Comparative observations on corneas,with special reference to Bowman's layer and Descemet's membrane in mammals and amphibians

Corneas of tadpole, mouse, rat, guinea pig, rabbit, cat, cattle, and human were examined by TEM and SEM in a comparative study. The differences between species were noted mainly by using TEM. Bowman's layer showed a tendency to be well developed in higher mammals. Tadpoles lack a Bowman's layer, lower mammals have a thin Bowman's layer, and higher mammals have a thick Bowman's layer. The boundary between the substantia propria and Descemet's membrane was distinct in higher mammals. On the other hand, there are no differences in thickness of the collagen fibrils that constitute Bowman's layer and those of the substantia propria. NaOH digestion was utilized for SEM preparation. SEM imaging revealed a textured appearance of the epithelial side of Bowman's layer. In Descemet's membrane, fibrous long spacing (FLS) fiber-like structures, which are arranged in parallel to the endothelium, were observed by both TEM and SEM. To our knowledge, this is the first report of SEM observations of FLS fiber-like structures on the endothelial surface of Descemet's membrane. SEM at a plane normal to the plane of the cornea showed that Descemet's membrane has a piled laminar structure. Descemet's membrane is closely associated with the collagen layer of the substantia propria. Collagen fibrils invading from the substantia propria into Descemet's membrane were observed with both TEM and SEM.     

Crystal structure of the antigen-binding fragment of apoptosis-inducing mouse anti-human Fas monoclonal antibody HFE7A.

Binding of Fas ligand to Fas induces apoptosis. The Fas-Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. The mouse anti-human Fas monoclonal antibody HFE7A (m-HFE7A), which induces apoptosis, has been humanized based on a structure predicted by homology modeling. A version of humanized HFE7A is currently under development for the treatment of autoimmune diseases such as rheumatoid arthritis. For a deeper understanding of the protein engineering aspect of antibody humanization, for which information on the three-dimensional structure is essential, we determined the crystal structure of the m-HFE7A antigen-binding fragment (Fab) by X-ray crystallography at 2.5 A resolution. The main-chain conformation of the five loops in the six complementarity-determining regions (CDRs) was correctly predicted with root-mean-square deviations of 0.30-1.04 A based on a comparison of the crystal structure with the predicted structure. The CDR-H3 conformation of the crystal structure, which was not classified as one of the canonical structures, was completely different from that of the predicted structure but adopted the conformation which followed the "H3-rules." The results of charge distribution analysis of the antigen-binding site suggest that electrostatic interactions may be important for its binding to Fas.     

The metabolic pathway of 4-aminophenol in Burkholderia sp. strain AK-5 differs from that of aniline and aniline with C-4 substituents

Burkholderia sp. strain AK-5 utilized 4-aminophenol as the sole carbon, nitrogen, and energy source. A pathway for the metabolism of 4-aminophenol in strain AK-5 was proposed based on the identification of three key metabolites by gas chromatography-mass spectrometry analysis. Strain AK-5 converted 4-aminophenol to 1,2,4-trihydroxybenzene via 1,4-benzenediol. 1,2,4-Trihydroxybenzene 1,2-dioxygenase cleaved the benzene ring of 1,2,4-trihydroxybenzene to form maleylacetic acid. The enzyme showed a high dioxygenase activity only for 1,2,4-trihydroxybenzene, with K(m) and V(max) values of 9.6 micro M and 6.8 micro mol min(-1) mg of protein(-1), respectively.     

Multiple four-stranded conformations of human telomere sequence d(CCCTAA) in solution

By detailed NMR analysis of a human telomere repeating unit, d(CCCTAA), we have found that three distinct tetramers, each of which consists of four symmetric single-strands, slowly exchange in a slightly acidic solution. Our new finding is a novel i-motif topology (T-form) where T4 is intercalated between C1 and C2 of the other duplex. The other two tetramers have a topology where C1 is intercalated between C2 and C3 of the other parallel duplex, resulting in the non-stacking T4 residues (R-form), and a topology where C1 is stacked between C3 and T4 of the other duplex (S-form). From the NMR denaturation profile, the R-form is the most stable of the three structures in the temperature range of 15–50°C, the S-form the second and the T-form the least stable. The thermodynamic parameters indicate that the T-form is the most enthalpically driven and entropically opposed, and its population is increased with decreasing temperature. The T-form structure determined by restrained molecular dynamics calculation suggests that inter-strand van der Waals contacts in the narrow grooves should contribute to the enthalpic stabilization of the T-form.     

Complete nucleotide sequence and functional analysis of the genes for 2-aminophenol metabolism from Pseudomonas sp. AP-3

A 13.9-kb region, which contained the 2-aminophenol 1,6-dioxygenase genes (amnBA) reported before, was cloned from the 2-aminophenol-assimilating bacterium Pseudomonas sp. AP-3. The complete nucleotide sequence of this region was determined and six genes were found downstream of amnBA. The eight genes together were designated amnBACFEDHG. Each gene was similar to the corresponding gene operating in the meta-cleavage pathway, except for amnB, amnA, and amnD. The four 2-aminophenol-metabolizing enzymes, 2-aminomuconic 6-semialdehyde dehydrogenase, 2-aminomuconate deaminase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase, were purified and characterized. NH2-terminal amino acid sequences of each purified enzyme agreed with those deduced from amnC, amnF, amnE, and amnD, respectively. These genes were therefore assigned as the genes encoding these respective proteins. The tight clustering of the amn genes, which were all transcribed in the same direction, raised the possibility that these genes formed a single operon. The organization of the amn genes was entirely different from that of the atd, dmp, and xyl genes reported in the meta-cleavage pathway, although these latter genes clustered similarly.     

Novel scaffold for cathepsin K inhibitors

Pyrrolopyrimidine, a novel scaffold, allows to adjust interactions within the S3 subsite of cathepsin K. The core intermediate 10 facilitated the P3 optimization and identified highly potent and selective cathepsin K inhibitors 11-20.     

The genome size evolution of medaka (Oryzias latipes) and fugu (Takifugu rubripes)

Evolution of the genome size in eukaryotes is often affected by changes in the noncoding sequences, for which insertions and deletions (indels) of small nucleotide sequences and amplification of repetitive elements are considered responsible. In this study, we compared the genomic DNA sequences of two kinds of fish, medaka (Oryzias latipes) and fugu (Takifugu rubripes), which show two-fold difference in the genome size (800 Mb vs. 400 Mb). We selected a contiguous DNA sequence of 790 kb from the medaka chromosome LG22 (linkage group 22), and made a precise comparison with the sequence (387 kb) of the corresponding region of Takifugu. The sequence of 178 kb in total was aligned common between two fishes, and the remaining sequences (612 kb for medaka and 209 kb for fugu) were found abundant in various repetitive elements including many types of unclassified low copy repeats, all of which accounted for more than a half (54%) of the genome size difference. Furthermore, we identified a significant difference in the length ratio of the unaligned sequences that locate between the aligned sequences (USBAS), particularly after eliminating known repetitive elements. These USBAS with no repetitive elements (USBAS-nr) located within the intron and intergenic region. These results strongly indicated that amplification of repetitive elements and compilation of indels are major driving forces to facilitate changes in the genome size.     

Identification of novel plasmin inhibitors possessing nitrile moiety as warhead

Lysine-nitrile derivatives having a trisubstituted benzene, which belongs to a new chemical class, were prepared and tested for inhibitory activities against plasmin and the highly homologous plasma kallikrein and urokinase. The use of the novel chemotype in the development of plasmin inhibitors has been demonstrated by derivatives of compound 9.     

Automatic sleep/wake scoring from body motion in bed: validation of a newly developed sensor placed under a mattress

The purpose of this study was to formulate a "sleep/wake" scoring algorithm for processing activity measurements obtained using a newly developed nonwear actigraphy (NWA) device, and to test its validity. The NWA device has a highly sensitive pressure sensor and is placed under a mattress. It can continuously record the activity of a person lying on the mattress and identify an "in-bed/out-of-bed" state from the vibrations of the mattress. We formulated the sleep/wake scoring algorithm by using data obtained simultaneously by wrist actigraphy (Act) and the NWA device in 33 healthy participants. Agreement rate, sensitivity, and specificity with Act were 95.7%, 97.6%, and 75.8% (33 healthy people); the corresponding values were 85.9%, 89.1%, and 79.8% for 12 nursing home residents and 93.7%, 97.2%, and 60.8% for 60 nights for 6 healthy persons who slept 10 nights on their futons. Agreement rate, sensitivity, and specificity with polysomnography were in almost perfect agreement with Act (12 nights; 6 healthy persons who slept 2 nights). All our validation results indicate that the NWA device, placed under a mattress or a futon, can produce almost identical sleep/wake scores to Act. It is expected that the NWA device, a nonwear device for scoring sleep/wake and in-bed/out-of-bed, enables convenient long-term sleep-related evaluation in various fields, including hospital settings, home-care settings, and care facility settings such as nursing homes.