分子氮影响下的蓝藻Anabaena7120乙炔还原

经分子氮预处理的蓝藻乙炔还原活性下降。分子氮对乙炔还原的抑制作用可因CO_2的加入而削弱,氮浓度增高时,CO_2的此种有益作用即消失。在5％ CO_2条件下:(1)光照强度大时,经分子氮预处理的蓝藻乙炔还原活性比未经处理的(氮气中)高些,弱光下则削弱,暗中削弱更大;(2)它受到光合抑制剂的抑制较大;(3)对CO_2和O_2的敏感度比未经氮预处理者小;(4)分子氢对其支持与来经氮预处理的相差甚微,但对其受O_2损伤时的保护作用则大些;(5)MSX对分子氮预处理的蓝藻乙炔还原也有明显的抑制,且比单加CO_2的大。     

Use of restrained molecular dynamics in water to determine three-dimensional protein structure: prediction of the three-dimensional structure of Ecballium elaterium trypsin inhibitor II

Refinement of distance geometry (DG) structures of EETI-II (Heitz et al.: Biochemistry 28:2392-2398, 1989), a member of the squash family trypsin inhibitor, have been carried out by restrained molecular dynamics (RMD) in water. The resulting models show better side chain apolar/polar surface ratio and estimated solvation free energy than structures refined "in vacuo." The consistent lower values of residual NMR constraint violations, apolar/polar surface ratio, and solvation free energy for one of these refined structures allowed prediction of the 3D folding and disulfide connectivity of EETI-II. Except for the few first residues for which no NMR constraints were available, this computer model fully agreed with X-ray structures of CMTI-I (Bode et al.: FEBS Lett. 242:285-292, 1989) and EETI-II complexed with trypsin that appeared after the RMD simulation was completed. Restrained molecular dynamics in water is thus proved to be highly valuable for refinement of DG structures. Also, the successful use of apolar/polar surface ratio and of solvation free energy reinforce the analysis of Novotny et al. (Proteins 4:19-30, 1988) and shows that these criteria are useful indicators of correct versus misfolded models.     

The effects of truncating long-range forces on protein dynamics

This paper considers the effects of truncating long-range forces on protein dynamics. Six methods of truncation that we investigate as a function of cutoff criterion of the long-range potentials are (1) a shifted potential; (2) a switching function; (3) simple atom-atom truncation based on distance; (4) simple atom-atom truncation based on a list which is updated periodically (every 25 steps); (5) simple group-group truncation based on distance; and (6) simple group-group truncation based on a list which is updated periodically (every 25 steps). Based on 70 calculations of carboxymyoglobin we show that the method and distance of long range cutoff have a dramatic effect on overall protein behavior. Evaluation of the different methods is based on comparison of a simulation's rms fluctuation about the average coordinates, the rms deviation from the average coordinates of a no cutoff simulation and from the X-ray structure of the protein. The simulations in which long-range forces are truncated by a shifted potential shows large rms deviations for cutoff criteria less than 14 A, and reasonable deviations and fluctuations at this cutoff distance or larger. Simulations using a switching function are investigated by varying the range over which electrostatic interactions are switched off. Results using a short switching function that switches off the potential over a short range of distances are poor for all cutoff distances. A switching function over a 5-9 A range gives reasonable results for a distance-dependent dielectric, but not using a constant dielectric. Both the atom-atom and group-group truncation methods based on distance shows large rms deviation and fluctuation for short cutoff distances, while for cutoff distances of 11 A or greater, reasonable results are achieved. Although comparison of these to distance-based truncation methods show surprisingly larger rms deviations for the group-group truncation, contrary to simulation studies of aqueous ionic solutions. The results of atom-atom or group-group list-based simulations generally appear to be less stable than the distance-based simulations, and require more frequent velocity scaling or stronger coupling to a heat bath.     

Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning,ultrastructural localization,and post-translational phosphorylation

Summary Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with³²P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.     

Rates of synonymous substitution in plant nuclear genes

Summary The rate of synonymous nucleotide substitution in nuclear genes of higher plants has been estimated. The rate varies among genes by a factor of up to two, in a manner that is not immediately explicable in terms of base composition or codon usage bias. The average rate, in both monocots and dicots, is about four times higher than that in chloroplast genes. This leads to an estimated absolute silent substitution rate of 6 × 10^–9 substitutions per site per year that falls within the range of average rates (2–8 × 10^–9) seen in different mammalian nuclear genomes.     

Intra- and interspecies analyses of the carcinoembryonic antigen (CEA) gene family reveal independent evolution in primates and rodents

Summary Various rodent and primate DNAs exhibit a stronger intra- than interspecies cross-hybridization with probes derived from the N-terminal domain exons of human and rat carcinoembryonic antigen (CEA)-like genes. Southern analyses also reveal that the human and rat CEA gene families are of similar complexity. We counted at least 10 different genes per human haploid genome. In the rat, approximately seven to nine different N-terminal domain exons that presumably represent different genes appear to be present. We were able to assign the corresponding genomic restriction endonuclease fragments to already isolated CEA gene family members of both human and rat. Highly similar subgroups, as found within the human CEA gene family, seem to be absent from the rat genome. Hybridization with an intron probe from the human nonspecific cross-reacting antigen (NCA) gene and analysis of DNA sequence data indicate the conservation of noncoding regions among CEA-like genes within primates, implicating that whole gene units may have been duplicated. With the help of a computer program and by calculating the rate of synonymous substitutions, evolutionary trees have been derived. From this, we propose that an independent parallel evolution, leading to different CEA gene families, must have taken place in, at least, the primate and rodent orders.     

Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.     

Simulated and measured nitrogen conditions in a manured and fertilised soil

The governing factors for soil nitrogen dynamics were identified with a simulation model. In addition, the model was used to interpret measurements from a plot fertilisation experiment in southwest Sweden.Simulated moisture and temperature conditions were the driving variables for the simulation of soil nitrogen dynamics and leaching during a 6-year period. The results of the simulation were compared with monthly observations on two plots with grain crops, one with liquid manure and commercial fertilisers applied and one with commercial fertilisers only.Simulated temporal variations of the nitrate and ammonium storages generally agreed with observations. The dominant role of the crops as a determinant of soil nitrogen conditions was demonstrated. A higher leaching loss from the plot with application of commerical fertilisers only occurred both in simulations and measurements compared to the plot with application of both commercial fertilisers and manure. The main reason was the higher N-application in the former treatment.The effect of water flows in macropores was interpreted as a delay of simulated leaching compared to observed leaching on some occasions in summer and early autumn. No direct effect of the macropores on the yearly rates of leaching could be seen.     

Evidence for a composite phylogenetic origin of the plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm

The nucleotide sequence and the 5 flanking region of the rbcL gene coding for the large subunit of ribulose bisphosphate-1,5-carboxylase/oxygenase of Pylaiella littoralis, a brown alga, has been determined and the deduced amino-acid sequence has been compared to those of various photosynthetic and chemoautotrophic Eubacteria, of a red alga and of green plastids (Euglena gracilis, green algae and higher plants). Unlike the rbcL genes of green plastids which are more closely related to those of cyanobacteria the P. littoralis rbcL gene is more closely related to that of a -purple bacterium, as was found for the rbcS gene of another chromophytic alga [Boczar et al., Proc Natl Acad Sci USA 86: 4996–4999, 1989]. Matrix data of homology between the rbcL gene of P. littoralis and the same gene of other organisms are presented. Based on our previous report, the gene coding for the 16S rRNA from P. littoralis is closely related to that of E. gracilis (Markowicz et al., Curr Genet 14: 599–608, 1988). We suggest that the large plastid DNA molecule of P. littoralis is a phylogenetically composite genome which probably resulted from mixed endosymbiosis events, or from a horizontal transfer of DNA.     

A note on sequence-dependence of DNA structure

A circular dichroism study was conducted on the solution structure of several different oligonucleotides, whose X-ray structures have been solved. It is suggested that in aqueous solution the oligonucleotides can form structures that maintain geometrical elements which are typical of B-DNA, A-DNA, and their intermediate forms. It is shown that 5'GGATGGGAG:5'CTCCCATCC, which forms an A-DNA helix in the crystal state (McCall et al. 1986), in aqueous solution maintains an A-DNA like structure at temperatures below 10 degrees C. At temperatures between 10 degrees C and 25 degrees C it shows a tendency to form an intermediate structure between A-DNA and B-DNA. Also, it is shown that TFE does not cause a transition from B-DNA to A-DNA helix in short DNA fragments, but instead disrupts the helix.