The effect of metal binding on the structure of annexin V and implications for membrane binding.

The structure of annexin V, crystallised in the presence of two calcium or barium ions for each protein molecule, was solved by molecular replacement to 0.24 nm resolution. The two metal ions are found in domains I and IV, i.e. on the same side of the channel that lies in the centre of the molecule. The structures of the barium and calcium form are extremely close, the only differences localised in the metal-binding sites that lie on the surface of the molecule. The occupancies of the metal ions, however, are lower for barium than for calcium, expressing the lower affinity of the protein for the former. The packing of the annexin molecules in the crystal asymmetric unit may represent a model for the calcium driven association of membrane-bound annexins that leads to membrane fusion.     

Phyto- and protozooplankton biomass during austral summer in surface waters of the Weddell Sea and vicinity

Summary Phyto- and protozooplankton were sampled in the upper 10 m of the water column in austral summer during a cruise of RV Polarstern from January 6 to February 20 1985 in the eastern Bransfield Strait vicinity and in the northern, southeastern (off Vestkapp, twice: I and II) and southern Weddell Sea (Vahsel Bay across the Filchner Depression to Gould Bay). The plankton assemblages are discussed in relation to physical, chemical and biological factors in the different geographical areas in summer. Phytoplankton biomass (Phytoplankton carbon, PPC) ranged from 4–194 g carbon/l and consisted on average of 65% diatoms and 35% autotrophic flagellates. Whereas in the northwest phytoplankton assemblages were dominated by small nanoflagellates (78% of PPC), higher biomass of diatoms (54–94% of PPC) occurred at the other sampling sites. In general autotrophic flagellates and small pennate diatoms dominated at oceanic stations; in neritic areas large centric diatoms prevailed. Chlorophyll a concentrations ranged from 0.25–3.14/g chl a/l with a mean of 1.13/gmg chlorophyll a/l and an average phytoplankton carbon/chlorophyll a ratio of 39. Protozooplankton biomass (Protozooplankton carbon, PZC) ranged from 0–67 g carbon/l and consisted of 49% ciliates, 49% heterotrophic dinoflagellates and 2% tintinnids. Heterotrophic dinoflagellates were more important in the northern investigation areas (58%–84% of PZC). Ciliates dominated the protozooplankton in the southeast and south (56%–65% of PZC); higher abundances of tintinnids were observed only in the south (11% of PZC). The most remarkable feature of the surface waters was the high protozooplankton biomass: protozooplankton amounted to 25% on an average of the combined biomass of PPC plus PZC for the entire investigation period. Protozoan biomass in the southeastern and southern Weddell Sea occasionally exceeded phytoplankton biomass. Temperature, salinity, and inorganic nutrients were generally lower in the southern regions; at most of these stations a meltwater layer occurred in the upper meters of the water column. We suggest that this physical regime allows a well developed summer system with a high proportion of heterotrophic microplankton. In the eastern Bransfield Strait, in the northern Weddell Sea and close to the coast off Vestkapp (I), however, early summer conditions occurred with less protozooplankton contribution.Contribution no. 427 from the Alfred-Wegener-Institute for Polar and Marine Research     

Plasma determination of the novel anticonvulsant d,l-3-hydroxy-3-ethyl-3-phenylpropionamide and preliminary pharmacokinetic studies in the rat

A sensitive gas chromatographic method with flame ionization detection was developed for the analysis in plasma of the novel anticonvulsant d,l-3-hydroxy-3-ethyl-3-phenylpropionamide (HEPP), using d,l-2-hydroxy-2-ethyl-2-phenylacetamide as the internal standard. HEPP was extracted from alkalinized plasma into dichloromethane and quantified after derivatization with bis(trimethylsilyl)-trifluoroacetamide. Standard curves were linear from 0.5 to 50 and from 2 to 100 μg/ml of plasma, using 1.5 and 5 μg of the internal standard, respectively. The lower limit of detection at a signal-to-noise ratio of 3 standard deviations was 0.33 μg/ml of sample. The sensitivity, accuracy and reproducibility of the method were shown to be satisfactory for pharmacokinetic studies of HEPP. After intraperitoneal administration of 50 mg/kg to Wistar rats, the principal kinetic parameters were: absorption half-life = 0.04 h; volume of distribution = 1.32 l/kg; clearance = 4.40 ml/min; peak concentration = 50 μg/ml; peak time = 0.25 h; mean residence time = 4.55 h.     

Visna virus synthesized in absence of host-cell division and DNA synthesis

Visna virus is similar to the avian and the murine oncornaviruses. Oncornavirus replication is dependent upon the provirus being integrated into the host cell's DNA but integration and subsequent oncornavirus synthesis is blocked when the host cells are prevented from synthesizing cellular DNA or dividing. The synthesis of visna virus is restricted in vivo and may be dependent upon the host cell's ability to synthesize cellular DNA or divide. Treatment of sheep choroid plexus (SCP) cells with ultraviolet light or with mitomycin C prior to infection irreversibly inhibited plexus (ScP) cells with ultraviolet light or with mitomycin C prior to infection irreversibly inhibited both cell division and cellular nucleic acid synthesis but did not inhibit visna virus synthesis. Similarly, the synthesis of visna virus in cultures of SCP cells which had been prevented from dividing by being deprived of serum and in cultures of SCP cells which were incapable of synthesizing host cell nucleic acids by being treated with miracil D or sodium hexachloroiridate was equivalent to the synthesis of visna virus in cultures of SCP cells which were allowed to both synthesize cellular nucleic acids and divide. The synthesis of visna virus in the presence of ethidium bromide further demonstrated that integration of the visna provirus into the host cell's DNA is not required for visna virus synthesis to occur.     

Biosynthesis of bacterial glycogen: purification and properties of Salmonella typhimurium LT-2 adenosine diphosphate glucose pyrophosphorylase.

The adenosine diphosphate glucose pyrophosphorylase from a Salmonella typhimurium LT-2 mutant, JP102, derepressed in the glycogen biosynthetic enzymes was purified to homogeneity. The enzyme was found to be identical with the parent wild-type enzyme with respect to regulatory properties, immunological reactivity, and kinetic constants for the allosteric effectors and for the substrate, adenosine triphosphate. The JP102 enzyme was composed of four identical subunits, each with a molecular weight of about 48,000. This was supported by the findings that (i) gel electrophoresis under denaturing conditions showed only one component; (ii) digestion with carboxypeptidase B released stoichiometric amounts of arginine, and (iii) amino-terminal sequencing showed a single sequence for the first 27 residues. The properties of the purified S. typhimurium enzyme were compared with the properties of the previously purified Escherichia coli B enzyme.     

Assays for the fidelity of DNA polymerases in cell-free extracts of Escherichia coli are complicated by contaminating nucleoside triphosphatases.

In the presence of DNA and a divalent cation, an enzyme activity in cell-free extracts of Escherichia coli readily hydrolyses dATP to dADP. dGTP is degraded to a smaller extent, dCTP and dTTP being hardly affected. The artificial template primers poly(dC) . oligo(dG) and poly(dT) . oligo(dA) are also effective cofactors for this triphosphatase activity. As a consequence, assays measuring the misincorporation, by cell-free extracts, of dATP and dGTP into these defined templates are difficult to interpret, since the triphosphate substrate is being rapidly degraded during the polymerase reaction. A partial characterization of the dATPase activity was performed, demonstrating that the optimal conditions for its activity resemble those commonly used for assaying polymerase activity. Thus in crude extracts both polymerase and dATPase compete for the same substrate. The inclusion of an ATP-generating system in the reaction mixture maintains the levels of deoxynucleoside triphosphates and changes the kinetics of misincorporation of dAMP into poly(dC) . oligo(dG). No reproducible difference in such misincorporation has been found between lysates prepared from tif-1 cells grown at either permissive or restrictive temperature.     

Blood glucose monitoring and computer regulation by means of an artificial endocrine pancreas.

Extracorporeal blood glucose regulation has been implemented by a closed-loop system (artificial endocrine pancreas, "aep") of our own construct. Simple, highly flexible algorithms have been developed that allow smooth repair of a deranged glucose imbalance up to a period of 125 hours.     

5-Formyl-2'-deoxyuridine: cytostatic and antiviral properties and possible modes of action.

5-Formyl-2'-deoxyuridine (fdUrd) was prepared by a new method starting from thymidine and investigated for its influence both on proliferation of cultured mammalian cells and virus replication in vitro. The compound was found to have strong cytostatic and antiviral properties: 50% inhibition of proliferation of BHK 21/C13 cells or Ehrlich ascites tumour cells (EAT) was obtained at 4 - 10(-6) and 6 - 10(-6) M, respectively, while the treatment of pseudorabies virus with the same concentration resulted in about 1.5 log reduction of virus yield. A concentration of 1 - 10(-4) M inhibited cell proliferation by 80 to 100% while the virus yield was reduced by more than 3 orders of magnitude. All inhibitions can be prevented by thymidine.--DNA synthesis of EAT cells in vitro, as estimated by incorporation of [32P]-phosphate or low concentrations of [3H]-thymidine, was inhibited. Further biochemical experiments have provided indirect evidence that the compound is phosphorylated by thymidine and thymidylate phosphorylating enzymes. An inhibition of cell free DNA synthesis was found to be depending on a given period of preincubation with the compound (supposed to be needed for the formation of fdUrd 5'-triphosphate). This suggests that the 5'-triphosphate of fdUrd is an inhibitor of DNA polymerases and--by analogy with experiments with 5-formyluridine-5'-triphosphate and RNA polymerases [14]--may be used as an affinity label for this group of enzymes. It is concluded that the described cytostatic and antiviral effects of fdUrd are due to an intracellular "lethal" synthesis of the relevant phosphates which inhibit thymidylate synthetase (as had been found earlier to occur with the chemically prepared nucleotide in cell free extracts [1, 2]) and DNA synthesizing enzymes.