Cellular expression of murine Ym1 and Ym2, chitinase family proteins,as revealed by in situ hybridization and immunohistochemistry

Ym is one of the chitinase family proteins, which are widely distributed in mammalian bodies and can bind glycosaminoglycans such as heparin/heparan sulfate. Ym1 is a macrophage protein produced in parasitic infections, while its isoform, Ym2, is upregulated in lung under allergic conditions. In the present study, we revealed the distinct cellular expression of Ym1 and Ym2 in normal mice by in situ hybridization and immunohistochemistry. Ym1 was principally expressed in the lung, spleen, and bone marrow, while Ym2 was found in the stomach. Ym1-expressing cells in the lung were alveolar macrophages, and the immunoreactivity for Ym1 was localized in rough endoplasmic reticulum. In the spleen, Ym1-expressing cells gathered in the red pulp and were electron microscopically identified as immature neutrophils. In the bone marrow, immature neutrophils were intensely immunoreactive, but lost this immunoreactivity with maturation. Moreover, needle-shaped crystals in the cytoplasm of macrophages, which formed erythroblastic islands, also showed intense Ym1 immunoreactivity. Ym2 expression was restricted to the stratified squamous epithelium in the junctional region between forestomach and glandular stomach. The function of Ym1 and Ym2 is still unclear; however, the distinct cellular localization under normal conditions suggests their important roles in hematopoiesis, tissue remodeling, or immune responses as an endogenous lectin.     

Effect of phytate-removal and deamidation of soybean proteins on calcium absorption in the in situ rats

Soybean proteins were deamidated by cation-exchange resins after phytate, the inhibitor for calcium absorption from the small intestine, was removed in order to provide the enhancement function of calcium absorption to soybean proteins. About 92% of the phosphorus was removed from the soybean proteins by anion-exchange-resin treatment, indicating that most of the phytate was removed. About 70% of the acid amide was deamidated by cation-exchange-resin treatment, and phytate-removed and deamidated soybean proteins (PrDS) having high calcium binding properties were obtained. PrDS were hydrolyzed by digestive enzymes and their calcium-binding properties and the enhancement function of the calcium absorption from the small intestine of rats were examined. As a result, PrDS retained their high calcium binding properties even after hydrolysis by digestive enzymes. In situ experiments showed that PrDS and their hydrolysates enhanced the calcium absorption from the intestine.     

Cloning, nucleotide sequence, and overexpression of the L-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli

The gene encoding L-rhamnose isomerase (L-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the L-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of L-RhI from E. coli are conserved in that from P. stutzeri. The L-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of L-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant L-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant L-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60 degrees C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.     

Molecular dynamics and interactions for creation of stimulation-induced stabilized rafts from small unstable steady-state rafts

We have evaluated the sizes and lifetimes of rafts in the plasma membrane from the existing literature, with a special attention paid to their intrinsically broad distributions and the limited time and space scales that are covered by the observation methods used for these studies. Distinguishing the rafts in the steady state (reserve rafts) from those after stimulation or unintentional crosslinking of raft molecules (stabilized receptor-cluster rafts) is critically important. In resting cells, the rafts appear small and unstable, and the consensus now is that their sizes are smaller than the optical diffraction limit (250 nm). Upon stimulation, the raft-preferring receptors are clustered, inducing larger, stabilized rafts, probably by coalescing small, unstable rafts or cholesterol-glycosphingolipid complexes in the receptor clusters. This receptor-cluster-induced conversion of raft types may be caused by suppression of alkyl chain isomerization and the lipid lateral diffusion in the cluster, with the aid of exclusion of cholesterol from the bulk domain and the boundary region of the majority of transmembrane proteins. We critically inspected the possible analogy to the boundary lipid concept. Finally, we propose a hypothesis for the coupling of GPI-anchored receptor signals with lipid-anchored signaling molecules in the inner-leaflet raft.     

Induction of Haploid Androgenesis in Pacific Oyster by UV Irradiation

Androgenesis, development from paternal but not maternal chromosomes, can be induced in some organisms including fish, but has not been induced previously in mollusk. In this study we investigated the induction of haploid androgenesis in the Pacific oyster by ultraviolet irradiation and observed nuclear behavior in the androgenetic eggs. Irradiation for 90 seconds at a UV intensity of 72 erg/mm² per second (6480 erg/mm²) was the optimal dose to achieve haploid androgenesis. The fertilization and development rates of D-shaped larvae decreased with increasing exposure time, and the development of the genetically inactivated eggs terminated before reaching the D-shaped stage. Cytologic observations showed that UV irradiation did not affect germinal vesicle breakdown or chromosomal condensation but caused various nuclear behavioral patterns during meiosis and first mitosis: 21.7% of eggs extruded all maternal chromosomes as 2 or 3 polar bodies, and 59.1% of eggs formed one female pronucleus. The maternally derived nucleus did not participate, or partially participated, in the first karyokinesis. The cytologic evidence demonstrates that the male genome is directing development in haploids produced by UV irradiation.     

Non-destructive on-chip cell sorting system with real-time microscopic image processing

Studying cell functions for cellomics studies often requires the use of purified individual cells from mixtures of various kinds of cells. We have developed a new non-destructive on-chip cell sorting system for single cell based cultivation, by exploiting the advantage of microfluidics and electrostatic force. The system consists of the following two parts: a cell sorting chip made of poly-dimethylsiloxane (PDMS) on a 0.2-mm-thick glass slide, and an image analysis system with a phase-contrast/fluorescence microscope. The unique features of our system include (i) identification of a target from sample cells is achieved by comparison of the 0.2-μm-resolution phase-contrast and fluorescence images of cells in the microchannel every 1/30 s; (ii) non-destructive sorting of target cells in a laminar flow by application of electrostatic repulsion force for removing unrequited cells from the one laminar flow to the other; (iii) the use of agar gel for electrodes in order to minimize the effect on cells by electrochemical reactions of electrodes, and (iv) pre-filter, which was fabricated within the channel for removal of dust contained in a sample solution from tissue extracts. The sorting chip is capable of continuous operation and we have purified more than ten thousand cells for cultivation without damaging them. Our design has proved to be very efficient and suitable for the routine use in cell purification experiments.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

Absence of the candidate male sex-determining gene dmrt1b(Y) of medaka from other fish species

Although the sex-determining genes are known in mammals, Drosophila, and C. elegans, little is known in other animals. Fishes are an attractive group of organisms for studying the evolution of sex determination because they show an amazing variety of mechanisms, ranging from environmental sex determination and different forms of hermaphroditism to classical sex chromosomal XX/XY or WZ/ZZ systems and modifications thereof. In the fish medaka, dmrt1b(Y) has recently been found to be the candidate male sex-determining gene. It is a duplicate of the autosomal dmrt1a gene, a gene acting in the sex determination/differentiation cascade of flies, worms, and mammals. Because in birds dmrt1 is located on the Z-chromosome, both findings led to the suggestion that dmrt1b(Y) is a "non-mammalian Sry" with an even more widespread distribution. However, although Sry was found to be the male sex-determining gene in the mouse and some other mammalian species, in some it is absent and has obviously been replaced by other genes that now fulfil the same function. We have asked if the same might be true of the dmrt1b(Y) gene. We find that the gene duplication generating dmrt1b(Y) occurred recently during the evolution of the genus Oryzias. The gene is absent from all other fish species studied. Therefore, it may not be the male-sex determining gene in all fishes.