Molecular cloning and expression profile of Xenopus calcineurin A subunit(1)

We have cloned a cDNA encoding a catalytic subunit of calcineurin (CnA) expressed in Xenopus oocytes. The deduced amino acid sequence indicates 96.3% and 96.8% identities with the mouse and human CnAalpha isoforms, respectively. Xenopus CnA (XCnA) RNA and protein are expressed as maternal and throughout development. Recombinant XCnA protein interacted with calmodulin in the presence of Ca(2+). Deletion of calmodulin binding domain and auto-inhibitory domain revealed calcium independent phosphatase activity, thereby showing that XCnA is likely to be modulated by both calmodulin and calcium.     

Conversion of cholic acid and chenodeoxycholic acid into their 7-oxo derivatives by Bacteroides intestinalis AM-1 isolated from human feces

Secondary bile acid-producing bacteria were isolated from human feces to improve our appreciation of the functional diversity and redundancy of the intestinal microbiota. In total, 619 bacterial colonies were isolated using a nutrient-poor agar medium and the level of secondary bile acid formation was examined in each by a liquid culture, followed by thin-layer chromatography. Of five strains analyzed by 16S rRNA gene sequencing and biochemical testing, one was identified as Bacteroides intestinalis AM-1, which was not previously recognized as a secondary bile-acid producer. GC-MS revealed that B. intestinalis AM-1 converts cholic acid (CA) and chenodeoxycholic acid into their 7-oxo derivatives, 7-oxo-deoxycholic acid (7-oxo-DCA) and 7-oxo-lithocholic acid, respectively. Thus, B. intestinalis AM-1 possesses 7α-hydroxysteroid dehydrogenase (7α-HSDH) activity. In liquid culture, B. intestinalis AM-1 showed a relatively higher productivity of 7-oxo-DCA than Escherichia coli HB101 and Bacteroides fragilis JCM11019^T, which are known to possess 7α-HSDH activity. The level of 7α-HSDH activity was higher in B. intestinalis AM-1 than in the other two strains under the conditions tested. The 7α-HSDH activity in each of the three strains is not induced by CA; instead, it is regulated in a growth phase-dependent manner.     

Sense-overlapping lncRNA as a decoy of translational repressor protein for dimorphic gene expression

Long noncoding RNAs (lncRNAs) are vastly transcribed and extensively studied but lncRNAs overlapping with the sense orientation of mRNA have been poorly studied. We analyzed the lncRNA DAPALR overlapping with the 5´ UTR of the Doublesex1 (Dsx1), the male determining gene in Daphnia magna. By affinity purification, we identified an RNA binding protein, Shep as a DAPALR binding protein. Shep also binds to Dsx1 5´ UTR by recognizing the overlapping sequence and suppresses translation of the mRNA. In vitro and in vivo analyses indicated that DAPALR increased Dsx1 translation efficiency by sequestration of Shep. This regulation was impaired when the Shep binding site in DAPALR was deleted. These results suggest that Shep suppresses the unintentional translation of Dsx1 by setting a threshold; and when the sense lncRNA DAPALR is expressed, DAPALR cancels the suppression caused by Shep. This mechanism may be important to show dimorphic gene expressions such as sex determination and it may account for the binary expression in various developmental processes.     

Non-fucosylated therapeutic antibodies: the next generation of therapeutic antibodies

Therapeutic antibody IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are of the complex biantennary type, composed of a trimannosyl core structure with the presence or absence of core fucose, bisecting N-acetylglucosamine (GlcNAc), galactose, and terminal sialic acid, which gives rise to structural heterogeneity. Both human serum IgG and therapeutic antibodies are well known to be heavily fucosylated. Recently, antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, has been found to be one of the critical effector functions responsible for the clinical efficacy of therapeutic antibodies such as anti-CD20 IgG1 rituximab (Rituxan^®) and anti-Her2/neu IgG1 trastuzumab (Herceptin^®). ADCC is triggered upon the binding of lymphocyte receptors (FcγRs) to the antibody Fc region. The activity is dependent on the amount of fucose attached to the innermost GlcNAc of N-linked Fc oligosaccharide via an α-1,6-linkage, and is dramatically enhanced by a reduction in fucose. Non-fucosylated therapeutic antibodies show more potent efficacy than their fucosylated counterparts both in vitro and in vivo, and are not likely to be immunogenic because their carbohydrate structures are a normal component of natural human serum IgG. Thus, the application of non-fucosylated antibodies is expected to be a powerful and elegant approach to the design of the next generation therapeutic antibodies with improved efficacy. In this review, we discuss the importance of the oligosaccharides attached to the Fc region of therapeutic antibodies, especially regarding the inhibitory effect of fucosylated therapeutic antibodies on the efficacy of non-fucosylated counterparts in one medical agent. The impact of completely non-fucosylated therapeutic antibodies on therapeutic fields will be also discussed.     

Wnt signalling regulates paxillin ubiquitination essential for mesodermal cell motility

Gastrulation movements are critical for establishing the three germ layers and the architecture of vertebrate embryos. During Xenopus laevis gastrulation, mesodermal tissue migrates on the blastocoel roof and elongates along the antero-posterior axis. During this process, cells in the dorsal mesoderm are polarized and intercalate with each other, which is defined as convergent extension and is known to be regulated by the non-canonical Wnt pathway. Here, we show that paxillin plays an essential role in this process. Paxillin is a focal-adhesion associated protein implicated in the regulation of actin cytoskeletal organization and cell motility, but its role in Xenopus embryogenesis has not yet been clarified. We demonstrate that the Wnt pathway controls the ubiquitination and stability of paxillin, and that this regulatory mechanism is essential for convergent extension movements. We identified a RING finger protein XRNF185, which physically binds to paxillin and the proteasome. XRNF185 destabilizes paxillin at focal adhesions and promotes mesodermal cell migration during convergent extension. We propose a mechanism to regulate gastrulation movements that involves paxillin ubiquitination and stability controlled by Wnt signalling.     

Biosynthetic origin of the carbon skeleton and nitrogen atom of pamamycin-607, a nitrogen-containing polyketide

The biosynthesis of pamamycin-607 (PM-607), a sixteen-membered macrodiolide compound, was studied with 13C- and 15N-labeled precursor units in Streptomyces alboniger. Feeding experiments with 13C-labeled acetate or propionate indicate that the carbon skeleton of PM-607 was derived from six acetate, four propionate and three succinate units. MS analyses of 15N-labeled PM-607 suggest that the nitrogen atom in PM-607 was derived from the alpha-amino group of an amino acid.     

Polymorphisms of TNFalpha, IL1beta, and IL1RN genes in chronic obstructive pulmonary disease

It is recognized that genetic factors play a role in the susceptibility to COPD. COPD is characterized by airflow limitation. Chronic inflammation causes small airway disease and parenchymal destruction, leading to the airflow limitation. Polymorphisms in pro-inflammatory cytokine genes may confer a risk for the development of COPD. A case-control association study was performed in Japanese population (88 COPD patients and 61 controls) and Egyptian population (106 patients and 72 controls). Genotype and allele frequencies of the TNFalpha -308 G/A and +489 G/A polymorphisms, the IL1beta -511 C/T, -31 T/C, and +3954 C/T polymorphisms, and a VNTR polymorphism in intron 2 of the IL1RN gene were investigated. In addition, pairwise haplotype frequencies were analyzed. When studied independently, none of the polymorphisms were associated with the development of COPD in both populations. However, in the Egyptian population, the distributions of the haplotype (IL1beta -31 T/C : IL1beta +3954 C/T) were significantly different between the COPD patients and the controls (p(corr)=0.0037). Our findings suggest that this haplotype within the IL1beta gene may be involved in the pathogenesis of COPD and that the genetic factors of COPD susceptibility might be different between different populations.