Nuclear sex-determining genes cause large sex-ratio variation in the apple snail Pomacea canaliculata

Evolutionary maintenance of genetic sex-ratio variation is enigmatic since genes for biased sex ratios are disadvantageous in finite populations (the "Verner effect"). However, such variation could be maintained if a small number of nuclear sex-determining genes were responsible, although this has not been fully demonstrated experimentally. Brood sex ratios of the freshwater snail Pomacea canaliculata are highly variable among parents, but population sex ratios are near unity. In this study, the effect of each parent on the brood sex ratio was investigated by exchanging partners among mating pairs. There were positive correlations between sex ratios of half-sib broods of the common mother (r = 0.42) or of the common father (r = 0.47). Moreover, the correlation between full-sib broods was very high (r = 0.92). Thus, both parents contributed equally to the sex-ratio variation, which indicates that nuclear genes are involved and their effects are additive. Since the half-sib correlations were much stronger than the parent-offspring regressions previously obtained, the variation was caused by zygotic sex-determining genes rather than by parental sex-ratio genes. The number of relevant genes appears to be small.     

Constitutively active parathyroid hormone receptor signaling in cells in osteoblastic lineage suppresses mechanical unloading-induced bone resorption

Multiple signaling pathways participate in the regulation of bone remodeling, and pathological negative balance in the regulation results in osteoporosis. However, interactions of signaling pathways that act comprehensively in concert to maintain bone mass are not fully understood. We investigated roles of parathyroid hormone receptor (PTH/PTHrP receptor) signaling in osteoblasts in unloading-induced bone loss using transgenic mice. Hind limb unloading by tail suspension reduced bone mass in wild-type mice. In contrast, signaling by constitutively active PTH/PTHrP receptor (caPPR), whose expression was regulated by the osteoblast-specific Col1a1 promoter (Col1a1-caPPR), suppressed unloading-induced reduction in bone mass in these transgenic mice. In Col1a1-caPPR transgenic (Tg) mice, hind limb unloading suppressed bone formation parameters in vivo and mineralized nodule formation in vitro similarly to those observed in wild-type mice. In addition, serum osteocalcin levels and mRNA expression levels of type I collagen, Runx2 and Osterix in bone were suppressed by unloading in both wild-type mice and Tg mice. However, in contrast to unloading-induced enhancement of bone resorption parameters in wild-type mice, Col1a1-caPPR signaling suppressed, rather than enhanced, osteoclast number and osteoclast surface as well as urinary deoxypyridinoline excretion upon unloading. Col1a1-caPPR signaling also suppressed mRNA expression levels of RANK and c-fms in bone upon unloading. Although the M-CSF and monocyte chemoattractant protein 1 (MCP-1) mRNA levels were enhanced in control Tg mice, these levels were suppressed in unloaded Tg mice. These results indicated that constitutive activation of PTH/PTHrP receptor signaling in osteoblastic cells suppresses unloading-induced bone loss specifically through the regulation of osteoclastic activity.     

Screening procedure for the analysis of distigmine bromide in serum by high-performance liquid chromatography-electrospray ionization mass spectrometry

A screening procedure was developed for the identification and quantification of distigmine bromide in serum samples by using liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS). In this method, distigmine bromide was analyzed in 0.5 mL serum by using pancuronium bromide as the internal standard, and gradient elution was performed using a reversed-phase column and a mixture of 10 mM-ammonium formate and methanol as the mobile phase. A highly sensitive assay could be performed with simple solid phase extraction using a cation exchange cartridge column by carrying out selected ion monitoring analysis in the positive ion detection mode. The procedure was validated in terms of linearity (0.9973 at 2.5 ng/mL). The inter- and intra-day precisions (coefficient of variation; CV%) were <8.5% and < 9.7%, respectively. The analytes were evaluated for stability and were found to be stable in serum for 1 week at 4 degrees C and 4 weeks at -30 degrees C, and successfully applied to in the analysis of two overdose cases. This method is sensitive and useful for the detection, quantification, and confirmation of distigmine bromide in serum.     

Possible molecular evolution of biomembranes: from single-chain to double-chain lipids

We have studied a possible evolution process permitting a 'primitive' membrane to evolve towards a membrane structure with an outer wall, similar to that of bacteria. We have investigated whether a polysaccharide bearing hydrophobic phytyl or cholesteryl chains coats giant vesicles made of single- or double-chain lipids. Phytyl-pullulan 5b was found to bind to the surface of vesicles made of either single- or double-chain lipids. In contrast, cholesteryl-pullulan 5a only coated the surface of vesicles made of double-chain lipids. These results indicate that there must be a close match between the size and shape of membrane constituents and the hydrophobic molecules to be inserted. This process could, thus, provide a selection mechanism of lipid-membrane constituents during the course of biomembrane evolution. The presence of the above 'hydrophobized' polysaccharides on the surface of different giant vesicles was identified by lectin binding. Both concanavalin A and annexin V were shown by fluorescence microscopy to bind spontaneously to vesicles made of double-chain lipids. Our experiments exemplify that self-organization of amphiphiles into closed vesicles in aqueous solution automatically leads to the coating of vesicles by 'hydrophobized' polysaccharides, which then permit lectin binding. This is a possible mechanism for the evolution of primitive membranes towards 'proto-cells'.     

Isolation and partial characterization of two glycoproteins from human liver metastases of sigmoid colon carcinoma

1. Perchloric acid-soluble glycoprotein fraction (PASF) extracted from human liver metastases (LM) of sigmoid colon carcinoma was chromatographed on a DEAE-cellulose column. The main fraction (DEAE-nonadsorbed fraction) passed through the column was then subjected to Sephacryl S-200 superfine gel filtration and separated into 12 fractions. 2. Among 12 fractions, only both Fractions 3 and 4 were demonstrated to be chemically and immunologically homogeneous glycoproteins, respectively, by a combination of chemical composition analysis, SDS-PAGE and EITB assay using antisera against the DEAE-nonadsorbed fractions of PASFs from human LMs, normal liver (NL) and normal sigmoid colon (NSC). Each of Fractions 3 and 4 reacted with anti-LM serum to give one immuno complex on a nitrocellulose sheet in EITB assay, but did not react with anti-NL and -NSC sera. 3. Apparent molecular weights of 80,900 and 62,100, respectively, were found for Fractions 3 and 4. Both the fractions, respectively, had abnormal sugar compositions. Fraction 3 contained sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine, but lacked glucose and mannose, and Fraction 4 contained sialic acid, fucose, galactose and N-acetylglucosamine, but lacked glucose, mannose and N-acetylgalactosamine, as sugar components.     

Totally synthetic polymer with lectin-like function: induction of killer cells by the copolymer of 3-acrylamidophenylboronic acid with N,N-dimethylacrylamide

Lectin-like function was demonstrated in this study for a novel water-soluble polymer with phenylboronic acid residues (poly (AAPBA-DMAm)), which induced appreciable proliferation of murine spleen lymphocytes with an increased expression of interleukin-2 (IL-2) receptor on their surface. Consequently, boosted proliferation of lymphocytes with cytotoxic action to YAC-1 cells was achieved by concurrent addition of IL-2 with poly(AAPBA-DMAm) in the medium, indicating this boronate-containing polymer to be worked as an effective immuno-adjuvant for the induction of lymphokine-activated killer (LAK) cells. Flow-cytofluorimetry study revealed that poly(AAPBA-DMAm) competitively inhibited the cellular binding of N-acetylneuraminic acid-specific lectin (Limax Flavus Agglutinin) in a concentration-dependent manner, suggesting that phenylboronate moiety in the polymer may recognize N-acetylneur- aminic acid (sialic acid) residues existing on the plasma-membrane surface of lymphocytes to induce their proliferation.