Inactivation of cloned Na channels expressed in Xenopus oocytes

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine

Kidney regeneration: common themes from the embryo to the adult

The vertebrate kidney has an inherent ability to regenerate following acute damage. Successful regeneration of the injured kidney requires the rapid replacement of damaged tubular epithelial cells and reconstitution of normal tubular function. Identifying the cells that participate in the regeneration process as well as the molecular mechanisms involved may reveal therapeutic targets for the treatment of kidney disease. Renal regeneration is associated with the expression of genetic pathways that are necessary for kidney organogenesis, suggesting that the regenerating tubular epithelium may be “reprogrammed” to a less-differentiated, progenitor state. This review will highlight data from various vertebrate models supporting the hypothesis that nephrogenic genes are reactivated as part of the process of kidney regeneration following acute kidney injury (AKI). Emphasis will be placed on the reactivation of developmental pathways and how our understanding of the resulting regeneration process may be enhanced by lessons learned in the embryonic kidney.Fil: Cirio, Maria Cecilia. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de Groh, Eric D.. University of Pittsburgh; Estados UnidosFil: de Caestecker, Mark P.. Vanderbilt University; Estados UnidosFil: Davidson, Alan J.. The University of Auckland; Nueva ZelandaFil: Hukriede, Neil A.. University of Pittsburgh; Estados Unido

Zebrafish scl functions independently in hematopoietic and endothelial development

AbstractThe SCL transcription factor is critically important for vertebrate hematopoiesis and angiogenesis, and has been postulated to induce hemangioblasts, bipotential precursors for blood and endothelial cells. To investigate the function of scl during zebrafish hematopoietic and endothelial development, we utilized site-directed, anti-sense morpholinos to inhibit scl mRNA. Knockdown of scl resulted in a loss of primitive and definitive hematopoietic cell lineages. However, the expression of early hematopoietic genes, gata2 and lmo2, was unaffected, suggesting that hematopoietic cells were present but unable to further differentiate. Using gene expression analysis and visualization of vessel formation in live animals harboring an lmo2 promoter-green fluorescent protein reporter transgene (Tg(lmo2:EGFP)), we show that angioblasts were specified normally in the absence of scl, but later defects in angiogenesis were evident. While scl was not required for angioblast specification, forced expression of exogenous scl caused an expansion of both hematopoietic and endothelial gene expression, and a loss of somitic tissue. In cloche and spadetail mutants, forced expression of scl resulted in an expansion of hematopoietic but not endothelial tissue. Surprisingly, in cloche, lmo2 was not induced in response to scl over-expression. Taken together, these findings support distinct roles for scl in hematopoietic and endothelial development, downstream of hemangioblast development

A zebrafish model of conditional targeted podocyte ablation and regeneration

Podocytes are specialized cells that contribute critically to the normal structure and function of the glomerular filtration barrier. Their depletion plays an important role in the pathogenesis of glomerulosclerosis. Here, we report generation of a genetic model of conditional podocyte ablation and regeneration in zebrafish using a bacterial nitroreductase strategy to convert a prodrug, Metronidazole, into a cytotoxic metabolite. A transgenic zebrafish line was generated that expresses a green fluorescence protein (GFP) and the nitroreductase fusion protein under the control of the podocin promoter Tg(podocin:nitroreductase-GFP). Treatment of these transgenic zebrafish with Metronidazole results in podocyte apoptosis, a loss of nephrin and podocin expression, foot process effacement, and a leaky glomerular filtration barrier. Following Metronidazole washout, proliferating cells were detected in the glomeruli of recovering transgenic fish with a restoration of nitroreductase-GFP fluorescence, nephrin and podocin expression, a reestablishment of normal foot process architecture and glomerular barrier function. Thus, our studies show that zebrafish podocytes are capable of regenerating following depletion and establish the Tg(podocin:NTR-GFP) fish as a new model to study podocyte injury and repair

Alkene anti-dihydroxylation with malonoyl peroxides

Malonoyl peroxide 1, prepared in a single step from the commercially available diacid, is an effective reagent for the anti-dihydroxylation of alkenes. Reaction of 1 with an alkene in the presence of acetic acid at 40 °C followed by alkaline hydrolysis leads to the corresponding diol (35-92%) with up to 13:1 anti-selectivity. A mechanism consistent with experimental findings is proposed that accounts for the selectivity observed

The College News, 1945-02-14, Vol. 31, No. 15

Bryn Mawr College student newspaper. Merged with The Haverford News in 1968 to form the Bi-college News (with various titles from 1968 on). Published weekly (except holidays) during the academic year

Exposure to the antimicrobial peptide LL-37 produces dendritic cells optimized for immunotherapy

Immunization of patients with autologous, ex vivo matured dendritic cell (DC) preparations, in order to prime antitumor T-cell responses, is the focus of intense research. Despite progress and approval of clinical approaches, significant enhancement of these personalized immunotherapies is urgently needed to improve efficacy. We show that immunotherapeutic murine and human DC, generated in the presence of the antimicrobial host defense peptide LL-37, have dramatically enhanced expansion and differentiation of cells with key features of the critical CD103 + /CD141 + DC subsets, including enhanced cross-presentation and co-stimulatory capacity, and upregulation of CCR7 with improved migratory capacity. These LL-37-DC enhanced proliferation, activation and cytokine production by CD8 + (but not CD4 + ) T cells in vitro and in vivo. Critically, tumor antigen-presenting LL-37-DC increased migration of primed, activated CD8 + T cells into established squamous cell carcinomas in mice, and resulted in tumor regression. This advance therefore has the potential to dramatically enhance DC immunotherapy protocols

The opposites task: Using general rules to test cognitive flexibility in preschoolers

A brief narrative description of the journal article, document, or resource. Executive functions play an important role in cognitive development, and during the preschool years especially, children's performance is limited in tasks that demand flexibility in their behavior. We asked whether preschoolers would exhibit limitations when they are required to apply a general rule in the context of novel stimuli on every trial (the "opposites" task). Two types of inhibitory processing were measured: response interference (resistance to interference from a competing response) and proactive interference (resistance to interference from a previously relevant rule). Group data show 3-year-olds have difficulty inhibiting prepotent tendencies under these conditions, whereas 5-year-olds' accuracy is near ceiling in the task. (Contains 4 footnotes and 1 table.