Risk of aphakic glaucoma after pars plana-lensectomy with and without removal of the peripheral lens capsule.

BACKGROUND: The etiology of aphakic glaucoma is unclear. It has been suggested that remaining lens epithelium releases cytokines transducing trabecular meshwork cells. Therefore, we compared two cohorts of children undergoing lensectomy. In cohort 1, the entire lens including its capsule was removed, in cohort 2 the peripheral lens capsule was left intact, also to facilitate secondary intraocular lens implantation later on. METHODS: We included children with uni- or bilateral congenital cataract who underwent lensectomy during the first year of life with subsequent contact lenses fitting. Group 1 comprised 41 eyes, group 2 comprised 33 eyes. In group 1, the median age at surgery was 4.0 months in unilateral and 3.0 months in bilateral cases 1, in group 2, 8.1 months and 2.4 months, respectively. The mean follow-up was 12.8 years in group 1 and 9.3 years in group 2. All cases were analyzed for the prevalence of aphakic glaucoma, for visual acuity and for compliance in visual rehabilitation (contact lens/occlusion therapy). RESULTS: We found no significant difference in glaucoma prevalence between group 1 and group 2 (p = 0.68). The overall glaucoma rate was 26% after the mean follow-up of 11 years in both groups. In unilateral cases, the median visual acuity was logMAR 0.7 in both groups. In bilateral cases it was logMAR 0.4 in group 1 and logMAR 0.2 in group 2 (p = 0.05). CONCLUSIONS: Leaving the peripheral lens capsule intact had no negative effect on the incidence of glaucoma and on resulting visual acuity.

Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates.

Antibodies are indispensable tools for basic research as well as diagnostic and therapeutic applications. Consequently, the development of alternative manufacturing strategies which circumvent the hurdles connected to conventional antibody production technologies is of enormous interest. To address this issue, we demonstrate the synthesis of complex antibody formats, in particular immunoglobulin G (IgG) and single-chain variable fragment Fc fusion (scFv-Fc), in a microsome-containing cell-free system based on translationally active chinese hamster ovary (CHO) cell lysates. To mimic the environment for antibody folding and assembly present in living cells, antibody genes were fused to an endoplasmic reticulum (ER)-specific signal sequence. Signal-peptide induced translocation of antibody polypeptide chains into the lumen of ER microsomes was found to be the prerequisite for antibody chain assembly and functionality. In this context, we show the rapid synthesis of antibody molecules in different reaction formats, including batch and continuous-exchange cell-free (CECF) reactions, depending on the amount of protein needed for further analysis. In addition, we demonstrate site-specific and residue-specific labeling of antibodies with fluorescent non-canonical amino acids. In summary, our study describes a novel antibody production platform which combines the highly efficient mammalian protein folding machinery of CHO cells with the benefits of cell-free protein synthesis.

Bryophyte DNA sequences from faeces of an arctic herbivore, barnacle goose (Branta leucopsis).

We tested DNA extraction methods and PCR conditions for the amplification of bryophyte DNA from barnacle goose (Branta leucopsis) faeces collected from Spitsbergen (Svalbard). Both the Qiagen stool kit and a silica-based extraction method received sufficient DNA from fresh and older droppings, as indicated by successful amplification of the plastid psbA-trnH spacer. Standard Taq polymerase outperformed two hot start polymerases. Sequencing of cloned PCR products revealed at least ten moss and two angiosperm sequences. This first example of identifying bryophyte DNA from faeces will allow analysing moss diets of arctic herbivores with a DNA barcoding approach.

Molecular evolution of the trnTUGU-trnFGAA region in Bryophytes.

Structure, variability, and molecular evolution of the trnT-F region in the Bryophyta (mosses and liverworts) is analyzed based on about 200 sequences of the trnT-L spacer and trnL 5' exon, 1000 sequences of the trnL intron, and 800 sequences of the trnL 3' exon and trnL-F spacer, including comparisons of lengths, GC contents, sequence similarities, and functional elements. Mutations occurring in the trnL 5' and 3' exons, including compensatory base pair changes, and a transition in the trnL anticodon in Takakia lepidozioides, are discussed. All three non-coding regions display a mosaic structure of highly variable elements (V1 - V3 in the trnT-L spacer, V4/V5 corresponding to stem-loop regions P6/P8 in the trnL intron, and V6/V7 in the trnL-F spacer) and more conserved elements. In the trnL intron this structure is a consequence of the defined secondary structure necessary for correct splicing, whereas in both spacers conserved regions are restricted to promoter elements. At least the highly variable regions in the trnT-L spacer and stem-loop region P8 of the trnL intron seem to evolve independently in the major bryophyte lineages and are therefore not suitable for high taxonomic level phylogenetic reconstructions. In mosses, a trend of length reduction towards the more derived lineages is observed in all three non-coding regions. GC contents are mostly linked to sequence variability, with the conserved regions being more GC rich and the more variable AT rich. The lowest GC values (< 10 %) are found in the trnT-L spacer of mosses. In addition to two putative sigma (70)-type promoters in the trnT-L spacer, a third putative promoter is present in the trnL-F spacer, although trnL and trnF are assumed to be co-transcribed. Consensus sequences are provided for the -35 and -10 sequences of the major bryophyte lineages. The third promoter is part of a hairpin secondary structure, whose loop region is highly homoplastic in mosses due to an inversion occurring independently in non-related taxa, even at the intraspecific level.

Sequence, molecular properties, and chromosomal mapping of mouse lumican.

PURPOSE: Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization. METHODS: Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross. RESULTS: Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology. CONCLUSIONS: This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene.

Characterization and chromosomal localization of the cornea-specific murine keratin gene Krt1.12.

Keratins are a group of water-insoluble proteins constituting paired acidic and basic keratin molecules that form 10-nm intermediate filaments in epithelial cells. Expression of the K3/K12 keratin pair characterizes the cornea-type differentiation. However, the mechanism that regulates this cornea-specific K12 expression remains unknown. To provide a better understanding of the cornea-specific expression, we have cloned the K12 cDNA (Liu, C.-Y., Zhu, G., Westerhausen-Larson, A., Converse, R., Kao, C. W.-C., Sun, T.-T., and Kao, W. W.-Y. (1993) Curr. Eye Res. 12, 963-974). In present studies, the murine K12 keratin gene (Krt1.12) was isolated and characterized. The murine Krt1.12 gene spans 6,567 base pairs of genomic DNA, and the mRNA encoding K12 keratin is distributed into eight exons. Chromosome mapping reveals that murine Krt1.12 is located within the Krt1 complex of mouse chromosome 11. In addition to the production of authentic K12 mRNA, the Krt1.12 gene gives rise to several alternate poly(A)+ RNAs by the use of alternative splicing in intron 2, an alternative promoter in intron 1, and/or both. Sequence analysis indicates that the transcripts derived from alternative splicing and/or the alternative promoter do not have a long open reading frame for keratin or keratin-like molecules. It is not known whether these alternate K12 poly(A)+ RNAs have any biological functions, e.g. regulation of K12 gene expression.