Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3

The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P-815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c-kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells.     

A rapid dot-blot method for species identification of bloodstains

A very simple and rapid test for species identification is reported. Extracts of bloodstains were applied to a synthetic porous membrane and dried. The membrane was then quenched with glycine buffered saline containing BSA and Tween 20. A suspension of colloidal gold particles (GP) coated with rabbit antiserum to human IgG was poured onto, gently whirled and aspirated through the membrane. Spots from the human and monkey bloodstains became red, whereas those from other species of animals remained unstained. This test was completed within 3 to 4 min, and the antibody-coated GP reagent was prepared within 20 min using a very small quantity of antiserum. Cellulose acetate membranes of 0.45 microns or more in pore size were appropriate to this test.     

66/77-kV C-GIS using composite insulation in SF6 gas

A second-generation model of cubicletype gas-insulated switchgear (C-GIS) with composite insulation incorporating SF₆ gas has been developed. The design does not require a gas process in field assembly; it has high reliability and its installation is more rapid; and a further reduction in size is achieved. The design principles are described in detail.     

Circularly polarised printed antenna fed by coplanar waveguide

A new structure of the circularly polarised printed antenna fed by coplanar waveguide (CPW) is proposed. FDTD analysis predicts the radiation of the circularly polarised wave from the antenna. The validity of this analysis is established by comparing with experimental results     

Gelation of poly(vinyl alcohol) solutions at low temperatures (20 to −78°C) and properties of gels

The flow points of atactic poly(vinyl alcohol) (a-PVA) gels with H₂O/dimethyl sulfoxide (DMSO) = 90/10 (v/v) chilled at 20 to ?78°C for 24 h depended on the chilling temperature and were 0–30°C for gels with the initial polymer concentrations (C_i) of 2–5 g/dL, whereas those for H₂O/DMSO = 50/50 chilled at 0 to ?78°C were independent of the chilling temperature and were 70–75°C. Syneresis occurred after eight cycles of freezing (?24°C) and thawing (20°C) for a-PVA hydrogels at concentrations above C_i = 4 g/dL and two such cycles for syndiotacticity-rich PVA (s-PVA) hydrogels at concentrations above C_i = 1 g/dL. The extent of syneresis per one cycle for s-PVA hydrogels was higher than that for a-PVA hydrogels at the initial cycles. In the a-PVA hydrogels with an initial polymer concentration of ca. 30 g/dL, syneresis was expected not to occur even after 20 cycles. If all the free water in the gels is assumed to have transuded by syneresis after 20 cycles, the residual water is bound water and is estimated to be six water molecules per one vinyl alcohol monomer unit. © 1994 John Wiley & Sons, Inc.     

An absolute requirement of fructose 1,6-bisphosphate for the Lactobacillus caseiL-lactate dehydrogenase activity induced by a single amino acid substitution

Lactobacillus casei allosteric L-lactate dehydrogenase (L-LDH)absolutely requires fructose 1,6-bisphosphate [Fru(1,6)P2] forits catalytic activity under neutral conditions, but exhibitsmarked catalytic activity in the absence of Fru(1,6)P₂ underacidic conditions through the homotropic activation effect ofsubstrate pyruvate. In this enzyme, a single amino acid replacement,i.e. that of His205 conserved in the Fru(1,6)P₂-binding siteof certain allosteric L-LDHs of lactic acid bacteria with Thr,did not induce a marked loss of the activation effect of Fru(1,6)P₂or divalent metal ions, which are potent activators that improvethe activation function of Fru(1,6)P₂ under neutral conditions.However, this replacement induced a great loss of the Fru(1,6)P₂-independentactivation effect of pyruvate or pyruvate analogs under acidicconditions, consequently indicating an absolute Fru(1,6)P₂ requirementfor the enzyme activity. The replacement also induced a significantreduction in the pH-dependent sensitivity of the enzyme to Fru(1,6)P₂,through a slight decrease and increase of the Fru(1,6)P₂ sensitivityunder acidic and neutral conditions, respectively, indicatingthat His205 is also largely involved in the pH-dependent sensitivityof L.casei L-LDH to Fru(1,6)P₂. The role of His205 in the allostericregulation of the enzyme is discussed on the basis of the knowncrystal structures of L-LDHs.     

Tailoring of Surfaces with Ultrathin Layers for Controlled Binding of Biopolymers and Adhesion and Guidance of Cells

Various strategies are described for the bio-functionalization of solid substrates by design of interfacial architectures. The first approach is based on the self-assembly process of long-chain thiol molecules from solution to a (noble) metal surface. If some of these building blocks carry a binding site (ligand) for proteins (receptors, antibodies, etc.) the metal surface can be tailored for maximum specific binding while simultaneously minimizing nonspecific adsorption. The second concept is based on polymers that are covalently attached to (oxide) surfaces. The preparation of these (end-) grafted functional polymers involves either the binding of preformed macromolecules to corresponding sites at the surface of the support or the recently introduced “grafting-from” method, by which an initiator molecule is first covalently bound to the surface and then activated — either by heat or light — in the presence of suitable monomer units such that a polymer chain grows from the solid/solution interface. Finally, the functionalization of patterned surfaces by peptide chains that mimic the binding domains of cell adhesion proteins is summarized. It is demonstrated that not only the selective adhesion of neuronal cells can then be controlled, but also their development with the outgrowth of dendrites and axons.     

Oligomer-Rich Fraction from Polydisperse Sample of Poly(bis(β-phenoxyethoxy)phosphazene)

Poly[bis(-phenoxyethoxy)phosphazene] [PBPEP] had been shown in our previous paper to be a very useful polymer for investigating the crystallization mechanism of polymers, as the crystallization rate of PBPEP is extraordinarily small when isothermally crystallized from the melt. The crystallization of the low molecular weight oligomers of PBPEP was first studied in comparison to the high molecular weight polymers. The oligomer-rich fraction was obtained by fractionation of the as-polymerized sample, which had a broad molecular weight distribution. The fractions thus obtained were characterized by solution viscometry and size exclusion chromatography. The melting temperature and the growth rate of the spherulite from the melt were investigated by differential scanning calorimetry and optical microscopy. The growth rate was one or two orders of magnitude smaller in the oligomer-rich fraction than in the other high molecular weight fractions. A collapsed spherulite appeared in the oligomer-rich fraction at high crystallization temperatures. It is speculated that in the oligomer-rich fraction there is an excess free energy due to defects in the crystal phase. This defect is considerably larger in the oligomer-rich fraction than in the other fractions because a large quantity of short length chains is present.     

Fritless packed columns for capillary electrochromatography: separation of uncharged compounds on hydrophobic hydrogels

A novel column is described that does not require frits to keep packing material within a capillary. A continuous bed is prepared in situ in aqueous solution by radical copolymerization of N-isopropylacrylamide and 2-acrylamido-2-methylpropanesulfonic acid (the resultant gel is denoted poly(AMPS-co-IPAAm). N,N'-Methylenebisacrylamide is used for cross-linking. On the application of an electrical field, electroosmotic flow (EOF) is developed in the bed along the capillary, where fluid propulsion would be otherwise difficult to achieve. The resultant EOF transports neutral compounds through the column without forcing the gel out of the capillary. Examination of the fluid motion in the continuous bed using a video microscope system and an image processor shows a relatively flat flow profile of EOF. The bed functions as the stationary phase for reversed-phase capillary electrochromatography (CEC). This new approach is an alternative to packed capillary columns which have been used previously in CEC. A high efficiency is obtained for a steroid which is separated on a 4.0% total monomer concentration (T), 10.0% degree of cross-linking (C), and 10.0% mole fraction of AMPS in the total monomer (S), poly(AMPS-co-IPAAm) column. A mixture of polyaromatic hydrocarbons is separated on a 6.9% T, 5.8% C, and 5.5% S poly(AMPS-co-IPAAm) column. The capacity factor of benzo[a]pyrene increases from 0.63 to 1.91 as the acetonitrile content in a Tris-boric acid buffer is decreased from 45 to 30% (v/v). The run-to-run RSD of analyte migration time is less than 0.73%, and the day-to-day RSD is acceptable. Potential benefits of this approach are also mentioned.