Isolation and characterization of sex chromosome rearrangements generating male muscle dystrophy and female abnormal oogenesis in the silkworm, Bombyx mori

In deletion-mapping of W-specific RAPD (W-RAPD) markers and putative female determinant gene (Fem), we used X-ray irradiation to break the translocation-carrying W chromosome (W ^Ze ). We succeeded in obtaining a fragment of the W ^Ze chromosome designated as Ze ^W, having 3 of 12 W-RAPD markers (W-Bonsai, W-Yukemuri-S, W-Yukemuri-L). Inheritance of the Ze ^W fragment by males indicates that it does not include the Fem gene. On the basis of these results, we determined the relative positions of W-Yukemuri-S and W-Yukemuri-L, and we narrowed down the region where Fem gene is located. In addition to the Ze ^W fragment, the Z chromosome was also broken into a large fragment (Z₁) having the + ^sch (1-21.5) and a small fragment (Z₂) having the + ^od (1-49.6). Moreover, a new chromosomal fragment (Ze ^WZ₂) was generated by a fusion event between the Ze ^W and the Z₂ fragments. We analyzed the genetic behavior of the Z₁ fragment and the Ze ^WZ₂ fragment during male (Z/Z₁ Ze ^WZ₂) and female (Z₁ Ze ^WZ₂/W) meiosis using phenotypic markers. It was observed that the Z₁ fragment and the Z or the W chromosomes separate without fail. On the other hand, non-disjunction between the Ze ^WZ₂ fragment and the Z chromosome and also between the Ze ^WZ₂ fragment and the W chromosome occurred. Furthermore, the females (2A: Z/Ze ^WZ₂/W) and males (2A: Z/Z₁) resulting from non-disjunction between the Ze ^WZ₂ fragment and the W chromosome had phenotypic defects: namely, females exhibited abnormal oogenesis and males were flapless due to abnormal indirect flight muscle structure. These results suggest that Z₂ region of the Z chromosome contains dose-sensitive gene(s), which are involved in oogenesis and indirect flight muscle development.     

Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein

AIMS: The purpose of this paper was to screen candidate bacterial strains for the production of proteases suitable for application to the degradation of pathogenic forms of prion protein (PrP(Sc)). This paper describes the biochemical characteristics and proteolytic activity of the isolated protease. METHODS AND RESULTS: After screening more than 200 bacterial proteases for keratinolytic activity, we identified a Bacillus stain that produced a protease exhibiting high-degradation activity against a scrapie PrP(Sc). Sequence analysis indicated that this serine-protease belonged to the Subtilisin family and had optimum pH and temperature ranges of 9-10 and 60-70 degrees C. Western blotting analysis revealed that the protease was also capable of decomposing bovine spongiform encephalopathy-infected brain homogenate. In addition, the protease was demonstrated to degrade dried PrP(Sc) that had become firmly attached to a plastic surface considerably more effectively than proteinase K or PWD-1, a previously reported keratinase. CONCLUSIONS: These results indicate that the isolated protease exhibited higher activity for PrP(Sc) degradation compared with other proteases examined. SIGNIFICANCE AND IMPACT OF THE STUDY: This protease could be used under moderate conditions for the decontamination of precision instruments that are susceptible to PrP(Sc) contamination.     

Comparison of genetic diversity between narrowly endemic shrub Menziesia goyozanensis and its widespread congener M. pentandra (Ericaceae)

Menziesia goyozanensis, a Japanese kindof fool's huckleberry, is a shrub speciesendemic only to Mt. Goyo in northern Japan.This species is listed as `criticallyendangered' in the Red Data Book of wild plantsin Japan, and only a single population has yetbeen discovered. The allozyme diversity andRAPD variation of Menziesia pentandrawere eight- and threefold greater than those ofM. goyozanensis, respectively, althoughwe examined only five populations representinga part of the distribution of M.pentandra. Because the environment surroundingthe population of M. goyoznaensis seemsto be stable at present, it is not necessary toplan an urgent ecological conservation practicefor the species. However, considering that thehabitat of M. goyozanensis is very smalland large vegetative disturbances occasionallyoccur in cool temperate forests, in which thespecies dwells, ex situ conservation isessential.     

Cardiac progenitor cells in brown adipose tissue repaired damaged myocardium

Cardiomyocyte (CM) regeneration is limited in adult life and is not sufficient to prevent myocardial infarction. Hence, the identification of a useful source of CM progenitors is of great interest for possible use in regenerative therapy. Mesenchymal stem cells in bone marrow, embryonic stem cells, and skeletal myoblasts are known sources of CM repletion; however, there are a number of critical problems for clinical application. In this study, we succeeded to identify CM progenitor cells in brown adipose tissue (BAT). Moreover, we showed that CM progenitor cells in BAT that existed in CD29-positive population could differentiate into CM with high efficiency. To confirm the in vivo effect of CD29(+)BAT-derived cells (BATDCs), we transplanted these cells into infarct border zone of an acute myocardial infarction model in rat. Results clearly indicated that implantation of CD29(+) BATDCs led to the reduction of the infarction area and improvement of left ventricular function by replacing newly developed CMs in comparison with that by CD29(+) white adipose tissue-derived cells or control saline. These findings suggest that BATDCs are one of the useful sources for a new strategy in CM regeneration.     

Expression of C gamma 4 T cell receptors and lack of isotype exclusion by dendritic epidermal T cell lines

Although four murine C gamma gene segments (C gamma 1, 2, 3, and 4) are known to exist, the large majority of expressed gamma-chains have been shown to be of the C gamma 1 isotype and no evidence exists for the expression of more than one receptor by gamma delta TCR-bearing cells. We investigated the nature of the TCR expressed on a number of murine dendritic epidermal T cell-derived cell lines by using both Northern blot and immunoprecipitation analyses. One of these CD3+ cell lines (T195) expresses C gamma 4, V gamma 1, and delta mRNA, and its CD3-associated TCR complex can be precipitated by both anti-C gamma 4 and anti-delta sera, indicating that this receptor is a C gamma 4/delta heterodimer. Furthermore, we show that two cell lines (Y245, Y93) express two distinct TCR gamma-chains, one derived from the C gamma 4 locus, whereas the second gamma-chain is probably derived from the C gamma 2 locus. Together with the previous demonstration of C gamma 1/delta TCR on a number of dendritic epidermal T cell lines (DETC), these results indicate that such DETC are capable of expressing a variety of gamma delta TCR and that, in some DETC, isotype exclusion of gamma-chain expression does not occur.     

Evaluation of adriamycin nephropathy by an in vivo electron paramagnetic resonance

A rat model for human minimal change nephropathy was obtained by the intravenous injection of adriamycin (ADR) at 5 mg/kg. By using an in vivo electron paramagnetic resonance (EPR) spectrometer operating at 700 MHz, the temporal changes in signal intensities of a nitroxide radical, 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), in the kidneys of rats with ADR nephropathy were investigated. The decay rate of the EPR signal intensity obtained in the kidney is indicative of the renal reducing ability. It was found that the reducing ability in the kidney declined on the 7th day after ADR administration and recovered after the 14th day. Impairment of the reducing ability occurred before the appearance of continuous urinary protein. The in vitro EPR study showed that this impairment of in vivo renal reducing ability is related to impairment of the reducing ability in the mitochondria.     

Morphogenetic roles of perlecan in the tooth enamel organ: an analysis of overexpression using transgenic mice

Perlecan, a heparan sulfate proteoglycan, is enriched in the intercellular space of the enamel organ. To understand the role of perlecan in tooth morphogenesis, we used a keratin 5 promoter to generate transgenic (Tg) mice that over-express perlecan in epithelial cells, and examined their tooth germs at tissue and cellular levels. Immunohistochemistry showed that perlecan was more strongly expressed in the enamel organ cells of Tg mice than in wild-type mice. Histopathology showed wider intercellular spaces in the stellate reticulum of the Tg molars and loss of cellular polarity in the enamel organ, especially in its cervical region. Hertwig's epithelial root sheath (HERS) cells in Tg mice were irregularly aligned due to excessive deposits of perlecan along the inner, as well as on the outer sides of the HERS. Tg molars had dull-ended crowns and outward-curved tooth roots and their enamel was poorly crystallized, resulting in pronounced attrition of molar cusp areas. In Tg mice, expression of integrin β1 mRNA was remarkably higher at E18, while expression of bFGF, TGF-β1, DSPP and Shh was more elevated at P1. The overexpression of perlecan in the enamel organ resulted in irregular morphology of teeth, suggesting that the expression of perlecan regulates growth factor signaling in a stage-dependent manner during each step of the interaction between ameloblast-lineage cells and mesenchymal cells.     

R-state haemoglobin with low oxygen affinity: crystal structures of deoxy human and carbonmonoxy horse haemoglobin bound to the effector molecule L35

Although detailed crystal structures of haemoglobin (Hb) provide a clear understanding of the basic allosteric mechanism of the protein, and how this in turn controls oxygen affinity, recent experiments with artificial effector molecules have shown a far greater control of oxygen binding than with natural heterotropic effectors. Contrary to the established text-book view, these non-physiological compounds are able to reduce oxygen affinity very strongly without switching the protein to the T (tense) state. In an earlier paper we showed that bezafibrate (BZF) binds to a surface pocket on the alpha subunits of R state Hb, strongly reducing the oxygen affinity of this protein conformation. Here we report the crystallisation of Hb with L35, a related compound, and show that this binds to the central cavity of both R and T state Hb. The mechanism by which L35 reduces oxygen affinity is discussed, in relation to spectroscopic studies of effector binding.     

Mitochondrial DNA sequence variation is associated with free-living activity energy expenditure in the elderly

The decline in activity energy expenditure underlies a range of age-associated pathological conditions, neuromuscular and neurological impairments, disability, and mortality. The majority (90%) of the energy needs of the human body are met by mitochondrial oxidative phosphorylation (OXPHOS). OXPHOS is dependent on the coordinated expression and interaction of genes encoded in the nuclear and mitochondrial genomes. We examined the role of mitochondrial genomic variation in free-living activity energy expenditure (AEE) and physical activity levels (PAL) by sequencing the entire (~16.5 kilobases) mtDNA from 138 Health, Aging, and Body Composition Study participants. Among the common mtDNA variants, the hypervariable region 2 m.185G>A variant was significantly associated with AEE (p=0.001) and PAL (p=0.0005) after adjustment for multiple comparisons. Several unique nonsynonymous variants were identified in the extremes of AEE with some occurring at highly conserved sites predicted to affect protein structure and function. Of interest is the p.T194M, CytB substitution in the lower extreme of AEE occurring at a residue in the Qi site of complex III. Among participants with low activity levels, the burden of singleton variants was 30% higher across the entire mtDNA and OXPHOS complex I when compared to those having moderate to high activity levels. A significant pooled variant association across the hypervariable 2 region was observed for AEE and PAL. These results suggest that mtDNA variation is associated with free-living AEE in older persons and may generate new hypotheses by which specific mtDNA complexes, genes, and variants may contribute to the maintenance of activity levels in late life.