Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein

NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P(270)KKRKAP(276)) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.     

Oxidative modification of aldose reductase induced by copper ion. Definition of the metal-protein interaction mechanism

Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys(303) appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys(298)-Cys(303). However, a Cys(80)-Cys(303) disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys(80)-Cys(303) disulfide to the more stable product Cys(298)-Cys(303) is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys(80)-Cys(303) disulfide. However, no evidence of conditions favoring the formation of the Cys(298)-Cys(303) disulfide was observed. Our proposal is that the generation of the Cys(298)-Cys(303) disulfide, either directly or by rearrangement of the Cys(80)-Cys(303) disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Giemsa-based cytological assessment of area,shape and nucleus:cytoplasm ratio of goblet cells of rabbit bulbar conjunctiva

Goblet cells were visualized in impression cytology specimens from bulbar conjunctiva of the rabbit eye using Giemsa staining. Highly magnified images were used to generate outlines of the goblet cells and their characteristic eccentric nuclei. Using sets of 10 cells from 15 cytology specimens, I found that the longest dimension of the goblet cells averaged 16.7 ± 2.3 μm, the shortest dimension averaged 14.4 ± 1.8 μm and the nucleus averaged 6.3 ± 0.8 μm. The goblet cells were ellipsoid in shape and the longest:shortest cell dimension ratio averaged 1.169 ± 0.091. The goblet cell areas ranged from 108 to 338 μm² (average 193 ± 50 μm²). The area could be predicted reliably from the longest and shortest dimensions (r² = 0.903). The areas of goblet cell nuclei were 15–58 μm² (average 33 ± μm²) and the nucleus:cytoplasm area fraction was predictably greater in smaller goblet cells and less in the larger goblet cells (Spearman correlation = 0.817). The nuclei were estimated to occupy an average of 9.5% of the cell volume. The differences in size, shape and nucleus:cytoplasm ratio may reflect differences in goblet cell maturation.