Joro spider toxin (JSTX) is one of the most potent antagonists of glutamatergic AMPA/KA (a-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate) receptor channels in invertebrates and vertebrates. A differential blocking effect on certain ypes of glutamatergic synapses-e.g., parallel and climbing fiber synaptic inputs to rat cerebeilar Purkinje neurons-has been shown by using a synthetic analog of the spider toxin. By investigating the molecular basis of the JSTX action on the recombinant AMPA/KA receptors GluRlGluR4 and GluR6 expressed in Xenopus oocytes, we found that submicromolar concentrations ofJSTX exert a subunit-specific block. Thus, receptor subunits forming a receptor channel with a linear current-voltage (I-V) relationship (GluRI/2, GluR2/3, and GluR6) were not affected, while receptor subunits with rectifying I-V relationships (GluRl, GluR3, GluR4, and GluRl/3) were reversibly blocked by JSTX. By using receptor-subunit mutants obtained by site-directed mutagenesis, we have identified a single amino acid position (glutamine in the proposed second transmembrane domain) that is critical for the JSTX block. Since this site has previously been shown to control the I-V relationship of the AMPA/KA receptor channel and to participate in the regulation of the channel's permeability for calcium ions, our findings suggest that JSTX binds close to the central pore region of the channel.Joro spider toxin (JSTX) (Fig. la), the toxin of the spider Nephila clavata, and the closely related arthropod toxins
Sapoviruses are one of the major agents of acute gastroenteritis in childhood. They form a tight genetic cluster (genus) in the Caliciviridae family that regroups both animal and human pathogenic strains. No permissive tissue culture has been developed for human sapovirus, limiting its characterization to surrogate systems. We report here on the first extensive characterization of the key enzyme of replication, the RNAdependent RNA polymerase (RdRp) pol synthesizes a double-stranded RNA or labels the template 3 terminus by terminal transferase activity. Initiation of RNA synthesis occurs de novo on heteropolymeric templates or in a primerdependent manner on polyadenylated templates. Strikingly, this mode of initiation of RNA synthesis was also described for norovirus, but not for lagovirus, suggesting structural and functional homologies in the RNAdependent RNA polymerase of human pathogenic caliciviruses. This first experimental evidence makes sapovirus 3D pol an attractive target for developing drugs to control calicivirus infection in humans.
Our results suggest that IL-6, IL-1β, and TNF-α only in combination induced osteoclaststimulating activity represented by the RANKL/OPG ratio in osteoblasts. Dexamethasone further increased this effect in osteoblasts, while decreasing cytokine expression. The results in osteoclasts support a direct and osteoblast-mediated effect on bone resorption. Our in vitro results differentiate for the first time the effect of cytokines on bone turnover as measured in adult CD patients from the additional dexamethasone effect on osteoblasts as part of the pathophysiology of osteoporosis.
Chronic inflammatory bowel diseases can be successfully treated with antibodies against the acute phase mediator TNF-a. The process of activation and of extravasation of inflammatory cells from the blood into the 'stressed' tissue site is controlled by cytokines and chemokines, which attract leukocytes and by adhesion molecules, which mediate their attachment and transmigration toward the affected cell(s). The changes in the gene expression of adhesion molecules taking place in those cells before attachment have been less investigated. Changes of PECAM-1, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) gene expression were studied in phytohaemagglutinin (PHA)-and lipolysaccharide (LPS)-treated human peripheral blood leukocytes (PBLs), granulocytes and the human monocyte cell line U-937. Cells were treated either with PHA or with LPS in the presence or absence of infliximab and incubated with TNF-a, IFN-g and/or transforming growth factor beta (TGF-b) and treated as above. Activation of PBLs by PHA or LPS treatment triggered a sharp upregulation of ICAM-1, VCAM-1 gene expression and a time-dependent downregulation of PECAM-1 gene expression reaching a minimum 4 h from start of the experiment. The anti-TNF-a antibody infliximab, by neutralizing TNF-a and IFN-g production, completely reversed PECAM-1 mRNA downregulation and ICAM-1 and VCAM-1 upregulation. Immunostaining of PBLs cytospins with antibodies against PECAM-1 and ICAM-1 confirmed RT-PCR and western blot results. PBLs IFN-g or TNF-a treatment downregulated PECAM-1 in parallel with the upregulation of ICAM-1 and VCAM-1 gene expression, whereas TGF-b upregulated PECAM-1-and downregulated ICAM-1 and VCAM-1 gene expression counteracting the effect of TNF-a or IFN-g. Similar results were obtained in human U937 cells and in granulocyte cultures by TNF-a or IFN-g treatment. Taken together, these results suggest that infliximab, blocking TNF-a and IFN-g production, exerts its anti-inflammatory effect through inhibiting downregulation of PECAM-1 gene expression and upregulation of ICAM-1 and VCAM-1 expression in leukocytes of the peripheral blood. These results also suggest that TGF-b may thus be of therapeutic importance as an anti-inflammatory agent.
Hypoparathyroidism patients suffer a variety of complaints often leading to reduced quality of life. Currently, no specific standard instrument exists to measure corresponding disease manifestations. We therefore aimed to develop a disease‐characteristic questionnaire for hypoparathyroid patients. We used an analytical‐empirical approach for questionnaire construction based on retrospective analysis of four well‐established but non‐disease‐specific questionnaires (Symptom Checklist 90, revised [SCL‐90‐R]; Giessen Complaint List [GBB]; Short‐Form‐36 Health Survey [SF‐36]; von Zerssen Symptom List [B‐L Zerssen]) and two additional unpublished or local questionnaires (SHGdQ and GPQ) in a German hypoparathyroidism self‐help group (n = 60). Retrospective data were compared with corresponding general population norms. The new questionnaire was administered prospectively over 1 year to patients with postoperative hypoparathyroidism and two control groups to validate specificity. Exploratory factor analysis (EFA) and reliability testing were applied to identify relevant scales and reduce overlapping items. In the self‐help group, SCL‐90‐R revealed elevated symptom load in four complaint areas (p = 0.003 to p < 0.001). The SF‐36 mental summary score (p < 0.001) and further scales were lowered. In the GBB, four of five scales (p = 0.009 to p < 0.001) were elevated. In the B‐L Zerssen, 6 of 24 items revealed complaint areas. Based on these findings, the new 40‐item “Hypoparathyroid Patient Questionnaire” (HPQ 40) was developed, tested prospectively, and further analyzed. EFA revealed five scales (pain and cramps, gastrointestinal symptoms, depression and anxiety, neurovegetative symptoms, loss of vitality), all with Cronbach's alpha >0.7. The questionnaire was revised accordingly and shortened to 28 questions to avoid redundancy. We present a new disease‐characteristic questionnaire for hypoparathyroidism patients. Prospective testing revealed five major complaint areas and promising psychometric properties. This questionnaire can be tested for usefulness in further clinical trials. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
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