Mechanism of endotoxin-induced reduction in the number of beta-adrenergic receptors in dog livers: role of phospholipase A

The role of phospholipase A on the endotoxin-induced reduction in the number of beta-adrenergic receptors in dog liver plasma membranes was investigated. The results show that digestion of control liver plasma membranes with exogenous phospholipase A2 (0.2 unit/200 micrograms protein) decreased the specific binding of (-)-[3H]dihydroalprenolol by 37.3% (P less than 0.01) and reduced the number of receptor sites by 31.7% (P less than 0.05). These decreases in the specific binding and the number of beta-adrenergic receptors were completely reversible by the addition of phosphatidylcholine (0.2 mM). Endotoxin administration (2 hr postendotoxin) decreased the specific binding by 36% (P less than 0.05) and reduced the number of beta-adrenergic receptors by 33% (P less than 0.05), and these decreases were completely reversible by the addition of 0.2 mM phosphatidylcholine. Digestion of control liver membranes with exogenous phospholipase A2 decreased phosphatidylcholine and phosphatidylethanolamine levels by 50.6 and 51.2%, respectively, but increased lysophosphatidylcholine and lysophosphatidylethanolamine levels by 12- and 8.4-fold, respectively. Endotoxin administration decreased phosphatidylcholine and phosphatidylethanolamine contents by 21.4 and 23.8%, respectively, but increased lysophosphatidylcholine and lysophosphatidylethanolamine contents by 2.1- and 1.4-fold, respectively. In addition, endotoxin administration increased endogenous phospholipase A activity by 73.5%. Based on these results, it is suggested that the decreases in the specific binding and the number of beta-adrenergic receptors in dog livers during endotoxic shock are a result of phospholipase A activation.     

Expression of mosquitocidal crystal protein genes in non-insecticidal Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis NTB-1 isolated from soil samples in Korea produces ovoidal parasporal inclusions with proteins of approximately 24–40 kDa in size. Although serological study indicated that the isolate has a flagella (H) antigen identical with subsp. israelensis , it seemed to be non-insecticidal against Lepidoptera and Coleoptera as well as Diptera. To investigate the activity of non-insecticidal B. thuringiensis transformed with insecticidal crystal protein genes, cryIVD and cytA genes of B. thuringiensis subsp. morrisoni PG-14, highly toxic to mosquito larvae, were introduced into the isolate NTB-1. The expression of mosquitocidal crystal protein genes in NTB-1 was characterized by SDS–PAGE analysis and electron microscopy. The results showed that crystalline inclusions of host, CryIVD and CytA were stably expressed in the transformant. However, the mosquitocidal activity of transformant was similar to that of B. thuringiensis subsp. kurstaki Cry⁻ B harbouring cryIVD and cytA genes, demonstrating that a synergistic effect by an interaction of both introduced insecticidal and resident non-insecticidal crystal proteins was not observed.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.     

Limits of artificial selection under unbalanced mating systems

Summary The influence of unbalanced mating systems — factorial mating (FM) and random loss of families after a full diallel crossing (RS) — on the ultimate probability of gene fixation (u()) and the time required to fix or lose a gene (t()) are investigated. The average u() of these systems is smaller than that of random mating, and the range of u() for a given initial parental genotype combination is very large (close to one for most initial genotypic combinations). The average u() of different parental genotypic combinations of a given gene frequency are different. These systems accelerate the t().This article was written and prepared by U.S. Government employees on official time, and it is therefore in the public domain     

A fluorescent derivative of ribosomal protein S18 which permits direct observation of messenger RNA binding.

70 S Escherichia coli ribosomes were reacted with the fluorescent dye N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid for 10 min under mild conditions. The resulting ribosomes were fully active. 30 S subunits isolated from these particles were also fully active. They contain approximately 0.7 eq of fluorescent dye. Nearly all of it is attached to protein S18. Competitive reaction with N-ethylmaleimide implies that the fluorescent dye is located at cysteine 10 of the protein. The labeled 30 S particles will recombine with 50 S subunits to form stable 70 S particles. Thus the procedures we have developed allow the large scale preparation of an active fluorescent conjugate of the 70 S ribosome. The fluorescence of the 70 S particles is sensitive to the binding of mRNA, showing both quenching and a shift in emission spectra. Thus it affords a simple way to quantitate mRNA binding directly. In pilot studies without tRNA, the binding constant of the initiation triplet codon adenylyl-(3' leads to 5')-uridylyl-(3' leads to 5')-guanosine to 70 S ribosome was found to be an order of magnitude larger than that of polyuridylic acid.     

Immunohistochemical localization of 36 and 29 kDa proteins in sparganum.

Antigenic proteins of 36 and 29 kDa were localized in Spirometra mansoni plerocercoid (sparganum) immunohistochemically by avidin biotin complex (ABC) staining. When polyclonal antibodies such as BALB/c mouse serum immunized with crude saline extract of sparganum or confirmed sparganosis sera were reacted as primary antibodies, the positive chromogen (3-amino, 9-ethylcarbazole) reactions were recognized at syncytial tegument, tegumental cells, muscle and parenchymal cells and lining cells of excretory canals. A monoclonal antibody (MAb) which was reacting to 36 and 29 kDa proteins in the extract of the worm was localized at the syncytial tegument and tegumental cells. The present results suggested that the potent antigenic proteins of 36 and 29 kDa in sparganum were produced at the tegumental cells and transported to the syncytial tegument.     

Ectoprotein kinase activity of the isolated rat adipocyte.

Intact adipocytes exhibit ectoprotein kinase activity as reflected by their ability to catalyze the transfer of the terminal phosphate of (γ-³²P) ATP to histone added to a cell suspension. This activity is substrate, time and cell number dependent. Lineweaver-Burk plots gave Km and Vmax values for ATP of 5 × 10^?5 M and 7.14 pmoles/min/1.5 × 10⁵ cells. Cyclic AMP but not cyclic GMP in μM concentrations stimulates ectoprotein kinase activity. The controlled tryptic digestion of intact cells results in reduction of ectoprotein kinase activity. This activity is not due to leakage of intracellular protein kinases during the preparative procedure nor to penetration of histone into the cells. Additional phosphoproteins not accessible to endogenous protein kinase activity are also localized on the external surface of the intact fat cell.     

The amino acid sequence of chick skin collagen α1-CB7: The presence of a previously unrecognized triplet

Because alignment of the amino acid sequences of chick skin collagen α2-CB3 (1) with the relevant portion of chick skin collagen α1-CB7 (2) suggested that a Gly-X-Y triplet may have been missed in the latter, the peptide TM-2, produced by tryptic digestion of maleylated α1-CB7, was reinvestigated. Cleavage by trypsin at the unblocked lysine at position 18, and isolation of the resulting COOH-terminal peptide, showed this to be a 15-residue peptide containing a previously unrecognized Gly-Pro-Hyp triplet. Sequencing of the peptide showed this to occupy positions 4 through 6, or 56 through 58 of α1-CB7. The latter thus has 271 instead of 268 residues, and the α1[I] chain 1055 instead of 1052.