A new type of RNase T2 ribonuclease in two Basidiomycetes fungi, Lentinus edodes and Irpex lacteus

Two new RNase T2 Ribonucleases, RNase Le37 and Irp3, with a molecular mass of 45 kDa, have been isolated from Basidiomycetes fungi, Lentinus edodes and Irpex lacteus, respectively. The ribonucleases consisted of three domains: an RNase active domain, a Ser/Thr rich domain similar to that of many fungal glycanhydrolases, and a C-terminal 10 kDa domain similar to that of RNase Rny1 in yeast. The locations of hydrophobic amino acids and Pro in the 10 kDa domain of the two basidiomycetous enzymes are very similar to those of RNase Rny1, indicating that these domains may have similar roles.     

Amino acids and peptides. LVII. Synthetic peptide with a sequence of ribonuclease from Sulfolobus solfataricus, SSR(1-62), does not function as an RNase

The 62 residue peptide, SSR(1-62), whose sequence corresponds to that of ribonuclease (RNase) from Sulfolobus solfataricus, and its related peptides, SSR(1-22) and SSR(10-62), were chemically synthesized and their RNase activity and DNA-binding activity were examined. The RNase activity assay using yeast RNA or tRNA(fMet) as substrate showed that the synthetic peptide SSR(1-62) did not hydrolyze yeast RNA or tRNA(fMet). These data were not consistent with previous reports that both the native peptide isolated from S. solfataricus [Fusi et al. (1993) Eur. J. Biochem. 211, 305-311] and the recombinant peptide expressed in Escherichia coli [Fusi et al. (1995) Gene 154, 99-103] were able to hydrolyze tRNA(fMet). However, the synthetic SSR(1-62) exhibited DNA-binding activity. In the presence of synthetic SSR(1-62), the cleavage of DNA (plasmid pUCRh2-4) by restriction endonuclease (EcoRI) was not observed, suggesting that synthetic SSR(1-62) bound to DNA protected DNA from its enzymatic digestion. Neither SSR(1-22) nor SSR(10-62) prevented DNA from being cleaved by a restriction enzyme. These findings strongly suggest the importance of not only the N-terminal region of SSR(1-62) but also the C-terminal region for DNA-binding. Circular dichroism spectroscopy of synthetic SSR(1-62) indicated a beta-sheet conformation, in contrast with synthetic SSR(1-22), which exhibited an unordered conformation.     

Detection of Escherichia coli O157:H7 in the Beef Marketed in Malaysia

Twelve strains of Escherichia coli O157:H7 were isolated from 9 of 25 beef samples purchased from retail stores in Malaysia. These strains produced Shiga toxin 2 with or without Shiga toxin 1 and had the eae gene and a 60-MDa plasmid. The antibiograms and the profiles of the arbitrarily primed PCR of the strains were diverse, suggesting that the strains may have originated from diverse sources.     

Effect of Na+ on Ca2+-Binding on the Plasma Membrane of Barley Mesophyll Cells: an Electrophoretic Study

Characteristics of Ca²⁺-binding on the plasma membrane of barleymesophyll cells were studied by a microelec-trophoretic method.The results indicated that Na⁺ ions could cause nonspecificreduction but not specific reduction in the total amount ofCa²⁺ bound to the plasma membrane of (salt-tolerant) barleymesophyll cells. (Received October 13, 1997; Accepted January 23, 1998)     

Stimulation of Macrophages with the β-Glucan Produced by Aureobasidium pullulans Promotes the Secretion of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL)

A β-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a β-(1,3)-linked main chain with β-(1,6)-linked glucose side residues. Various β-glucans consisting of β-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial β-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.     

Crystallization of a complex between ribonuclease Ms and 3'-guanylic acid

The crystals of a complex between ribonuclease Ms, the extracellular ribonuclease from Aspergillus saitoi, and 3'-guanylic acid were obtained from 2-methyl-2,4-pentanediol solution by vapor diffusion technique in the hanging drop mode. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with dimensions a = 47.0 A, b = 62.8 A, c = 37.9 A. The crystals diffract strongly up to at least 2.0 A resolution.     

Structure determination of a nucleoside Q precursor isolated from E. coli tRNA: 7-(aminomethyl)-7-deazaguanosine.

A precursor of modified nucleoside Q isolated from E. coli methyl-deficient tRNA was determined to be 7-(aminomethyl)-7-deazaguanosine. The structure was deduced by means of its chromatographic and electrophoretic mobilities, and UV and mass spectra, in addition to comparison with the synthesized authentic compound. The same molecule is also found in tRNA of an E. coli mutant selected for deficient synthesis of modified nucleosides.     

A Novel Isourazole Herbicide, Fluthiacet-Methyl, is a Potent Inhibitor of Protoporphyrinogen Oxidase after Isomerization by Glutathione S-Transferase

A novel isourazole herbicide, fluthiacet-methyl (methyl [[2-chloro-4-fluoro-5-[(5,6,7,8-tetrahydro-3-oxo-lH,3H-[l,3,4]thiadiazolo[3,4-a]pyridazin-l-ylidene)amino]phenyrjthio]acetate;experimental code name, KIH-9201) promoted the leakage of electrolytesfrom cotyledons of velvetleaf (Abtilon theophtasti Medic) andcotton (Gossypium hirsutum L.) plants that are sensitive tothis compound. It induced the accumulation of protoporphyrinIX in cotyledons of cotton and inhibited Chl biosynthesis incotyledons of velvetleaf and cotton at low concentrations (I₅₀values, 10–12 nM). Fluthiacet-methyl was converted toits urazole by glutathione S-transferase that had been partiallypurified from velvetleaf. The urazole inhibited protoporphyrinogenoxidase (Protox, EC 1.3.3.4 [EC] ) from some plants, including velvetleaf,at low concentrations (I₅₀ values, 5.1–11 nM), whereasfluthiacet-methyl was not as potent. The effects in vivo (electrolyteleakage and inhibition of Chi biosynthesis) of fluthiacet-methylwere correlated with the inhibition of Protox activity by theurazole and not with the action of fluthiacet-methyl itself.From these results, it is concluded that fluthiacet-methyl inhibitsProtox activity after conversion to the corresponding urazoleby glutathione S-transferase. It is in this way that fluthiacet-methylexerts its effect as a light-dependent peroxidizing herbicide. (Received November 1, 1994; Accepted March 6, 1995)     

The guanine nucleotide-binding regulatory proteins (G proteins) in myocardium with ischemia

Guanine nucleotide-binding regulatory proteins (G proteins) play a major role in the regulation of a number of physiological processes, such as stimulation or Inhibition of adenylate cyclase activity or gaiting of ionic channels. Myocardial ischemia could induce the changes in receptor-G protein signal transduction system in the heart. Therefore, this article will focus on the role and alterations of G proteins (especially, Gs and Gi) in myocardial ischemia. The Gi protein rapidly loses functional activity during very early myocardial ischemia. In contrast to Gi protein, the function of Gs protein during this phase has not been evaluated. Moreover, the changes in Gs protein after 30 min of ischemia are contradictory. However, the sensitization of the adenylate cyclase activity in the very early phase of acute ischemia is gradually replaced by a decrease in adenylate cyclase activity with prolonged ischemia. The decrease in the function and amount of Gs protein may be one of the factors that induce these changes. The function of Gs protein was also decreased in the canine hearts with ischemia and reperfusion. In contrast to ischemia and reperfusion, there are no significant alterations in G proteins and modulation of adenylate cyclase in the stunned myocardium. It has become increasingly evident that Gi protein may play an important role in the cardioprotective effects of preconditioning. When -adrenoceptor densities are reduced in chronic myocardial ischemia, decreased in the amount and function of Gi protein and increased amount of Gs protein may play the role in preservation of the adenylate cyclase activity. These alterations in G proteins may play the important role in the myocardial function during myocardial ischemia.     

Kinetic studies on the cleavage of oligouridylic acids and poly U by bovine pancreatic ribonuclease A

Kinetic parameters, Km and Vmax for the transesterification of oligouridylic acid, (Up)nU greater than p (n=0-4), by RNase A were measured spectrophotometrically at pH 7.0 and 25 degrees C. The kinetic parameters, pKm and log Vmax increased with increase in the chain length (n), and seemed to be almost constant with substrates having n greater than or equal to 2. The contribution of each subsite to the binding was estimated according to Hiromi's theory. The subsite affinities for (B1, R1, P1)+(B2, R2, P2) and (B3, R3, P3) are 8.03 kcal and 0.72 kcal/mol, respectively, and those for (B4, R4, P4) and (B5, R5, P5) are less than 0.5 kcal/mol. Therefore, we postulate that the size of the RNase A active site is about 3 nucleotides in length. Transesterification of poly U by RNase A was followed spectrophotometrically. The reaction is markedly influenced by ionic strength. At lower ionic strength, the v0-S curve of poly U cleavage was sigmoidal and cooperative, and it became less cooperative at higher ionic strength. Since the estimated Vmax value for poly U cleavage at ionic strength of 0.1 was more than 20 times larger than that of oligouridylic acids cleavage, we propose a non-specific interaction of poly U anion with cationic groups on the surface of the enzyme, modulating the conformation of active site, and thus increasing the activity at low ionic strength. The interaction decreases at higher ionic strength due to the interaction of counter anions with the non-specific sites.