Birefringence Imaging Directly Reveals Architectural Dynamics of Filamentous Actin in Living Growth ConesV⃞

We have investigated the dynamic behavior of cytoskeletal fine structure in the lamellipodium of nerve growth cones using a new type of polarized light microscope (the Pol-Scope). Pol-Scope images display with exquisite resolution and definition birefringent fine structures, such as filaments and membranes, without having to treat the cell with exogenous dyes or fluorescent labels. Furthermore, the measured birefringence of protein fibers in the thin lamellipodial region can be interpreted in terms of the number of filaments in the bundles. We confirmed that birefringent fibers are actin-based using conventional fluorescence-labeling methods. By recording movies of time-lapsed Pol-Scope images, we analyzed the creation and dynamic composition of radial fibers, filopodia, and intrapodia in advancing growth cones. The strictly quantitative information available in time-lapsed Pol-Scope images confirms previously deduced behavior and provides new insight into the architectural dynamics of filamentous actin.

Architectural limits on split genes

Exon/intron architecture varies across the eukaryotic kingdom with large introns and small exons the rule in vertebrates and the opposite in lower eukaryotes. To investigate the relationship between exon and intron size in pre-mRNA processing, internally expanded exons were placed in vertebrate genes with small and large introns. Both exon and intron size influenced splicing phenotype. Intron size dictated if large exons were efficiently recognized. When introns were large, large exons were skipped; when introns were small, the same large exons were included. Thus, large exons were incompatible for splicing if and only if they were flanked by large introns. Both intron and exon size became problematic at ≈500 nt, although both exon and intron sequence influenced the size at which exons and introns failed to be recognized. These results indicate that present-day gene architecture reflects at least in part limitations on exon recognition. Furthermore, these results strengthen models that invoke pairing of splice sites during recognition of pre-mRNAs, and suggest that vertebrate consensus sequences support pairing across either introns or exons.

The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerization.

The histones of all eukaryotes show only a low degree of primary structure homology, but our earlier crystallographic results defined a three-dimensional structural motif, the histone fold, common to all core histones. We now examine the specific architectural patterns within the fold and analyze the nature of the amino acid residues within its functional segments. The histone fold emerges as a fundamental protein dimerization motif while the differentiations of the tips of the histone dimers appear to provide the rules of core octamer assembly and the basis for nucleosome regulation. We present evidence for the occurrence of the fold from archaebacteria to mammals and propose the use of this structural motif to define a distinct family of proteins, the histone fold superfamily. It appears that evolution has conserved the conformation of the fold even through variations in primary structure and among proteins with various functional roles.

Carbohydrate gluing, an architectural mechanism in the supramolecular structure of an annelid giant hemoglobin.

We report a carbohydrate-dependent supramolecular architecture in the extracellular giant hemoglobin (Hb) from the marine worm Perinereis aibuhitensis; we call this architectural mechanism carbohydrate gluing. This study is an extension of our accidental discovery of deterioration in the form of the Hb caused by a high concentration of glucose. The giant Hbs of annelids are natural supramolecules consisting of about 200 polypeptide chains that associate to form a double-layered hexagonal structure. This Hb has 0.5% (wt) carbohydrates, including mannose, xylose, fucose, galactose, glucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). Using carbohydrate-staining assays, in conjunction with two-dimensional polyacrylamide gel electrophoresis, we found that two types of linker chains (L1 and L2; the nomenclature of the Hb subunits followed that for another marine worm, Tylorrhynchus heterochaetus) contained carbohydrates with both GlcNAc and GalNAc. Furthermore, two types of globins (a and A) have only GlcNAc-containing carbohydrates, whereas the other types of globins (b and B) had no carbohydrates. Monosaccharides including mannose, fucose, glucose, galactose, GlcNAc, and GalNAc reversibly dissociated the intact form of the Hb, but the removal of carbohydrate with N-glycanase resulted in irreversible dissociation. These results show that carbohydrate acts noncovalently to glue together the components to yield the complete quaternary supramolecular structure of the giant Hb. We suggest that this carbohydrate gluing may be mediated through lectin-like carbohydrate-binding by the associated structural chains ("linkers").

Comparative analysis of the relationship between anterior and posterior corneal shape analyzed by Scheimpflug photography in normal and keratoconus eyes

Background To analyze and compare the relationship between anterior and posterior corneal shape evaluated by a tomographic system combining the Scheimpflug photography and Placido-disc in keratoconus and normal healthy eyes, as well as to evaluate its potential diagnostic value. Methods Comparative case series including a sample of 161 eyes of 161 subjects with ages ranging from 7 to 66 years and divided into two groups: normal group including 100 healthy eyes of 100 subjects, and keratoconus group including 61 keratoconus eyes of 61 patients. All eyes received a comprehensive ophthalmologic examination including an anterior segment analysis with the Sirius system (CSO). Antero-posterior ratios for corneal curvature (k ratio) and shape factor (p ratio) were calculated. Logistic regression analysis was used to evaluate if some antero–posterior ratios combined with other clinical parameters were predictors of the presence of keratoconus. Results No statistically significant differences between groups were found in the antero–posterior k ratios for 3-, 5- and 7-mm diameter corneal areas (p ≥ 0.09). The antero–posterior p ratio for 4.5- and 8-mm diameter corneal areas was significantly higher in the normal group than in the keratoconus group (p < 0.01). The k ratio for 3, 5, and 7 mm was significantly higher in the keratoconus grade IV subgroup than in the normal group (p < 0.01). Furthermore, significant differences were found in the p ratio between the normal group and the keratoconus grade II subgroup (p ≤ 0.01). Finally, the logistic regression analysis identified as significant independent predictors of the presence of keratoconus (p < 0.01) the 8-mm anterior shape factor, the anterior chamber depth, and the minimal corneal thickness. Conclusions The antero-posterior k and p ratios are parameters with poor prediction ability for keratoconus, in spite of the trend to the presence of more prolate posterior corneal surfaces compared to the anterior in keratoconus eyes.