The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses

The proper intracellular distribution of mitochondria is assumed to be critical for normal physiology of neuronal cells, but direct evidence for this idea is lacking. Extension or movement of mitochondria into dendritic protrusions correlates with the development and morphological plasticity of spines. Molecular manipulations of dynamin-like GTPases Drp1 and OPA1 that reduce dendritic mitochondria content lead to loss of synapses and dendritic spines, whereas increasing dendritic mitochondrial content or mitochondrial activity enhances the number and plasticity of spines and synapses. Thus, the dendritic distribution of mitochondria is essential and limiting for the support of synapses. Reciprocally, synaptic activity modulates the motility and fusion/fission balance of mitochondria and controls mitochondrial distribution in dendrites.     

Highly potent PDE4 inhibitors with therapeutic potential

Based on the hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system (CNS), design and synthesis of a hydrophilic analogue is considered to be one approach to improving the side-effect profile of Ariflo 1. Water-soluble piperidine derivatives were found to possess therapeutic potential.     

A polyclonal antibody directed against syringylpropane epitopes of native lignins

With a view to visualizing the ultrastructural distribution of syringyl lignins in secondary plant cell walls, a polyclonal antibody raised from a synthetic DHP polymer consisting only of syringyl propane units was prepared. To test the reactivity of the antiserum, a mini-dot-blot immunoassay reducing the amounts of substrates and antiserum was developed. A characteristic attribute of the S-antiserum appears to be its specific recognition of sequences of three or more consecutive syringyl units. On ultra-thin sections of model plants of Arabidopsis thaliana, Populus and tobacco, the antiserum allowed us to demonstrate a higher concentration of syringyl epitopes in fibres than in vessels. Variations in the distribution pattern of these epitopes between the three plants examined suggest that the synthesis of syringyl lignins in angiosperms depends on the species.     

Highly potent PDE4 inhibitors with therapeutic potential

The hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system prompted us to design and synthesize a hydrophilic piperidine analog to improve the side effect profile of Ariflo 1, which is an orally active second-generation PDE4 inhibitor. During evaluation of various water-soluble piperidine analogs, 2a-b, 11b-14b, and 17a showed therapeutic potential in cross-species comparison studies. The following three findings were obtained: (1) The hydroxamic acid group, a well known metal chelator, caused a marked increase of inhibitory activity. (2) Water-soluble piperidine analogs lacked the configurational isomerism of Ariflo 1 without loss of inhibitory activity. (3) Replacement of the 4-methoxy residue with a difluoromethoxy residue led to an increase of in vivo potency. Structure-activity relationships are presented. Single-dose rat pharmacokinetic data for 11b, 12b, and 17a are also presented.     

Changes in the fabric and compliance tensors of cancellous bone due to trabecular surface remodeling, predicted by a digital image-based model

Fabric and compliance tensors of a cube of cancellous bone with a complicated three-dimensional trabecular structure were obtained for trabecular surface remodeling by using a digital image-based model combined with a large-scale finite element method. Using mean intercept length and a homogenization method, the fabric and compliance tensors were determined for the trabecular structure obtained in the computer remodeling simulation. The tensorial quantities obtained indicated that anisotropic structural changes occur in cancellous bone adapting to the compressive loading condition. There were good correlations between the fabric tensor, bone volume fraction, and compliance tensor in the remodeling process. The result demonstrates that changes in the structural and mechanical properties of cancellous bone are essentially anisotropic and should be expressed by tensorial quantities.     

The novel gene HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 of rice encodes a putative coiled-coil protein required for homologous chromosome pairing in meiosis

We have identified and characterized a novel gene, PAIR1 (HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1), required for homologous chromosome pairing and cytokinesis in male and female meiocytes of rice (Oryza sativa). The pair1 mutation, tagged by the endogenous retrotransposon Tos17, exhibited meiosis-specific defects and resulted in complete sterility in male and female gametes. The PAIR1 gene encodes a 492-amino acid protein, which contains putative coiled-coil motifs in the middle, two basic regions at both termini, and a potential nuclear localization signal at the C terminus. Expression of the PAIR1 gene was detected in the early stages of flower development, in which the majority of the sporocytes had not entered meiosis. During prophase I of the pair1 meiocyte, all the chromosomes became entangled to form a compact sphere adhered to a nucleolus, and homologous pairing failed. At anaphase I and telophase I, chromosome nondisjunction and degenerated spindle formation resulted in multiple uneven spore production. However, chromosomal fragmentation frequent in plant meiotic mutants was never observed in all of the pair1 meiocytes. These observations clarify that the PAIR1 protein plays an essential role in establishment of homologous chromosome pairing in rice meiosis.     

Hierarchical Chromatin Structure of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> Revealed by Atomic Force Microscopy

Many structural studies on higher eukaryotic chromatin have been carried out, but chromatin structure in fungi remains unclear. Schizosaccharomyces pombe has been used for investigations of chromosome function; however, the structural details of S. pombe chromatin have not been clarified owing to its small nucleus. We used atomic force microscopy for nano-scale imaging of chromatin isolated from S. pombe. Topographic images indicated that nuclear chromatin contained at least three hierarchical structures: large-scale chromatin fibers, spherical domains in the fibers, and nodules in the domains. The average diameters of the domain and the nodule were 363 ± 85.2 nm and 46.2 ± 9.30 nm. Each structure comprising the hierarchy was similar to higher eukaryotic chromatin thus far observed, despite definite differences in chromatin organization at the nucleosomal level. The presence of histone H1 suggested that there might be an alternative to compensate for histone H1 lacking in S. pombe.     

Expression and characterization of recombinant human antithrombin III in Pichia pastoris

Antithrombin III (ATIII) is a member of the serpin superfamily and a major regulator of the blood coagulation cascade. To express recombinant human ATIII (rATIII) in the methylotrophic yeast Pichia pastoris, we constructed an rATIII expression plasmid which contained the ATIII cDNA encoding mature protein region connected with the truncated mAOX2 promoter and the SUC2 secretion signal, introduced it into the P. pastoris genome, and screened for a single copy transformant. The secretion of rATIII from the transformant reached a level of 320 IU/L in the culture broth at 169 h. From the culture-supernatant, rATIII was purified to over 99% by heparin-affinity chromatography and other column chromatography methods. We characterized rATIII and compared it with human plasma-derived ATIII (pATIII). The purified rATIII possessed correct N-terminal amino acid sequence, and its molecular weight by SDS-PAGE of 56,000 Da was slightly different from the 58,000 Da of pATIII. Sequence and mass spectrometry analysis of BrCN fragments revealed that posttranslational modifications had occurred in rATIII. O-linked mannosylation was found at Ser 3 and Thr 9, and in some rATIII molecules, modification with O-linked mannosyl-mannose had probably occurred at Thr 386, close to the reactive center. Although the heparin-binding affinity of rATIII was 10-fold higher than that of pATIII, its inhibitory activity against thrombin was only half. As the conformation of rATIII and pATIII by circular dichroism spectroscopy was similar, O-glycosylation in the reactive center loop was assumed to be mainly responsible for the decreased inhibitory activity. pATIII can inactivate thrombin through formation of a stable thrombin-ATIII complex, but rATIII modified with O-glycosylation in the reactive center loop may act as a substrate rather than an inhibitor of thrombin.