Enzymes Associated with Defence against Toxic Oxygen Species in PCNB-Tolerant and Sensitive Soil Fungi

The specific activities of enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX) and glutathione reductase (GR), which are involved in protection against toxic species of oxygen, were determined in mycelia extracts of pentachloronitrobenzene (PCNB)-tolerant and susceptible soil fungi. The organisms assayed were the highly PCNB-sensitive Rhizoctonia solani and Rhizopus arrhizus; Sclerotium rolfsii and Trichoderma harzianum, which are moderately susceptible to PCNB, and the fungicide-tolerant Fusarium oxysporum f. sp. melonis and Pythium aphanidermatum. No GPOX activity was detected in the six examined fungi. Significant differences in the specific activities of the other enzyme systems among the fungi were evident. Remarkably low levels of CAT activities were measured in R. solani. Except for T. harzianum, no meaningful differences regarding SOD, CAT and GR activities with age of the fungi cultures were observed. The electrophoretic patterns of SOD and CAT displayed dissimilarities among the fungi under study. P. aphanidermatum is more polymorphic with respect to both SOD and CAT enzyme systems as compared to the other fungi. The SOD of F. oxysporum f. sp. melonis, R. arrhizus and T. harzianum is a cuprozinc enzyme, while the mangano-SOD species was detected in S. rolfsii, R. solani and T. harzianum.     

Thromboxane receptor antagonism combined with thromboxane synthase inhibition. 6. 4-substituted 3-pyridinylalkanoic acids.

(3-Pyridinyl)alkanoic acids substituted at the 4-position with an (arylsulfonamido)alkyl group were synthesized and found to behave as platelet thromboxane receptor antagonists (TxRAs) and thromboxane synthase inhibitors (TxSIs). The compounds behaved as agonists at the vascular receptor for thromboxane A₂.     

Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line

Intracellular Ca²⁺ mobilization events were assessed in mouse L cells, which contain native prostaglandin E₁ receptors and transfected human ₂ adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca²⁺]_i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca²⁺]_i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the -adrenergic antagonist, propranolol, and is a result of intracellular Ca²⁺ release. [Ca²⁺]₁ in these cells was also increased by prostaglandin E₁, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP₁, IP₂, and IP₃. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP₃ production which leads to an elevation in [Ca²⁺];. We propose that this cyclic AMP dependent activation of the IP₃ generating system occurs at a post-receptor site.Abbreviations cAMP Adenosine Cyclic 3-5-Monophosphate - [Ca²⁺]_i intracellular [Ca²⁺]_i - 8 Br cAMP 8 Bromo Adenosine Cyclic 3-5-Monophosphate - DAG Diacylglycerol - EGTA] [Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid - BSA Bovine Serum Albumin - HBSS-H Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4 - HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - PIP₂ Phosphatidylinositol 4,5-bisphosphate - IP₂ Inositol 4 Phosphate - IP₂ Inositol 4,5 Bisphosphate - IP₃ Inositol Trisphosphate - PGE₁ Prostaglandin E₁ - PBS Phosphate Buffered Saline Solution     

Adenosine deaminase activity in relation to the appearance of early and late Epstein-Barr virus antigens induced in lymphoblastoid cells

Summary Induction of Epstein-Barr virus (EBV) capsid antigen synthesis in 59.6% of P3HR-1 cells was followed by a decrease to 70% in adenosine deaminase (ADA) activity. In Daudi cells synthesizing EBV early antigen, ADA activity did not decrease.     

Amino acid analysis utilizing phenylisothiocyanate derivatives

Advances in liquid chromatography have brought about the development of new techniques in amino acid analysis which take full advantage of precolumn derivatization procedures. Using phenylisothiocyanate as the reagent, detection limits under 1 pmol can be routinely achieved, allowing the analysis of submicrogram protein samples. Analysis times as short as 10 min for samples after hydrolysis and 1 h for physiologic samples are possible. Accurate, reproducible quantitation of amino acids can be obtained from complex matrices such as plasma, urine, feed, and food samples. This level of performance and flexibility gives the analyst the first realistic alternative to ion-exchange analysis without compromising desirable features of the traditional methodology.     

Primary structure of the site on bovine hormone-sensitive lipase phosphorylated by cyclic AMP-dependent protein kinase

The primary structure of a region on hormone-sensitive lipase was determined to be: Lys-Thr-Glu-Pro-Met-Arg-Arg-Ser- Val-Ser-Glu-Ala-Ala-Leu-Thr-Gln-Pro-Glu-Gly-Pro-Leu-Gly-Thr-Asp-Ser-Leu-Lys. Ser-8 was the only residue in the intact protein phosphorylated by cyclic AMP-dependent protein kinase. However, Ser-10 also appeared to be present in a phosphorylated form, suggesting that it is a target for a distinct protein kinase in vivo.     

Complete nucleotide sequence of the Streptomyces lividans plasmid pIJ101 and correlation of the sequence with genetic properties.

The complete nucleotide sequence of the multicopy Streptomyces plasmid pIJ101 has been determined and correlated with previously published genetic data. The circular DNA molecule is 8,830 nucleotides in length and has a G+C composition of 72.98%. The use of a computer program, FRAME, enabled identification in the sequence of seven open reading frames, four of which, tra (621 amino acids [aa]), spdA (146 aa), spdB (274 aa), and kilB (177 aa), appear to be genes involved in plasmid transfer. At least two of the above genes are predicted to be transcribed by known promoters that are regulated in trans by the products of the korA (241 aa) and korB (80 aa) loci on the plasmid. The segment of the plasmid capable of autonomous replication contains one large open reading frame (rep; 450 aa) and a noncoding region presumed to be the origin of replication. Four other small (less than 90 aa) open reading frames are also present on the plasmid, although no function can be attributed to them. The sequence of the pIJ101 replication segment present in several widely used cloning vectors (e.g., pIJ350 and pIJ702) has also been determined, so that the complete nucleotide sequences of these vectors are now known.     

Site-directed mutagenesis of the charged residues near the carboxy terminus of the colicin E1 ion channel

Colicin E1 was altered by oligonucleotide-directed mutagenesis at the site of three charged residues on the COOH side of the 35-residue hydrophobic segment in the channel-forming domain. Asp-509 is one of five conserved acidic residues in the channel domain of colicins A, B, E1, Ia, and Ib and is the first charged residue following the hydrophobic segment, followed by the basic residues Lys-510 and Lys-512. Asp-509 and Lys-512 were changed to amber and ochre stop codons, respectively, while Lys-510 was mutated to a Met codon. Proteins truncated after residue 508 or 511, and missing the last 14 or 11 residues, were obtained from a nonsuppressing cell strain harboring the mutant plasmid while full-length colicin molecules with single residue changes at Asp-509 to Leu, Ser, and Gln, and Lys-512 to Tyr, were obtained by using appropriate suppressor strains. The truncated colicins displayed (i) a low cytotoxicity, approximately 1% of intact wild-type colicin, (ii) 10-fold less in vitro channel activity with liposomes, and (iii) reduced labeling of the colicin in liposomes by a phospholipid photoaffinity probe, showing that one or more of the residues following Asn-511 is necessary for both in vivo and in vitro activity and insertion into the bilayer. (iv) The truncated mutants also displayed an altered conformation at pH 6 that allowed greater binding and activity with liposomes at this pH relative to wild type. The cytotoxicity of single residue substitutions at Asp-509 showed a range of cytotoxicities, wild type greater than Ser-509 greater than Gln-509 greater than Leu-509, although none of these changes greatly affected the in vitro channel activity or pH dependence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine

The hydrophobic, photoactivatable probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to label acetylcholine receptor rich membranes purified from Torpedo californica electric organ. All four subunits of the acetylcholine receptor (AChR) were found to incorporate label, with the gamma-subunit incorporating approximately 4 times as much as each of the other subunits. Carbamylcholine, an agonist, and histrionicotoxin, a noncompetitive antagonist, both strongly inhibited labeling of all AChR subunits in a specific and dose-dependent manner. In contrast, the competitive antagonist alpha-bungarotoxin and the noncompetitive antagonist phencyclidine had only modest effects on [125I]TID labeling of the AChR. The regions of the AChR alpha-subunit that incorporate [125I]TID were mapped by Staphylococcus aureus V8 protease digestion. The carbamylcholine-sensitive site of labeling was localized to a 20-kDa V8 cleavage fragment that begins at Ser-173 and is of sufficient length to contain the three hydrophobic regions M1, M2, and M3. A 10-kDa fragment beginning at Asn-339 and containing the hydrophobic region M4 also incorporated [125I]TID but in a carbamylcholine-insensitive manner. Two further cleavage fragments, which together span about one-third of the alpha-subunit amino terminus, incorporated no detectable [125I]TID. The mapping results place constraints on suggested models of AChR subunit topology.