Double mutant cycle analysis of aspartate 69, 97, and 103 to asparagine mutants in the m2 muscarinic acetylcholine receptor

Double mutant cycles provide a method for analyzing the effects of a mutation at a defined position in the protein structure on the properties of an amino acid at a second site. This approach was used to map potential interactions between aspartates 69, 97, and 103 in the m2 muscarinic acetylcholine receptor transmembrane helices 2 and 3. Receptors containing single and double aspartate to asparagine mutants were expressed in Chinese hamster ovary cells and their effects on ligand binding, signal transduction, and thermal stability determined. Analysis of the double mutant cycles showed that the mutations had approximately additive effects on ligand binding, signal transduction, and thermal stability. Ligand binding and thermal inactivation results support the conclusion that aspartate-103 is the ligand amine counterion. Effector coupling properties of the mutant receptors showed that aspartate-103 was also required for signal transduction activity. The mutation of aspartate-69 to asparagine completely eliminated signal transduction by the agonists acetylcholine, carbachol, and pilocarpine but not oxotremorine M, which caused reduced but significant inhibition of adenylyl cyclase and stimulation of phospholipase C. In contrast, adenylyl cyclase stimulation by the asparagine-69 mutant was elicited only by acetylcholine and carbachol but not by oxotremorine M. The variation in agonist-dependent effector coupling properties provides evidence that the asparagine-69 mutant can exist in activated receptor states that are different from the wild-type m2 muscarinic receptor.     

Haemoglobins of the shark, Heterodontus portusjacksoni II. Amino acid sequence of the alpha-chain

The amino acid sequence of the alpha-chain of the principal haemoglobin from the shark, H. portusjacksoni has been determined. The chain has 148 residues and is acetylated at the amino terminal. The soluble peptides obtained by tryptic and chymotryptic digestion of the protein or its cyanogen bromide fragments were isolated by gel filtration, paper ionophoresis and paper chromatography. The amino acid sequences were determined by the dansyl-Edman procedure. The insoluble "core" peptide from the tryptic digestion contained 34 residues and required cleavage by several prosteases before the sequence was established. Compared with human alpha-chain there are 88 amino acid differences including the additional seven residues which appear on the amino terminal of the shark chain. There is also one deletion and one insertion. The chain contains no tryptophan but has four cysteinyl residues which is the highest number of such residues recorded for a vertebrate globin. In the alpha1beta1 contact sites there are four changes in the oxyhaemoglobin form and six deoxy form. Nine of the 16, alpha1beta1 contact sites show variation while three of the haem contact sites have changed in comparison to the residues known to be involved in these interactions in horse haemoglobin alpha-chain. Use of the sequence data to estimate a time of divergence of the shark from the main vertebrate line yielded the value of 410 +/- 46 million years. The data, in general, support the palaeontological view that bony fishes arose before the elasmobranchs.     

热轧带肋钢筋22MnSiV的研制

系统的研究了热轧带肋钢筋22MnSiV各项性能，其各项性能不仅达到GB1449－1998中HRB400的要求，还有其特性，脆性转变温度低于－40℃，抗形变时效，高应变低周疲劳性能好，因此，特别适用于地震区，寒冷地区，也适用于钢筋混凝土结构。     

现代转炉炼钢技术及对攀钢炼钢发展的建议

回顾了转炉炼钢技术的发展历程及中个时期的主要技术进步，介绍了当代转炉炼钢的重要技术，结合具体实际情况，提出了攀钢炼钢技术发展的建议。     

Dexamethasone adjunctive therapy for bacterial meningitis in children: a retrospective study in Brazil

Contribution of a case report of megacalycosis in a nine and a half-year old male child, in whom the disorder was diagnosed by our team when he was only four. The intense colic pains experienced by the child together with the isotopic renogram non responsive to seguril typical of obstruction, raised the diagnosis of superimposed stenosis of the pyeloureteral attachment. As a result, an Anderson-Hynes pyeloplasty was performed. Post-operative status after four years is absolutely normal from both a clinical and scintiscan point of view.