ELKS, a protein structurally related to the active zone-associated protein CAST, is expressed in pancreatic beta cells and functions in insulin exocytosis: interaction of ELKS with exocytotic machinery analyzed by total internal reflection fluorescence microscopy

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic beta cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic beta cells.     

Enhanced gene expression in lung by a stabilized lipoplex using sodium chloride for complex formation

BACKGROUND: In this study, we investigated the in vivo gene transfection efficacy of a 'surface charge regulated' (SCR) lipoplex, dispersed in the presence of an essential amount of NaCl during lipoplex formation. METHODS: SCR lipoplexes were prepared and their physicochemical properties were analyzed. After intravenous (i.v.) administration, transfection efficacy, distribution characteristics, and liver toxicity were evaluated in mice. RESULTS: At NaCl concentrations of 10 mM, the particle sizes of the SCR lipoplexes were about 120 nm and were compatible with a conventional lipoplex. However, fluorescent resonance energy transfer analysis revealed that cationic liposomes in the SCR lipoplexes increased fusion. After i.v. administration, the transfection activity in the lung of the SCR lipoplex (10 mM NaCl solution in the lipoplex) was approximately 10-fold higher than that of the conventional lipoplex. Pharmacokinetic studies demonstrated a higher distribution in lung by the SCR lipoplex. When the gene expression levels of the SCR lipoplex and conventional lipoplex were compared, the SCR lipoplex at a dose of 30 microg was compatible with that of the conventional lipoplex at a dose of 50 microg. A significantly higher serum alanine aminotransferase (ALT) activity and TNFalpha concentration was observed by the conventional lipoplex (pDNA dose; 50 microg), but this was not the case for the SCR lipoplex (pDNA dose; 30 microg). CONCLUSIONS: We demonstrated that the SCR lipoplex could enhance the transfection efficacy in the lung without increasing the liver toxicity. Hence, the information will be valuable for the future use, design, and development of lipoplexes for in vivo applications.     

Intersubunit interaction induced by subunit rearrangement is essential for the catalytic activity of the hyperthermophilic glutamate dehydrogenase from Pyrobaculum islandicum

The specific activity of recombinant Pyrobaculum islandicum glutamate dehydrogenase (pis-GDH) expressed in Escherichia coli is much lower than that of the native enzyme. However, when the recombinant enzyme is heated at 90 degrees C or exposed to 5 M urea, the activity increases to a level comparable to that of the native enzyme. Small-angle X-ray scattering measurements revealed that the radius of gyration (R(g,z)) of the hexameric recombinant enzyme was reduced to 47 A from 55 A by either heat or urea, and that the final structure of the active enzyme is the same irrespective of the mechanism of activation. Activation was accompanied by a shift in the peaks of the Kratky plot, though the molecular mass of the enzyme was unchanged. The activation-induced decline in R(g,z) followed first-order kinetics, indicating that activation of the enzyme involved a transition between two states, which was confirmed by singular-value decomposition analysis. When the low-resolution structure of the recombinant enzyme was restored using ab initio modeling, we found it to possess no point symmetry, whereas the heat-activated enzyme possessed 32-point symmetry. In addition, a marked increase in the fluorescence emission was observed with addition of ANS to the inactive recombinant enzyme but not the active forms, indicating that upon activation hydrophobic residues on the surface of the recombinant protein moved to the interior. Taken together, these data strongly suggest that subunit rearrangement, i.e., a change in the quaternary structure of the hexameric recombinant pis-GDH, is essential for activation of the enzyme.     

Nonaggregating refolding of ribonuclease A using reverse micellar dialysis

A hydrophilic ultrafiltration membrane, regenerated cellulose, facilitates the size-selectable permeability of hydrophilic solutes in reverse micellar solution. By using an ultrafiltration membrane with a molecular weight cutoff of 3,500, we demonstrate a nonaggregating protein refolding technique based on the dialysis of reverse micellar solution. This realizes concurrent removal of denaturants, urea and 2-mercaptoethanol, and the supply of redox reagents, reduced and oxidized glutathione (GSH, GSSG), to promote renaturation of proteins. Two mg/ml ribonuclease A (RNase A) was refolded completely without any dilution and aggregation for 60 h. The refolding behavior of RNase A is strongly influenced by the ratio of GSH and GSSG. Moreover, we recovered 90% of the refolded RNase A from AOT reverse micellar solution with acetone precipitation and beta-cyclodextrin washing. These findings should facilitate the production of a continuous protein refolding membrane reactor.     

Inhibition of the RhoA/Rho kinase system attenuates catecholamine biosynthesis in PC 12 rat pheochromocytoma cells

The small GTPase, RhoA, and its downstream effecter Rho-kinase (ROK) are reported to be involved in various cellular functions, such as myosin light chain phosphorylation during smooth muscle contraction and exocytosis. Indeed, growing evidence suggests that the RhoA/Rho-kinase pathway plays an important role in regulating exocytosis in these cells. However, it is not known whether the RhoA/Rho-kinase pathway has an effect on catecholamine synthesis. Using the rat pheochromocytoma cell line, PC12, we examined the effects of either Rho-kinase inhibitor (Y27632) or RhoA inhibitor (C3 toxin) on nicotine-induced catecholamine biosynthesis. We show that nicotine (10 microM) induces a significant, though transient, increase in RhoA activation in these cells. Treatment with either Y27632 (1 microM) or C3 toxin (10 microg/ml) significantly inhibited the nicotine-induced increase of tyrosine hydroxylase (TH) mRNA and the corresponding enzyme activity. TH catalyzes the rate-limiting step in the biosynthesis of catecholamine. Y27632 significantly inhibited nicotine-induced phosphorylation of TH at Ser40 as well as Ser19, which are known to be phosphorylated by Ca(2+)/calmodulin kinase II. Furthermore, Y27632 (10 microM) as well as C3 toxin (10 microg/ml) significantly inhibited the nicotine-induced increase of TH at the protein level. Thus, we propose that activation of RhoA, and its downstream effecter Rho-kinase, is a prerequisite for catecholamine biosynthesis in PC12 cells. At the concentrations used in our experiments, Y27632 does not affect cAMP/PKA activity or PKC activity, indicating that the inhibitory effect of Y27632 can be attributed to the inhibition of Rho-kinase activity as observed in chromaffin cells. In contrast, neither Y27632 (10 microM) nor C3 toxin (10 microg/ml) significantly altered catecholamine secretion in PC12 cells. In conclusion, we have demonstrated that inhibition of the Rho/Rho-kinase pathway in chromaffin cells lowers TH activity, probably through CaMKII inhibition. By contrast, neither Y27632 nor C3 toxin affect the secretion of catecholamine.     

Sclerosing polycystic adenosis of the left parotid gland: report of a case with fine needle aspiration cytology

BACKGROUND: Sclerosing polycystic adenosis (SPCA) of major salivary glands is a rare recently described entity. We report a case of SPCA of the left parotid gland, including the cytologic and histopathologic findings. CASE: A 20-year-old man presented with a left parotid mass that had been growing slowly for 3 years. Fine needle aspiration cytology showed many syncytial cell clusters of variable size and some ductal structures with an inflammatory background. The cells forming syncytial clusters were large and polygonal, with abundant, eosinophilic, granular or lacelike cytoplasm. Apocrine differentiation with decapitation secretion was commonly seen. The ductal cells had a relatively high nuclear/cytoplasmic ratio, with granular cytoplasm. Grossly, the 5-cm lesion was a discrete, pale, cystic nodule embedded within the parotid gland parenchyma. Microscopically, the lesion was a nonencapsulated, circumscribed mass of sclerotic and hyalinized, collagenous tissue with lymphoplasmacytic infiltration. Sclerosing adenosis and cystic ducts with frequent apocrinelike cells were commonly seen. Some acinar cells contained eosinophilic, intracytoplasmic granules of various sizes. CONCLUSION: The presence of syncytial clusters with apocrine metaplasia and ductal structures in a lymphoplasmacytic background should suggest a diagnosis of SPCA of a major salivary gland.     

Novel archaeal alanine:glyoxylate aminotransferase from Thermococcus litoralis

A novel alanine:glyoxylate aminotransferase was found in a hyperthermophilic archaeon, Thermococcus litoralis. The amino acid sequence of the enzyme did not show a similarity to any alanine:glyoxylate aminotransferases reported so far. Homologues of the enzyme appear to be present in almost all hyperthermophilic archaea whose whole genomes have been sequenced.     

Six4, a putative myogenin gene regulator, is not essential for mouse embryonal development

Six4 is a member of the Six family genes, homologues of Drosophila melanogaster sine oculis. The gene is thought to be involved in neurogenesis, myogenesis, and development of other organs, based on its specific expression in certain neuronal cells of the developing embryo and in adult skeletal muscles. To elucidate the biological roles of Six4, we generated Six4-deficient mice by replacing the Six homologous region and homeobox by the beta-galactosidase gene. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of the heterozygous mutant embryos revealed expression of Six4 in cranial and dorsal root ganglia, somites, otic and nasal placodes, branchial arches, Rathke's pouch, apical ectodermal ridges of limb buds, and mesonephros. The expression pattern was similar to that of Six1 except at the early stage of embryonic day 8.5. Six4-deficient mice were born according to the Mendelian rule with normal gross appearance and were fertile. No hearing defects were detected. Six4-deficient embryos showed no morphological abnormalities, and the expression patterns of several molecular markers, e.g., myogenin and NeuroD3 (neurogenin1), were normal. Our results indicate that Six4 is not essential for mouse embryogenesis and suggest that other members of the Six family seem to compensate for the loss of Six4.