Activation of cyclic nucleotide formation in murine neuroblastoma N1E-115 cells by modified human thrombins

The activity of alpha-thrombin and chemically modified derivatives of this enzyme in stimulating cGMP formation in murine neuroblastoma clone N1E-115 cells is reported. The rank order potency of the compounds falls into three classes: 1) alpha-thrombin is the most potent and effective; 2) the catalytically active enzymes gamma-thrombin, trypsin, and nitro-alpha-thrombin are approximately 50-fold less potent than alpha-thrombin; and 3) active site blocked derivatives of alpha-thrombin are 100 to 1000-fold less potent than alpha-thrombin. Native alpha-thrombin consistently produces the most effective response, usually 1.5 to 3-fold greater than any of the other compounds tested. Preincubation of cells with quinacrine, an inhibitor of phospholipase A2, or with the lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid prior to thrombin challenge resulted in a concentration-dependent attenuation of the response. Indomethacin was without effect in modifying the response. These results suggest that thrombin stimulation of neuroblastoma cells involves the release of arachidonic acid and its metabolism by lipoxygenase. These results clearly demonstrate the activity of alpha-thrombin in stimulating a receptor-mediated response in cultured nerve cells. The results are discussed in relation to the interaction of endogenous alpha-thrombin with nerve cells following invasive trauma and to the possible presence of endogenous proteinases with a neurotransmitter-like function.     

Initiation of DNA synthesis by human thrombin: relationships between receptor binding, enzymic activity, and stimulation of 86Rb+ influx

Stimulation of amiloride-sensitive sodium (Na+) influx and the subsequent activation of NA+, K+-ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human alpha-thrombin was converted to gamma-thrombin, nitro-alpha-thrombin, and diisopropylphospho (DIP)-alpha-thrombin. These derivatives retain either the capacity to bind cell surface alpha-thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of alpha-thrombin or the various thrombin derivatives, only alpha-thrombin stimulates 86Rb+ influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP-alpha-thrombin that saturate the alpha-thrombin receptors (up to 2 micrograms/ml) do not stimulate either the early or late influx of 86Rb+, indicating that DIP-alpha-thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either gamma-thrombin or nitro-alpha-thrombin, however, stimulate both early and late 86RB+ uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86Rb+ influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.     

Collapse and viscoelasticity of diseased human arteries

A laboratory study of the hydrostatic collapse of diseased tibial arteries demonstrated hysteresis in the pressure-flow behaviour which resembled that seen in the stress-strain relations of the arterial tissue. The pressures at which the vessels collapsed were found to be considerably lower than expected on the basis of theoretical elastic models. Also, the pressures at which the vessels reopened were consistently lower than the pressures at which they collapsed. These findings were explained on the basis of viscoelasticity. The difference between collapse and opening pressure may provide insight into the mechanical properties of vessels, and a clue to errors in non-invasive measurements of blood pressure which depend upon collapse of arteries.     

CHANGES IN HEPATIC STRUCTURE IN RATS PRODUCED BY BREATHING PURE OXYGEN

The livers of rats exposed to pure oxygen were examined electron microscopically to study toxic effects of oxygen in a metabolically sensitive organ. Pressures of 1/3 (258 mm Hg), 1 (760 mm Hg), and 3 (2280 mm Hg) atmospheres were used, with exposures up to 90 days with the lowest pressures. The first changes in the hepatocytes were loss of glycogen and enlargement of mitochondria with development of mitochondria with bizarre shapes which were seen after 3 days at 258 mm, 1 day at 760 mm, and 3 hours at 2280 mm. These changes were followed by formation of increased numbers of cristae, membranes surrounding mitochondria, autophagic vacuoles, and polyribosome clusters. After 2 weeks at 258 mm, which is the pressure of the atmosphere of space cabins, numerous mitochondrial myelin figures appeared but the mitochondrial enlargement had begun to regress. After 90 days at 258 mm, the liver cells appeared almost normal except that many pigment granules had accumulated in the pericanalicular zones. The changes were non-specific and seemed to parallel biochemical alterations recorded elsewhere. They are not considered the result of toxicity but rather of adaptation. These atmospheres, which are used in clinical medicine and in space travel, appear to have no permanent deleterious effects on the liver in rats under the conditions of this experiment.     

Structure of the hirulog 3-thrombin complex and nature of the S' subsites of substrates and inhibitors.

The X-ray crystallographic structure of the human alpha-thrombin complex with hirulog 3 (a potent, noncleavable hirudin-based peptide of the "hirulog" class containing a beta-homoarginine at the scissile bond), which is isomorphous with that of the hirugen-thrombin crystal structure, was solved at 2.3-A resolution by starting with a model for thrombin derived from the hirugen-thrombin complex and was refined by restrained least squares methods (R = 0.132). Residues of hirulog 3 were well-defined in the electron density, which included most of the pentaglycine linker and the C-terminal helical turn that was disordered in a related structure of thrombin with hirulog 1. The interactions of D-Phe1'-Pro2'-beta-homoArg3' with the active site of thrombin were essentially identical to those of related structures of PPACK- (D-Phe-Pro-Arg chloromethyl ketone) and hirulog 1-thrombin, with the guanidinium function of the arginyl P1 residue forming a hydrogen-bonding ion pair with Asp189 of the S1 site. A noticeable shift in the CA atom of beta-homoArg3' due to the methylene insertion displaces the scissile bond from attack by Ser195, thus imparting proteolytic stability to the beta-homoArg hirulog derivative. Resolution of the pentaglycine spacer, linking N- and C-terminal functional domains into a single oligopeptide bivalent inhibitor, permitted delineation of corresponding S' subsites of thrombin. The position of Gly4' (P1') is stabilized by three hydrogen bonds with His57, Lys60F, and Ser195, while the conformational angles maintained in a strained, nonallowed configuration for non-glycyl amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dinuclear silver(I) complexes of 24-membered bibracchial tetraimine Schiff base macrocycles derived from 2,5-diformylthiophene

The cyclocondensation of 2,5-diformylthiophene and the amines N,N-bis-(2-aminoethyl)-2-phenylethylamine, N,N-bis-(2aminoethyl)-t-butyl-amine and N,N-bis-(2-aminoethyl)-t-butyl-amine in the presence of silver(I) salts yields homodinuclear bibracchial tetraimine Schiff base macrocyclic complexes. The structures of two such complexes are also reported. The complex Ag₂L⁴(NO₃)(PF₆) (2) crystallises in the triclinic space group , No. 2) and has unit-cell dimensions a = 12.834(6), B = 13.183(6), C = 14.588(7) Å, = 64.86(4), β = 79.77(4), γ = 69.44(3)° with Z = 2; there is a monodentate and singly bridging nitrate anion present and the Ag---Ag separation is 4.161 Å. The complex Ag₂L⁴(CH₃CN)₂(BF₄)₂·CH₃CN (9) crystallises in the triclinic space group , No. 2) and has unit-cell dimensions a = 9.297(4), B = 12.985(3), C = 21.770(5) Å, = 91.570(10), β = 92.33(3), γ = 97.92(3) ° with Z = 2; there is a strongly bonded acetonitrile molecule coordinated to each silver atom and the Ag---Ag separation is 4.920 Å.     

The impact of phytoplankton on spectral water transparency in the Southern Ocean: implications for primary productivity

Spectral water transparency in the Northern Weddell Sea was studied during Austral spring. The depth of the 1-% surface irradiance level (euphotic depth) varied between 35 and 109 m and was strongly influenced by phytoplankton biomass. Secchi depths were non-linearly related to euphotic depth. In phytoplankton-poor water, the most penetrating spectral region was restricted to a relatively narrow waveband in the blue (488 nm), but the range was broader, between 488 and 525 nm when phytoplankton were abundant. Water transparency in the red spectral range was always low and only to a small extent affected by phytoplankton. Two independent procedures were used to quantify the impact of phytoplankton on spectral water transparency: (1) Regression analysis of spectral in situ vertical light attenuation coefficients in the sea, against coincident chlorophyll concentrations. This method gave chlorophyll-specific light attenuation coefficients; the y-intercept could be interpreted as a measure of light attenuation by pure water plus non-algal material. (2) Spectra of in vivo light absorption derived by spectroscopy, using phytoplankton enriched to varying degrees onto filters. Thus chlorophyll-specific absorption cross-sections were determined. Estimates obtained by both procedures were in close agreement. By integrating over the spectrum of underwater irradiance, in situ chlorophyll-specific absorption cross sections of phytoplankton suspensions, related to all photosynthetically active radiation, were calculated. Light absorption by phytoplankton for photosynthesis is accomplished mainly in the blue spectral range. Also dissolved and particulate organic matter contributed to the attenuation of blue light. Because in water poor in phytoplankton, underwater irradiance was progressively restricted to blue light, chlorophyll-specific absorption cross-sections of phytoplankton, averaged over the spectrum of photosynthetically active irradiance, increased with water depth. In water with elevated phytoplankton biomass, overall light attenuation was generally enhanced. However, because the spectral composition of underwater light changed relatively little with depth, except immediately below the water surface, light absorption cross-sections of phytoplankton changed little below 10 m depth. Vertical differences in the proportions of underwater light absorbed by the phytoplankton community here were mainly dependent on biomass variations. Because of the comparatively small attenuation of blue light by non-algal matter, the efficiency of light harvesting by phytoplankton at any given concentration of chlorophyll in Antractic waters is greater than in other marine regions. At the highest phytoplankton biomass observed by us, as much as 70% of underwater light was available for phytoplankton photosynthesis. When phytoplankton were scarce, <10% of underwater light was harvested by phytoplankton.Contribution within the European Polarstern Study (EPOS), supported by the Deutsche Forschungsgemeinschaft, Grant Ti 115/16-1 to MMT, the European Science Foundation, and by the Alfred Wegener Institut für Polar-und Meeresforschung, Bremerhaven