Pharmacologic effects on mouse isolated atria of immunoglobulins directed against class I histocompatibility antigens

Sera from alloimmunized mice exert potent inotropic and chronotropic effects on mouse isolated atria. In this report, we present data showing that both total immunoglobulins and purified IgG from alloimmunized mice were able to exert per se these effects. The pharmacologic effects of IgG were parallel to its cytotoxic titer but not to its immunofluorescence titer. The specificity of the inotropic and chronotropic effects was studied by using several interstrain immunizations and target atria. It was observed that only the sera from mice immunized with H-2-disparate cells were able to exert pharmacologic effects on atria; these effects were evident not only on atria from the immunizing strain, but also on atria from other strains having identical H-2 but different backgrounds. Neither normal sera nor sera from animals immunized against non-H-2 differences were active. The effect of sera, total immunoglobulins, and purified IgG were blocked by propranolol, suggesting the involvement of beta-adrenoreceptor in the reaction.     

Intranasal Immunization with Yersinia enterocolitica O:8 Cellular Extract Protects against Local Challenge Infection

Yersinia enterocolitica is enteropathogenic for humans and rodents. Immune protection from oral and respiratory pathogens may be most effectively elicited following intranasal (i.n.) vaccination. An experimental murine intranasal challenge model was used to evaluate the immunogenicity of a Y. enterocolitica O:8 cellular extract (CE) in mucosa. This antigenic preparation has demonstrated to induce protection by subcutaneous immunization. Mice were immunized intranasally with two doses of CE. Immunized and nonimmunized animals were challenged with 5×10⁶ colony-forming units (CFU) by nasal infection. Antibodies in serum and bronchoalveolar lavage (b.a.l.) fluid were assessed before and 48 hr after challenge. The CFU were determined by analysis of lung homogenate samples. The CE immunization induced significant b.a.l.-specific IgA and IgG, and serum-specific IgG, IgA and IgM. Histopathological studies 24 and 48 hr postchallenge demonstrated that immunization protected against progressive lesions resulting from Y. enterocolitica invasion of the pulmonary mucosa. The CFU in the lungs showed that CE immunization led to significant clearance as compared to the bacterial level in nonimmunized controls. From the results obtained, it can be concluded that CE can induce local and systemic immunity and protect against nasal infection.     

Rab35 and its GAP EPI64C in T cells regulate receptor recycling and immunological synapse formation

Upon antigen recognition, T-cell receptor (TCR/CD3) and other signaling molecules become enriched in a specialized contact site between the T cell and antigen-presenting cell, i.e. the immunological synapse (IS). Enrichment occurs via mechanisms that include polarized secretion from recycling endosomes, but the Rabs and RabGAPs that regulate this are unknown. EPI64C (TBC1D10C) is an uncharacterized candidate RabGAP we identified by mass spectrometry as abundant in human peripheral blood T cells that is preferentially expressed in hematopoietic cells. EPI64C is a Rab35-GAP based both on in vitro Rab35-specific GAP activity and findings in transfection assays. EPI64C and Rab35 dominant negative (DN) constructs each impaired transferrin export from a recycling pathway in Jurkat T-cells and induced large vacuoles marked by transferrin receptor, TCR, and SNAREs implicated in TCR-polarized secretion. Rab35 localized to the plasma membrane and to intracellular vesicles where it substantially colocalized with TfR and with TCR. Rab35 was strongly recruited to the IS. Conjugate formation was impaired by transfection with Rab35-DN or EPI64C and by EPI64C knock down. TCR enrichment at the IS was impaired by Rab35-DN. Thus, EPI64C and Rab35 regulate a recycling pathway in T cells and contribute to IS formation, most likely by participating in TCR transport to the IS.     

Fast-acting excitatory amino acids are involved in the enhancement of the aversiveness of the electrical stimulation of the inferior colliculus by systemic injections of muscimol

Gradual increases in the electrical stimulation of the inferior colliculus produces progressive aversive responses from vigilance, through freezing, until escape. These responses are probably mediated by excitatory amino acids (EAA) mechanisms as microinjection of glutamate into the inferior colliculus can trigger freezing responses while microinjections of NMDA cause a mixture of immobility and escape responses. Moreover, it has been shown that the neural substrates for defensive behavior in this structure are regulated by GABA-benzodiazepine mechanisms. Indeed, these responses are depressed by muscimol and midazolam locally injected into the inferior colliculus. In this work we were interested in knowing how GABAergic mechanisms interact with the EAA-mediated neural substrates of aversion generated at the inferior colliculus level. We found that while intraperitoneal injections of muscimol caused the expected antiaversive effects, unexpectedly systemic injections of muscimol enhanced the aversive reactions induced by electrical stimulation of the inferior colliculus of rats. Local injections into the central nucleus of the inferior colliculus of GDEE-an AMPA/kainate receptor antagonist-inhibited whereas AP7-a NMDA receptor antagonist-did not influence these responses. It is suggested that systemic injections of muscimol inhibit GABAergic inputs to the inferior colliculus. The removal of these inhibitory influences reduce the well-known tonic inhibitory control exerted by GABAergic mechanisms on the neural substrates of aversion of the inferior colliculus. Activation of these neural substrates by fast-acting AMPA/kainate receptors trigger the initial steps of the defense reaction in the central nucleus of the inferior colliculus.     

LH response (in vivo and in vitro) to an LHRH agonist administered to domestic male cats

We investigated plasma luteinizing hormone (LH) concentration in domestic male cats challenged with Luteinizing Hormone Releasing Hormone Analog (LHRH-A) [des Gly 10, (DTrp6)-LHRH ethylamide] that mediates the function of the hypothalamic-pituitary-gonadal axis (HPG). Plasma LH concentrations in cats treated daily with LHRH (10 microg/100 microl/kg/day, subcutaneously-s.c.) for 19 days (LHRH group) and in controls treated with saline (NaCl-0.9%, same volume-SAL group) were chronically studied. LHRH administration (s.c.) for 15 days induced a significant fall (P < 0.05) in plasma LH concentrations during the chronic study. After the 15th day of treatment the groups were divided once more into animals treated with LHRH (10 microg/100 microl/kg) or saline (i.v.), and a time course study (300 min) was performed (acute study). Next, four groups of cats were compared in an acute study involving the s.c./i.v. administration of SAL/SAL, SAL/LHRH, LHRH/SAL, and LHRH/LHRH. The responses of the SAL animals challenged by acute i.v. administration of LHRH (group SAL/LHRH) were significantly higher (P < 0.01) than those of animals treated with LHRH (sc) (group LHRH/LHRH). LH release was also significantly increased in the latter group (P < 0.05), although the effect was short lasting, being recorded only at the first observation (45 min). An in vitro study with the pituitaries was also performed on day 20. Mean (+/-SEM) LH concentrations in the culture medium containing pituitaries with LHRH (10(-7) M) or saline were determined. In vitro analysis of these pituitaries demonstrated a significantly reduced response (P < 0.05) by animals treated sc with LHRH for 19 days. This study represents a source of data for the domestic cat going beyond its own physiology. Serving as a model, this animal provide important information for the study of reproductive physiology in other members of its family (Felidae), almost all of them threatened with extinction.     

Chelation of ThIV, EuIII and NdIII by dianionic N,N'-bis(pyridoxylideneiminato)ethylene, (Pyr2en)2-. On the search of feasible modelings for heavy metals damage inhibition in living beings

The neutral Schiff base N,N'-bis(pyridoxylideneiminato)ethylene {H(2)pyr(2)en} reacts with Th(NO(3))4.4H2O, NdCl3.6H2O and EuCl3.6H2O to give [Th(pyr(2)en)2(H2O)] (1), [Nd(pyr(2)en)(Hpyr(2)en)].12H2O (2) and [Eu(pyr(2)en)(Hpyr(2)en)] (3). In the three not yet reported bimolecular chelate systems the endo hydroxyl groups of the rings undergo deprotonation confirming the remarkable ability of the pyridoxal-containing ligand H(2)pyr(2)en to yield stable heavy metal chelates with unusual coordination polyhedra. Complexes 2 and 3 show a coordination number 8 for Nd and Eu, achieving a distorted quadratic antiprism. In complex 1 the additional water molecule increases the coordination number of Th to 9 producing a capped square antiprism. The synthesis and structural elucidation of the title complexes starting from a probably non-toxic metabolite like H(2)pyr(2)en should represent a useful contribution to the research on models of prevention and therapy of damage caused by radioactive and heavy elements.     

Mitochondrial ATPase activity and membrane fluidity changes in rat liver in response to intoxication with Buckthorn (Karwinskia humboldtiana)

Background

Karwinskia humboldtiana (Kh) is a poisonous plant of the rhamnacea family. To elucidate some of the subcellular effects of Kh toxicity, membrane fluidity and ATPase activities as hydrolytic and as proton-pumping activity were assessed in rat liver submitochondrial particles. Rats were randomly assigned into control non-treated group and groups that received 1, 1.5 and 2 g/Kg body weight of dry powder of Kh fruit, respectively. Rats were euthanized at day 1 and 7 after treatment.

Results

Rats under Kh treatment at all dose levels tested, does not developed any neurologic symptoms. However, we detected alterations in membrane fluidity and ATPase activity. Lower dose of Kh on day 1 after treatment induced higher mitochondrial membrane fluidity than control group. This change was strongly correlated with increased ATPase activity and pH gradient driven by ATP hydrolysis. On the other hand, membrane fluidity was hardly affected on day 7 after treatment with Kh. Surprisingly, the pH gradient driven by ATPase activity was significantly higher than controls despite an diminution of the hydrolytic activity of ATPase.

Conclusions

The changes in ATPase activity and pH gradient driven by ATPase activity suggest an adaptive condition whereby the fluidity of the membrane is altered.     

AUF1/hnRNP D is a novel protein partner of the EBER1 noncoding RNA of Epstein-Barr virus

Epstein-Barr virus (EBV)–infected cells express two noncoding RNAs called EBV-encoded RNA (EBER) 1 and EBER2. Despite their high abundance in the nucleus (about 10⁶ copies), the molecular function of these noncoding RNAs has remained elusive. Here, we report that the insertion into EBER1 of an RNA aptamer that binds the bacteriophage MS2 coat protein allows the isolation of EBER1 and associated protein partners. By combining MS2-mediated selection with stable isotope labeling of amino acids in cell culture (SILAC) and analysis by mass spectrometry, we identified AUF1 (AU-rich element binding factor 1)/hnRNP D (heterogeneous nuclear ribonucleoprotein D) as an interacting protein of EBER1. AUF1 exists as four isoforms generated by alternative splicing and is best known for its role in destabilizing mRNAs upon binding to AU-rich elements (AREs) in their 3′ untranslated region (UTR). Using UV crosslinking, we demonstrate that predominantly the p40 isoform of AUF1 interacts with EBER1 in vivo. Electrophoretic mobility shift assays show that EBER1 can compete for the binding of the AUF1 p40 isoform to ARE-containing RNA. Given the high abundance of EBER1 in EBV-positive cells, EBER1 may disturb the normal homeostasis between AUF1 and ARE-containing mRNAs or compete with other AUF1-interacting targets in cells latently infected by EBV.     

Myosin 1G Is an Abundant Class I Myosin in Lymphocytes Whose Localization at the Plasma Membrane Depends on Its Ancient Divergent Pleckstrin Homology (PH) Domain (Myo1PH)

Class I myosins, which link F-actin to membrane, are largely undefined in lymphocytes. Mass spectrometric analysis of lymphocytes identified two short tail forms: (Myo1G and Myo1C) and one long tail (Myo1F). We investigated Myo1G, the most abundant in T-lymphocytes, and compared key findings with Myo1C and Myo1F. Myo1G localizes to the plasma membrane and associates in an ATP-releasable manner to the actin-containing insoluble pellet. The IQ+tail region of Myo1G (Myo1C and Myo1F) is sufficient for membrane localization, but membrane localization is augmented by the motor domain. The minimal region lacks IQ motifs but includes: 1) a PH-like domain; 2) a “Pre-PH” region; and 3) a “Post-PH” region. The Pre-PH predicted α helices may contribute electrostatically, because two conserved basic residues on one face are required for optimal membrane localization. Our sequence analysis characterizes the divergent PH domain family, Myo1PH, present also in long tail myosins, in eukaryotic proteins unrelated to myosins, and in a probable ancestral protein in prokaryotes. The Myo1G Myo1PH domain utilizes the classic lipid binding site for membrane association, because mutating either of two basic residues in the “signature motif” destroys membrane localization. Mutation of each basic residue of the Myo1G Myo1PH domain reveals another critical basic residue in the β3 strand, which is shared only by Myo1D. Myo1G differs from Myo1C in its phosphatidylinositol 4,5-bisphosphate dependence for membrane association, because membrane localization of phosphoinositide 5-phosphatase releases Myo1C from the membrane but not Myo1G. Thus Myo1PH domains likely play universal roles in myosin I membrane association, but different isoforms have diverged in their binding specificity.     

Recycling of the Ca2+-activated K+ Channel,KCa2.3, Is Dependent upon RME-1, Rab35/EPI64C,and an N-terminal Domain

Regulation of the number of Ca²⁺-activated K⁺ channels at the endothelial cell surface contributes to control of the endothelium-derived hyperpolarizing factor response, although this process is poorly understood. To address the fate of plasma membrane-localized KCa2.3, we utilized an extracellular epitope-tagged channel in combination with fluorescence and biotinylation techniques in both human embryonic kidney cells and the human microvascular endothelial cell line, HMEC-1. KCa2.3 was internalized from the plasma membrane and degraded with a time constant of 18 h. Cell surface biotinylation demonstrated that KCa2.3 was rapidly endocytosed and recycled back to the plasma membrane. Consistent with recycling, expression of a dominant negative (DN) RME-1 or Rab35 as well as wild type EPI64C, the Rab35 GTPase-activating protein, resulted in accumulation of KCa2.3 in an intracellular compartment. Expression of DN RME-1, DN Rab35, or wild type EPI64C resulted in a decrease in steady-state plasma membrane expression. Knockdown of EPI64C increased cell surface expression of KCa2.3. Furthermore, the effect of EPI64C was dependent upon its GTPase-activating proteins activity. Co-immunoprecipitation studies confirmed an association between KCa2.3 and both Rab35 and RME-1. In contrast to KCa2.3, KCa3.1 was rapidly endocytosed and degraded in an RME-1 and Rab35-independent manner. A series of N-terminal deletions identified a 12-amino acid region, Gly²⁰⁶–Pro²¹⁷, as being required for the rapid recycling of KCa2.3. Deletion of Gly²⁰⁶–Pro²¹⁷ had no effect on the association of KCa2.3 with Rab35 but significantly decreased the association with RME-1. These represent the first studies elucidating the mechanisms by which KCa2.3 is maintained at the plasma membrane.