Hyaline cartilage formation and enchondral ossification modeled with KUM5 and OP9 chondroblasts

What is it that defines a bone marrow‐derived chondrocyte? We attempted to identify marrow‐derived cells with chondrogenic nature and immortality without transformation, defining “immortality” simply as indefinite cell division. KUM5 mesenchymal cells, a marrow stromal cell line, generated hyaline cartilage in vivo and exhibited enchondral ossification at a later stage after implantation. Selection of KUM5 chondroblasts based on the activity of the chondrocyte‐specific cis‐regulatory element of the collagen α2(XI) gene resulted in enhancement of their chondrogenic nature. Gene chip analysis revealed that OP9 cells, another marrow stromal cell line, derived from macrophage colony‐stimulating factor‐deficient osteopetrotic mice and also known to be niche‐constituting cells for hematopoietic stem cells expressed chondrocyte‐specific or ‐associated genes such as type II collagen α1, Sox9, and cartilage oligomeric matrix protein at an extremely high level, as did KUM5 cells. After cultured OP9 micromasses exposed to TGF‐β3 and BMP2 were implanted in mice, they produced abundant metachromatic matrix with the toluidine blue stain and formed type II collagen‐positive hyaline cartilage within 2 weeks in vivo. Hierarchical clustering and principal component analysis based on microarray data of the expression of cell surface markers and cell‐type‐specific genes resulted in grouping of KUM5 and OP9 cells into the same subcategory of “chondroblast,” that is, a distinct cell type group. We here show that these two cell lines exhibit the unique characteristics of hyaline cartilage formation and enchondral ossification in vitro and in vivo. J. Cell. Biochem. 100: 1240–1254, 2007. © 2006 Wiley‐Liss, Inc.     

Mutations in the helix termination motif of mouse type I IRS keratin genes impair the assembly of keratin intermediate filament

Two classical mouse hair coat mutations, Rex (Re) and Rex wavy coat (Re(wc)), are linked to the type I inner root sheath (IRS) keratin genes of chromosome 11. An N-ethyl-N-nitrosourea-induced mutation, M100573, also maps close to the type I IRS keratin genes. In this study, we demonstrate that Re and M100573 mice bear mutations in the type I IRS gene Krt25; Re(wc) mice bear an additional mutation in the type I IRS gene Krt27. These three mutations are located in the helix termination motif of the 2B alpha-helical rod domain of a type I IRS keratin protein. Immunohistological analysis revealed abnormal foam-like immunoreactivity with an antibody raised to type II IRS keratin K71 in the IRS of Re/+ mice. These results suggest that the helix termination motif is essential for the proper assembly of types I and II IRS keratin protein complexes and the formation of keratin intermediate filaments.     

Odorant transfer characteristics of white bread during baking

The potent odorants in the crust and crumb of white bread were identified and quantified by gas chromatography-mass spectrometry and gas chromatography/olfactometry. The weight loss ratio of the samples baked at 220 °C was controlled in the range of 0-28%. The odorants were classified into 5 types by the transfer characteristics: i) All amounts of odorant transferred from the crust to external space (type-I). ii) All transferred from the crust to the crumb and external space (type-II). iii) Certain amount remaining in the crust and the rest transferred to the crumb and external space (type-III). iv) All transferred from the crumb to external space (type-IV). v) Certain amount remaining in the crumb and the rest transferred to the crust and external space (type-V). The odorants of type-IV were not apparent after the crust had formed. The results indicate that the crust could be a barrier to prevent the odorants from being transferred to external space.     

IKKε regulates cell elongation through recycling endosome shuttling

1. Download : Download high-res image (412KB)
2. Download : Download full-size image

Highlights? Recycling endosomes shuttle along the proximal-distal axis of growing bristles ? IKK? locally antagonizes Rab11 effector Nuf at the bristle tip by phosphorylation ? Nuf-endosome pathway is independent of actin cytoskeleton or DIAP1-caspase pathway ? Mammalian IKK-related kinases antagonize Rab11-FIP3 through phosphorylation     

New insights into the phylogenetic distribution and evolutionary origins of the septins

Until recently, it had appeared that the septin family of proteins was restricted to the opisthokont eukaryotes (the fungi and animals and their close relatives the microsporidia and choanoflagellates). It has now become apparent that septins are also present in several other widely divergent eukaryotic lineages (chlorophyte algae, brown algae, and ciliates). This distribution and the details of the non-opisthokont septin sequences appear to require major revisions to hypotheses about the origins and early evolution of the septins.     

Molecular properties of rod and cone visual pigments from purified chicken cone pigments to mouse rhodopsin in situ.

We have investigated the molecular properties of rod and cone visual pigments to elucidate the differences in the molecular mechanism(s) of the photoresponses between rod and cone photoreceptor cells. We have found that the cone pigments exhibit a faster pigment regeneration and faster decay of meta-II and meta-III intermediates than the rod pigment, rhodopsin. Mutagenesis experiments have revealed that the amino acid residues at positions 122 and 189 in the opsins are the determinants for these differences. In order to study the relationship between the molecular properties of visual pigments and the physiology of rod photoreceptors, we used mouse rhodopsin as a model pigment because, by gene-targeting, the spectral properties of the pigment can be directly correlated to the physiology of the cells. In the present paper, we summarize the spectroscopic properties of cone pigments and describe our studies with mouse rhodopsin utilizing a high performance charge coupled device (CCD) spectrophotometer.     

Generation of knock-in mice carrying third cones with spectral sensitivity different from S and L cones

Red-green color vision in primates is unique in the sense that it is mediated by two photoreceptor cells that are indistinguishable in all aspects except for their visual pigments. In order to generate an animal model for investigation of the interaction between red-green inputs at the molecular level, we applied knock-in technology and X-chromosome inactivation machinery to make a mouse model with cone cells possessing visual pigments with different spectral sensitivities. We introduced a S308A point mutation into the Green opsin gene allele on the X-chromosome. This manipulation generated a 24 nm red-shift of absorption maximum in the cone pigment with negligible functional differences in other molecular properties. Amplitudes of responses in ERG and ganglion cell recordings of homozygotes were similar to those of wild-types, although the spectral sensitivities differed. Heterozygotes showed variable spectral sensitivities of ganglion cell responses due to the different integration of the native and the S308A cone inputs on the dendritic fields. In situ hybridization experiments showed that cone cells with respective pigments formed patch-like clusters of specific L cone-types, approximately 30 mum in diameter, which were randomly distributed in the dorsal region of the retinas. Since the patch-like clustering was arranged by X-inactivation, such clustering could be present in the peripheral retinas of New World monkeys with polymorphic L pigments, indicating that our mice would be a suitable model to study evolution of the mammalian color vision system.