A ubiquitin ligase HRD1 promotes the degradation of Pael receptor, a substrate of Parkin

It has been proposed that in autosomal recessive juvenile parkinsonism (AR-JP), a ubiquitin ligase (E3) Parkin, which is involved in endoplasmic reticulum-associated degradation (ERAD), lacks E3 activity. The resulting accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R), a substrate of Parkin, leads to endoplasmic reticulum stress, causing neuronal death. We previously reported that human E3 HRD1 in the endoplasmic reticulum protects against endoplasmic reticulum stress-induced apoptosis. This study shows that HRD1 was expressed in substantia nigra pars compacta (SNC) dopaminergic neurons and interacted with Pael-R through the HRD1 proline-rich region, promoting the ubiquitylation and degradation of Pael-R. Furthermore, the disruption of endogenous HRD1 by small interfering RNA (siRNA) induced Pael-R accumulation and caspase-3 activation. We also found that ATF6 overexpression, which induced HRD1, accelerated and caused Pael-R degradation; the suppression of HRD1 expression by siRNA partially prevents this degradation. These results suggest that in addition to Parkin, HRD1 is also involved in the degradation of Pael-R.     

Approach for functional analysis of glycan using RNA interference

The elucidation of the biological role of glycan is one of the most important issues to be resolved following the genome project. RNA interference is becoming an efficient reverse genetic tool for studying gene function in model organisms, including C.elegans and Drosophila melanogaster. Our molecular evolutionary study has shown that a prototype of glycosyltransferases, which synthesize a variety of glycan structures in the Golgi apparatus, was conserved between mammals and Drosophila. For analyses of the basic physiological functions of glycans, we established the Drosophila inducible RNAi knockdown system and applied it to one glycosyltransferase and one transporter, proteoglycan UDP-galactose: beta-xylose beta1,4galactosyltransferase I and the PAPS-transporter, respectively. If on the silencing of each gene induced ubiquitously under the control of a cytoplasmic actin promoter, the RNAi knockdown fly died, then the protein was indispensable for life. The expression of the target gene was disrupted specifically and the degree of interference was well correlated with the phenotype. The inducible RNAi knockdown fly obtained using the GAL4-UAS system will pave the way for the functional analysis of glycans.     

Reduced pain hypersensitivity and inflammation in mice lacking microsomal prostaglandin e synthase-1

We examined the in vivo role of membrane-bound prostaglandin E synthase (mPGES)-1, a terminal enzyme in the PGE2-biosynthetic pathway, using mPGES-1 knockout (KO) mice. Comparison of PGES activity in the membrane fraction of tissues from mPGES-1 KO and wild-type (WT) mice indicated that mPGES-1 accounted for the majority of lipopolysaccharide (LPS)-inducible PGES in WT mice. LPS-stimulated production of PGE2, but not other PGs, was impaired markedly in mPGES-1-null macrophages, although a low level of cyclooxygenase-2-dependent PGE2 production still remained. Pain nociception, as assessed by the acetic acid writhing response, was reduced significantly in KO mice relative to WT mice. This phenotype was particularly evident when these mice were primed with LPS, where the stretching behavior and the peritoneal PGE2 level of KO mice were far less than those of WT mice. Formation of inflammatory granulation tissue and attendant angiogenesis in the dorsum induced by subcutaneous implantation of a cotton thread were reduced significantly in KO mice compared with WT mice. Moreover, collagen antibody-induced arthritis, a model for human rheumatoid arthritis, was milder in KO mice than in WT mice. Collectively, our present results provide unequivocal evidence that mPGES-1 contributes to the formation of PGE2 involved in pain hypersensitivity and inflammation.     

Selective changes in gangliosides of human milk during lactation: a molecular indicator for the period of lactation

Gangliosides of human milk from women at various periods of lactation were analyzed. GD3 in colostrum, particularly in the early period of lactation, was the major ganglioside, and the molar ratio of GM3 to GD3 was 0.2-0.3 in the milk at 2-6 days postpartum. In contrast, milk from women at 60-390 days postpartum contained GM3 as the major ganglioside and the molar ratio of GM3 to GD3 was more than 3. Milk at 8-40 days postpartum represented an intermediate stage in terms of the ratio of GM3 to GD3. The selective change in the molar ratio of gangliosides was observed as a phenomenon common to all human milk from different individuals at different periods of lactation, indicating that the periods of lactation can be defined on the basis of the ratio. Since glycolipids in human milk are preferentially localized in the milk fat globule membrane, which is derived from the plasma membrane of epithelial cells in the mammary gland, the changes in the ganglioside composition reported in this communication may reflect a qualitative change of the cells in the mammary gland.     

An Electron Spin Resonance Study for Real-time Detection of Ascorbyl Free Radicals After Addition of Dimethyl Sulfoxide in Murine Hippocampus or Plasma During Kainic Acid-Induced Seizures

Electron spin resonance (ESR)-silent ascorbate solutions generate a detectable, likely concentration-dependent signal of ascorbyl free radicals (AFR) immediately upon addition of a molar excess of dimethyl sulfoxide (DMSO). We aimed to perform quantitative ESR analysis of AFR in real time after addition of DMSO (AFR/DMSO) to evaluate ascorbate concentrations in fresh hippocampus or plasma following systemic administration of kainate in mice. Use of a special tissue-type quartz cell allowed immediate detection of AFR/DMSO ESR spectra in fresh tissues from mice. AFR/DMSO content was increased significantly in fresh hippocampus or plasma obtained during kainate-induced seizures of mice, reaching maximum levels at 90 min after intraperitoneal administration of 50 mg/kg kainic acid. This suggests that oxidative injury of the hippocampus resulted from the accumulation of large amounts of ascorbic acid in the brain after kainic acid administration. AFR/DMSO content measured on an ESR spectrometer can be used for real-time evaluation of ascorbate content in fresh tissue. Due to the simplicity, good performance, low cost and real-time monitoring of ascorbate, this method may be applied to clinical research and treatment in the future.     

Gait analysis using gravitational acceleration measured by wearable sensors

A novel method for measuring human gait posture using wearable sensor units is proposed. The sensor units consist of a tri-axial acceleration sensor and three gyro sensors aligned on three axes. The acceleration and angular velocity during walking were measured with seven sensor units worn on the abdomen and the lower limb segments (both thighs, shanks and feet). The three-dimensional positions of each joint are calculated from each segment length and joint angle. Joint angle can be estimated mechanically from the gravitational acceleration along the anterior axis of the segment. However, the acceleration data during walking includes three major components; translational acceleration, gravitational acceleration and external noise. Therefore, an optimization analysis was represented to separate only the gravitational acceleration from the acceleration data. Because the cyclic patterns of acceleration data can be found during constant walking, a FFT analysis was applied to obtain some characteristic frequencies in it. A pattern of gravitational acceleration was assumed using some parts of these characteristic frequencies. Every joint position was calculated from the pattern under the condition of physiological motion range of each joint. An optimized pattern of the gravitational acceleration was selected as a solution of an inverse problem. Gaits of three healthy volunteers were measured by walking for 20 s on a flat floor. As a result, the acceleration data of every segment was measured simultaneously. The characteristic three-dimensional walking could be shown by the expression using a stick figure model. In addition, the trajectories of the knee joint in the horizontal plane could be checked by visual imaging on a PC. Therefore, this method provides important quantitive information for gait diagnosis.     

Modulation of rat cecal microbiota by administration of raffinose and encapsulated Bifidobacterium breve

To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.     

Normal specification of the extraembryonic lineage after somatic nuclear transfer

To examine the establishment and maintenance of trophectoderm (TE) lineage in somatic cloned blastocysts, the expression of Cdx2, a key molecule for specification of TE fate, was immunohistochemically examined simultaneously with Oct4 expression. Cloned mouse embryos were made by nuclear transfer using cumulus cells, tail-tip fibroblasts, and embryonic stem cells. After 96 h of culture, the rates of Oct4-expressing blastocysts were as low as 50% and 60% for cumulus and fibroblast clones, respectively. However, regardless of Oct4 expression, the majority of those cloned blastocysts (> 90%) normally expressed Cdx2. Thus, even though somatic cloned embryos have reduced potential to produce the inner cell mass lineage, the TE lineage can be established and maintained.     

Regulated Expression of a Gene-Fusion Product Derived from the Gene for Phytochrome I from Pisum sativum and the uidA Gene from E. coli in Transgenic Petunia hybrida

The 5'-upstream region (2.4 kb) of the gene for phytochromeI from Pisum sativum (phyl) was fused to the uidA gene fromEscherichia coli that encodes ß-glucuronidase (GUS).The resulting PHY-GUS fusion was introduced into Petunia hybridaand was used as a reporter of the expression of the phyI genewhich was recognized by GUS activity. The PHY-GUS fusion wasexpressed at a relatively high level when transgenic plantswere grown in the dark, while leaves and stems of light-grownplants showed background activity. Flowers of light-grown plantswere shown to have significant levels of GUS activity but rootsdid not have such activity. When light-grown transgenic plantswere transferred to the dark, they expressed the activity atlevels that corresponded to those of dark-grown plants. Lighttreatment prior to growth in darkness revealed red/far-red reversibilityof recovery of the activity. Thus, the 2.4-kb fragment fromthe 5' region of the phyI gene carries the information necessaryfor the light-repressible autoregulation. (Received March 30, 1991; Accepted May 20, 1991)     

New Neplanocin Analogues. V. A Potent Adenosylhomocysteine Hydrolase Inhibitor Lacking Antiviral Activity. Synthesis And Antiviral Activity Of 6″-Carboxylic Acid Derivatives Of Neplanocin A 1

Abstract

The 6′-carboxylic acid derivative of neplanocin A 3 was synthesized from NPA, and was converted to the corresponding methyl ester 4 and amides 5 and 6. These were evaluated for their anti-RNA-virus activities. Of the derivatives synthesized, only 5 was active against RNA viruses within the concentration range of 0.14-4.88 μg/mL. Compounds 3 and 5 showed a potent inhibitory effect on S-adenosylhomocysteine (AdoHcy) hydrolase from rabbit erythrocytes. Although a close correlation between the inhibitory effect of adenosine analogues on AdoHcy hydrolase and their antiviral potency has been demonstrated, 3 did not show any anti-RNA-virus activities.