Purification and characterization of periplasmic alpha-amylase from Xanthomonas campestris K-11151.

Xanthomonas campestris K-11151, isolated from soil, produced a periplasmic alpha-amylase of a new type. The enzyme was purified to homogeneity, as shown by several criteria. The purified enzyme showed almost the same activities on alpha-, beta-, and gamma-cyclodextrins, soluble starch, and amylose. Moreover, it was active on branched cyclodextrins, pullulan, and maltose but not on glycogen. Kinetic analysis showed that alpha-cyclodextrin was the best substrate among the cyclodextrins. The substrate specificity suggested that this enzyme had the combined activities of alpha-amylase, cyclodextrinase, and neopullulanase.     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

A new yeast gene, HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest

A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).     

Effects of Far-Red Light on K+ Fluxes in a Colorless Mutant of Chlorella

In a colorless mutant of Chlorella kessleri, far-red light significantlyenhanced the K⁺ efflux. This effect was abolished by the K⁺channel-blocker tetraethylammonium acetate. Using cyanine dyeto monitor membrane potential, we deduced that the K⁺ effluxunder far-red light was probably accompanied by hyperpolarizationof the plasmalemma. (Received August 30, 1993; Accepted November 16, 1993)     

Purification and Characterization of Membrane Protein (90 kDa) from Mycoplasma salivarium,Which Binds Immunoglobulin (Ig) G Fc Fragment

A 90 kDa protein of Mycoplasma salivarium was released from cell membranes of the organism with Triton X-100 and purified by ion-exchange chromatography and chromatofocusing. The protein was eluted at pH 5.5 by chromatofocusing. The protein was shown to react with the Fc fragments of IgG from human and nine different animal species and did not distinguish between IgG from different species, while protein A, tested for comparative purposes, displayed a strong specificity for human and swine IgG. Furthermore, the protein reacted with antigen specific goat IgG (specific for gamma chains of human IgG), sheep red blood cells (SRBC) sensitized with rabbit antiserum to SRBC, that is, the Fc part of rabbit IgG, and concanavalin A as well. These findings may suggest that the protein is a lectin which binds the carbohydrate moiety of the Fc part of IgG.     

A physical map and clone bank of the black pine (Pinus thunbergii) chloroplast genome

Black pine chloroplast DNA is 119,707 bp long. The physical map is shown and the genes are listed. Plasmid clones covering the entire DNA sequence have been ordered and available upon request.