Oral/aboral ectoderm differentiation of the sea urchin embryo depends on a planar or secretory signal from the vegetal hemisphere

A monoclonal antibody that recognizes oral ectoderm and esophagus of sea urchin larvae was newly produced. Distribution of the antigen, named Hpoe, was examined by indirect immunofluorescence microscopy. Hpoe did not exist in eggs and appeared during the cleavage stage. In hatched blastulae, Hpoe was detected on the apical surface of all cells. As embryogenesis progressed, Hpoe disappeared from the primary mesenchyme, archenteron and aboral ectoderm. Hpoe reappeared in foregut at the prism stage and was restricted to the oral ectoderm and esophagus at the pluteus stage. Using this antigen as a molecular marker of oral/aboral ectoderm differentiation, the role of the vegetal hemisphere in ectoderm differentiation was examined. All animal hemispheres isolated from 16-cell stage embryos, mesenchyme blastulae, early gastrulae and mid gastrulae developed into epithelial balls and every cell expressed Hpoe. These epithelial balls failed in oral/aboral ectoderm differentiation. Twenty millimolar LiCI-treated whole embryos developed into exo-gastrulae but Hpoe restriction in ectoderm occurred in these exo-gastrulae. These results show that oral/aboral ectoderm differentiation requires an inductive interaction from the vegetal hemisphere and indicate that the inductive interaction depends on a planar or secretory signal, rather than the contact of the esophagus and ectoderm.     

Production of the constant domain of murine T-cell receptor beta-chain in Escherichia coli

T-cell antigen receptor is a heterodimer of disulfide-linked alpha- and beta-chains. Although the essential features of T-cell receptor seem to be rather similar to those of immunoglobulin, the amount of T-cell receptor expressed on the surface of a T-cell is not large enough to be analyzed physcio-chemically. In this study, the DNA fragment encoding 120 amino acids from the 116th to the 235th of the murine beta-chain which corresponds to the presumed constant domain was inserted into an expression vector in E. coli. A large amount of this 18 kDa protein was observed to be synthesized in E. coli, and might be a good source for the three dimensional analysis of the T-cell receptor molecule.     

The laser method for efficient introduction of foreign DNA into cultured cells

A minute hole upon a cultured cell, perforated with a finely focused laser beam, was found to repair itself within a short period of time. The procedure constitutes a new way of introducing exogenous gene materials dissolved in medium into cells. The 'laser-aided' DNA transfection is better than the existing methods because it allows the treatment of a large number of cells in a shorter time, and an improved success rate.     

Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions

Chimeric genes composed of immunoglobulin (Ig)-derived variable (V) regions and T-cell receptor (TCR)-derived constant (C) regions were constructed. The VL and VH genes showing anti-phosphorylcholine (PC) activity were used in this study. Two pairs of chimeric genes, VL-C beta and VH-C alpha genes, and VL-C alpha and VH-C beta genes, were inserted into an expression vector containing both Ecogpt and neo genes, and transfected into EL4 cells. Cells which express both chimeric receptor molecules were established. The activity of the transformants to the antigen was examined by using stopped-flow fluorometry. An increase in the concentration of cytoplasmic calcium ion was observed after addition of Staphylococcus pneumoniae R36A bacteria grown in the choline-containing medium which express PC molecules, but not after the PC-negative bacteria grown in the ethanolamine-containing medium.     

Spin-label studies of the oligomeric structure of band 3 protein in erythrocyte membranes and in reconstituted systems

A spin-labeled fatty acid (16-doxylstearic acid), linked by an ester bond to a maleimide or a nitrene residue, was covalently attached to band 3 of erythrocyte membranes. The electron spin resonance spectrum of the spin-labeled protein was examined at different temperatures in: (a) whole erythrocyte ghosts; (b) ghosts depleted of spectrin and actin; (c) alkaline-treated ghosts; (d) vesicles made with purified band 3 reassociated with dimyristoylphosphatidylcholine. Most spectra are composite with a major component corresponding to a large overall splitting. The determination of the percentage of the immobilized component was carried out by pairwise subtraction. At low temperatures (1–7°C), the highest fraction of immobilized component was found in dimyristoylphosphatidylcholine vesicles (approx. 100%); alkaline-treated membranes had approx. 75% of the immobilized component at the same temperature; whole erythrocyte, spectrin/actin-depleted and spectrin/actin/ankyrin-depleted ghosts gave identical results (approx. 60% of immobilized component). The immobilized fraction decreased in all samples with increasing temperature or addition of a nonsolubilizing concentration of dodecyl octaethylene glycol monoether. In dimyristoylphosphatidylcholine vesicles, however, the modification in the ratio of the two components was obtained only above the lipid transition temperature (23°C). The strong immobilization of the spin-labeled lipid chain at all temperatures suggested trapping of the lipid chain between proteins. At low temperature, in dimyristoylphosphatidylcholine vesicles or in alkaline-treated ghosts, lipid-protein segregation is likely to take place. In whole erythrocyte ghosts, on the other hand, the large contribution of the motionally restricted component at physiological temperature indicates the oligomeric nature of band 3. Partial dissociation of the oligomers occurs as the temperature is increased, but the presence or absence of cytoskeletal proteins has no influence on the state of oligomerization of band 3.     

Enzymatic copolymerization to hybrid glycosaminoglycans: a novel strategy for intramolecular hybridization of polysaccharides

Hybrid glycosaminoglycans (GAGs) having an intramolecularly hybridized structure of hyaluronan-chondroitin (3a) and hyaluronan-chondroitin 4-sulfate (3b) have been synthesized via enzymatic copolymerization catalyzed by hyaluronidase (HAase). N-Acetylhyalobiuronate (GlcAbeta(1-->3)GlcNAc)-derived oxazoline (1) was copolymerized with N-acetylchondrosine (GlcAbeta(1-->3)GalNAc)-derived oxazoline (2a) by HAase catalysis at pH 7.5 and 30 degrees C, giving rise to copolymer 3a with Mn 7.4 x 103 in a 50% yield. Also, HAase-catalyzed copolymerization of monomer 1 with N-acetylchondrosine oxazoline having a sulfate group at C4 on GalNAc (2b) was carried out to produce copolymer 3b with Mn 1.4 x 104 in a 60% yield. The copolymer compositions were controllable by varying the comonomer feed ratio. These hybrid GAGs were successfully digested by the catalysis of hyaluronan lyase, clearly exhibiting that the products are not a blend of different homopolymers but an intramolecularly hybridized GAG.     

Calcium signals regulate the functional differentiation of thymic iNKT cells

How natural or innate‐like lymphocytes generate the capacity to produce IL‐4 and other cytokines characteristic of type 2 immunity remains unknown. Invariant natural killer T (iNKT) cells differentiate in the thymus into NKT1, NKT2, and NKT17 subsets, similar to mature, peripheral CD4⁺ T helper cells. The mechanism for this differentiation was not fully understood. Here, we show that NKT2 cells required higher and prolonged calcium (Ca²⁺) signals and continuing activity of the calcium release‐activated calcium (CRAC) channel, than their NKT1 counterparts. The sustained Ca²⁺ entry via CRAC pathway in NKT2 cells was apparently mediated by ORAI and controlled in part by the large mitochondrial Ca²⁺ uptake. Unique properties of mitochondria in NKT2 cells, including high activity of oxidative phosphorylation, may regulate mitochondrial Ca²⁺ buffering in NKT2 cells. In addition, the low Ca²⁺ extrusion rate may also contribute to the higher Ca²⁺ level in NKT2 cells. Altogether, we identified ORAI‐dependent Ca²⁺ signaling connected with mitochondria and cellular metabolism, as a central regulatory pathway for the differentiation of NKT2 cells.     

p51/p63 Controls subunit alpha3 of the major epidermis integrin anchoring the stem cells to the niche

p51/p63, a member of the tumor suppressor p53 gene family, is crucial for skin development. We describe here identification of ITGA3 encoding integrin alpha(3) as a target of its trans-activating function, proposing that p51/p63 allows epidermal stem cells to express laminin receptor alpha(3)beta(1) for anchorage to the basement membrane. When activated by genotoxic stress or overexpressed ectopically in non-adherent cells, p51/p63 transduced a phenotype to attach to extracellular matrices, which was accompanied by expression of ITGA3. Motifs matching the p53-binding consensus sequence were located in a scattered form in intron 1 of human ITGA3, and served as p51/p63-responsive elements in reporter assays. In addition to the trans-activating ability of the TA isoform, we detected a positive effect of the DeltaN isoform on ITGA3. The high level alpha(3) production in human keratinocyte stem cells diminished upon elimination of p51/p63 by small interfering RNA or by Ca(2+)-induced differentiation. Furthermore, a chromatin immunoprecipitation experiment indicated a physical interaction of p51/p63 with intron 1 of ITGA3. This study provides a molecular basis for the standing hypothesis that p51/p63 is essential for epidermal-mesenchymal interactions.     

Assay method for mitochondrial sterol 27-hydroxylase with 7alpha-hydroxy-4-cholesten-3-one as a substrate in the rat liver

Mitochondrial sterol 27-hydroxylase (EC 1.14.13.15) is an important enzyme, not only in the formation of bile acids from cholesterol intermediates in the liver but also in the removal of cholesterol by side chain hydroxylation in extrahepatic tissues. The enzyme has been assayed by complicated methods using radiolabeled substrates or deuterium-labeled tracers. These methods may be inaccurate for measuring enzyme activity, because the amount of electron-transferring proteins may be insufficient for maximal velocity. To solve this problem, after solubilization of the enzyme from rat liver mitochondria with n-octyl-beta-d-glucopyranoside (OGP), we measured the enzyme activity by incubating the solubilized enzyme with saturated amounts of electron-transferring proteins. In our assay system, using 7alpha-hydroxy-4-cholesten-3-one (HCO) as a substrate, we could easily measure the product, 7alpha,27-dihydroxy-4-cholesten-3-one, with HPLC monitoring absorbance at 240 nm. The product formation was proportionate to the time up to 5 min and the protein concentration up to 0.5 mg of protein/ml. The maximal velocity of the enzyme was 1.1 nmol/min/mg of protein, which was 4- to 16-fold higher than previously reported values. A simple and accurate assay method for sterol 27-hydroxylase in rat liver mitochondria is herein described.     

Sulfhydryl groups responsible for extreme lability of human cholesterol 7alpha-hydroxylase identified by site-directed mutagenesis

Cholesterol 7alpha-hydroxylase (cholesterol-NADPH oxidoreductase, EC 1.14.13.17, 7alpha-hydroxylating) is known to have extremely sensitive sulfhydryl group(s). It is believed that a cysteine residue that has a sulfhydryl group plays an important role in the decrease of this enzyme activity. The amino acid sequences of cholesterol 7alpha-hydroxylase of five different mammalian species, human, rat, rabbit, hamster and mouse, revealed that these mammalian species contain eight cysteine residues that are well conserved. To identify which cysteine residues are responsible for the extremely high lability, we used the technique of the site-directed mutagenesis. Eight mutated genes of human cholesterol 7alpha-hydroxylase in which one codon for a cysteine residue was changed to that for alanine were prepared and expressed in COS-1 cells. The protein mass and enzyme activity of cholesterol 7alpha-hydroxylse obtained from these eight mutated genes were determined. While all mutated genes expressed the enzyme mass, two mutated genes did not express protein capable of catalyzing 7alpha-hydroxylation of cholesterol: in one mutant a codon for the 7th cysteine residue (Cys 444) was substituted to that for alanine and in the other mutant a codon for the 8th cysteine residue (Cys 476) was changed similarly. These results suggest that the 7th and 8th cysteine residues are important for expression of the enzyme activity. Based on the fact that Cys 444 exists in the heme binding region, Cys 476 was suggested to be responsible for enzyme lability.