Immunohistochemical localization of G protein betagamma subunits in the lateral wall of the rat cochlea

The role of G protein-mediated signal transduction in the production of endolymph, an extracellular fluid of unusual ionic composition, is beginning to be understood. The identity of Galpha subunits in the stria vascularis and the spiral ligament of the lateral wall of the cochlear duct is well established. However, little is known about the presence of betagamma subunits. This study used immunohistochemistry to investigate the distribution of G protein betagamma subunits in the lateral wall of the cochlea. Temporal bones of 6- to 8-week-old rats were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for embedding in paraffin wax. The dewaxed, midmodiolar sections of the cochlea were incubated with subunit-specific polyclonal antibodies. The results show that the pattern of immunoreactivity varies for the G protein beta1-4 and gamma1-3, 5 and 7 subunits in the stria vascularis and spiral ligament. In the stria vascularis, immunoreactivity was detected for beta2, beta3, beta4, gamma1, gamma2 and gamma7 subunits. All five types of fibrocytes in the spiral ligament exhibited positive staining for gamma2 and gamma7. However, immunoreactivity for beta1-4 subunits was variable. Immunoreactivity for gamma3 and gamma5 subunits was not detected in the lateral cochlear wall. The expression pattern of G protein betagamma subunits in lateral wall provides a basis for interpreting the functions of G protein-coupled receptors in cochlear fluid homeostasis.     

Lectin binding patterns in nonsensory regions of rat cochlea during postnatal development

The distribution of glycoconjugates was examined in the nonsensory regions of the rat cochlea during postnatal development using biotin-conjugated lectins. Temporal bones of rats at postnatal d 1 and at wk 2, 4 and 6 were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for paraffin wax embedding. The dewaxed sections were incubated with 7 biotinylated lectins, followed by avidin-biotin-peroxidase complex. A different staining pattern was observed in the stria vascularis, spiral ligament and spiral limbus in the age groups examined. The staining intensity varied between lectins and the reaction product exhibited limited disparity. The staining intensity for WGA increased with age in all the 3 nonsensory regions. The staining patterns for the other lectins differed in the various nonsensory regions examined indicating tissue specificity. The limited variations in the lectin binding patterns after 2nd wk of postnatal life also indicate that the changes in the carbohydrate moieties are established during the fetal period of cochlear development and limited changes take place during postnatal maturation of the nonsensory regions.     

Mitotic activity and specification of fibrocyte subtypes in the developing rat cochlear lateral wall

Spiral ligament fibrocytes (SLFs) in the mammalian cochlear lateral wall participate in K⁺ recycling; they are classified into five subtypes based on their morphology, distribution, and function. Regeneration of SLFs is a potential therapeutic strategy for correcting several types of hearing loss, prompting us to investigate how SLF subtypes are established during development. We compared transitional SLF-type marker expression with mitotic activity to evaluate proliferation-differentiation relationships in SLFs from postnatal rat cochleae. I.p. injection of 5-bromo-2′-deoxyuridine (BrdU) demonstrated that the overall mitotic activity of SLFs decreased significantly between postnatal day 7 (P7) and P10. For all developmental periods, BrdU incorporation was weakest in the area where type I SLFs reside. The onset of expression of markers for type II/IV SLFs followed the reduced mitotic activity of the cells, whereas that of aquaporin-1, a marker for type III SLFs, was already detectable at P7, when the type III SLFs were still proliferating vigorously. Distribution of BrdU⁺ cells increased in the area of type I SLFs between P7 and P10, suggesting migration of SLFs from adjacent areas. We conclude that the time course of development of SLFs is subtype-specific.     

人内耳蜗神经管的应用解剖学

目的：为临床人工耳蜗植人术提供解剖学依据。方法：观测30例(60侧)硅胶蜗神经管的长度和直径。观测15例(30侧)CT蜗神经管的长度和直径。结果：蜗神经管为位于内耳道底至耳蜗底之间的一个粗短的圆柱型管道。硅胶蜗神经管的长度为(1．69±0．34)mm,平均直径为(2．53±0．27)mm。CT蜗神经管平均直径为(2．20±0．30)mm,直径的正常参考值为(2．08～2．23)mm(95%可信区间)。结论：首次测得了CT蜗神经管直径的正常参考值,该数值可作为人工耳蜗植入术前评估的解剖学依据。     

Multiple expression of glucose transporters in the lateral wall of the cochlear duct studied by quantitative real-time PCR assay

We have investigated the gene expression of the facilitated glucose transporter (GLUT), H⁺-coupled myo-inositol cotransporter (HMIT), and Na⁺ glucose cotransporter (SGLT) in the lateral wall of the cochlear duct by conventional RT-PCR and quantitative real-time PCR. The isoforms GLUT1, -3, -4, -5, -8, -10, -12 and HMIT were detected in both the stria vascularis and the spiral ligament, whereas no SGLT isoforms could be detected in these tissues. Quantitative real-time PCR analysis revealed significant differences in the gene expression of GLUT1, -4, -5, -10, and HMIT isoforms between the stria vascularis and the spiral ligament. This result reflects the tissue-dependent distributions of GLUT isoforms. These findings strongly suggest that a number of GLUT isoforms participate in glucose transport in the stria vascularis and the spiral ligament.     

Expression of functionally active α4β1 integrin by thymic epithelial cells

We have investigated the expression and function of the VLA-4 heterodimer α₄β₁, a member of the β₁ integrin subfamily, on human thymic epithelial cells (TEC) derived from cortical epithelium. The expression of the α₄ integrin chain was studied in four different cloned TEC lines derived from either fetal or post-natal human thymus by both flow cytometry and immunoprecipitation techniques with anti-α₄ MoAbs. All different cell lines assayed expressed significant levels of α₄, as revealed by their reactivity with MoAbs specific for distinct α₄epitopes. The α₄ subunit expressed by TEC was associated to β₁ but not to β₇ chain, and displayed the characteristic 80/70 kD pattern of proteolytic cleavage. The VLA-4 integrin in these cells was constitutively active in terms of adhesiveness to both fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In addition, this heterodimer localized to punctate regions of the cell in the area of contact with the substratum, named point contacts assessed by staining with the anti-β₁ activation epitope 15/7 MoAb. According to the cortical origin of the TEC lines expressing VLA-4, human thymus sections stained with different anti-α₄ antibodies revealed the presence of cortical, and in smaller numbers medullary epithelial cells bearing α₄ integrin. The expression of α₄ in the thymus was also found in both adult and fetal rats, in which epithelial cells were also specifically stained. Altogether, our data show that VLA-4 is an additional component of the integrin repertoire of TEC, and suggest that it could have an important role in thymus epithelial cell–thymocyte interactions.     

Integrins αvβ3 and α5β1 Mediate Attachment of Lyme Disease Spirochetes to Human Cells

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin α_IIbβ₃ was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins α_vβ₃ and α₅β₁, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified α_vβ₃ and α₅β₁. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified α_vβ₃ and α₅β₁ was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to α_vβ₃ was also shown by using a transfected cell line that expresses this receptor but not α_IIbβ₃. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both α₅β₁ and α_vβ₃. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.     

Expression patterns of FGF receptors in the developing mammalian cochlea

Many studies have shown the importance of the fibroblast growth factor (FGF) family of factors in the development of the mammalian cochlea. There are four fibroblast growth factor receptors (FGFR1–4) and all four are expressed in the cochlea during development. While there are examples in the literature of expression patterns of some of the receptors at specific stages of cochlear development there has been no systematic study. We have assembled a full analysis of the patterns of receptor expression during cochlear development for all four Fgfrs using in situ hybridization. We have analyzed the expression patterns from embryonic day 13.5 through postnatal ages. We find that Fgfr1, 2, and 3 are expressed in the epithelium of the cochlear duct and Fgfr4 is limited in its expression to the mesenchyme surrounding the duct. We compare the receptor expression pattern to markers of the sensory domain (p27^kip1) and the early hair cells (math1). Developmental Dynamics 239:1019–1026, 2010. © 2010 Wiley‐Liss, Inc.     

Sequential Expression of Transforming Growth Factors α and β1 by Eosinophils During Cutaneous Wound Healing in the Hamster

Transforming growth factor-α (TGF-α) and TGF-β₁ have been proposed as important regulators of processes critical to successful wound healing. Although various cells present in wounds represent potential sources of either TGF-α and/or TGF-β, including macrophages, neutrophils, keratinocytes, fibroblasts, and endothelial cells, we recently identified eosinophils as an additional potential source of these cytokines. We therefore used in situ hybridization and immunohistochemistry to determine whether eosinophils represent significant sources of TGF-α and/or TGF-β₁ in skin wounds in the hamster. We found that these wounds developed a prominent infiltration of eosinophils, and that eosinophils were a cellular source of both TGF-α and TGF-β₁, mRNAs. TGF-α and TGF-β₁ proteins were detectable both within eosinophils and extracellularly. Moreover, there was a sequential pattern of TGF-α and TGF-β₁ expression by infiltrating eosinophils, with the onset of eosinophil-associated TGF-α expression preceding that of TGF-β₁. This sequential pattern of TGF expression suggests that eosinophils may help to regulate critical biological processes during wound healing.     

Streptozotocin-induced diabetes in mice lacking αβ T cells

Multiple low-dose streptozotocin (MD-STZ) is widely used for the experimental induction of diabetes, but, as non-obese diabetic (NOD)-scid/scid mice have been found to display enhanced susceptibility to MD-STZ, whether or not the model is genuinely autoimmune and T cell-mediated has been unclear. Mice bearing a targeted mutation of the T cell receptor (TCR) α-chain were therefore used to assess whether TCR αβ⁺ cells are involved in the diabetogenic effects of MD-STZ injections. Young NOD mice lacking TCR αβ cells, when given five daily injections of 40 mg/kg STZ, developed diabetes at low frequency (2/12), despite the widespread destruction of pancreatic islet cells. By comparison, most normal control mice became hyperglycaemic (12/23). We conclude that whilst much of the tissue destruction observed in this model is due to the direct toxic effect of STZ, a significant amount is also due to the action of TCR αβ cells tipping the balance between tolerable and clinically damaging action on islet cells.     

Homing Receptor α4β7 Integrin Expression Predicts Digestive Tract Involvement in Mantle Cell Lymphoma

Appropriate staging and evaluation of residual disease is critical to improving the treatment of patients with lymphoma. The specific expression of homing receptors may determine the preferential dissemination pattern of tumoral cells. We investigated the expression of the mucosal homing receptor α4β7 on tumoral cells from peripheral lymph node in patients with newly diagnosed mantle cell lymphoma (MCL) to check whether it is associated with gastrointestinal involvement. Expression of the α4β1 integrin and the peripheral lymph node addressin CD62L were also examined. Thirteen MCL patients presenting with peripheral lymphadenopathy were studied. Expression of the mucosal homing receptor integrin α4β7 by peripheral lymph node lymphoma cells was found to be frequent (5/13) and associated with gastrointestinal involvement (5/7). In contrast, lymphoma cells from patients without gastrointestinal involvement did not express α4β7 (6/6) (P = 0.03). These data suggest that α4β7 integrin is expressed by a subset of MCLs and that its expression may predict digestive tract involvement in MCL, furnishing a basis for recognizing two distinct clinical and phenotypic forms, ie, “digestive homing (or digestive primitive)” versus “peripheral” MCL. Further studies on more patients will be needed to understand the impact of biological differences on the prognosis of these two clinical forms.     

Mammary Epithelial Cell-Cycle Progression via the α2β1 Integrin : Unique and Synergistic Roles of the α2 Cytoplasmic Domain

The alpha(2)beta(1) integrin supports cell-cycle progression of mammary epithelial cells adherent to type I collagen matrices. Integrin collagen receptors containing the alpha(2) cytoplasmic domain stimulated expression of cyclin E and cyclin-dependent kinase (cdk)2, resulting in cyclin E/cdk2 activation in the absence of growth factors other than insulin. Integrin collagen receptors in which the alpha(2) cytoplasmic domain was replaced by the alpha(1) cytoplasmic domain or an alpha(2) subunit cytoplasmic domain truncated after the GFFKR sequence failed to stimulate cyclin E/cdk2 activation or entry into S phase in the absence of growth factors. Although overexpression of cyclins D or E or cdk2 in cells expressing the integrin collagen receptor with the alpha(1)-integrin cytoplasmic domain did not restore G(1) progression when mammary epithelial cells adhered to type I collagen, co-expression of cyclin E and cdk2 did rescue the ability of the transfectants to enter S phase. Activation of cyclin E/cdk2 complex by mammary epithelial cells required synergy between adhesion mediated by an integrin collagen receptor containing the alpha(2)-integrin subunit cytoplasmic domain and the insulin receptor.     

Primary Role of Interleukin-1α and Interleukin-1β in Lipopolysaccharide-Induced Hypoglycemia in Mice

Within a few hours of its injection into mice, lipopolysaccharide (LPS) induces hypoglycemia and the production of various cytokines. We previously found that interleukin-1α (IL-1α), IL-1β, and tumor necrosis factor alpha (TNF-α) induce hypoglycemia and that the minimum effective dose of IL-1α or IL-1β is about 1/1,000 that of TNF-α. In the present study, we examined the contribution made by IL-1 to the hypoglycemic action of LPS. Nine other cytokines tested were all inactive at inducing hypoglycemia. LPS produced hypoglycemia in mice deficient in either IL-1α or IL-1β but not in mice deficient in both cytokines (IL-1α and -1β knockout [IL-1α/β KO] mice). IL-1α, IL-1β, and TNF-α induced hypoglycemia in IL-1α/β KO mice, as they did in normal control mice. The LPS-induced elevation of serum cortisol was weaker in IL-1α/β KO mice than in control mice, and, in the latter, serum cortisol was markedly raised while blood glucose was declining. IL-1α decreased blood glucose both in NOD mice (which have impaired insulin production) and in KK-Ay mice (insulin resistant). These results suggest that (i) cortisol may not be involved in mediating the resistance of IL-1α/β KO mice to the hypoglycemic action of LPS, (ii) as a mediator, IL-1 is a prerequisite for the hypoglycemic action of LPS, (iii) IL-1α and IL-1β perform mutual compensation, and (iv) IL-1 plays a role as the primary stimulator of the many anabolic reactions required for the elaboration of immune responses against infection.     

Processing of Mycobacterium tuberculosis Bacilli by Human Monocytes for CD4+ αβ and γδ T Cells: Role of Particulate Antigen

Mycobacterium tuberculosis readily activates both CD4⁺ and Vδ2⁺ γδ T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented by M. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing of M. tuberculosis antigens to human CD4 and γδ T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vδ2⁺ γδ T cells. For γδ T cells, recognition of M. tuberculosis-infected monocytes was dependent on Vδ2⁺ T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4⁺ T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing of M. tuberculosis bacilli for Vδ2⁺ γδ T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for γδ T cells was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4⁺ T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing of M. tuberculosis bacilli for CD4⁺ and γδ T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4⁺ T cells. Chloroquine and ammonium chloride insensitivity of processing of M. tuberculosis bacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4⁺ and γδ T cells.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is spread readily from person to person by inhalation of aerosolized mycobacteria (8). A hallmark of M. tuberculosis infection is the ability of most healthy individuals to control the infection by mounting an acquired immune response, in which antigen-specific T cells and mononuclear phagocytes arrest the growth of M. tuberculosis bacilli and maintain control over dormant bacilli within granulomas (reviewed in reference 25). This protective cellular immune response results in conversion of the tuberculin skin test from negative to positive and probably in increased resistance to reinfection with tubercle bacilli.CD4⁺ αβ-T-cell-receptor (αβ TCR)-bearing T cells (CD4⁺ T cells) are readily activated by mycobacterial antigens and have a dominant role in the protective immune response to M. tuberculosis in humans (2, 34). These CD4⁺ T cells not only secrete cytokines but also serve directly as cytotoxic effector cells against M. tuberculosis-infected macrophages (6). In addition to CD4⁺ T cells, M. tuberculosis antigens activate other human T-cell subsets such as γδ TCR⁺ T cells (γδ T cells) (15, 16, 18). Vδ2⁺ and Vγ9⁺ γδ T cells are particularly responsive to live M. tuberculosis (15). A role for both γδ and CD4⁺ T cells in protective immunity to acute M. tuberculosis infection has been demonstrated in murine models (20, 21, 26, 27). A recent study of humans suggests that Vγ9⁺ and Vδ2⁺ γδ T-cell numbers and function are reduced in tuberculosis patients (23).Functional comparisons of human CD4⁺ and γδ T-cell responses of healthy tuberculin-positive persons demonstrate that both T-cell subsets have similar cytotoxic effector functions for M. tuberculosis-infected monocytes and produce large amounts of gamma interferon (IFN-γ), with γδ T cells being slightly more efficient producers of IFN-γ than CD4⁺ T cells (37). Despite similarities in function, these two T-cell subsets differ in the mycobacterial antigens recognized by their TCRs and the manners in which antigens are presented to them by M. tuberculosis-infected mononuclear phagocytes. CD4⁺ T cells recognize a wide diversity of mycobacterial peptides in the context of class II major histocompatibility complex (MHC) molecules, which include secreted as well as somatic antigens (6, 13, 33, 37). In contrast, Vγ9⁺ and Vδ2⁺ γδ T cells, the dominant γδ TCR subsets activated by M. tuberculosis, recognize mycobacterial antigens in a non-MHC-restricted manner and the repertoire of antigens includes small phosphate-containing antigens such as TUBag’s (5, 9, 19, 22, 29, 36).Both blood monocytes and alveolar macrophages infected with M. tuberculosis are efficient antigen-presenting cells for mycobacterial antigen-specific CD4⁺ and γδ T cells (1, 5). However, little is known about how M. tuberculosis-infected mononuclear phagocytes process antigens for these two T-cell subsets. M. tuberculosis bacilli are taken up by mononuclear phagocytes through a variety of surface receptors, including complement receptor 4, mannose receptor, and complement receptor 3 (17, 31, 32). Within mononuclear phagocytes, the mycobacteria reside within phagosomes and modulate the phagosome by preventing fusion with acidic lysosomal compartments (7). Although the vacuolar membranes surrounding the phagosome acquire endosomal markers, the vesicular proton ATPase is actively excluded, resulting in an elevated pH of 6.3 to 6.5 compared to the normal lysosomal pH of 4.5 (7, 35). The elevated pH in the phagosome does not appear to inhibit the ability of mycobacterial antigens to be processed and presented to CD4⁺ and Vδ2⁺ γδ T cells. This study was undertaken to gain insight into the mechanisms used by monocytes infected with live M. tuberculosis bacilli to process mycobacterial antigens for presentation to both CD4⁺ and γδ T cells.     

Antagonism between Goα and Gqα in Caenorhabditis elegans: the RGS protein EAT-16 is necessary for Goα signaling and regulates Gqα activity

To elucidate the cellular role of the heterotrimeric G protein G(o), we have taken a molecular genetic approach in Caenorhabditis elegans. We screened for suppressors of activated GOA-1 (G(o)alpha) that do not simply decrease its expression and found mutations in only two genes, sag-1 and eat-16. Animals defective in either gene display a hyperactive phenotype similar to that of goa-1 loss-of-function mutants. Double-mutant analysis indicates that both sag-1 and eat-16 act downstream of, or parallel to, G(o)alpha and negatively regulate EGL-30 (G(q)alpha) signaling. eat-16 encodes a regulator of G protein signaling (RGS) most similar to the mammalian RGS7 and RGS9 proteins and can inhibit endogenous mammalian G(q)/G(11) in COS-7 cells. Animals defective in both sag-1 and eat-16 are inviable, but reducing function in egl-30 restores viability, indicating that the lethality of the eat-16; sag-1 double mutant is due to excessive G(q)alpha activity. Analysis of these mutations indicates that the G(o) and G(q) pathways function antagonistically in C. elegans, and that G(o)alpha negatively regulates the G(q) pathway, possibly via EAT-16 or SAG-1. We propose that a major cellular role of G(o) is to antagonize signaling by G(q).