Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells

The gene expression patterns of primordial germ cells (PGCs) and embryonic stem cells were analyzed by a modified serial analysis of gene expression. During the process, we cloned a novel gene, PGC7, which was preferentially expressed in PGCs. Immunohistochemical analysis revealed that PGC7 was specifically expressed in early pre-implantation embryos, PGCs and oocytes. These results suggest that PGC7 might play an important role in the development of PGCs and oocytes.     

Pyrazole derivatives as new potent and selective 20-hydroxy-5,8,11,14-eicosatetraenoic acid synthase inhibitors

Improvement of the physical properties of pyrazole derivative 1, which we reported previously as a potent and selective 20-HETE synthase inhibitor (IC(50) 5.7 nM), is described. Introduction of a sufficient substituted-amino group on the side chain enhanced the water-solubility of 1 (0.014 mg/mL at pH 6.8). Among the products, 2-piperazinoethoxy derivatives 3e and 6b showed solubility suitable for injection and potent inhibitory activity toward 20-HETE synthase (IC(50) 21.2 and 14.0 nM, respectively).     

Ancient Origin of the Null Allele se 428 of the Human ABO-Secretor Locus (FUT2)

In human populations, a null allele having several nucleotide differences from the wild-type allele is segregating at the FUT2 locus (the ABO-Secretor locus) encoding α(1,2)fucosyltransferase. To estimate the age of the most recent common ancestor (MRCA) of these two alleles, we sequenced FUT2 homologues from chimpanzee, gorilla, orangutan, and green monkey. Since we did not detect acceleration or any heterogeneity in the substitution rate at this locus among these species, the age of the MRCA was estimated to be around 3 MYA, assuming the divergence time of human and chimpanzee to be 5 MYA. We developed a simple test to examine whether or not the old age of the MRCA of the FUT2 is consistent with that expected for two divergent neutral alleles sampled from a random mating population. An application of the test to the data at FUT2 indicated that the age of the MRCA is too old to be explained by the simple neutral assumptions, although our test depends on accurate estimation of the divergence time of human and chimpanzee in units of twice the human population size. Various possibilities including balancing selection are discussed to explain this old age of the MRCA. Received: 9 May 1999 / Accepted: 20 September 1999     

Antiestrogenic activity of vitamin A in in vivo uterotrophic assay

All-trans-retinoic acid (ATRA), the primary active metabolite of vitamin A, was examined for its antiestrogenic activity in rats using an in vivo uterotrophic assay. All rats were ovariectomized 2 weeks prior to receiving 5 mg/kg/day ATRA or 0.3 micro g/kg/day ethynyl estradiol (EE) subcutaneously once a day for 3 consecutive days. Rats were sacrificed 1, 3, 6, 12 or 24 h after the last treatment. EE increased uterine weight and the coinjection of ATRA with EE significantly suppressed this effect 3 and 24 h after treatment. mRNA expression was examined during this 24-h period and the mRNA expression levels of estrogen receptor alpha (ER alpha), retinoic acid receptor beta (RAR beta), retinoid X receptor gamma (RXR gamma) and cellular retinol-binding protein I (CRBP I) were found to have significantly increased in the ATRA+EE group compared with those in the EE group. This is the first report on the antiestrogenic activity of ATRA determined using an in vivo adult rat uterotrophic assay. The up-regulation of RAR or RXR mRNA expression level was probably responsible for the antiestrogenic activity of ATRA.     

Intestinal Dysbiosis and Lowered Serum Lipopolysaccharide-Binding Protein in Parkinson’s Disease

Background

The intestine is one of the first affected organs in Parkinson’s disease (PD). PD subjects show abnormal staining for Escherichia coli and α-synuclein in the colon.

Methods

We recruited 52 PD patients and 36 healthy cohabitants. We measured serum markers and quantified the numbers of 19 fecal bacterial groups/genera/species by quantitative RT-PCR of 16S or 23S rRNA. Although the six most predominant bacterial groups/genera/species covered on average 71.3% of total intestinal bacteria, our analysis was not comprehensive compared to metagenome analysis or 16S rRNA amplicon sequencing.

Results

In PD, the number of Lactobacillus was higher, while the sum of analyzed bacteria, Clostridium coccoides group, and Bacteroides fragilis group were lower than controls. Additionally, the sum of putative hydrogen-producing bacteria was lower in PD. A linear regression model to predict disease durations demonstrated that C. coccoides group and Lactobacillus gasseri subgroup had the largest negative and positive coefficients, respectively. As a linear regression model to predict stool frequencies showed that these bacteria were not associated with constipation, changes in these bacteria were unlikely to represent worsening of constipation in the course of progression of PD. In PD, the serum lipopolysaccharide (LPS)-binding protein levels were lower than controls, while the levels of serum diamine oxidase, a marker for intestinal mucosal integrity, remained unchanged in PD.

Conclusions

The permeability to LPS is likely to be increased without compromising the integrity of intestinal mucosa in PD. The increased intestinal permeability in PD may make the patients susceptible to intestinal dysbiosis. Conversely, intestinal dysbiosis may lead to the increased intestinal permeability. One or both of the two mechanisms may be operational in development and progression of PD.     

Comparison of the in vitro apparent permeability and stability of opioid mimetic compounds with that of the native peptide

Three dimethyl-L-tyrosine (Dmt) based peptide analogues were identified in a previous study as excellent agonists for the mu-opioid receptor showing very low K(i) values and good in vivo antinociceptive activity upon intracerebroventricular administration to mice. This activity decreased markedly when the compounds were delivered subcutaneously or orally. To establish the cause of this decrease of activity the apparent permeability across Caco-2 cell monolayers of each compound and their relative stability to the digestive enzymes present in the cell line has been determined and compared to that of the native peptide endomorphin 2. The compounds' permeabilities clearly correlate with their increasing lipophilicity suggesting that the analogues cross the monolayer via passive diffusion and the results show that the compound with high K(i) value for the mu-receptor (K(i)mu=0.114 nM) exhibited the highest permeability suggesting that this may be the better lead compound despite the lower binding affinity than that of compound 2 or 3.     

Measuring context dependency in birdsong using artificial neural networks

Context dependency is a key feature in sequential structures of human language, which requires reference between words far apart in the produced sequence. Assessing how long the past context has an effect on the current status provides crucial information to understand the mechanism for complex sequential behaviors. Birdsongs serve as a representative model for studying the context dependency in sequential signals produced by non-human animals, while previous reports were upper-bounded by methodological limitations. Here, we newly estimated the context dependency in birdsongs in a more scalable way using a modern neural-network-based language model whose accessible context length is sufficiently long. The detected context dependency was beyond the order of traditional Markovian models of birdsong, but was consistent with previous experimental investigations. We also studied the relation between the assumed/auto-detected vocabulary size of birdsong (i.e., fine- vs. coarse-grained syllable classifications) and the context dependency. It turned out that the larger vocabulary (or the more fine-grained classification) is assumed, the shorter context dependency is detected.     

Splicing Reporter Mice Revealed the Evolutionally Conserved Switching Mechanism of Tissue-Specific Alternative Exon Selection

Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of “splice code”. So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this “balance” model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the “switch-like” mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved “splice code,” in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions.