ALBERT

Notes: Summary We have compared the amino acid sequences of cytochromec's from 45 species of organisms representing all five kingdoms, including one species each for the Protista and Monera. We have made a phylogeny for these data by reconstructing probable ancestral sequences which generate the present descendants through a minimum number of mutations. Several trials with different data sets produced the same minimal configuration. Assuming the occurrence of no major shifts in mutation acceptance rate, we find an early differentiation between prokaryote and eukaryote stocks. Afterward the eukaryote stem gave rise first to the protozoan flagellate branch and later to the multicellular green plant branch; after this the fungi and multicellular animal stems diverged from each other. A probable ancestral sequence was estimated for each kingdom of multicellular organisms. The basic eukaryote ancestor was probably a non-photosynthetic, heterotrophic flagellate. The photosynthetic apparatus could have been a later symbiotic acquisition in the plant ancestry. The dicotyledons had differentiated into two stocks before the emergence of a monocotyledon line as did the Ascomycetes before the emergence of the Basidiomycetes. The mollusc and chordate lines may have had a common acoelomate ancestor at the divergence of the arthropod stock. The numbers of mutations on all of the branches of the phylogenetic tree were calculated as well as the numbers of mutations and repeated mutations at each amino acid position.

Notes: A versatile crystal cooling device is described for macromolecular crystallographic applications in the 290 to 80 K temperature range. It utilizes a fluctuation-free cold-nitrogen-gas supply, an insulated Mylar crystal cooling chamber and a universal ball joint, which connects the cooling chamber to the goniometer head and the crystal. The ball joint is a novel feature over all previous designs. As a result, the device can be used on various rotation cameras, precession cameras and diffractometers. The lubrication of the interconnecting parts with graphite allows the cooling chamber to remain stationary while the crystal and goniometer rotate. The construction allows for 360° rotation of the crystal around the goniometer axis and permits any settings on the arcs and slides of the goniometer head (even if working at 80 K). There are no blind regions associated with the frame holding the chamber. Alternatively, the interconnecting ball joint can be tightened and fixed. This results in a set up similar to the construction described by Bartunik & Schubert [J. Appl. Cryst. (1982), 15, 277–231], where the cooling chamber rotates with the crystal. The flexibility of the systems allows for the use of the device on most cameras or diffractometers. This device has been installed at the protein crystallographic stations of the Synchrotron Radiation Source at Daresbury Laboratory and in the Laboratory of Molecular Biophysics, Oxford. Several data sets have been collected with processing statistics typical of data collected without a cooling chamber. Tests using the full white beam of the synchrotron also look promising.

Notes: [Auszug] The structure of the segment 1 domain of gelsolin, a protein that fragments actin filaments in cells, is reported in complex with actin. Segment 1 binds monomer using an apolar patch rimmed by hydrogen bonds in a cleft between actin domains. On the actin filament model it binds tangentially, ...

Notes: Abstract Presently the sequences of more than 150 different kinds of proteins and nucleic acids are known from the many thousands thought to exist in all living creatures. Some few of these have occpied much the same functional niche within the living cell from near the beginning of life. In three of these latter, sequence evidence pointing to duplications of genetic material in a primitive ancestor is available and in the fourth other evidence suggests it. Such a duplication, shared by the many descendant species, permits us to locate the point of earliest time on an evolutionary tree and to infer the actual order of subsequent evolutionary events. The amounts of change which have occurred in each descendant line can be estimated with good confidence. Some inferences can be made of the structure of the ancestral duplicated sequence, the evolutionary mechanisms which have been operative on it, and the functional capacity of the organism in which it originated. We will describe new, sensitive, objective methods for establishing the probable common ancestry of very distantly related sequences and the quantitative evolutionary change which has taken place. These methods will be applied to the four families, and evolutionary trees will be derived where possible. Of the three families containing duplications of genetic material, two are nucleic acids: transfer RNA and 5S ribosomal RNA. Both of these structures are functional in the synthesis of coded proteins, and prototypes must have been present in the cell at the inception of the fundamental coding process that all living things share. There are many types of tRNA which recognise the various nucleotide triplets and the 20 amino acids. These types are thought to have arisen as a result of many gene duplications. Relationships among these types will be discussed. The 5S ribosomal RNA, presently functional in both eukaryotes and prokaryotes, is very likely descended from an early form incorporating almost a complete duplication of genetic material. The amount of evolution in the various lines can again be compared. The other two families containing duplications are proteins: ferredoxin and cytochrome c. Ferredoxin from photosynthetic and nonphotosynthetic bacteria shows clear evidence of a duplication of genetic material. This duplication is very possibly shared by the ferredoxin from plant plastids and the related adrenodoxin from mammalian mitochondria. If so, a chronology of the detalls of evolution of these groups can be inferred. From these examples of protein and nucleic acid sequence, we conclude that the amount of change in the bacterial lines is less than that in the eukaryote lines. Even though mutant bacteria are easily produced in the laboratory, though their evolutionary adaptation to new drugs is very rapid, and though new virulent strains often appear spontaneously, nevertheless the sequences of ancient structures in the wild types have changed less than those in the eukaryote lines. Cytochrome c sequences from many eukaryotes and the closely related cytochrome c2 fromRhodospirillum rubrum are known. Other types of cytochrome, such as c551 and c553, are probably related to these through gene duplication. Knowledge of enough of these structures to establish an early duplication will provide a time orientation for the cytochrome c evolutionary tree. This quantitative tree now contains sequences from animals, fungi, green plants, protozoa, and bacteria, examples from all five biological kingdoms.

Notes: A rat model was used to investigate the effect of net surface charge on polymer biocompatibility and its potential to modify and stimulate the inflammatory response. Poly(ether)urethane was taken as the base material and the net charge altered by introducing sulphonate ionic groups to the polymer backbone. Three differently charged poly(ether)urethanes were made with 10, 20, and 30% sulphonate substitution, giving a range of negative charge, with unmodified poly(ether)urethane used as a control. The polymers were implanted intramuscularly into rats for 2 days, and for 1, 2, and 12 weeks. After explantation, the cellular infiltration in the tissue surrounding the implants was evaluated using immunohistochemistry to stain for specific cell types: macrophages, neutrophils, lymphocytes, and the cytokine TNFα. In situ hybridization was used to detect expression of mRNA encoding TNFα. Stained sections were analyzed and the cellular response quantified using image analysis. Initially macrophages and neutrophils were observed around all the materials, but neutrophils were absent in all samples at 12 weeks. The 2-day time point had significantly more macrophages than the later time points. By 2 weeks the 20%-charged polymer elicited significantly less neutrophil infiltration than the other three polymers. In all samples where macrophages were observed, cells staining positive for TNFα protein and message also were observed. No T or B lymphocytes were observed in the infiltrates around the materials at any time point. The results indicate that surface charge can influence the early phase acute inflammatory response to an implanted material. © 1996 John Wiley and Sons, Inc.