Ligand-independent pathway that controls stability of interferon alpha receptor

Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination, and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance.     

The molecular basis of multidrug resistance in cancer: the early years of P-glycoprotein research

The discovery and characterization of P-glycoprotein, an energy-dependent multidrug efflux pump, as a mechanism of multidrug resistance in cancer is generally accepted as a significant contribution to the ongoing effort to end death and suffering from this disease. The historical reflections of Victor Ling and Michael Gottesman concerning the early years of this research highlight the important contributions of the multidisciplinary teams involved in these studies, and illustrate how technological developments in biochemistry and molecular and cell biology enabled this discovery.     

Cloning and characterization of CD300d, a novel member of the human CD300 family of immune receptors

Herein we present the cloning and molecular characterization of CD300d, a member of the human CD300 family of immune receptors. CD300d cDNA was cloned from RNA obtained from human peripheral blood mononuclear cells, and RT-PCR revealed the gene to be expressed in cells of myeloid lineage. The cloned cDNA encoded for a type I protein with a single extracellular Ig V-type domain and a predicted molecular mass of 21.5 kDa. The short cytoplasmic tail is lacking in any known signaling motif, but there is a negatively charged residue (glutamic acid) within the transmembrane domain. CD300d forms complexes with the CD300 family members, with the exception of CD300c. Contrary to other activating members of the CD300 family of receptors, surface expression of CD300d in COS-7-transfected cells required the presence of an immunoreceptor tyrosine-based activating motif-bearing adaptor (FcεRγ). Accordingly, we found that CD300d was able to recruit FcεRγ. Unexpectedly, we could not detect CD300d on the surface of cells expressing FcεRγ, suggesting the existence of unknown mechanisms regulating the trafficking of this molecule. The presence of other CD300 molecules also did not modify the intracellular expression of CD300d. In fact, the presence of CD300d decreased the levels of surface expression of CD300f but not CD300c. Our data suggest that the function of CD300d would be related to the regulation of the expression of other CD300 molecules and the composition of CD300 complexes on the cell surface.     

Influence of oocyte donor and embryo recipient conditions on cloning efficiency in dogs

To determine factors that affect the efficiency of dog cloning by somatic cell nuclear transfer, the present study was performed to investigate 1) the effects of surgical history (non-operated/operated) and parity (nullipara/multipara) on the recovery of in vivo canine oocytes; 2) the effects of surgical history and parity of recipients on the pregnancy and delivery; and 3) the effects of synchronization state (AA, advanced asynchrony; SY, synchrony; RA, retarded asynchrony) between oocytes donor and recipient on the pregnancy and delivery. Oocyte recovery rate was significantly higher in non-operated dogs compared to operated dogs (93.8 vs. 89.6%, P < 0.05) and not different between nulliparous dogs and multiparous dogs. Delivery rate was also significantly higher in non-operated dogs compared to operated dogs (2.8 vs. 1.0%, P < 0.05) and in nulliparous dogs than multiparous dogs (3.0 vs. 1.7%, P < 0.05). Even though SY showed increased pregnancy and delivery rate (20.0% and 3.0%) compared to AA (15.0% and 2.0%) and RA (0.0% and 0.0%), there was no significant difference. In conclusion, we recommend non-operated dogs as experimental dogs and nulliparous dogs as recipient dogs to increase delivery rate after transfer of somatic cell nuclear transferred embryos, but further study is needed to find out appropriate synchrony status at the transfer.     

Differences in (-)citronellal binding to various odorant receptors

To test the hypothesis that olfactory receptors (ORs) recognize different molecular features of odor molecules termed "odotypes", we studied receptor-ligand interactions of two human and two mouse ORs, recognizing (-)citronellal. Structurally similar receptors provide identical binding pockets (OLFR43, OR1A1, and OR1A2), and have comparable EC(50) values. Other ORs with lower sequence identity bind (-)citronellal in a different way, leading to different EC(50) values.     

Mutations that modulate receptor-hormone congruency as a cause of the primate GH receptor species specificity

Peptide hormones depend on reliable recognition by their receptors. Any mutation that compromises recognition of hormone and receptor molecules is dangerous, the carrier animal would not procreate and the mutation would be lost. Although, most of the hormones from one mammalian species are active when injected into another, the incompatibility of human GH receptor toward nonprimate GHs is a notable exception. It is reported that the coevolution of GH and GHR in primates includes two crucial steps (Mol. Biol. Evol. 18 (2001) 945). The first was mutation of GH His→Asp at position 171 that happened before the split of Old world and New world monkeys. The second event was Leu→Arg change at position 43 in the GH receptor molecule that happened in the ancestor of Old world monkeys. The proposed model is based on the possibility that certain mutations can modify the surface of one of interacting molecules to form a confined empty space, a niche in the otherwise congruent hormone/receptor interface. Although affinity between molecules is probably slightly reduced, recognition and function are not compromised in this special case. Further mutations of hormone and receptor molecules are allowed under the condition that they remain confined to the niche space. Mutations that do not compromise hormone function can be passed to offsprings. If the consequent mutation of one molecule change its shape to fill the niche space, further mutations without function loss will become less probable. Without the niche space, the phase of fast evolution is closed and both genes become conserved. In this setting, accumulated mutations before the niche closing mutation are the cause of species specificity. To become a dominant variety, carrier animals must possess survival advantage in comparison to the carriers of other less advantageous mutations.     

Characterization of human-dog social interaction using owner report

Dog owners were surveyed for observations of social behaviors in their dogs, using questions adapted from the human Autism Diagnostic Observation Schedule (ADOS) pre-verbal module. Using 939 responses for purebred and mixed-breed dogs, three factors were identified: initiation of reciprocal social behaviors (INIT), response to social interactions (RSPNS), and communication (COMM). There were small or no effects of sex, age, breed group or training. For six breeds with more than 35 responses (Border Collie, Rough Collie, German Shepherd, Golden Retriever, Labrador Retriever, Standard Poodle), the behaviors eye contact with humans, enjoyment in interactions with human interaction, and name recognition demonstrated little variability across breeds, while asking for objects, giving/showing objects to humans, and attempts to direct humans’ attention showed higher variability across these breeds. Breeds with genetically similar backgrounds had similar response distributions for owner reports of dog response to pointing. When considering these breeds according to the broad categories of “herders” and “retrievers,” owners reported that the “herders” used more eye contact and vocalization, while the “retrievers” used more body contact. Information regarding social cognitive abilities in dogs provided by owner report suggest that there is variability across many social cognitive abilities in dogs and offers direction for further experimental investigations.     

Embryo production and possible species preservation by nuclear transfer of somatic cells isolated from bovine semen

Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important when an animal has died and the only viable genetic material available is frozen semen. Usefulness of somatic cells obtained from fresh (cultured) and frozen (isolated, not cultured) bovine semen for nuclear transfer was evaluated. Twelve ejaculates were collected from nine bulls representing three breeds: Charolais, Brahman, and crossbred Rodeo bull. All samples were processed immediately and cell growth was obtained from seven of the twelve ejaculates (58.3%). Cells from three bulls (with the best growth rates) were evaluated by optical microscopy and used in cloning experiments. In culture, these cells exhibited classic epithelial morphology and expressed cytokeratin and vimentin, indicating they were of epithelial origin. When cells from the three bulls were used as donor cells, 15.9% (18/113), 34.5% (29/84), and 14.4% (13/90) of the fused embryos developed into blastocysts, respectively. Of the blastocyst stage embryos, 38.9% (7/18), 72.4% (21/29), and 61.5% (8/13) hatched, respectively. Somatic cells isolated (not cultured) from frozen bovine semen were also used in the cloning experiments. Although cleavage occurred, no compact morulae or blastocysts were obtained. In conclusion, epithelial cell growth was obtained from fresh bovine ejaculates with relatively high efficiency. Somatic cells from semen can be used as nucleus donors to produce cloned blastocyst-stage embryos.     

Monocyte and interferon based therapy for the treatment of ovarian cancer

Cytokines and cells of the innate immune system have been shown to be critical regulators in the elimination, equilibrium and escape of malignant cells. Despite in vitro and in vivo evidence, components of the innate immune system have shown limited efficacy in the treatment of ovarian cancer. Intraperitoneal immunotherapies are a promising field that has not yet been fully explored in ovarian cancer. Cytokine immunotherapy using interferon alpha (IFN-α) and interferon gamma (IFN-γ) has predominantly been used intraperitoneally in ovarian cancer, with promising results. Early studies also showed that autologous monocytes infused into the peritoneum have anti-tumor properties. Combination therapies have been shown to be more effective in treating cancer than mono-therapies. Based on these observations the combination of cell therapy with cytokine therapy may provide a unique strategy for the treatment of chemotherapy resistant solid cancers.     

T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements

Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3ε complexed with the mitogenic anti-CD3ε antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3ε from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3ε. Saturation mutagenesis of surface-exposed CD3ε residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3ε is highly exposed and reveals that no large new CD3ε interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.     

A phase-II study of pegylated interferon alfa-2b for patients with metastatic renal cell carcinoma and removal of the primary tumor

Twenty-two patients with metastatic renal cell carcinoma and removal of the primary tumor were treated with subcutaneous pegylated interferon alfa-2b (PEG-Intron) to evaluate toxicity and efficacy. Start dose was 3.0 g/kg/week, escalated to 6.0 g/kg/week. After 2 months, therapy was extended in case of response or stable disease (SD) until progressive disease (PD) or relapse for a maximum of 2 years. National Cancer Institute common toxicity criteria (NCI-CTC) were monitored every 2–4 weeks. After 2 months, nine patients did not continue (8 PD, 1 SD with grade 4 CTC) and 13 extended treatment [three partial response (PR), 10 SD], of these, 11 progressed. One patient with PR developed a durable complete response later. Overall response rate was 13.6% (3/22). Median overall survival is 13 months (range 3–35 months). Dosage was escalated to 6 g/kg/week in three patients . NCI-CTC grade 2 and 3 required dose attenuation in 12 patients during escalation, and reduction in 10 during the trial. Three patients discontinued because of grade 4 CTC (two fatigue, one hyperglycemia). Fatigue was the major dose-limiting toxicity. These results suggest an efficacy and toxicity of PEG-Intron comparable to standard interferon alfa-2b in patients with mRCC and removal of the primary tumor.Author disclosure declaration: None of the authors has a relationship with pharmaceutical companies, biomedical device manufacturers or other corporations whose products or services are related to the subject matter of the submission, nor do the authors have financial interests such as investments, licensing, or other commercial interest in any drugs, goods, or services in connection with the matter under consideration.     

Fibroblast growth factors and their receptors in the central nervous system

Fibroblast growth factors (FGFs) and their receptors constitute an elaborate signaling system that participates in many developmental and repair processes of virtually all mammalian tissues. Among the 23 FGF members, ten have been identified in the brain. Four FGF receptors (FGFRs), receptor tyrosine kinases, are known so far. Ligand binding of these receptors greatly depends on the presence of heparan sulfate proteoglycans, which act as low affinity FGFRs. Ligand binding specificity of FGFRs depends on the third extracellular Ig-like domain, which is subject to alternative splicing. Activation of FGFRs triggers several intracellular signaling cascades. These include phosphorylation of src and PLC leading finally to activation of PKC, as well as activation of Crk and Shc. SNT/FRS2 serves as an alternative link of FGFRs to the activation of PKC and, in addition, activates the Ras signaling cascade. In the CNS, FGFs are widely expressed; FGF-2 is predominantly synthesized by astrocytes, whereas other FGF family members, e.g., FGF-5, FGF-8, and FGF-9, are primarily synthesized by neurons. During CNS development FGFs play important roles in neurogenesis, axon growth, and differentiation. In addition, FGFs are major determinants of neuronal survival both during development and during adulthood. Adult neurogenesis depends greatly on FGF-2. Finally, FGF-1 and FGF-2 seem to be involved in the regulation of synaptic plasticity and processes attributed to learning and memory.     

Suppressor of cytokine signaling (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha receptor (IFNAR1)-associated tyrosine kinase Tyk2

Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN α receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2.     

The evolution of the type I interferon gene family in mammals

A phylogenetic analysis of mammalian type I interferon (IFN) genes showed: (1) that the three main subfamilies of these genes in mammals (IFN-, IFN-, and IFN-) diverged after the divergence of birds and mammals but before radiation of the eutherian orders and (2) that IFN- diverged first. Although apparent cases of interlocus recombination among mouse IFN- genes were identified, the hypothesis that coding regions of IFN- genes have been homogenized within species by interlocus recombination was not supported. Flanking regions as well as coding regions of IFN- were more similar within human and mouse than between these species; and reconstruction of the pattern of nucleotide substitution in IFN- coding regions of four mammalian species by the maximum parsimony method suggested that parallel substitutions have occurred far more frequently between species than within species. Therefore, it seems likely that IFN- genes have duplicated independently within different eutherian orders. In general, type I IFN genes are subject to purifying selection, which in the case of IFN- and IFN- is strongest in the putative receptor-binding domains. However, analysis of the pattern of nucleotide substitution among IFN- genes suggested that positive Darwinian selection may have acted in some cases to diversify members of this subfamily at the amino acid level.     

Recent developments in the study of orchid mycorrhiza

Orchids are mycoheterotrophic during their seedling stage and in many species the dependency on fungi as a carbohydrate source is prolonged into adulthood. The mycobionts in orchid mycorrhiza belong in at least 5 major taxonomic groups of basidiomycetes. Traditional records have mainly focused on saprotrophic mycobionts but the participation of both ectomycorrhizal and parasitic fungi in orchid mycorrhiza has been corroborated. There is an increasing evidence of specific relationships between orchids and fungi, though usually not on a species-to-species level. Physiological compatibility demonstrated under artificial conditions, as in vitro, may be much broader, however. Recent development of field sowing techniques has improved the possibilities of evaluating orchid-fungal relations in an ecological context. Although the general nutrient flow in orchid mycorrhiza is well known, some questions remain regarding breakdown processes of fungi within orchid tissues, especially the ptyophagic syndrome that has recently been illustrated at the ultrastructural level for the first time.     

Tyrosine phosphorylation of protein kinase D2 mediates ligand-inducible elimination of the Type 1 interferon receptor

Type 1 interferons (including IFNα/β) activate their cell surface receptor to induce the intracellular signal transduction pathways that play an important role in host defenses against infectious agents and tumors. The extent of cellular responses to IFNα is limited by several important mechanisms including the ligand-stimulated and specific serine phosphorylation-dependent degradation of the IFNAR1 chain of Type 1 IFN receptor. Previous studies revealed that acceleration of IFNAR1 degradation upon IFN stimulation requires activities of tyrosine kinase TYK2 and serine/threonine protein kinase D2 (PKD2), whose recruitment to IFNAR1 is also induced by the ligand. Here we report that activation of PKD2 by IFNα (but not its recruitment to the receptor) depends on TYK2 catalytic activity. PKD2 undergoes IFNα-inducible tyrosine phosphorylation on specific phospho-acceptor site (Tyr-438) within the plekstrin homology domain. Activated TYK2 is capable of facilitating this phosphorylation in vitro. Tyrosine phosphorylation of PKD2 is required for IFNα-stimulated activation of this kinase as well as for efficient serine phosphorylation and degradation of IFNAR1 and ensuing restriction of the extent of cellular responses to IFNα.     

Sister chromatid differential staining and NOR activity of BrdU-resistant sublines

Summary Two 30 g/ml BrdU-resistant sublines and two 60 g/ml BrdU-resistant sublines are induced from a Chinese hamster cell line Wg3h (HGPRT^–) by one-step and two-step selections, respectively. By inoculating the cells into BrdU-free medium or by adding more BrdU into the culture medium for 26–27 h, it was found that the two BrdU-resistant sublines analysed have very clear sister chromatid differential (SCD) staining patterns. This indicates that some of the nuclear DNA of the BrdU-resistant cells incorporate with BrdU to reach a kinetic balance. Frequencies of sister chromatid exchange (SCE) of the resistant cells are twice to four times as high as those of the Wg3h cells, depending on which BrdU-resistant subline is analysed. The SCE frequencies of the resistant cells also increase with the BrdU concentration in the medium. Analysis of silver-stained nucleolar organizer regions (NORs) indicates that the NOR activity of three out of the four BrdU-resistant sublines is significantly suppressed, i.e., averages of the Ag-NOR number and number of the chromosomes bearing Ag-NORs per cell decrease significantly. The degree of suppression for different BrdU-resistant sublines may be quite different. The suppressed NOR activity of the resistant cells can gradually be restored when the cells are inoculated into BrdU-free medium, but the recovery speed is far lower than that of the Wg3h cells. The suppression of the NOR activity of the BrdU-resistant sublines should be due to BrdU toxicity.