Carbohydrate specificity of IgM autoantibodies to CD45 in Systemic Lupus Erythematosus

Patients with SLE develop IgM autoantibodies to different isoforms of CD45, the major surface membrane protein tyrosine phosphatase on lymphocytes and other nucleated hemopoietic cells. Because such autoantibodies could have a potential role in the development of immune dysfunction in this disorder, we performed a series of experiments to characterize their antigenic specificity further. Blots of recombinantE. coli fusion proteins encoded by exons 3–7 of the p220 and p180 isoforms were uniformly non-reactive with SLE IgM, suggesting that anti-CD45 autoantibodies in SLE are directed against conformational and/or carbohydrate epitopes, rather than linear polypeptide epitopes. This issue was examined further using chemically and enzymatically modified CD45 purified from T cells by lectin affinity chromatography as substrates. Treatment of CD45 with 25 mM sodium-m-periodate, sufficient to abrogate binding to various lectins, abolished the reactivity with SLE anti-CD45 autoantibodies. On the other hand, digestion of CD45 with neuraminidase enhanced the binding of anti-CD45 autoantibodies from some of the SLE sera. This result probably reflects decreased steric hindrance or charge repulsion because the binding of mouse monoclonal antibodies directed against linear polypeptide epitopes of CD45 was similarly enhanced. Digestion of CD45 with N-glycosidase F had no effect on autoantibody staining. Taken together, these data suggest that IgM anti-CD45 autoantibodies in SLE recognize non-sialylated carbohydrate determinants in the highly O-glycosylated polymorphic domains of CD45.Abbreviations SLE systemic lupus erythematosus - SBA soybean agglutinin - RCAI Ricinus communis agglutinin - SNL Sambucus nigra lectin - MBP maltose binding protein - mAb monoclonal antibody - WGA wheat germ agglutinin     

Analysis of the biosynthetic process of cellulose and curdlan using C-labeled glucoses

In order to elucidate the biosynthetic process of cellulose and curdlan, ¹³C-labeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 31749), from culture media containing -(1-¹³C)glucose, -(2-¹³C)glucose, -(4-¹³C)glucose, or -(6-¹³C)glucose as the carbon source, and their structures were determined by ¹³C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-1, C-3 and C-5, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-1 and C-3, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-1 to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Distribution of Vacuolar H+-Pyrophosphatase and a Membrane Integral Protein in a Variety of Green Plants

Vacuolar membranes isolated from several species including fernand moss exhibited pyro-phosphate-dependent H⁺ transport activity.On immunoblot analysis, H⁺ -pyrophosphatase was detected inthe vacuolar membranes. A membrane integral protein of 23,000daltons was not found in the membranes of Chara, Conocephalum,or Kalanchoë. Thus, H⁺-pyrophosphatase may be a universalenzyme among green plants, but the 23-kDa protein is not a commonprotein of central vacuoles. (Received September 10, 1993; Accepted November 29, 1993)     

Lipofection of Synthetic Oligodeoxyribonucleotide Having a Palindromic Sequence of AACGTT to Murine Splenocytes Enhances Interferon Production and Natural Killer Activity

A synthetic 22-mer oligodeoxyribonucleotide having an AACGTT palindrome, AAC-22, induced interferon (IFN) production and augmented the natural killer (NK) activity in murine splenocytes, whereas its analogue, ACC-22, having an ACCGGT palindrome, did not. The binding of AAC-22 to splenocytes was not different from that of ACC-22. Lipofection of AAC-22 to splenocytes remarkably enhanced IFN production and NK cell activity, whereas that of ACC-22 caused little enhancement. These results strongly suggest that the prerequisite for IFN production is not the binding of AAC-22 to the cell surface receptors, but its penetration into the spleen cells.     

The induction of recessive mutations in mouse primordial germ cells with N-ethyl-N-nitrosourea

A specific-locus test was carried out to examine the mutagenic activity of N-ethyl-N-nitrosourea (ENU) on mouse primordial germ cells (PGC). Embryos of C3H/He mice were treated transplacentally with 30 or 50 mg ENU per kg of maternal body weight on day 8.5, 10.5, or 13.5 of gestation (G8.5 day, G10.5 day, or G13.5 day). Male and female mice that had been treated with ENU in embryonic stages were mated with female or male tester PW mice to detect recessive mutations induced in PGC.

ENU induced recessive mutations at a relatively high rate in PGC at these developmental stages. The most sensitive stage was G10.5 day. On G8.5 day, the induced mutation rate in males and females was not significantly different. Cluster mutations, which originate from the limited number of PGC and cell killing, were more frequently induced at an earlier developmental stage. The induced mutation rate per unit dose of ENU (1 mg/kg) was higher in G8.5 and G10.5 day PGC than in stem-cell spermatogonia. It can be concluded that mouse PGC are more sensitive than stem-cell spermatogonia to the induction of recessive mutations by ENU.     

Establishment of a human anaplastic thyroid cancer cell line secreting granulocyte colony-stimulating factor in response to cytokines

Summary A human anaplastic thyroid cancer cell line K-119, derived from a 77-yr-old woman who had developed marked neutrophilia and underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike and polygonal cells in shape are stably proliferating since the beginning of its culture 2 yr ago. The cells grow rapidly and the population doubling time is 26 h. The chromosomes show many abnormalities and many marker chromosomes have been observed. Heterotransplantation of the cells into nude mice has resulted in the formation of tumors that are histologically interpreted as anaplastic cancer. The most noteworthy characteristics of the cell line are the many Ki-67-positive cells (86.3%) and that the cell line spontaneously secretes granulocyte colony-stimulating factor (G-CSF) and releases increased amounts of G-CSF in response to the stimulation of tumor necrosis factor, interleukin 1α, and interleukin 1β. The conditioned medium obtained from K-119 cells contains an autocrine factor stimulating the proliferation of themselves.     

Partial characterization of the glucose transport activity in the golgi-rich fraction of fat cells

The glucose transport activity solubilized from the basal and plus insulin forms of the Golgi-rich fraction of adipocytes was partially characterized, and the results were compared with those of the activity obtained from the plus insulin form of the plasma membrane-rich fraction. The transport activity was determined in a cell-free, reconstituted, system. Prior to reconstitution, the activities in the three preparations were all (a) stable at 0°C for at least 4 h, but not at 37°C or above; (b) most stable at pH 7–9, and (c) less stable in Tes than in Tris buffer. After reconstitution, the three activities were all (d) stable at 0°C, (e) most active at pH 5.5, (f) mildly stimulated by divalent cations, (g) unaffected by insulin or 1 mM of several SH-blocking agents, (h) inhibited by heavy metal ions, 10–100 mM of monovalent salts, organic solvents, several sugar isomers, and specific sugar-transport inhibitors. The rates of d-glucose uptake by the three liposome preparations were all inhibited more strongly by 2-deoxy-d-glucose or $\text{3}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ than by d-glucose. These data indicate that the general properties of the glucose transport activity in the Golgi-rich fraction are similar to those of the activity in the plasma membrane-rich fraction.     

Comparison of stimulus-response (V-log I) functions in five types of lepidopteran compound eyes (46 species)

Summary Stimulus intensity-response relations (V-log I curves) were electrophysiologically (ERG) determined for the compound eyes of 46 lepidopteran species belonging to five different groups: butterflies (22 species), hesperids (3 species), diurnal sphingids (2 species), diurnal moths (3 species) and nocturnal moths (16 species). The V-log I curves were fitted to the Naka and Rushton equation, in whichn represents the slope of the linear part of each curve. The slopes so determined range fromn=0.35 (the shallowest slope) in nocturnal moths with the greatest dynamic range ton=0.54 (the steepest slope) in diurnal moths andn=0.53 in butterflies both of which have narrow dynamic range. Hesperids (n=0.41) and diurnal sphingids (n=0.38) have intermediate values between butterflies and nocturnal moths.The ratio of rhabdom to retinula volume is significantly higher in nocturnal moths (70–75%), however, those of butterflies and of diurnal moths are very small (2–5%), and hesperids and diurnal sphingids show intermediate ratio (ca. 25%).The slopes of V-log I curves are inversely proportional to the ratio of rhabdom to retinula volume in the various eye types. In all groups except diurnal moths, the light intensities which produce maximal and saturated responses are nearly the same, therefore the nocturnal moths which have the lowest threshold to light increase their sensitivity to dim light mainly by decreasing the slopes of V-log I curves.     

Effect of N-ethylmaleimide on leucine transport in chang liver cells. I. Stimulatory effect on Na+-independent transport at low concentrations of leucine

Pretreatment of Chang liver cells with $\text{N-}\text{ethylmaleimide}$ (0.5 or 1 mM) stimulated Na⁺-independent uptake of leucine at low concentrations (?1 mM). The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the uptake of leucine measured in Na⁺-replete medium was completely blocked by the addition of $\text{b-2-}\text{aminobicyclo}\text{[2,2,1]}\text{heptane}\text{-2-}\text{carboxylate}$ (5 mM), which shows that the L system participates in the stimulation. The Na⁺-dependent uptake of glycine was depressed by $\text{N-}\text{ethylmaleimide}$ pretreatment. The stimulation of the Na⁺-independent component of leucine uptake continued for at least 30 min after $\text{N-}\text{ethylmaleimide}$ treatment, while the inhibition of glycine uptake was progressive with time and the Na⁺-dependent uptake of leucine became depressed later, after the treatment. It has been demonstrated that treatment of cells with $\text{N-}\text{ethylmaleimide}$ is capable of increasing the Na⁺-independent influx of leucine and at the same time slightly decreasing the efflux of it. These results suggest that $\text{N-}\text{ethylmaleimide}$ attacks the Na⁺-independent system of amino acid transport at the reactive SH groups(s) of relevant protein(s) in favor of specific activation of that system in this cell.