Green fluorescent protein tag for studies of drug-induced translocation of nucleolar protein RH-II/Gu

We have constructed a human osteogenic sarcoma cell line, U-2 OS/GFP-Gu, that expresses nucleolar RNA helicase RH-II/Gu tagged with green fluorescent protein (GFP). The presence of a GFP tag does not inhibit RNA helicase, RNA folding and ATPase activities of RH-II/Gu protein. The derived cell line responds to cytotoxic agents like the parental cell line U-2 OS. In the presence of either actinomycin D or toyocamycin, the GFP-RH-II/Gu fusion protein translocates from the nucleolus to the nucleoplasm in the same way as the translocation of endogenous RH-II/Gu. The drug-induced translocation of GFP-RH-II/Gu is easily monitored by direct observation of live cells in vivo. This cell line can be used to screen cytotoxic drugs and to study the mechanisms of drug-induced translocation of RH-II/Gu. The cellular localization of RH-II/Gu during the cell cycle-dependent formation of the nucleolus is readily monitored. Real-time results are obtained more quickly without the disadvantages associated with cell fixation and immunofluorescence-based staining.     

Strengthening immature teeth during and after apexification

Idiopathic arterial calcification of infancy is a rare condition characterized by extensive arterial calcification and stenoses of large and medium sized arteries. We report the sonographic and magnetic resonance angiographic findings of this entity and correlate them with the findings at autopsy.     

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.     

Toxic shock syndrome complicating orthopaedic manipulation of bone. A report of two cases

We studied prospectively 81 consecutive patients undergoing hip surgery using the Hardinge (1982) approach. The abductor muscles of the hip in these patients were assessed electrophysiologically and clinically by the modified Trendelenburg test. Power was measured using a force plate. We performed assessment at two weeks, and at three and nine months after operation. At two weeks we found that 19 patients (23%) showed evidence of damage to the superior gluteal nerve. By three months, five of these had recovered. The nine patients with complete denervation at three months showed no signs of recovery when reassessed at nine months. Persistent damage to the nerve was associated with a positive Trendelenburg test.     

Cloning and localization of two multigene receptor families in goldfish olfactory epithelium

Goldfish reproduction is coordinated by pheromones that are released by ovulating females and detected by males. Two highly potent pheromones, a dihydroxyprogesterone and a prostaglandin, previously have been identified, and their effects on goldfish behavior have been studied in depth. We have cloned goldfish olfactory epithelium cDNAs belonging to two multigene G-protein coupled receptor families as a step toward elucidating the molecular basis of pheromone recognition. One gene family (GFA) consists of homologs of putative odorant receptors (approximately 320 residues) found in the olfactory epithelium of other fish and mammals. The other family (GFB) consists of homologs of putative pheromone receptors found in the vomeronasal organ (VNO) of mammals and also in the nose of pufferfish. GFB receptors (approximately 840 residues) are akin to the V2R family of VNO receptors, which possess a large extracellular N-terminal domain and are homologs of calcium-sensing and metabotropic glutamate receptors. In situ hybridization showed that the two families of goldfish receptors are differentially expressed in the olfactory epithelium. GFB mRNA is abundant in rather compact cells whose nuclei are near the apical surface. In contrast, GFA mRNA is found in elongated cells whose nuclei are positioned deeper in the epithelium. Our findings support the hypothesis that the separate olfactory organ and VNO of terrestrial vertebrates arose in evolution by the segregation of distinct classes of neurons that were differentially positioned in the olfactory epithelium of a precursor aquatic vertebrate.