Mice lacking connexin45 conditionally in cardiac myocytes display embryonic lethality similar to that of germline knockout mice without endocardial cushion defect

The gap junction protein connexin45-deficient (Cx45-KO) mice die shortly after the hearts begin to beat. In addition to the heart defect, they also show defective vascular development which may be closely related with the cardiac phenotype. Therefore, we created mice whose floxed-Cx45 locus could be removed conditionally. We utilized cardiac alpha-actin-Cre transgenic mice to investigate the specific cardiac muscular function of Cx45 in vivo. The resultant conditional mutants were lethal, showing conduction block similar to that of the Cx45-KO mice. Unlike Cx45-KO, development of the endocardial cushion was not disrupted in the conditional mutants. X-gal staining was detected in the embryonic cardiac myocytes as a hallmark of Cre-loxP mediated floxed-Cx45 deletion. These results reconfirm the requirement of Cx45 for developing cardiac myocytes. These also indicate that establishing the first contractions is a crucial task for the early hearts.     

Thermostabilized ovalbumin that occurs naturally during development accumulates in embryonic tissues

We have reported that ovalbumin accumulates without digestion in various tissues during embryonic development of the chicken. There are different types of ovalbumin with respect to thermal stability and one of them, which was named "HS-ovalbumin" in the present study, was found to have a T(m) value of 83 degrees C and to be present dominantly in albumen, egg yolk, amniotic fluid, and serum of fertilized eggs. HS-ovalbumin, arising physiologically from its native form (N-ovalbumin), is reminiscent of the previously described intermediate form appearing during the production processes of the so-called S-ovalbumin, which disappeared shortly in fertilized eggs. We showed that HS-ovalbumin is distinguishable from S-ovalbumin by a monoclonal antibody and also from N-ovalbumin by the stability to heating. At the late stages of development, ovalbumin of amniotic fluid seems to be swallowed through pharynx, carried in the intestine through stomach, and absorbed in the blood. Analyses by monoclonal antibody and heat treatment indicated that the HS-form occupies the largest fraction of ovalbumin that accumulates in the embryonic tissues. The current findings suggest that HS-ovalbumin is crucial for embryogenesis.     

A unique role of an amino terminal 16-residue region of long-type GATA-6

Human GATA-6 mRNA utilizes two Met-codons in frame as translational initiation codons in cultured mammalian cells. An internal ribosome entry site (IRES) is not present in front of the coding region for short-type GATA-6. The 5'-upstream sequence with a short upstream open reading frame (uORF) did not affect the production of either long- or short-type GATA-6. Introduction of a canonical Kozak sequence around the upstream Met-codon resulted in predominant synthesis of long-type GATA-6, suggesting that the translation of short-type GATA-6 could be due to leaky scanning of the Met-codon by ribosomes. We found that at least the sequence comprising the 90th to 139th nucleotide bases from the first letter of the upstream Met-codon plays a positive role in the expression of long-type GATA-6. This was confirmed by insertion of the corresponding sequence in frame at the site of deletion (the 38th to 304th nucleotide residues). However, insertion of the sequence comprising the 92nd to 141st bases did not suppress the negative effect of the deletion. These results suggest that the translation of this region (Glu-31-Cys-46) could be critical for the apparent production of long-type GATA-6. We also demonstrated that long-type GATA-6 is potentially more active than the short-type.     

Changes of mRNA expression of vascular endothelial growth factor, angiopoietins and their receptors during the periovulatory period in eCG/hCG-treated immature female rats

Angiogenic factors can induce the perifollicular capillary network in the theca interna that shows marked changes in and around the preovulatory luteinizing hormone (LH) surge. To get more information on their functional crosstalk, the aim of the present study was to investigate the manner of mRNA expression of vascular endothelial growth factors (VEGFs) 120, 164, angiopoietin (Ang)-1, Ang-2 and their specific receptors during the periovulatory phase. We used an established equine and human chorionic gonadotropins (eCG/hCG)-derived experimental model capable of stimulating naturally occurring follicular maturation, ovulation and corpus luteum (CL) formation. On day 28 postpartum, immature female rats were administrated s.c. with 10 IU of eCG to promote follicular development, followed 48 hr later by i.p. administration of 20 IU of hCG. Ovaries were dissected at 0, 6, 12, 18 and 24 hr after hCG treatment, and were obtained on day 30 in the untreated control. After induction of follicular growth by the eCG treatment, each mRNA expression of VEGF 120, VEGF 164, Neuropilin-1 and Flt-1 significantly increased. The peaks in mRNA expressions of VEGF120 and VEGF164 were both found at 18 hr after hCG treatment. Flk-1 mRNA expression maintained up to 6 hr after hCG treatment, and then decreased at 12, 18 and 24 hr after hCG treatment. Ang-2 mRNA expression increased in the ovaries at 6 and 12 hr after hCG treatment. Tie-2 mRNA expression decreased at 24 hr after the treatment of gonadotropins. Our findings suggest that ovarian vascular formation during the periovulatory period including preovulatory follicles, ovulation and CL formation may develop via crosstalk of the VEGF-Flt-1 and Ang-Tie2 systems.     

Detection of phosphatidylcholine oxidation products in rat heart using quadrupole time-of-flight mass spectrometry

An improved technique for the analysis of phosphatidylcholine (PC) and lyso-phosphatidylcholine (lyso-PC) oxidation products was developed using quadrupole time of flight (Q-TOF) mass spectrometry with electrospray ionization. We separated these products using an HPLC C(8) column with a gradient of methanol and 10mM aqueous ammonium acetate. Monohydroxides, oxo derivatives, and trihydroxides of palmitoyl-linoleoyl (C16:0/C18:2) PC, stearoyl-linoleoyl (C18:0/C18:2) PC, and oleoyl-linoleoyl (C18:1/C18:2) PC were detected mainly as MH(+) and [M+Na](+) ions in the heart of the intact rat. Using standard synthetic PC-OH (C16:0/C18:2-OH), the lipid extract component was identified as (C16:0/C18:2-OH) PC based on the product ions of ESI-MS-MS and, the PC-OH concentration was quantitated. Four oxidatively modified 1-lyso-phosphatidylcholines (lyso-PCs) were also detected. This is the first report showing the presence of monohydroxides, oxo derivatives, and trihydroxides of (C16:0/C18:2)PC, (C18:0/C18:2)PC, and (C18:1/C18:2) PC in the rat heart.     

The RNA-binding protein SUP-12 controls muscle-specific splicing of the ADF/cofilin pre-mRNA in C. elegans

Tissue-specific alternative pre-mRNA splicing is essential for increasing diversity of functionally different gene products. In Caenorhabditis elegans, UNC-60A and UNC-60B, nonmuscle and muscle isoforms of actin depolymerizing factor (ADF)/cofilin, are expressed by alternative splicing of unc-60 and regulate distinct actin-dependent developmental processes. We report that SUP-12, a member of a new family of RNA recognition motif (RRM) proteins, including SEB-4, regulates muscle-specific splicing of unc-60. In sup-12 mutants, expression of UNC-60B is decreased, whereas UNC-60A is up-regulated in muscle. sup-12 mutations strongly suppress muscle defects in unc-60B mutants by allowing expression of UNC-60A in muscle that can substitute for UNC-60B, thus unmasking their functional redundancy. SUP-12 is expressed in muscle and localized to the nuclei in a speckled pattern. The RRM domain of SUP-12 binds to several sites of the unc-60 pre-mRNA including the UG repeats near the 3'-splice site in the first intron. Our results suggest that SUP-12 is a novel tissue-specific splicing factor and regulates functional redundancy among ADF/cofilin isoforms.     

Remarkably different structures and reaction mechanisms of ketoreductases for the opposite stereochemical control in the biosynthesis of BIQ antibiotics

Two ketoreductases, RED1 and RED2, are involved in the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2) and dihydrogranaticin in S. violaceoruber Tu22, respectively. They are responsible for the stereospecific reductions of the bicyclic intermediate to give (S)- or (R)-DNPA, although there is no similarity between their amino acid sequences. Biotransformation using synthetic analogous substrates revealed that the substrate specificities are quite different. Homology modelling studies and site directed mutagenesis showed remarkable differences in three-dimensional structures and catalytic mechanisms between RED1 and RED2.     

The Rhox5 homeobox gene regulates the region-specific expression of its paralogs in the rodent epididymis

The mechanisms by which the region-specific expression patterns of clustered genes evolve are poorly understood. The epididymis is an ideal organ to examine this, as it is a highly segmented tissue that differs significantly in structure between closely related species. Here we examined this issue through analysis of the rapidly evolving X-linked reproductive homeobox (Rhox) gene cluster, the largest known homeobox gene cluster in metazoans. In the mouse, we found that most Rhox genes are expressed primarily in the caput region of the epididymis, a site where sperm mature and begin acquiring forward motility. This region-specific expression pattern depends, in part, on the founding member of the Rhox cluster--Rhox5--as targeted mutation of Rhox5 greatly diminishes the expression of several other family members in the caput region. In the rat, Rhox5 expression switches from the caput to the site of sperm storage: the cauda. All Rhox genes under the control of Rhox5 in the mouse epididymis display a concomitant change in their regional expression in the rat epididymis. Our results lead us to propose that widespread changes in the region-specific expression pattern of genes over evolutionary time can be the result of alterations of one or only a few master regulatory genes.