Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors

Summary Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids. Offprint requests to: R. Schröder     

The GTPase stimulatory activities of the neurofibromatosis type 1 and the yeast IRA2 proteins are inhibited by arachidonic acid.

Three proteins, GTPase activating protein (GAP), neurofibromatosis 1 (NF1) and the yeast inhibitory regulator of the RAS-cAMP pathway (IRA2), have the ability to stimulate the GTPase activity of Ras proteins from higher animals or yeast. Previous studies indicate that certain lipids are able to inhibit this activity associated with the mammalian GAP protein. Inhibition of GAP would be expected to biologically activate Ras protein. In these studies arachidonic acid is shown also to inhibit the activity of the catalytic fragments of the other two proteins, mammalian NF1 and the yeast IRA2 proteins. In addition, phosphatidic acid (containing arachidonic and stearic acid) was inhibitory for the catalytic fragment of NF1 protein, but did not inhibit the catalytic fragments of GAP or IRA2 proteins. These observations emphasize the biochemical similarity of these proteins and provide support for the suggestion that lipids might play an important role in their biological control, and therefore also in the control of Ras activity and cellular proliferation.     

Modified calcium-dependent regulatory function of troponin C central helix mutants.

Mutations have been made in the exposed region of the avian troponin C central helix, the D/E linker, which change its length and the orientation of the Ca2(+)-binding domains relative to each other. The region 87Glu-Asp-Ala-Lys-Gly-Lys-Ser-Glu-Glu-Glu97 has been altered in five deletion (d) mutants: dEDA, dKG, dKGK, dSEEE, and dKEDAKGK. The recombinant troponin Cs were expressed in Escherichia coli, purified, and assayed for function. All mutants retained basic troponin C function. They all bound Ca2+ to the low and high affinity sites, and they all were able to confer Ca2+ sensitivity on the regulated actomyosin ATPase. However, the regulatory function of all mutants except dSEEE was defective in one part of the Ca2+ switch or the other. In certain conditions dKGK and dKEDAKGK failed to inhibit fully whereas dEDA and dKG failed to activate the regulated actomyosin ATPase fully. The following general conclusions have been made. (a) The length of the D/E linker per se (assuming the linker is helical) and the orientation of the two Ca2(+)-binding domains relative to each other are not crucial for regulation. (b) The conserved charge cluster 95Glu-Glu-Glu97, in a region of troponin C known to bind to troponin I and postulated to be required for regulation, appears to be unimportant for function. (c) Deletion of 88Glu-Asp-Ala90 resulted in a troponin C that could not activate the actomyosin (or S1) ATPase over the level of actomyosin alone, thus defining a role for troponin C in this aspect of thin filament regulation. The results have been interpreted in terms of the crystallographic structure of troponin C and related to results with analogous calmodulin mutants.     

Composition and ecological drivers of the kwongan scrub and woodlands in the northern Swan Coastal Plain,Western Australia

The nature of community patterns and environmental drivers in kwongan mediterranean‐type shrubland on nutrient‐poor soils occurring in Western Australia remain poorly examined. We aimed to determine whether (i) classification of the kwongan vegetation of the northern Swan Coastal Plain would be ecologically informative and (ii) which environmental drivers underpin the plant community patterns. The study area was positioned on the northern Swan Coastal Plain, locality of Cooljarloo (30°39′ S, 115°22′ E), situated 170 km north of Perth, Western Australia. Compositional (518 species × 337 relevés) and environmental data set (29 variables × 87 relevés) describing time since last fire, soil chemical and physical properties, and terrain characteristics were analysed using classification and ordination techniques. OptimClass assisted in the selection of a robust data transformation, resemblance function and clustering algorithm to identify the vegetation patterns. Major ecological drivers of the vegetation patterns were detected using distance‐based redundancy analysis (db‐RDA). Classification revealed major groupings of Wet Heath and Banksia Woodland distinguishable by the high prevalence of myrtyoid and proteoid taxa, respectively. On floristic‐sociological grounds, we recognised four Wet Heath and two Banksia Woodland communities. The Wet Heath was constrained to areas of higher litter depth (db‐RDA axis 1: 9%). Soil chemical and physical properties explained the highest proportion (17%) of the compositional variance, while the terrain‐ and fire‐related variables explained 2% and <0.001%, respectively. While fire explained little compositional variance overall, a separate db‐RDA analysis found that it may play an important pattern‐structuring role within Banksia Woodlands. Fine‐scale compositional patterns correspond only to a small extent to environmental data; the substantial unexplained variance may be due to slow‐acting neutral and stochastic processes.     

New bradykinin analogs in contraction of rat uterus

In this study, we evaluated 20 of our previously synthesized peptides on isolated rat uterus by Holton's procedure with minor modifications, and compared their activity with that assessed previously by their ability to inhibit vasodepressor response to exogenous bradykinin (BK) in conscious rats. We used [D-Arg(0), Hyp(3), Thi(5, 8), (D-Phe)(7)]BK, the B(2) antagonist of Vavrek and Stewart as a model when designing our analogs. We observed that, in the case of the rat uterus test, the activity of peptides modified by acylation of the N-terminus with various bulky groups depends substantially on the chemical character of the substituent. We also learned that, contrary to previous examples, acylation of the N-terminus of antagonists, which contain a sterically restricted fragment in the C-terminal part, may not improve their antagonistic potencies. Besides an improved characterization of a series BK analogs, our studies have provided new information on the structure-activity relationship, which in turn may be of value in the design of more potent and selective bradykinin antagonists. The results of our studies appear to support the hypothesis of others about the presence of different subtypes of B(2) receptors in rat uterus and blood vessels.     

The platinum complexes with histamine: Pt(II)(Hist)Cl2, Pt(II)(Iodo-Hist)Cl2 and Pt(IV)(Hist)2Cl2

The Pt(II) and Pt(IV) complexes with histamine were calculated by using more than 20 DFT functionals and various basis sets. Based on the comparison between the X-ray and theoretical geometrical parameters of the Pt(II)(Hist)Cl₂ complex the MPW1PW91, OPW91 and SVWN5 functionals combined with the 6-311G^∗∗ basis set for non-metallic and SDD (ECP) basis set for platinum were found to yield the most satisfactory agreement. The structure of the Pt(II) complex with iodohistamine important for pharmacy, so far isolated only in minute amounts, was predicted by using the MPW1PW91 functional. Comparison of the theoretical NMR chemical shifts of the Pt(II)(Hist)Cl₂ complex with those found experimentally have shown that the theoretical ¹H and ¹³C NMR chemical shifts are in plausible agreement with the experimental ones, whereas the theoretical ¹⁹⁵Pt chemical shifts fit the experimental values only when the relativistic approach is applied within the ZORA formalism. We confirmed suitability of the three selected functionals for reproduction of the experimental structure of Pt complexes at fourth oxidation state by using the cis- and ions as models. Finally, with the selected theoretical methods, the structures and stabilities of four Pt(IV)(Hist)₂Cl₂ complex isomers were predicted.