The human amylase-encoding genes amy2 and amy3 are identical to AMY2A and AMY2B

Inspection of the published nucleotide sequences reveals that the human amylase-encoding genes, amy2 and amy3, must be identical to the genes AMY2A and AMY2B, respectively.     

Structure and protein environment of the retinal chromophore in light- and dark-adapted bacteriorhodopsin studied by solid-state NMR

Our previous solid-state 13C NMR studies on bR have been directed at characterizing the structure and protein environment of the retinal chromophore in bR568 and bR548, the two components of the dark-adapted protein. In this paper, we extend these studies by presenting solid-state NMR spectra of light-adapted bR (bR568) and examining in more detail the chemical shift anisotropy of the retinal resonances near the ionone ring and Schiff base. Magic angle spinning (MAS) 13C NMR spectra were obtained of bR568, regenerated with retinal specifically 13C labeled at positions 12-15, which allowed assignment of the resonances observed in the dark-adapted bR spectrum. Of particular interest are the assignments of the 13C-13 and 13C-15 resonances. The 13C-15 chemical resonance for bR568 (160.0 ppm) is upfield of the 13C-15 resonance for bR548 (163.3 ppm). This difference is attributed to a weaker interaction between the Schiff base and its associated counterion in bR568. The 13C-13 chemical shift for bR568 (164.8 ppm) is close to that of the all-trans-retinal protonated Schiff base (PSB) model compound (approximately 162 ppm), while the 13C-13 resonance for bR548 (168.7 ppm) is approximately 7 ppm downfield of that of the 13-cis PSB model compound. The difference in the 13C-13 chemical shift between bR568 and bR548 is opposite that expected from the corresponding 15N chemical shifts of the Schiff base nitrogen and may be due to conformational distortion of the chromophore in the C13 = C14-C15 bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular endothelial cells synthesize a plasma membrane protein indistinguishable from the platelet membrane glycoprotein IIa

To define the role of membrane components that function in endothelial cell physiology and to characterize them biochemically, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained producing antibodies which bound to endothelial cells and also to platelets. The antibody of one of these clones, CLB-HEC 75, was studied in more detail. This antibody is directed against a single protein which is synthesized constitutively by endothelial cells and is expressed on the surface of both endothelial cells and platelets. The CLB-HEC 75 antigen was isolated from Nonidet P-40-solubilized endothelial cells and platelets by immunoprecipitation and exhibited an apparent molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 145,000 in the presence of 2-mercaptoethanol. Two-dimensional polyacrylamide gel electrophoresis and crossed immunoelectrophoresis revealed that the mobility of the CLB-HEC 75 antigen relative to platelet glycoproteins Ib, IIa, IIb, and IIIa fits previously defined criteria for platelet membrane glycoprotein IIa. The CLB-HEC 75 antigen isolated from endothelial cells co-migrated under all conditions tested with the antigen from platelets. These results indicate that endothelial cells share a plasma membrane protein indistinguishable from platelet membrane glycoprotein IIa. This protein may be a component involved in the interaction of endothelial cells with their environment including coagulation factors, platelets, and the subendothelial matrix. CLB-HEC 75 may serve as a useful tool for studying these processes.     

Electroimmunoassay of rat apolipoproteins A-I, A-IV, and E. A procedure for sample treatment to increase the sensitivity in diluted fractions

Methods for the quantitative determination of rat apolipoproteins A-I, A-IV, and E by electroimmunoassay are described. Apolipoproteins present in diluted samples of biological fluids (approx. 2 ml) were concentrated by precipitation with deoxycholate and trichloroacetic acid. The protein pellets were solubilized in 0.1 ml of 0.5 M NaOH and these samples were delipidated with tetramethylurea and assayed. This protocol enables the measurement of apolipoprotein concentrations that are at least 10 times lower than normally detectable; 0.2 micrograms of apolipoprotein A-IV, 0.2 micrograms of apolipoprotein A-I, and 0.8 micrograms of apolipoprotein E can be easily detected in samples of 2 ml.     

Decades of population genetic research reveal the need for harmonization of molecular markers: the grey wolf Canis lupus as a case study

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Death rate in a small air-lift loop reactor of vero cells grown on solid microcarriers and in macroporous microcarriers

The death rate of Vero cells grown on Cytodex-3 microcarriers was studied as a function of the gas flow rate in a small air-lift loop reactor. The death rate may be described by first-order death-rate kinetics. The first-order death-rate constant as calculated from the decrease in viable cells, the increase in dead cells and the increase in LDH activity is linear proportional to the gas flow rate, with a specific hypothetical killing volume in which all cells are killed of about 2·10^–3 m³ liquid per m³ of air bubbles. In addition, an experiment was conducted in the same air-lift reactor with Vero cells grown inside porous Asahi microcarriers. The specific hypothetical killing volume calculated from this experiment has a value of 3·10^–4 m³ liquid per m³ of air bubbles, which shows that the porous microcarriers were at least in part able to protect the cells against the detrimental hydrodynamic forces generated by the bubbles.     

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida,strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

The consistency of large concerted motions in proteins in molecular dynamics simulations.

A detailed investigation is presented into the effect of limited sampling time and small changes in the force field on molecular dynamics simulations of a protein. Thirteen independent simulations of the B1 IgG-binding domain of streptococcal protein G were performed, with small changes in the simulation parameters in each simulation. Parameters studied included temperature, bond constraints, cut-off radius for electrostatic interactions, and initial placement of hydrogen atoms. The essential dynamics technique was used to reveal dynamic differences between the simulations. Similar essential dynamics properties were found for all simulations, indicating that the large concerted motions found in the simulations are not particularly sensitive to small changes in the force field. A thorough investigation into the stability of the essential dynamics properties as derived from a molecular dynamics simulation of a few hundred picoseconds is provided. Although the definition of the essential modes of motion has not fully converged in these short simulations, the subspace in which these modes are confined is found to be reproducible.     

Interstellar dust,chirality, comets and the origins of life: Life from dead stars?

The physical, chemical and astrophysical processes by which chiral prebiotic molecules can be produced in interstellar dust and later delivered safely to the earth are considered. A laboratory analog experiment on the irradiation by circularly polarized UV light of mirror image molecules at the low temperatures of interstellar dust demonstrates that a substantial degree of chirality can be produced by irradiation of the dust by circularly polarized light from pulsars whose mean brightness and distribution in the Milky Way provide the energetic photons. The chirality is then preserved by cold aggregation of the dust into low density fragile nuclei. The thermal evolution of comets following them from birth through billions of years in the Oort cloud and back to the inner solar system results in preservation of dust organics in largely pristine form — even including effects of radiogenic heating. Physical justification for the cushioned transfer of fragments of the fluffy comets impacting the earth's atmosphere provides a conceptual basis for depositing significant concentrations of interstellar prebiotic molecules. Chiral amplification in water on the earth is presumed to be enhanced by this local concentration. If chiral molecules are discovered in comet nucleus material which will some day be returned to the laboratory, we may have in our hands the same building blocks from which we evolved.