Extracellular zinc triggers ERK-dependent activation of Na+/H+ exchange in colonocytes mediated by the zinc-sensing receptor

Extracellular zinc promotes cell proliferation and its deficiency leads to impairment of this process, which is particularly important in epithelial cells. We have recently characterized a zinc-sensing receptor (ZnR) linking extracellular zinc to intracellular release of calcium. In the present study, we addressed the role of extracellular zinc, acting via the ZnR, in regulating the MAP kinase pathway and Na+/H+ exchange in colonocytes. We demonstrate that Ca2+ release, mediated by the ZnR, induces phosphorylation of ERK1/2, which is highly metal-specific, mediated by physiological concentrations of extracellular Zn2+ but not by Cd2+, Fe2+, Ni2+, or Mn2+. Desensitization of the ZnR by Zn2+, is followed by approximately 90% inhibition of the Zn2+ -dependent ERK1/2 phosphorylation, indicating that the ZnR is a principal link between extracellular Zn2+ and ERK1/2 activation. Application of both the IP3 pathway and PI 3-kinase antagonists largely inhibited Zn2+ -dependent ERK1/2 phosphorylation. The physiological significance of the Zn2+ -dependent activation of ERK1/2 was addressed by monitoring Na+/H+ exchanger activity in HT29 cells and in native colon epithelium. Preincubation of the cells with zinc was followed by robust activation of Na+/H+ exchange, which was eliminated by cariporide (0.5 microm); indicating that zinc enhances the activity of NHE1. Activation of NHE1 by zinc was totally blocked by the ERK1/2 inhibitor, U0126. Prolonged acidification, in contrast, stimulates NHE1 by a distinct pathway that is not affected by extracellular Zn2+ or inhibitors of the MAP kinase pathway. Desensitization of ZnR activity eliminates the Zn2+ -dependent, but not the prolonged acidification-dependent activation of NHE1, indicating that Zn2+ -dependent activation of H+ extrusion is specifically mediated by the ZnR. Our results support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways that affect pH homeostasis in colonocytes. Furthermore activation of both, ERK and NHE1, by extracellular zinc may provide the mechanism linking zinc to enhanced cell proliferation.     

MHC class I-independent recognition of NK-activating receptor KIR2DS4

Natural killer cells are capable of killing tumor and virus-infected cells. This killing is mediated primarily via the natural cytotoxicity receptors, including NKp46, NKp44, NKp30, and by the NKG2D receptor. Killer cell Ig-like receptors (KIRs) are mainly involved in inhibiting NK killing (inhibitory KIRs) via interaction with MHC class I molecules. Some KIRs, however, have been found to enhance NK killing when interacting with MHC class I molecules (activating KIRs). We have previously demonstrated that KIR2DS4, an activating KIR, recognizes the HLA-Cw4 protein. The interaction observed was weak and highly restricted to HLA-Cw4 only. These findings prompted us to check whether KIR2DS4 might have additional ligand(s). In this study, we show that KIR2DS4 is able to also interact with a non-class I MHC protein expressed on melanoma cell lines and on a primary melanoma. This interaction is shown to be both specific and functional. Importantly, site-directed mutagenesis analysis reveals that the amino acid residues involved in the recognition of this novel ligand are different from those interacting with HLA-Cw4. These results may shed new light on the function of activating KIRs and their relevance in NK biology.     

G protein-activated K+ channels: a reporter for rapid activation of G proteins by lysophosphatidic acid in Xenopus oocytes

Threshold concentrations of lysophosphatidic acid (LPA) or acetylcholine (ACh) induce pertussis toxin (PTX)-sensitive rapid desensitization of responses to LPA in Xenopus oocytes. To demonstrate that threshold [LPA] rapidly activates Gi/o proteins, we used the G protein-activated K+ channel (GIRK) as a reporter. Low [LPA] induced IK+ in <3 s of the agonist addition with little or no activation of chloride current. Depletion of Galphao/Galphao1 each decreased the LPA-induced IK+ by approximately 40-50%, while PTX completely abolished it. This is the first direct evidence showing the activation of GIRK by LPA, and the involvement of G proteins of the Go family in rapid desensitization of LPA responses.     

Highly efficient transformation and regeneration of aspen plants through shoot-bud formation in root culture

The natural capacity of aspen (Populus tremula L.) roots for direct shoot-bud regeneration was harnessed to establish a highly efficient transformation and regeneration procedure that does not require a pre-selection stage on antibiotics. Aspen stem segments were transformed using wildtype Agrobacterium rhizogenes (LBA9402) with the binary p35SGUSINT plasmid carrying the genes coding for -glucuronidase (GUS) and neomycin phosphotransferase II. High levels of transient GUS expression were found in the basal cut surface of 87% of the segments, and 98% of these formed well-developed adventitious roots. Proliferating root cultures were established in liquid culture, and GUS expression was found in 75% of the roots. Shoot-bud regeneration in root cultures was very high: 99% of the roots yielded shoot-buds (4.3 buds per root), of which 91% expressed GUS. Southern blot analysis and polymerase chain reaction confirmed the transgenic nature of the plants expressing GUS. Kanamycin resistance of transformants was tested with respect to callus growth and bud regeneration. Callus from transgenic plants exhibited a high growth rate in the presence of up to 100 g/l kanamycin, and bud regeneration from transformed roots occurred in the presence of up to 30 g/l kanamycin. Callus and buds from control (non-transformed) plants failed to proliferate or regenerate, respectively, in the presence of kanamycin at concentrations above 10 g/l. Ninety-four independent clones from different transformation events were established, of which 52 were phenotypically true-to-type.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzylaminopurine - GUS -glucuronidase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - EtOH ethanol - CTAB cetyltrimethylammonium bromide - SDS sodium dodecyl sulfate - NOS nopaline synthase - CaMV cauliflower mosaic virus     

Physiotherapy scoliosis-specific exercises – a comprehensive review of seven major schools

In recent decades, there has been a call for change among all stakeholders involved in scoliosis management. Parents of children with scoliosis have complained about the so-called “wait and see” approach that far too many doctors use when evaluating children’s scoliosis curves between 10° and 25°. Observation, Physiotherapy Scoliosis Specific Exercises (PSSE) and bracing for idiopathic scoliosis during growth are all therapeutic interventions accepted by the 2011 International Society on Scoliosis Orthopaedic and Rehabilitation Treatment (SOSORT). The standard features of these interventions are: 1) 3-dimension self-correction; 2) Training activities of daily living (ADL); and 3) Stabilization of the corrected posture. PSSE is part of a scoliosis care model that includes scoliosis specific education, scoliosis specific physical therapy exercises, observation or surveillance, psychological support and intervention, bracing and surgery. The model is oriented to the patient. Diagnosis and patient evaluation is essential in this model looking at a patient-oriented decision according to clinical experience, scientific evidence and patient’s preference. Thus, specific exercises are not considered as an alternative to bracing or surgery but as a therapeutic intervention, which can be used alone or in combination with bracing or surgery according to individual indication. In the PSSE model it is recommended that the physical therapist work as part of a multidisciplinary team including the orthopeadic doctor, the orthotist, and the mental health care provider - all are according to the SOSORT guidelines and Scoliosis Research Society (SRS) philosophy. From clinical experiences, PSSE can temporarily stabilize progressive scoliosis curves during the secondary period of progression, more than a year after passing the peak of growth. In non-progressive scoliosis, the regular practice of PSSE could produce a temporary and significant reduction of the Cobb angle. PSSE can also produce benefits in subjects with scoliosis other than reducing the Cobb angle, like improving back asymmetry, based on 3D self-correction and stabilization of a stable 3D corrected posture, as well as the secondary muscle imbalance and related pain. In more severe cases of thoracic scoliosis, it can also improve breathing function.This paper will discuss in detail seven major scoliosis schools and their approaches to PSSE, including their bracing techniques and scientific evidence. The aim of this paper is to understand and learn about the different international treatment methods so that physical therapists can incorporate the best from each into their own practices, and in that way attempt to improve the conservative management of patients with idiopathic scoliosis. These schools are presented in the historical order in which they were developed. They include the Lyon approach from France, the Katharina Schroth Asklepios approach from Germany, the Scientific Exercise Approach to Scoliosis (SEAS) from Italy, the Barcelona Scoliosis Physical Therapy School approach (BSPTS) from Spain, the Dobomed approach from Poland, the Side Shift approach from the United Kingdom, and the Functional Individual Therapy of Scoliosis approach (FITS) from Poland.     

Wounding by bombardment yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.)

Highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.) was obtained by first wounding stem explants via microprojectile bombardment. When this was followed by cocultivation with disarmed Agrobacterium in the dark, the transformation frequency-based on transient GUS expression-increased to over 10-fold that of explants wounded by other means and cocultivated under constant light. Two cycles of regeneration/selection on kanamycin were employed to generate stably transformed carnation plants and eliminate chimeras: first, plantlets were regenerated from inoculated stem explants and then leaves from these plantlets were used to generate transgenes in a second selection cycle of adventitious shoot regeneration. Agrobacterium strain AGLO, carrying the binary vector pCGN7001 containing uidA and nptII genes, was used in the stable transformation experiments. The combination of wounding via bombardment, cocultivation in the dark and two cycles of kanamycin selection yielded an overall transformation efficiency of 1–2 transgenes per 10 stem explants for the three carnation varieties analyzed. Histochemical and molecular analyses of marker genes in T₀ and T₁ generations confirmed the transgenic nature of the selected plants.     

Breast cancer is marked by specific,Public T-cell receptor CDR3 regions shared by mice and humans

The partial success of tumor immunotherapy induced by checkpoint blockade, which is not antigen-specific, suggests that the immune system of some patients contain antigen receptors able to specifically identify tumor cells. Here we focused on T-cell receptor (TCR) repertoires associated with spontaneous breast cancer. We studied the alpha and beta chain CDR3 domains of TCR repertoires of CD4 T cells using deep sequencing of cell populations in mice and applied the results to published TCR sequence data obtained from human patients. We screened peripheral blood T cells obtained monthly from individual mice spontaneously developing breast tumors by 5 months. We then looked at identical TCR sequences in published human studies; we used TCGA data from tumors and healthy tissues of 1,256 breast cancer resections and from 4 focused studies including sequences from tumors, lymph nodes, blood and healthy tissues, and from single cell dataset of 3 breast cancer subjects. We now report that mice spontaneously developing breast cancer manifest shared, Public CDR3 regions in both their alpha and beta and that a significant number of women with early breast cancer manifest identical CDR3 sequences. These findings suggest that the development of breast cancer is associated, across species, with biomarker, exclusive TCR repertoires.