis senses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions of F. tularensis with host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis of F. tularensis subsp. holarctica live vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 in F. tularensis subsp. tularensis Schu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophages in vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuated in vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as an F. tularensis gene important for virulence and evasion of the host immune response.     

The amino-terminal residue of glycoprotein B is critical for neutralization of bovine herpesvirus 1

In order to address the neutralization epitope on bovine herpesvirus 1 (BHV1) glycoprotein B (gB), a panel of monoclonal antibodies (MAbs), a series of truncation forms of the glycoprotein and an MAb-escape mutant were used in this study. Immunocytochemistry on the truncations using MAbs against the glycoprotein revealed that the neutralization epitopes recognized by the MAbs lay between residues 1 and 52 of mature gB. Comparison of the sequences among the mutant, parent, and revertant viruses demonstrated that the amino-terminal residue of mature gB of the escape mutant was changed from Arg to Gln. These findings indicate that the amino-terminal residue of gB is critical for neutralization of BHV1.     

The CD40/CD40 ligand interaction is required for resistance to toxoplasmic encephalitis

Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-gamma), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-gamma when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-gamma or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L(-/-) mice infected with T. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-gamma but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L(-/-) mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-gamma, to activate macrophages in vitro to control replication of T. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.     

An amino-terminal fragment of SV40 T antigen transforms REF52 cells.

An SV40 mutant, T147D, encodes only the amino-terminal 147 amino acids of large T antigen and does not make small t antigen. We show here that a retrovirus which expresses this mutant T antigen transforms rat REF52 cells as efficiently as a retrovirus that expresses both the wild-type large and small T antigens. This cell line had previously been refractory to transformation by mutants that make short, amino-terminal fragments of T antigen.     

CAML is a p56Lck-interacting protein that is required for thymocyte development

Calcium modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein implicated in T cell signaling, although its mechanism and physiologic role in the immune system are unknown. We show here that CAML is essential for peripheral T cell development. Inactivation of CAML in mouse thymocytes lowered the numbers of double-positive and single-positive thymocytes, concomitant with reduced positive and enhanced negative selection. We found that CAML interacts with p56Lck and appears to regulate subcellular localization of the kinase in both resting and T cell receptor (TCR)-stimulated cells. CAML-deficient cells displayed enhanced p56lck and ZAP-70 phosphorylation and increased IL2 production and cell death after TCR stimulation, suggesting that CAML may act as a negative regulator of p56lck. Our data establish a novel role for CAML as an essential mediator of T cell survival during thymopoiesis and indicate that its loss deregulates p56Lck signaling.     

The bundle crossing region is responsible for the inwardly rectifying internal spermine block of the Kir2.1 channel

Inward rectifier potassium channels conduct K⁺ across the cell membrane more efficiently in the inward than outward direction in physiological conditions. Voltage-dependent and flow-dependent blocks of outward K⁺ currents by intracellular polyamines (e.g., spermine (SPM)) have been proposed as the major mechanisms underlying inward rectification. In this study, we show that the SPM blocking affinity curve is shifted according to the shift in K⁺ reversal potential. Moreover, the kinetics of SPM entry to and exit from the binding site are correlatively slowed by specific E224 and E299 mutations, which always also disrupt the flux coupling feature of SPM block. The entry rates carry little voltage dependence, whereas the exit rates are e-fold decelerated per ～15 mV depolarization. Interestingly, the voltage dependence remains rather constant among WT and quite a few different mutant channels. This voltage dependence offers an unprecedented chance of mapping the location (electrical distance) of the SPM site in the pore because these kinetic data were obtained along the preponderant direction of K⁺ current flow (outward currents for the entry rate and inward currents for the exit rate) and thus contamination from flow dependence should be negligible. Moreover, double mutations involving E224 and A178 or M183 seem to alter the height of the same asymmetrical barrier between the SPM binding site and the intracellular milieu. We conclude that the SPM site responsible for the inward rectifying block is located at an electrical distance of ～0.5 from the inside and is involved in a flux coupling segment in the bundle crossing region of the pore. With preponderant outward K⁺ flow, SPM is “pushed” to the outmost site of this segment (～D172). On the other hand, the blocking SPM would be pushed to the inner end of this segment (～M183–A184) with preponderant inward K⁺ flow. Moreover, E224 and E299 very likely electrostatically interact with the other residues (e.g., R228, R260) in the cytoplasmic domain and then allosterically keep the bundle crossing region in an open conformation appropriate for the flux coupling block of SPM.     

CD40L, but not CD40, is required for allergen-induced bronchial hyperresponsiveness in mice

Asthma is characterized by immunoglobulin (Ig) E production, infiltration of the respiratory mucosa by eosinophils (EOSs) and mononuclear cells, and bronchial hyperresponsiveness (BHR). Interaction of CD40 on B cells and antigen presenting cells, with its ligand (CD40L) expressed transiently on activated T cells, is known to augment both T cell-driven inflammation and humoral immune responses, especially IgE production. Considering both the prominent role of inflammation in asthma and the association of the disease with IgE, we hypothesized that CD40-CD40L interactions would be important in pathogenesis. To test this hypothesis, we subjected wild-type (WT) mice and animals lacking either CD40 or CD40L to repeated inhalation of Aspergillus fumigatus (Af ) antigen. Af-treated WT mice displayed elevated IgE levels, bronchoalveolar lavage and pulmonary tissue eosinophilic inflammation, and BHR. IgE production was markedly suppressed in both the CD40 -/- and CD40L -/- strains. However, pulmonary inflammation did not appear to be inhibited by either of these mutations. Paradoxically, development of BHR was prevented by the lack of CD40L but not by the absence of CD40. We conclude that CD40/CD40L interactions, although critical in the induction of IgE responses to inhaled allergen, are not required for the induction of EOS-predominant inflammation. CD40L, but not CD40, is necessary for the development of allergen-induced BHR.     

CD40 is required for the optimal induction of protective immunity to Mycobacterium avium

C57Bl/6 mice and mice deficient in the CD40 molecule were infected with three strains of Mycobacterium avium. Two of the M. avium strains proliferated more extensively in CD40-deficient (CD40-/-) mice than in control mice. The increased susceptibility to infection of CD40-/- mice was associated with the generation of poorer interleukin-12 (IL-12) p40 and interferon-gamma (IFN-gamma) responses as compared to the controls, suggesting a role for CD40 in the development of protective immunity. In contrast, direct triggering of CD40 on infected macrophages failed to induce any anti-mycobacterial activity in infected macrophages.     

The small-t protein of SV40 is required for loss of actin cable networks and plasminogen activator synthesis in transformed rat cells

Of a variety of properties characteristic of SV40 transformed rat embryo fibroblast cells we find that those acquired by minimally transformed cells (the ability to grow clonally and in medium supplemented with low levels of serum) depend only on the large-T protein of SV40 while those properties associated exclusively with fully transformed, tumorigenic cells (growth suspended in methyl cellulose or agar, loss of cytoplasmic actin networks, and synthesis of plasminogen activator) are more likely to occur in cells transformed by viruses which encode the small-t protein as well. In addition one of these properties of the fully transformed cells, PA synthesis, is at least partially independent of large-T activity. Thus the phenotypic difference between the two classes of transformants may be related to the presence or absence of small-t influence within the cell.     

RNAi reveals doublecortin is required for radial migration in rat neocortex

Mutations in the doublecortin gene (DCX) in humans cause malformation of the cerebral neocortex. Paradoxically, genetic deletion of Dcx in mice does not cause neocortical malformation. We used electroporation of plasmids encoding short hairpin RNA to create interference (RNAi) of DCX protein in utero, and we show that DCX is required for radial migration in developing rat neocortex. RNAi of DCX causes both cell-autonomous and non-cell autonomous disruptions in radial migration, and creates two disruptions in neocortical development. First, many neurons prematurely stop migrating to form subcortical band heterotopias within the intermediate zone and then white matter. Second, many neurons migrate into inappropriate neocortical lamina within normotopic cortex. In utero RNAi can therefore be effectively used to study the specific cellular roles of DCX in neocortical development and to produce an animal model of double cortex syndrome.     

The amino-terminal 147 amino acids of SV40 large T antigen transform secondary rat embryo fibroblasts.

T147D is an SV40 mutant that encodes only the amino-terminal 147 amino acids of large T antigen and does not make small t antigen. We have constructed a recombinant retrovirus that expresses the T147D mutant protein. We show here that this virus can transform the established rat cell line, F111, in an agar assay with high efficiency. More importantly, we demonstrate that this retrovirus transforms secondary rat embryo fibroblasts to anchorage independence as efficiently as a recombinant retrovirus that expresses both wild-type large and small T antigens. These data indicate that in rat cells, the amino-terminal 147 amino acids of T antigen are sufficient for transformation. Further, since the T147D protein does not bind p53, we conclude that the association between T antigen and p53 is not required for the transformation of rat cells to anchorage-independent growth.     

Co-stimulation by anti-immunoglobulin is required for B cell activation by CD40Llow T cells

During cognate B : T interactions, B cells encounter antigen (Ag) through surface immuno-globulin (sIg) and present antigenic peptides to T helper (Th) cells. However, most in vitro systems used to study contact events involved in the delivery of T help for B cells circumvent the requirement for T cell Ag specificity by using anti-CD3/T cell receptor (TcR) monoclonal antibodies (mAb) to activate T cells. To study the role of sIg engagement in the responsiveness of B cells to T help, we pre-treated small resting B cells with soluble anti-χ mAb prior to contact with an activated Th1 clone. By reducing the concentration of anti-TcR mAb we obtained low levels of CD40 ligand (CD40L^low) on Th cells, comparable to those expressed by lymph node T cells activated in vitro (ex vivo T cells). In contrast to untreated B cells, which did not respond to CD40L^low Th, anti-Ig-treated B cells responded strongly. Low buoyant density B cells also responded to CD40L^low Th cells. There was no B cell response to resting Th cells. mAb against CD54/intercellular adhesion molecule-1 or major histocompatibility complex (MHC) class II completely inhibited B cell responses to CD40L^low Th1 cells, equivalent to the effects of blocking CD40 interactions. This contrasts with mAb blocking responses to CD40L^highTh, where CD40 effects predominate. Our data show that sIg engagement is necessary for the induction of B cell response to CD40L^low Th cells. Anti-CD3-activated ex vivo T cells that were also CD40L^low did not provide help to small resting B cells, but did induce responses from sIg-stimulated B cells. Thus, our data support a requirement for sIg signaling in physiological B cell activation, and further confirm previous work showing CD40 ligation to be necessary but not sufficient for delivery of T help to B cells.     

OMA-1 is a P granules-associated protein that is required for germline specification in Caenorhabditis elegans embryos

In Caenorhabditis elegans, CCCH-type zinc-finger proteins have been shown to be involved in the differentiation of germ cells during embryonic development. Previously, we and others have identified novel redundant CCCH-type zinc-finger proteins, OMA-1 and OMA-2, that are involved in oocyte maturation. In this study, we report that the cytoplasmic expression level of OMA-1 protein was largely reduced after fertilization. In contrast to its cytoplasmic degradation, OMA-1 was found to accumulate exclusively on P granules in germline blastomeres during embryogenesis. A notable finding is that embryos with partially suppressed oma-1; oma-2 expression showed inappropriate germline specification, including abnormal distributions of PGL-1, MEX-1 and PIE-1 proteins. Thus, our results suggest that oma gene products are novel multifunctional proteins that participate in crucial processes for germline specification during embryonic development.     

Hyaluronan peroxidation is required for normal synovial function: An hypothesis

Despite widespread use of antioxidants, reactive oxygen species have important functions in normal tissues. Herein, we present an example of a physiological role for free radicals, and in particular, reactive oxygen species, that are suppressed by anti-oxidants. Free radicals catalyze the degradation of hyaluronan in synovial fluid, a tissue in which hyaluronidase activity is barely detectable. Articular cartilage requires a low oxygen environment. The process of hyaluronan peroxidation consumes significant amounts of molecular oxygen, thus keeping the tension of oxygen in the joint at a low but physiologically critical level. One concern is the change in physical activity between day and night, with periods of joint hyperemia and ischemia, respectively. Increased oxygen and the resulting oxidative stress would lead to chondrocyte dysfunction and cartilage damage. A mechanism for keeping oxygen levels low is required. We postulate that a mechanism indeed exists for the removal of excess oxygen. High-molar-mass hyaluronan turnover in synovial fluid utilizes peroxidative degradation, during which oxygen is massively consumed. The peroxidation itself may be initiated by hydrogen peroxide, which is produced by chondrocyte mitochondria, that can diffuse into the synovial fluid. The resulting decrease in available oxygen down-regulates hyaluronan peroxidation. This in turn prevents excessive oxygen consumption. It appears that free radicals and reactive oxygen species may be components of normal physiology, particularly in the synovial fluid of joints and articular cartilage. It is suggested therefore that indiscriminate use of anti-oxidants, vigorously promoted currently by health professionals and the health industry, be approached with caution.     

Cleavage of membrane-bound CD40 ligand is not required for inducing B cell proliferation and differentiation

The phycical interaction between the B cell surface molecule CD40 and its ligand, CD40L, is known to be crucial in the development and maintenance of humoral immunity. Recently it has been shown that the CD40L is processed and that its soluble cleavage products are released into the extracellular environment. To study the functions of soluble and membrane-bound human CD40L on human B cells, we generated an uncleavable CD40L cDNA deletion mutant. The activities of transfectants expressing either mutated or wild-type CD40L were then compared on human B cells. Both the soluble and the uncleavable membrane-bound CD40L were able to induce, in conjunction with interleukin-4, B cell proliferation and IgE synthesis. Therefore, membrane-bound and soluble CD40L exhibit the same pattern of activities on B cells and membrane CD40L cleavage is not a prerequisite for its function.     

An N-terminal variant of Trpv1 channel is required for osmosensory transduction

Body fluid homeostasis requires the release of arginine-vasopressin (AVP, an antidiuretic hormone) from the neurohypophysis. This release is controlled by specific and highly sensitive 'osmoreceptors' in the hypothalamus. Indeed, AVP-releasing neurons in the supraoptic nucleus (SON) are directly osmosensitive, and this osmosensitivity is mediated by stretch-inhibited cation channels. However, the molecular nature of these channels remains unknown. Here we show that SON neurons express an N-terminal splice variant of the transient receptor potential vanilloid type-1 (Trpv1), also known as the capsaicin receptor, but not full-length Trpv1. Unlike their wild-type counterparts, SON neurons in Trpv1 knockout (Trpv1(-/-)) mice could not generate ruthenium red-sensitive increases in membrane conductance and depolarizing potentials in response to hyperosmotic stimulation. Moreover, Trpv1(-/-) mice showed a pronounced serum hyperosmolality under basal conditions and severely compromised AVP responses to osmotic stimulation in vivo. These results suggest that the Trpv1 gene may encode a central component of the osmoreceptor.     

Expression of rat CD59: functional analysis confirms lack of species selectivity and reveals that glycosylation is not required for function.

This study reports the expression and functional characterization of the rat analogue of the human complement regulatory molecule CD59. We here describe the expression in chinese hamster ovary (CHO) cells of rat CD59 and a modified rat CD59 in which an N-glycosylation site at Asn-16 has been deleted by point mutation. The complement-inhibiting capacity of these two forms of rat CD59 has been analysed and compared. Expressed rat CD59 efficiently inhibited complement lysis of CHO cells when rat serum was used as a source of complement and also inhibited lysis by complement from all other species tested, confirming that rat CD59 displayed little or no species restriction of activity. Blocking of expressed rat CD59 with a monoclonal antibody abrogated the inhibition of lysis for all sources of complement, confirming that the expressed molecule was responsible for the protection. The glycosylation mutant had a much reduced molecular weight on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (12,000 MW as compared with 20,000-28,000 MW for unmutated), confirming that it was unglycosylated. However, the glycosylation mutant had complement-inhibitory activity which was at least as potent as that of the unmutated molecule, demonstrating that the large, N-linked carbohydrate moiety was not required for function.     

Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition

Development of the mammalian tooth has been intensively studied as a model system for epithelial/mesenchymal interactions during organogenesis, and progress has been made in identifying key molecules involved in this signaling. We show that activin βA is expressed in presumptive tooth-germ mesenchyme and is thus a candidate for a signaling molecule in tooth development. Analysis of tooth development in activin βA mutant embryos shows that incisor and mandibular molar teeth fail to develop beyond the bud stage. Activin βA is thus an essential component of tooth development. Development of maxillary molars, however, is unaffected in the mutants. Using tissue recombination experiments we show that activin is required in the mesenchyme prior to bud formation and that although activin signaling from mesenchyme to epithelium takes place, mutant epithelium retains its ability to support tooth development. Implantation of beads soaked in activin A, into developing mandibles, is able to completely rescue tooth development from E11.5, but not E12.5 or E13.5, confirming that activin is an early, essential mesenchyme signal required before tooth bud formation. Normal development of maxillary molars in the absence of activin shows a position specific role for this pathway in development of dentition. Functional redundancy with activin B or other TGFβ family members that bind to activin receptors cannot explain development of maxillary molars in the mutants since the activin-signaling pathway appears not to be active in these tooth germs. The early requirement for activin signaling in the mesenchyme in incisor and mandibular molar tooth germs must be carried-out in maxillary molar mesenchyme by other independent signaling pathways.