Localization and Regulation of Claudin-14 in Experimental Models of Hypercalcemia

Variations in the CLDN14 gene have been linked to increased risk of hypercalciuria and kidney stone formation. However, the exact cellular localization of CLDN14 and its regulation remain to be fully delineated. To this end, we generated a novel antibody that allowed the detection of CLDN14 in paraffin-embedded renal sections. This showed CLDN14 to be detectable in the kidney only after induction of hypercalcemia in rodent models. Protein expression in kidney is localized exclusively to the thick ascending limbs (TAL), mainly restricted to the cortical and upper medullary portion of the kidney. However not all cells in the TAL expressed the tight junction protein. In fact, CLDN14 was primarily expressed in cells also expressing CLDN16, but devoid of CLDN10. CLDN14 appeared in very superficial apical cell domains and near cell junctions in a belt-like formation along the apical cell periphery. In transgenic mice, Cldn14 promotor-driven LacZ activity did not show complete colocalization with CLDN14 protein nor was it increased by hypercalcemia, suggesting that LacZ activity cannot be used as a marker for CLDN14 localization and regulation in this model. In conclusion, CLDN14, showed a restricted localization pattern in the apical domain of select cells of the TAL.

A new method of producing NS5A antigen of hepatitis C virus

A task of creating a universal platform for engineering affordable recombinant producers of viral proteins conserving immunogenicity has not been solved yet. High toxicity of the viral proteins for the host cells, low yield and abnormal folding of the products often present severe obstacles to obtaining producers of the viral proteins. In this work, we report a new method of engineering and screening of deletion libraries from the viral antigen genes. This method allows selection of artificial derivatives of these genes adapted for expression in microbial producer cells. The method involves PCR amplification of the gene fragments using a system of randomized and adapter primers, which allows the spontaneous formation of duplexes from the random primers in the absence of the template DNA to be prevented. For selecting variants capable of in vivo expression, the obtained PCR products are cloned to a special vector of a direct phenotypical selection pQL30. It contains E. coli β-galactosidase gene with an inserted polylinker producing a frame-shift mutation. Using this screening method, an artificial variant of hepatitis C (HCV) NS5a gene with optimal biotechnological properties was established. 27 clinical specimens of 1670 bp long HCV1b NS5a fragments were used as a source gene. A PCR bank of the deletion derivatives was produced. 40 LacZ-positive clones based on pQL30 vector with a 50-700 bp long insertion were selected. The LacZ activity of the cell lysates and the immunogenicity of the products were tested. As a result, a single clone encoding a soluble protein with Mr = 114 kDa was selected. Its yield reached 0.3% of the total cell protein. It was highly reactive with sera of HCV 1b infected patients but not with sera of the healthy donors.

Targeted Gene Disruption of Kinesin-3 Family Motors Unveils a Negative Regulatory Mechanism for Mouse Erythroblast Enucleation

Abstract 371 Terminal differentiation of mammalian erythroid precursors involves enucleation, a process required for the production of reticulocytes. The signaling pathways and the molecular components mediating the final enucleation step are not well understood, although the resemblance of enucleation to asymmetric cell division has been suggested. The enucleation of erythroblasts can be replicated in vitro; however, the enucleation efficiency is not optimal under in vitro culture conditions. A functional role of both microfilaments and microtubules has been suggested in the regulation of erythroblast enucleation. We hypothesized that molecular motors known to regulate asymmetric cell division might also play a functional role in erythroblast enucleation. GAKIN (also called KIF13B) is a kinesin-3 motor implicated in the regulation of cell polarity pathways. Its Drosophila homologue Khc-73 regulates polarity formation during the asymmetric cell division of neuroblasts. We generated a mutant mouse line of GAKIN and examined the efficiency of erythroblast enucleation using the fetal liver in vitro erythroid precursor culture system originally developed by the Lodish group. The GAKIN mutant mouse line was produced from the embryonic stem (ES) cells containing a genomic insertion of the beta-galactosidase-neomycin gene. The beta-geo insertion was mapped within intron 38 of the GAKIN gene, thus removing the CAP-Gly domain located at the C-terminus of GAKIN. This allowed us to visualize specific tissue expression of GAKIN, in addition to the subcellular localization of a GAKIN-lacZ fusion protein by lacZ activity staining. The lacZ activity revealed that the GAKIN-lacZ fusion protein is expressed in bone marrow macrophages and erythroblasts, while also highly concentrated at contact sites between macrophages and erythroblasts. GAKIN mutant mice are viable and fertile, and complete blood analysis did not reveal any discernible phenotype. To test for the enucleation efficiency, erythroid precursors were isolated from fetal liver stage at day 14.5 embryos and enucleation was quantified by flow cytometry after 2 days of in vitro culture. The GAKIN-lacZ fusion protein appeared as a single dot representing the microtubule organizing center (MTOC) in the erythroid precursors isolated from fetal liver (Fig. 1). However, GAKIN mutant erythroblasts enucleated at the same efficiency as wild type erythroblasts under steady state conditions in vitro. Since the mammalian genome contains a close homologue of GAKIN/KIF13B, termed KIF13A, a possibility exists that KIF13A functionally compensates for GAKIN mutation in critical pathways. To address this issue, we generated KIF13A null mice using a similar gene disruption strategy. Again, the KIF13A mutant erythroblasts did not show any measurable change in enucleation efficiency under similar conditions. However, fetal liver erythroblasts isolated from GAKIN and KIF13A double mutant mice exhibited a significant enhancement of enucleation efficiency. This finding suggests that together, GAKIN and KIF13A negatively regulate the erythroblast enucleation by modulating the microtubule-based cytoskeleton. We propose that efficient erythroid enucleation in vivo involves signaling mechanisms inhibiting both GAKIN and KIF13A motors. Since erythrocytes from adult double mutant mice appear to be hematologically normal, our findings raise the possibility of enhancing the in vitro production of functional erythrocytes by inhibiting GAKIN and KIF13A activity in CD34 positive stem cells. Disclosures: No relevant conflicts of interest to declare.

GyrA Interacts with MarR To Reduce Repression of the marRAB Operon in Escherichia coli

ABSTRACT Bacterial two-hybrid studies of randomly cloned Escherichia coli DNA identified a physical interaction between GyrA, subunit A of gyrase, and MarR, a repressor of the marRAB operon. GyrA-His immobilized on Ni-nitrilotriacetic acid (NiNTA) resin bound MarR, while MarR alone did not bind. GyrA interfered with MarR binding to marO, as detected by electrophoretic mobility assays. In a strain bearing the marRAB operon and a marO-lacZ reporter, overexpression of GyrA increased LacZ activity, indicating decreased repression of marO-lacZ by MarR. These results were confirmed by an increased survival of cells treated with quinolones and other antibiotics when GyrA was overexpressed. This work, like a previous study examining TktA (12), shows that unrelated proteins can regulate MarR activity. The findings reveal an unexpected regulatory function of GyrA in antibiotic resistance.

Nuclear Factor 1 and T-Cell Factor/LEF Recognition Elements Regulate Pitx2 Transcription in Pituitary Development

ABSTRACT Pitx2, a paired-related homeobox gene that is mutated in Rieger syndrome I, is the earliest known marker of oral ectoderm. Pitx2 was previously shown to be required for tooth, palate, and pituitary development in mice; however, the mechanisms regulating Pitx2 transcription in the oral ectoderm are poorly understood. Here we used an in vivo transgenic approach to investigate the mechanisms regulating Pitx2 transcription. We identified a 7-kb fragment that directs LacZ expression in oral ectoderm and in many of its derivatives. Deletion analysis of transgenic embryos reduced this fragment to a 520-bp region that directed LacZ activity to Rathke's pouch. A comparison of the mouse and human sequences revealed a conserved nuclear factor 1 (NF-1) recognition element near a consensus T-cell factor (TCF)/LEF binding site. The mutation of either site individually abolished LacZ activity in transgenic embryos, identifying Pitx2 as a direct target of Wnt signaling in pituitary development. These findings uncover a requirement for NF-1 and TCF factors in Pitx2 transcriptional regulation in the pituitary and provide insight into the mechanisms controlling region-specific transcription in the oral ectoderm and its derivatives.