Ganglioside-embedding small bicelles for probing membrane-landing processes of intrinsically disordered proteins.

We successfully displayed a series of gangliosides on small bicelles with a uniform confined size, offering nanoscale standardized membrane mimics for spectroscopic characterization of weak encounter complexes formed between ganglioside clusters and amyloidogenic proteins. This enabled probing of initial membrane-landing processes of α-synuclein as a therapeutic target by NMR spectroscopy.

Transpupillary thermotherapy for atypical central serous chorioretinopathy.

BACKGROUND: Central serous chorioretinopathy (CSC) has been traditionally treated with laser photocoagulation. We thought that transpupillary thermotherapy (TTT) utilizing a lower temperature than that of conventional laser photocoagulation might minimize permanent retinal and choroidal damage. Studies suggest that undesirable effects on vision due to TTT are minimal even if it is applied to foveal and/or parafoveal lesions when TTT requires a larger irradiation spot. The aim of this study was to evaluate the efficacy of TTT in the management of atypical CSC. METHODS: We defined atypical CSC as bullous retinal detachment with diffuse or several leakages, severe leakage with fibrin formation under serous retinal detachment, or leakage within a pigment epithelium detachment. Eight consecutive patients with atypical CSC underwent visual acuity testing, ophthalmic examination, color photography, fluorescein angiography, and optical coherence tomography to evaluate the results of transpupillary thermotherapy. Retreatment of atypical CSC was based on ophthalmic examination, optical coherence tomography, and fluorescein angiography. TTT was performed on the leaking spots shown in fluorescein angiography, with a power of 50-250 mW, spot size of 500-1200 µm, and exposure time of 13-60 seconds to minimize retinal damage. RESULTS: In five of eight affected eyes, serous detachments completely resolved within 1 month after the initial TTT. One eye had persistent subretinal fluid and required a second TTT treatment. Two eyes showed no resolution of CSC and were treated by conventional photocoagulation. Initial best-corrected visual acuity (BCVA) ranged from 20/600 to 20/20 (mean, 20/40; median, 20/30). Final BCVA ranged from 20/200 to 20/20 (mean, 20/25; median, 20/20). BCVA improved in all cases. Only two eyes with persistent subretinal fibrin and existing retinal pigment epithelial alternations in macular area showed limited improvement of BCVA despite the absence of subretinal exudation. The presence of retinal attachment was confirmed by optical coherence tomography in six eyes (75%). CONCLUSIONS: TTT seems to be effective for the treatment of atypical CSC in the short term. Additional studies are necessary to evaluate the long-term effectiveness and safety.

Improved secretion of molecular chaperone-assisted human IgG in silkworm, and no alterations in their N-linked glycan structures.

Human 29IJ6 IgG was expressed in silkworm using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. The mean amounts of 296IJ6 IgG produced in larval hemolymph and whole pupae were 30.1 microg/larva and 78.0 microg/pupa, respectively. The use of molecular chaperones including calreticulin (CRT), calnexin (CNX), and immunoglobulin heavy chain binding protein (BiP, GRP78) improved the production of 296IJ6 IgG secretion in the larvae fivefold. The total yield of recombinant 29IJ6 IgG was 239 microg/mL when coexpressed with CRT. However, the overexpression of molecular chaperones had negative effects on secretion. The N-linked glycans of secreted 296IJ6 IgG in silkworm hemolymph were dominated by paucimannose structures. Small amounts of GlcNAc residues linked to the Manalpha1,3 branch were detected. When molecular chaperones were coexpressed, the compositions of N-linked glycans in the IgG1 produced were unchanged compared with those produced without them. This suggests that N-glycosylation is controlled by a regulatory function in the Golgi apparatus even though the post-translational modification of 296IJ6 IgG was assisted by the coexpression of molecular chaperones. Therefore, if the glycosylation pathways that coexpress N-acetylglucosaminyltransferase, galactosyltransferase, and sialyltransferase could be improved, silkworm larvae might prove a useful system for producing human antibodies.

Comparison of the N-linked glycosylation of human beta1,3-N-acetylglucosaminyltransferase 2 expressed in insect cells and silkworm larvae.

N-Glycosylation of human beta1,3N-acetylglucosaminyltransferase 2 (beta3GnT2) is essential for its biological function. beta3GnT2 fused to GFP(uv) (GFP(uv)-beta3GnT2) was produced by non-virus expression systems in stably transformed insect cells and silkworm larvae using a recombinant BmNPV bacmid, and purified for analysis of N-glycosylation. The N-glycan structure of beta3GnT2 was identified by glycoamidase A digestion, labeling with 2-aminopyridine (PA), and HPLC mapping. The paucimannosidic N-glycan structure (73.2%) was predominant in stably transformed Trichoplusia ni cells. In contrast, N-glycan with Gal (21.3%) and GlcNAc (16.2%) terminal residues linked to Manalpha(1,3) branch were detected on beta3GnT2 expressed in silkworm larvae. The presence of terminal Gal and bisecting GlcNAc residues such as Galbeta1, 4GlcNAcbeta1, 2Manalpha1,3(GlcNAcbeta1,4)(Manalpha1,6)Manbeta1, 4GlcNAc is not typical structure for lepidopteran insect N-glycosylation. Although allergenic alpha1,3-fucose residues have been found in T. ni cells, only alpha1,6-fucose residues were attached to the beta3GnT2 glycan in silkworm larvae. Therefore, silkworm larvae might be a useful host for producing human glycoproteins.

Accommodative loss after retinal cryotherapy.

PURPOSE: To investigate the effects of peripheral retinal cryotherapy on accommodative amplitude in patients with retinal lattice degeneration. DESIGN: Prospective, observational case series. METHODS: We studied 92 eyes in 69 patients (age range, 13 to 79 years) treated with cryotherapy for lattice degeneration between December 2001 and September 2004. Pretreatment and posttreatment accommodative amplitudes were measured. Acute accommodative loss was calculated from the difference between accommodative amplitudes before treatment and one week after treatment. We investigated the time course of accommodative amplitudes, acute accommodative loss in different age groups and in pretreatment accommodative amplitude groups, the influence of cryotherapy numbers on accommodative amplitude, and the influence of cryotherapy sites on accommodative amplitude. RESULTS: No significant difference was noted between pretreatment and posttreatment accommodative amplitudes in the overall subject cohort. Dividing subjects by age revealed significant decreases in accommodative amplitude only among patients in their 10s and 20s at one and three weeks after treatment. Accommodative amplitude was lowest among those in their 10s, followed by that among those in their 20s (P < .01). Accommodative amplitudes recovered to pretreatment level by six weeks. Acute accommodative loss was greatest in those in their 10s compared with other age groups (P < .01). A significant correlation was observed between acute accommodative loss and cryotherapy numbers (P = .03; r = 0.41). CONCLUSIONS: The decrease in accommodative amplitude was greatest at one week after treatment and recovered to pretreatment levels after six weeks. Accommodative amplitude showed the greatest decrease after cryotherapy among patients in their 10s and 20s. A decrease in accommodative amplitude was observed with increased numbers of cryotherapy spots administered.