Mucor hiemalis endo-beta-N-acetylglucosaminidase can transglycosylate a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide

We found that the recombinant endo-beta-N-acetylglucosaminidase of Mucor hiemalis (Endo-M) expressed in Candida boidinii had the transglycosylation activity of transferring a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide to the acceptor (p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside) in a good yield of 43%.     

Thermal equilibrium of two conformations in photosensitive nitrile hydratase probed by the FTIR band of nitric oxide bound to the non-heme iron center

Nitrile hydratase (NHase) from Rhodococcus N-771 is a novel enzyme that is inactive in the dark due to an enodogenous nitric oxide (NO) molecule bound to the non-heme iron center, and is activated by its photodissociation. FTIR spectra in the NO stretching region of the dark-inactive NHase were recorded in the temperature range of 270-80 K. Two NO peaks were observed at 1854 and 1846 cm-1 at 270 K, and both frequencies upshifted as the temperature was lowered, retaining the peak separation of 8-9 cm-1. The relative intensity of the lower-frequency peak increased with decreasing temperature up to ~120 K, whereas it was mostly unchanged below this temperature. This observation indicates that two distinct conformations with slightly different NO structures are thermally equilibrated in the dark-inactive NHase above ~120 K, and the interconversion is frozen-in at lower temperatures. The intensity ratio of the NO bands changed gradually upon increasing the pH from 5.5 to 11.0, but no specific pKa value was found. This result, together with the comparison of the light-induced FTIR difference spectra measured at pH 6.5 and 9.0, suggests that the protonation/deprotonation of a specific amino acid group in the active site of NHase is not a direct cause of the occurrence of the two conformations, although several protonatable groups in the protein may influence the energetics of the two conformers. From the previous observation that the isolated alpha subunit of NHase exhibited a single broad NO peak, it is suggested that interaction of the beta subunit forming the reactive cavity is essential for the double-minimum potential of the active-site structure. The frequencies and widths of the two NO bands changed upon addition of propionamide, 1,4-dioxane, and cyclohexyl isocyanide, indicating that these compounds are bound to the active pocket and change the interactions of the iron center or the dielectric environments around the NO molecule. Thus, the NO bands of NHase can also be a useful probe to monitor the binding of substrates and their analogues to the active pocket.     

Subcellular localization of fukutin and fukutin-related protein in muscle cells

Fukuyama-type congenital muscular dystrophy and congenital muscular dystrophy 1C are congenital muscular dystrophies that commonly display reduced levels of glycosylation of alpha-dystroglycan in skeletal muscle. The genes responsible for these disorders are fukutin and fukutin-related protein (FKRP), respectively. Both gene products are thought to be glycosyltransferases, but their functions have not been established. In this study, we determined their subcellular localizations in cultured skeletal myocytes. FKRP localizes in rough endoplasmic reticulum, while fukutin localizes in the cis-Golgi compartment. FKRP was also localized in rough endoplasmic reticulum in skeletal muscle biopsy sample. Our data suggest that fukutin and FKRP may be involved at different steps in O-mannosylglycan synthesis of alpha-dystroglycan, and FKRP is most likely involved in the initial step in this synthesis.     

Resistance of B16 melanoma cells to CD47-induced negative regulation of motility as a result of aberrant N-glycosylation of SHPS-1

The adhesion receptor SHPS-1 activates the protein-tyrosine-phosphatase SHP-2 and thereby promotes integrin-mediated reorganization of the cytoskeleton. SHPS-1 also contributes to cell-cell communication through association with CD47. Although functional alteration of SHPS-1 is implicated in cellular transformation, the role of the CD47-SHPS-1 interaction in carcinogenesis has been unclear. A soluble SHPS-1 ligand (CD47-Fc) has now been shown to bind to Melan-a non-tumorigenic melanocytes but not to syngeneic B16F10 melanoma cells. Treatment of B16F10 cells with 1-deoxymannojirimycin, which prevents N-glycan processing, restored the ability of SHPS-1 derived from these cells to bind CD47-Fc in vitro, indicating that aberrant N-glycosylation of SHPS-1 impairs CD47 binding in B16F10 cells. CD47-Fc inhibited the migration of Melan-a cells but not that of B16F10 cells. However, a monoclonal antibody that reacts with SHPS-1 on both Melan-a and B16F10 cells inhibited the migration of both cell types similarly. CD47 binding induced proteasome-mediated degradation of SHPS-1 in a tyrosine phosphorylation-independent manner. Furthermore, overexpression of SHPS-1 reduced the level of tyrosine phosphorylation of focal adhesion kinase, and this effect was reversed by CD47 binding. These results suggest that CD47 binds to and thereby down-regulates SHPS-1 on adjacent cells, resulting in inhibition of cell motility. Resistance to this inhibitory mechanism may contribute to the highly metastatic potential of B16 melanoma.     

73-kDa molecular chaperone HSP73 is a direct target of antibiotic gentamicin

Although gentamicin (GM) has been used widely as an antibiotic, the specific binding protein of the drug has not yet been understood sufficiently. Here we show that GM specifically associates with the 73-kDa molecular chaperone HSP73 and reduces its chaperone activity in vitro. In the present study, we investigated GM-specific binding proteins using a GM-affinity column and porcine kidney cytosol. After washing the column, only the 73-kDa protein was eluted from the column by the addition of 10 mm GM. None of the other proteins were found in the eluant. Upon immunoblotting, the protein was identical to HSP73. Upon CD spectrum analysis, the binding of GM to HSP73 resulted in a conformational change in the protein. Although HSP73 prevents aggregation of unfolded rhodanese in vitro, the chaperone activity of HSP73 was suppressed in the presence of GM. Using limited proteolysis of HSP73 by TPCK-trypsin, the GM binding site is a COOH-terminal for one third of the protein known to be a peptide-binding domain. During immunohistochemistry, HSP73 and GM were co-localized in enlarged lysosomes of rat kidneys with GM-induced acute tubular injury in vivo. Our results suggest that the specific association between HSP73 and GM may reduce the chaperone activity of HSP73 in vitro and/or in vivo, and this may have an interaction with GM toxicity in kidneys with GM-induced acute tubular injury.     

Cytologic characteristics of pulmonary papillary adenoma. A case report

BACKGROUND: Pulmonary papillary adenoma is a benign pulmonary neoplasm. Previously pulmonary papillary adenoma was described in terms of immunohistochemistry and ultrastructure. However, there are no previous reports describing the cytologic characteristics of pulmonary papillary adenoma. CASE: A 50-year-old male was admitted for evaluation of a coin lesion in the left upper lung field. Radiologic images showed a solid, round tumor approximately 25 mm in diameter in the left upper lung. Transbronchial needle aspiration biopsy (TBNA) was performed, and small numbers of atypical cells were collected. Adenocarcinoma was suggested clinically, and left upper segmentectomy was performed. The histologic diagnosis was pulmonary papillary adenoma. Imprint cytology of the cut surface of the tumor showed tumor cells arranged in sheets that contained scant or vesicular cytoplasm. The nuclei were oval or round, without obvious anisokaryosis, and their chromatin was fine, without hyperchromasia. Cytologically, the nuclei of the tumor cells in the imprint specimen (38.70 +/- 8.69 microns 2) were uniform in size and similar to the atypical cells in the TBNA specimen (38.29 +/- 11.56 microns 2) but significantly larger than the nuclei of the bronchial cells (23.61 +/- 5.98 microns 2) (P < .0001). CONCLUSION: The cytologic appearance of pulmonary papillary adenoma was characterized morphologically and morphometrically. The possibility of cytodiagnosis by TBNA was suggested.     

Ubiquitination-mediated regulation of biosynthesis of the adhesion receptor SHPS-1 in response to endoplasmic reticulum stress

Misfolding of proteins during endoplasmic reticulum (ER) stress results in the formation of cytotoxic aggregates. The ER-associated degradation pathway counteracts such aggregation through the elimination of misfolded proteins by the ubiquitin-proteasome system. We now show that SHP substrate-1 (SHPS-1), a transmembrane glycoprotein that regulates cytoskeletal reorganization and cell-cell communication, is a physiological substrate for the Skp1-Cullin1-NFB42-Rbx1 (SCF(NFB42)) E3 ubiquitin ligase, a proposed mediator of ER-associated degradation. SCF(NFB42) mediated the polyubiquitination of immature SHPS-1 and its degradation by the proteasome. Ectopic expression of NFB42 both suppressed the formation of aggresome-like structures and the phosphorylation of the translational regulator eIF2alpha induced by overproduction of SHPS-1 as well as increased the amount of mature SHPS-1 at the cell surface. An NFB42 mutant lacking the F box domain had no such effects. Our results suggest that SCF(NFB42) regulates SHPS-1 biosynthesis in response to ER stress.     

Urocortins and corticotropin releasing factor type 2 receptors in the hypothalamus and the cardiovascular system

In addition to urocortin (Ucn I), Ucn II and Ucn III were identified as endogenous ligands for corticotropin-releasing factor type 2 receptor (CRF2 receptor). CRF2 receptor is abundantly located in central hypothalamic ventromedial nucleus (VMH) and in peripheral cardiovascular system. In this mini-review, we focused on the roles of these urocortins and CRF2 receptor in the hypothalamus and the cardiovascular system. Ucn II mRNA was increased in the parvocellular part or the magnocellular part of the hypothalamic paraventricular nucleus (PVN) following immobilization stress or 3 days of water deprivation, respectively. Therefore, it is thought that Ucn II may modulate CRF and vasopressin synthesis in the PVN in a paracrine or autocrine fashion through PVN CRF2 receptor. The early and later phases of Ucn I-mediated feeding suppression may be CRF1 and CRF2 receptor-mediated events, respectively. Ucn II decreases food intake at a later phase, beyond 4 h post injection. A large dose of corticosterone increased plasma leptin and insulin levels as well as the levels of CRF2 receptor mRNA. Adrenalectomy, starvation, and immobilization each lowered plasma leptin and insulin levels and were associated with decrements in CRF2 receptor mRNA levels in the VMH. Peripheral injection of leptin increased VMH CRF2 receptor mRNA, as can induce reductions of food intake and body weight, indicating that circulating leptin is involved in the regulation of VMH CRF2 receptor mRNA expression. Therefore, it is also plausible that VMH CRF2 receptor transduces the anorexogenic effects of leptin as well as those of urocortins. The systemic administration of Ucn II decreases mean arterial pressure (arterial vascular tone) and causes tachycardia via vascular CRF2 receptor in rats, similar to the effects of Ucn I. Thus, CRF2 receptor seems to mediate cardioprotective effects of urocortins.     

Swi1 and Swi3 are components of a replication fork protection complex in fission yeast

Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3(+). The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Delta and swi3Delta mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Delta or swi3Delta cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.