Identification of a heparin-binding growth factor-1 nuclear translocation sequence by deletion mutation analysis.

We have shown previously that a deletion mutant of human heparin-binding growth factor (HBGF)-1, HBGF-1U, lacking the sequence Asn-Tyr-Lys-Lys-Pro-Lys-Leu is capable of initiating c-fos mRNA expression and polypeptide phosphorylation on tyrosine residues at concentrations that do not induce either DNA synthesis or cell proliferation (1). The fact that addition of the nuclear translocation signal from the yeast histone 2B protein to the HBGF-1U mutant caused reconstitution of the biological activity of HBGF-1 indicated that nuclear translocation may be an important component of the mitogenic signal induced by HBGF-1. In order to examine the nuclear translocation potential of HBGF-1 alpha, the deletion mutant HBGF-1U, and the yeast histone 2B-HBGF-1 chimera, HBGF-1U2, we expressed these forms of HBGF-1 in murine endothelial cells. Western blot and two-dimensional Western blot analysis of cytosol and nuclei demonstrate that although the three forms of HBGF-1 are readily detectable in the cytosol of the individual transfectants, HBGF-1 alpha and HBGF-1U2 but not HBGF-1U was detected in the nucleus. Furthermore, murine endothelial cells expressing HBGF-1 alpha and HBGF-1U2 exhibited an atypical cellular phenotype in vitro that was absent in the HBGF-1U transfectants. These data suggest that HBGF-1 contains a functional nuclear translocation sequence that may be responsible for the initiation of DNA synthesis, and these data further correlate the presence of the nuclear translocation sequence with an abnormal endothelial cell phenotype in vitro.     

How the pterosaur got its wings

Throughout the evolutionary history of life, only three vertebrate lineages took to the air by acquiring a body plan suitable for powered flight: birds, bats, and pterosaurs. Because pterosaurs were the earliest vertebrate lineage capable of powered flight and included the largest volant animal in the history of the earth, understanding how they evolved their flight apparatus, the wing, is an important issue in evolutionary biology. Herein, I speculate on the potential basis of pterosaur wing evolution using recent advances in the developmental biology of flying and non‐flying vertebrates. The most significant morphological features of pterosaur wings are: (i) a disproportionately elongated fourth finger, and (ii) a wing membrane called the brachiopatagium, which stretches from the posterior surface of the arm and elongated fourth finger to the anterior surface of the leg. At limb‐forming stages of pterosaur embryos, the zone of polarizing activity (ZPA) cells, from which the fourth finger eventually differentiates, could up‐regulate, restrict, and prolong expression of 5′‐located Homeobox D (Hoxd) genes (e.g. Hoxd11, Hoxd12, and Hoxd13) around the ZPA through pterosaur‐specific exploitation of sonic hedgehog (SHH) signalling. 5′Hoxd genes could then influence downstream bone morphogenetic protein (BMP) signalling to facilitate chondrocyte proliferation in long bones. Potential expression of Fgf10 and Tbx3 in the primordium of the brachiopatagium formed posterior to the forelimb bud might also facilitate elongation of the phalanges of the fourth finger. To establish the flight‐adapted musculoskeletal morphology shared by all volant vertebrates, pterosaurs probably underwent regulatory changes in the expression of genes controlling forelimb and pectoral girdle musculoskeletal development (e.g. Tbx5), as well as certain changes in the mode of cell–cell interactions between muscular and connective tissues in the early phase of their evolution. Developmental data now accumulating for extant vertebrate taxa could be helpful in understanding the cellular and molecular mechanisms of body‐plan evolution in extinct vertebrates as well as extant vertebrates with unique morphology whose embryonic materials are hard to obtain.     

Analytical theory of species abundance distributions of a random community model

We review the history and recent progress of the analytical theories of a random community models. In particular, we focus on a global stability analysis of replicator equations with random interactions and species abundance distributions based on statistical mechanics.     

A novel and mild isolation procedure of chlorosomes from the green sulfur bacterium Chlorobaculum tepidum

In this article, we developed a new and mild procedure for the isolation of chlorosomes from a green sulfur bacterium Chlorobaculum tepidum. In this procedure, Fenna-Matthews-Olson (FMO) protein was released by long cold treatment (6°C) of cells under the presence of a chaotrope (2?M NaSCN) and 0.6?M sucrose. Chlorosomes were released by an osmotic shock of the cold-treated cells after the formation of spheroplasts without mechanical disruption. Chlorosomes were finally purified by a sucrose step-wise density gradient centrifugation. We obtained two samples with different density (20 and 23% sucrose band, respectively) and compared them by SDS-PAGE, absorption spectroscopy at 80?K, fluorescence and CD spectroscopy at room temperature. Cells whose absorption maximum was longer than 750?nm yielded higher amount of the 20% sucrose fraction than those having an absorption maximum shorter than 750?nm.     

Ectodomain shedding of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, by TIMP-2- and TIMP-3-sensitive proteolysis

Neuroglycan C (NGC) is a transmembrane-type of chondroitin sulfate proteoglycan with an epidermal growth factor (EGF)-like module that is exclusively expressed in the CNS. Because ectodomain shedding is a common processing step for many transmembrane proteins, we examined whether NGC was subjected to proteolytic cleavage. Western blotting demonstrated the occurrence of a soluble form of NGC with a 75 kDa core glycoprotein in the soluble fraction of the young rat cerebrum. In contrast, full-length NGC with a 120 kDa core glycoprotein and its cytoplasmic fragment with a molecular size of 35 kDa could be detected in the membrane fraction. The soluble form of NGC was also detectable in culture media of fetal rat neurons, and the full-length form existed in cell layers. The amount of the soluble form in culture media was decreased by adding a physiological protease inhibitor such as a tissue inhibitor of metalloproteinase (TIMP)-2 or TIMP-3, but not by adding TIMP-1. Both EGF-like and neurite outgrowth-promoting activity of the NGC ectodomain may be regulated by this proteolytic processing.     

Characterization of Novel MSX1 Mutations Identified in Japanese Patients with Nonsyndromic Tooth Agenesis

Since MSX1 and PAX9 are linked to the pathogenesis of nonsyndromic tooth agenesis, we performed detailed mutational analysis of these two genes sampled from Japanese patients. We identified two novel MSX1 variants with an amino acid substitution within the homeodomain; Thr174Ile (T174I) from a sporadic hypodontia case and Leu205Arg (L205R) from a familial oligodontia case. Both the Thr174 and Leu205 residues in the MSX1 homeodomain are highly conserved among different species. To define possible roles of mutations at these amino acids in the pathogenesis of nonsyndromic tooth agenesis, we performed several functional analyses. It has been demonstrated that MSX1 plays a pivotal role in hard tissue development as a suppressor for mesenchymal cell differentiation. To evaluate the suppression activity of the variants in mesenchymal cells, we used the myoD-promoter, which is one of convenient reporter assay system for MSX1. Although the gene products of these MSX1 variants are stable and capable of normal nuclear localization, they do not suppress myoD-promoter activity in differentiated C2C12 cells. To clarify the molecular mechanisms underlying our results, we performed further analyses including electrophoretic mobility shift assays, and co-immunoprecipitation assays to survey the molecular interactions between the mutant MSX1 proteins and the oligonucleotide DNA with MSX1 consensus binding motif or EZH2 methyltransferase. Since EZH2 is reported to interact with MSX1 and regulate MSX1 mediated gene suppression, we hypothesized that the T174I and L205R substitutions would impair this interaction. We conclude from the results of our experiments that the DNA binding ability of MSX1 is abolished by these two amino acid substitutions. This illustrates a causative role of the T174I and L205R MSX1 homeodomain mutations in tooth agenesis, and suggests that they may influence cell proliferation and differentiation resulting in lesser tooth germ formation in vivo.     

Genetic control of a central pattern generator: rhythmic oromotor movement in mice is controlled by a major locus near Atp1a2

Fluid licking in mice is a rhythmic behavior that is controlled by a central pattern generator (CPG) located in a complex of brainstem nuclei. C57BL/6J (B6) and DBA/2J (D2) strains differ significantly in water-restricted licking, with a highly heritable difference in rates (h(2)≥0.62) and a corresponding 20% difference in interlick interval (mean ± SEM?=?116.3±1 vs 95.4±1.1 ms). We systematically quantified motor output in these strains, their F(1) hybrids, and a set of 64 BXD progeny strains. The mean primary interlick interval (MPI) varied continuously among progeny strains. We detected a significant quantitative trait locus (QTL) for a CPG controlling lick rate on Chr 1 (Lick1), and a suggestive locus on Chr 10 (Lick10). Linkage was verified by testing of B6.D2-1D congenic stock in which a segment of Chr 1 of the D2 strain was introgressed onto the B6 parent. The Lick1 interval on distal Chr 1 contains several strong candidate genes. One of these is a sodium/potassium pump subunit (Atp1a2) with widespread expression in astrocytes, as well as in a restricted population of neurons. Both this subunit and the entire Na(+)/K(+)-ATPase molecule have been implicated in rhythmogenesis for respiration and locomotion. Sequence variants in or near Apt1a2 strongly modulate expression of the cognate mRNA in multiple brain regions. This gene region has recently been sequenced exhaustively and we have cataloged over 300 non-coding and synonymous mutations segregating among BXD strains, one or more of which is likely to contribute to differences in central pattern generator tempo.     

Agonistic and antagonistic effects of C5a-chimera bearing S19 ribosomal protein tail portion on the C5a receptor of monocytes and neutrophils, respectively

C-terminus of S19 ribosomal protein (RP S19) endows the cross-linked homodimer with a dual effect on the C5a receptor in leucocyte chemoattraction; agonistic effect on the monocyte receptor, and antagonistic effect on the neutrophil receptor. C5a exhibits the uniform agonistic effect on this receptor of both cell types. We have currently prepared a recombinant C5a-chimeric protein bearing the C-terminus of RP S19 (C5a/RP S19 chimera) to be used as a substitute of the RP S19 dimer. In vitro, this chimera similarly inhibited the intracellular Ca(2+) mobilization of neutrophils induced by C5a to the RP S19 dimer did. In the guinea pig skin, 10(-7) M C5a/RP S19 chimera exhibited an inhibitory capacity to the neutrophil infiltration induced by 3 x 10(-7) M C5a without enhancing monocyte infiltration. In reverse passive Arthus reaction, the neutrophil infiltration associated with plasma extravasation was significantly reduced by the simultaneous administration of 10(-7) M C5a/RP S19 chimera with antibodies. The C5a/RP S19 chimera is a useful tool not only to examine the molecular mechanism that underlies the functional difference of the C5a receptor between monocytes and neutrophils, but also to prevent C5a-mediated hyper-response of neutrophils in acute inflammation.     

Hydrodynamics of cryogelation

Cryogel, prevalent in the plasma of rheumatoid arthritis patients, is a plasma fibronectin (pFN)-extra domain A containing FN [EDA(+)FN]-fibrinogen (Fbg) aggregate formed by the addition of heparin (Hep) at low temperature. Although EDA(+)FN is not usually present in normal plasma, its prevalence in rheumatic patients induces cryogelation. In this study, we determined the hydrodynamic radius (R_h) ratio (R_h/R_h30) of the cryogel component by dynamic light scattering in vitro. R_h/R_h30 was normalized to R_h at 30 °C (R_h30) at several temperatures. The R_h/R_h30 of Fbg was found to increase only by self-aggregation, whereas the R_h/R_h30 of FNs does not increase in response to temperature changes. The R_h/R_h30 of the Fbg/FN aggregate is increased by the addition of Hep, and the R_h/R_h30 (12.5) of the Hep-induced EDA(+)FN/Fbg aggregate is greater than that (2.5) of the pFN/Fbg aggregate. These results suggest that cryogelation requires Fbg self-aggregation and the interaction between EDA(+)FN and Hep.