A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.     

An establishment of ELISA for murine IL-6

Summary Murine interleukin-6 (mIL-6) was expressed inEscherichia coli as human growth hormone (hGH) fusion protein. The products were cleaved by thrombin to liberate mIL-6. Monoclonal and polyclonal antibodies specific to mIL-6 were prepared by immunizing rats with mIL-6 thus obtained. ELISA for the quantitation of mIL-6 was also established, which could detect mIL-6 in a quantity as low as 2 ng/ml.     

Selective inhibition of MAO-A in serotonergic synaptosomes by two amphetamine metabolites, p-hydroxyamphetamine and p-hydroxynorephedrine

The present study was carried out mainly to clarify whether the two amphetamine metabolites, p-hydroxyamphetamine (P-OHA) and p-hydroxynorephedrine (p-OHN) are taken up by mouse brain 5-hydroxytryptamine (5-HT) nerve terminals to inhibit type A monoamine oxidase (MAO-A) and then potentiate the abnormal behavior, head-twitch. Of the two metabolites, only intracerebroventricular p-OHA, at 80 μg/mouse, sufficient to cause a head-twitch response (HTR), appreciably inhibited MAO-A activity without affecting MAO-B activity in homogenates of the mouse striatum, hypothalamus and the rest of the forebrain; and p-OHN did not inhibit either type of MAO at the dose tested. Estimation of intra- and extrasynaptosomal MAO-A activity showed that both metabolites significantly inhibited only the intrasynaptosomal deamination of 5-HT by MAO-A with p-OHA being more potent. Taken together with our previous findings, these present results clearly indicate that p-OHA may accumulate in the 5-HT nerve terminals through the uptake system, and concomitantly inhibit MAO-A activity. These actions of p-OHA may increase intraneuronal 5-HT levels and then potentiate 5-HT release to cause interaction with the post-synaptic 5-HT receptors.     

Pyridoxal kinase immunoreactivity in rabbit brain

Murine polyclonal antibody against purified bovine brain pyridoxal kinase (EC 2.7.1.35) was generated and showed cross-reactivity with rabbit brain pyridoxal kinase. This antibody was used to immunohistochemically examine the distribution of pyridoxal kinase in the rabbit brain. The cytoplasm of neuronal cells and neuroglial cells in the cerebral cortex, hippocampal region, brain nuclei and cerebellar cortex showed positive staining with various degrees of intensity. The neuronal cells and surrounding fibers in some brain nuclei, such as the area tegmentalis ventralis or the substantia nigra, showed intense staining. The neuronal cells of the hippocampal region showed somewhat weak reactivity, but some with intense reactivity were found sparsely distributed and positive staining fiber networks of a very low density were also observed.     

Osteological and histological comparison of the development of the interphalangeal intercalary skeletal element between hyloid and ranoid anurans

Some frog species have a unique skeletal element, referred to as the intercalary element (IE), in the joints between the terminal and subterminal phalanges of all digits. IEs are composed of cartilage or connective tissue and have a markedly differ shape than the phalanges. IEs are highly related to the arboreal lifestyle and toe pads. The IE is found only in neobatrachian frogs among anurans, suggesting that it is a novelty of Neobatrachia. IEs are widely distributed among multiple neobatrachian lineages and are found in the suborders Hyloides and Ranoides (the two major clades in Neobatrachia). However, it is unclear whether the IEs found in multiple linages resulted from convergent evolution. Therefore, in this study, we aimed to examine how similar or different the developmental trajectories of the IEs are between Hyloides and Ranoides. To that end, we compared the osteological and histological developmental processes of the IEs of the hyloid frog Dryophytes japonicus and the ranoid frog Zhangixalus schlegelii. Both species shared the same IE-initiation site and level of tissue differentiation around the IE when it began to form in tadpoles, although the IE developments initiated at different stages which were determined by external criteria. These results suggest that similar mechanisms drive IE formation in the digits of both species, supporting the hypothesis that the IEs did not evolve convergently.     

Effect of Chlorine Substitution on the Biodegradability of Polychlorinated Biphenyls

Thirty-one isomers of polychlorinated biphenyl (PCB) were examined for biodegradability by two species of Alcaligenes and Acinetobacter. The following relationships between chlorine substitution and biodegradability of PCBs were observed. (i) Degradation decreased as chlorine substitution increased. PCB isomers containing more than four chlorines were less susceptible to degradation. (ii) PCBs containing two chlorines on either the ortho position of a single ring (i.e., 2,6-) or on both rings (i.e., 2,2′-) showed very poor degradability. (iii) PCBs containing all chlorine atoms on only a single ring were generally degraded faster than when the same number of chlorines were substituted on both rings. (iv) Preferential ring fission of the molecules occurred with nonchlorinated or lesser chlorinated rings. (v) The formation and accumulation of a yellow intermediate was always observed in 4′-chloro-substituted PCBs. (vi) Significant differences between the two organisms with respect to degradability were not observed except for 2,4,6-trichlorobiphenyl.     

Cortisol promotes stress tolerance via DAF-16 in Caenorhabditis elegans

In this study, we studied the effects of cortisol and cortisone on the age-related decrease in locomotion in the nematode Caenorhabditis elegans and on the tolerance to heat stress at 35 °C and to oxidative stress induced by the exposure to 0.1% H₂O₂. Changes in mRNA expression levels of C. elegans genes related to stress tolerance were also analyzed. Cortisol treatment restored nematode movement following heat stress and increased viability under oxidative stress, but also shortened worm lifespan. Cortisone, a cortisol precursor, also restored movement after heat stress. Additionally, cortisol treatment increased mRNA expression of the hsp-12.6 and sod-3 genes. Furthermore, cortisol treatment failed to restore movement of daf-16-deficient mutants after heat stress, whereas cortisone failed to restore the movement of dhs-30-deficient mutants after heat stress. In conclusion, the results suggested that cortisol promoted stress tolerance via DAF-16 but shortened the lifespan, whereas cortisone promoted stress tolerance via DHS-30.     

Autologous Bone-Marrow Mesenchymal Stem Cell Implantation and Endothelial Function in a Rabbit Ischemic Limb Model

Background

The purpose of this study was to determine whether autologous mesenchymal stem cells (MSCs) implantation improves endothelial dysfunction in a rabbit ischemic limb model.

Methods

We evaluated the effect of MSC implantation on limb blood flow (LBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent vasodilator, in rabbits with limb ischemia in which cultured MSCs were implanted (n = 20) or saline was injected as a control group (n = 20). LBF was measured using an electromagnetic flowmeter. A total of 10⁶ MSCs were implanted into each ischemic limb.

Results

Histological sections of ischemic muscle showed that capillary index (capillary/muscle fiber) was greater in the MSC implantation group than in the control group. Laser Doppler blood perfusion index was significantly increased in the MSC implantation group compared with that in the control group. LBF response to ACh was greater in the MSC group than in the control group. After administration of N^G-nitro-L-arginine, a nitric oxide synthase inhibitor, LBF response to ACh was similar in the MSC implantation group and control group. Vasodilatory effects of SNP in the two groups were similar.

Conclusions

These findings suggest that MSC implantation induces angiogenesis and augments endothelium-dependent vasodilation in a rabbit ischemic model through an increase in nitric oxide production.     

Changes in metabolism of cell wall polysaccharides in oat leaves during senescence: relevance to the senescence-promoting effect of methyl jasmonate

Changes in cell wall polysaccharides in oat (Avena sativa L.) leaf segments during senescence promoted by methyl jasmonate (JA-Me) were studied. During the incubation with water at 25 °C in the dark, the loss of chlorophyll of the segments excised from the primary leaves of 8-day-old green seedlings was found dramatically just after leaf excision, and leaf color completely turned to yellow after the 3- to 4-day incubation in the dark. Application of 10 µM JA-Me substantially promoted the loss of chlorophyll corresponding with the chloroplast degradation. Cell wall polysaccharides in oat leaf segments mainly consisted of hemicellulosic and cellulosic ones. During the process of leaf senescence, the amount of hemicellulosic I and II, and cellulosic polysaccharides decreased, but little in pectic polysaccharides. JA-Me significantly enhanced the decrease in cellulosic polysaccharides, but little in hemicellulosic ones. Arabinose, xylose and glucose were identified as main constituents of neutral sugars of hemicellulosic polysaccharides. The neutral sugar compositions of hemicellulosic polysaccharides changed little during leaf senescence both in the presence or absence of JA-Me. These facts suggest that JA-Me affects sugar metabolism relating to cellulosic polysaccharides during leaf senescence.