Total absence of protein 4.2 and partial deficiency of band 3 in hereditary spherocytosis

Unlike previously reported cases with total protein 4.2 deficiency due to mutations in the EPB42 gene, we describe a total deficiency in protein 4.2 with normal EPB42 alleles. Hereditary spherocytosis (HS) was observed in a Japanese woman (unsplenectomized) and her daughter (splenectomized). The mother showed a partial deficiency in band 3 and a proportional reduction in protein 4.2. She was heterozygous for a novel allele of the EPB3 gene, allele Okinawa, which contains the two mutations that define the Memphis II polymorphism (K56E, AAG-->GAG, and P854L, CCG-->CTG) and, additionally, the mutation: G714R, GGG-->AGG, located in a highly conserved position of transmembrane segment 9. The latter change was responsible for HS. In trans to allele Okinawa, the daughter displayed allele Fukuoka: G130R, GGA-->AGA, an allele known to alter the binding of protein 4.2 to band 3. The daughter presented with a more pronounced decrease of band 3, and lacked protein 4.2, resulting in aggravated haemolytic features. Although the father was not available for study, heterozygosity for allele Fukuoka has been documented in another individual who showed no clinical or haematological signs, and a normal content of band 3. We suggest that band 3 Okinawa binds virtually all the protein 4.2 in red cell precursors, band 3 Fukuoka being unable to do so, and that the impossibility of band 3 Okinawa incorporation into the membrane leads to degradation of the band 3 Okinawa protein 4.2 complex. In contrast, band 3 Fukuoka, free of bound protein 4.2, could then incorporate normally into the bilayer. Thus, protein 4.2 would not appear in the daughter's red cell membrane.     

Low-Temperature Sintering and High Thermal Conductivity of YLiO2-Doped AlN Ceramics

The present work indicates through thermodynamic considerations that YLiO₂ additive is beneficial for low-temperature sintering of AlN ceramics. Pressureless sintering of commercially available AIN powders with simultaneous additions of YLiO₂ and CaO resulted in materials with high thermal conductivity (170 W·m^–1·K^–1 after sintering at 1600°C for 6 h). It is demonstrated that improvement of thermal conductivity is possible at low firing temperature by use of sintering aids.     

A markedly disrupted skeletal network with abnormally distributed intramembrane particles in complete protein 4.1-deficient red blood cells (allele 4.1 Madrid): implications regarding a critical role of protein 4.1 in maintenance of the integrity of the red blood cell membrane

Electron microscopic (EM) studies were performed to clarify the interactions of membrane proteins in the red blood cell membrane structure in situ of a homozygous patient with total deficiency of protein 4.1 who carried a point mutation of the downstream translation initiation codon (AUG --> AGG) of the protein 4.1 gene [the 4.1 (-) Madrid; Dalla Venezia et al, J Clin Invest 90:1713, 1992]. Immunologically, as expected, protein 4.1 was completely missing in the red blood cell membrane structure in situ. A markedly disrupted skeletal network was observed by EM using the quick-freeze deep-etching method and the surface replica method, although the number of spectrin molecules was only minimally reduced (395 +/- 63/microm2; normal, 504 +/- 36/microm2). The number of basic units in the skeletal network was strikingly reduced (131 +/- 21/microm2; normal, 548 +/- 39/microm2), with decreased small-sized units (17 +/- 4/microm2; normal, 384 +/- 52/microm2) and increased large-sized units (64% +/- 14%; normal, 5% +/- 1%). Concomitantly, immuno-EM disclosed striking clustering of spectrin molecules with aggregated ankyrin molecules in the red blood cell membrane structure in situ. Although no quantitative abnormalities in the number and size distribution of the intramembrane particles were observed, there was a disappearance of regular distribution, with many clusters of various sizes, probably reflecting the distorted skeletal network. Therefore, protein 4.1 suggests by EM to play a crucial role in maintenance of the normal integrity of the membrane structure in situ not only of the skeletal network but also of the integral proteins.     

Enhancement of xylose uptake in 2-deoxyglucose tolerant mutant of Saccharomyces cerevisiae

Chemical mutation of Saccharomyces cerevisiae using ethyl methane sulfonate was performed to enhance its ability of xylose uptake for ethanol production from lignocellulose under microaerobic condition. Among the appeared mutants, the mutant no. 2 (M2) strain screened using inhibitory effects of 2-deoxyglucose (DOG) showed more than 4-fold high ability in xylose uptake compared with the wild type strain, under the presence of glucose. The catabolite repression by glucose was sufficiently reduced in M2 strain due to its tolerance to the high concentration of DOG (0.5%, wt./vol.). Metabolomic analyses of various sugars in the cell revealed that some of xylose was reduced to xylitol in M2 cell, providing the concentration gradient of xylose and more uptake of xylose. Xylulose-5-phosphate was significantly detected in the crude cell extract from M2 strain, indicating higher metabolic activity in pentose phosphate pathway. This was also confirmed by in vitro analyses of key enzymes involved in glucose and xylose metabolism, such as hexokinase, glucose-6-phosphate dehydrogenase and xylose reductase. Glucose uptake was moderately suppressed in the presence of trehalose-6-phosphate inhibiting the activation of hexokinase, resulting in more uptake of xylose through hexose transport system. To our knowledge, this study is the first report verifying that the mutation technique successfully enhances the xylose uptake by S. cerevisiae, particularly under the presence of glucose.     

Identification of a Point Mutation in the Granule-bound Starch Synthase I Gene (GBSSI) in a waxy Diploid Wheat Mutant and Design of Molecular Markers for Backcrossing

In cereals, granule-bound starch synthase I (GBSSI)-deficient mutants accumulate glutinous (amylose-free) starch in their storage tissues. The amylose-free starch produced by waxy (wx) mutants of hexaploid bread wheat (Triticum aestivum L.) is used in cakes and breads. However, wx mutants of diploid wheat (T. monococcum L.) have so far no commercial applications. In this study, we identified a mutation in exon 6 of GBSSI in a diploid wheat wx mutant that resulted in the replacement of Trp355 with a stop codon. Molecular markers were developed for the rapid screening of the mutation, which should allow the selection of heterozygous and homozygous plants during backcrossing. This will facilitate the improvement of the agricultural traits of the wx mutant and the generation of new amylose-free wx lines.     

Effect of additives on some properties of silicon oxynitride ceramics

Silicon oxynitride ceramics are formed by reaction sintering of silicon nitride and silica with certain metal oxide additives. The reaction rate during sintering and the subsequent properties of silicon oxynitride are affected by the quantity and kinds of additives. The reaction rate increased for addition of equal molar amounts of ZrO₂, ZrO₂ (+2.8 mol % Y₂O₃), AlO_1.5, LnO_1.5, CeO₂, MgO, in that order (where Ln=Nd, Sm, Gd, Dy, Er, Yb and Y). The lanthanide oxide (1.5 mol %)-doped silicon oxynitride ceramics had a high fracture toughness, because crack deflection occurred due to the precipitation of an intergranular crystalline phase with a high thermal expansion coefficient compared with silicon oxynitride. The oxidation rate was higher with an increasing quantity of additive. In samples containing an intergranular crystalline phase, stability of the crystalline phase is an important factor and could impair the oxidation resistance of silicon oxynitride ceramics.     

Experimental and analytical studies of pipe whip tests under PWR LOCA conditions

A series of pipe rupture tests has been performed at the Japan Atomic Energy Research Institute (JAERI) to demonstrate the safety of primary coolant circuits in the event of pipe rupture in nuclear power plants. Pipe whip tests and jet discharge tests have been conducted under boiling water reactor (BWR) and pressurized water reactor (PWR) loss-of-coolant accident (LOCA) conditions. The present paper describes the experimental and analytical results of the pipe whip tests performed under PWR LOCA conditions using 4, 6 and 8-inch test pipes. The tests were carried out at an initial pressure and temperature of 15.7 MPa and 325°C, respectively. Moreover, a dynamic analysis of pipe whip tests was carried out using the general purpose finite element program ADINA.     

Rapid analysis of aflatoxins in raw peanuts,corn, buckwheat and red pepper by a new mini-column cleanup and HPLC using post-column photochemical derivatization system

A rapid and clean method for the analysis of aflatoxins (AFs) was developed by using a new column and post-column photochemical derivatization HPLC with fluorescence detection. The new cleanup column consisted of magnesia and basic alumina poured on the top of a commercial multi-functional mini-column. It was extremely effective for the cleanup of AFs from raw peanut, corn, buckwheat and red pepper. Fluorescent substances, which interfered with the analysis of AFs from corn, were completely absorbed at the top of the magnesia layer. Recoveries of AFs (B1, B2, G1, G2) added to raw peanuts, corn, buckwheat and red pepper were over 80% at two levels of fortification (higher level: 10, 3, 10, 3 ng/g, respectively, lower level: 1.0, 0.3, 1.0, 0.3 ng/g, respectively). Coefficients of variation were smaller than 12%, except the lower fortified level for red pepper. Limits of detection for AFs in raw peanuts, corn and buckwheat were 0.3 ng/g for B1 and G1, and 0.1 ng/g for B2 and G2. Those in red pepper were 0.5 ng/g for B1, B2, G1 and G2.     

Phorbol myristate inhanced specific incorporation of arachidonic acid into phospholipids through lysophospholipid acyltransferase in cultured smooth muscle cells

The effect of stimulation of phospholipase with phorbol 12-myristate 13-acetate and lipopolysaccharide on 1-acyl-glycerophospholipid acyltransferase was studied in cultured rabbit aorta smooth muscle cells. The acyltransferase in smooth muscle cells without stimulation was active on a wide range of unsaturated fatty acids and was not arachidonic acid specific. Upon increase in phospholipase activity, acyltransferase activity only with arachidonic acid as substrate increased in a time-dependent fashion. Apparent acyltransferase activity was increased most upon increase in phospholipase activity when lysophosphatidylcholine was used as acceptor. These results suggest that arachidonic acid specific acyltransferase was induced in smooth muscle cells by increase in phospholipase activity. The role of this acyltransferase is postulated to be the specific incorporation of endogenously released arachidonic acid.