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[Intra- and extravascular distribution of coagulation proteins. Interaction with the vascular wall].

Blood proteins specific for the clotting process show the same distribution behavior into the extra vascular space like other plasma proteins. They participate in the macromolecular transport through the vessel wall into the interstitial space and the lymph fluid. The reported results were obtained by vital microscopic investigations of mesenteric vessels of rats using different quantitative techniques with labeled proteins. Fluorescein labeled fibrinogen, antithrombin III, and plasminogen in this technique behave differently according to their molecular weight differences. In humans the extravascular distribution of clotting factors was studied using the model of cantharides skin blister. Some clotting factors were found to be diminished to a different degree pointing to clotting phenomena during transport. - In contrast to most other plasma proteins fluorescein labeled fibrinogen is found to have an affinity to blood vessels, as can be shown using the vital microscopic technique and rat mesenteric vessels. With this, one can detect areas at the interendothelial cell borders of small veins where the fibrinogen turnover primarily takes place. Similar affinities have been detected with fibronectin and a polypeptide from F VIII digest. The results are interpreted as a morphological hint at the functional connection between blood coagulation and the permeability of vessels for macromolecules.

Animals↗

A mutant of protein phosphatase-1 that exhibits altered toxin sensitivity.

A chimeric mutant was constructed in which a 4-amino acid region (GEFD, residues 274-277) of rabbit muscle protein phosphatase-1 was replaced with the sequence YRCG corresponding to residues 267-270 of rabbit protein phosphatase-2A. This was based on the findings of a gene mutation in okadaic acid-resistant cells which results in a Cys-->Gly conversion in protein phosphatase-2A. The YRCG mutant of protein phosphatase-1 was expressed and purified. The properties of the mutant enzyme were investigated in terms of its sensitivity toward several toxin inhibitors (okadaic acid, microcystin, nodularin, calyculin A, and cantharidic acid), as well as inhibitor-2. The mutant enzyme exhibited a gain of function in the form of a 10-fold increased sensitivity toward okadaic acid that suggests this region is involved in toxin binding. Significant changes in sensitivity to inhibitor-2 and several of the other toxins were also observed, indicating that these may have a common binding region.

Amino Acid Sequence↗

[Diffusion of lipid fractions through the barrier of cantharidin blisters in hyperlipidemias under conditions of clofibrate and rutinosid interaction].

Diffusion of lipid fractions from blood to fluid in blister induced by cantharidal ointment, applied on the forearm skin, was studied in 54 patients, thereof 18 with normlipidemia, 13 having type II hyperlipidemia, 23 with type IV hyperlipidemia. Concentrations of triglycerides, phospholipids, free fatty acids, total cholesterol, HDL-cholesterol and LDL--cholesterol were studied in blood and fluid before treatment, 10 days after applying clofibrate, 20 days after clofibrate application, and 10 days following the application of rutinosid, i.e. upon completion of therapy. After the treatment it was observed that in all the patients the concentration of lipid fractions in blood was lowered, except for HDL-cholesterol, the level of which was elevated, as was the concentration of all the fractions in the blister fluid. Lipid concentration in serum, with the exception of free fatty acids, was invariably higher than in blister fluid. Free fatty acids, LDL-cholesterol and HDL-cholesterol diffused from blood to fluid in a greater percentage. Lipid fraction concentration in fluid depended mainly on the concentration of HDL-cholesterol and triglycerides in the blood. In normlipidemia, the highest percentage of lipid fractions was diffused to blister fluid; the percentage was lower in type IV hyperlipidemia, the lowest being in type II hyperlipidemia. Clofibrate hypolipemia action correlated best with with HDL-cholesterol and triglyceride activity. After the treatment, the elevated diffusion of all the fractions from blood to blister fluid was, in my opinion, consistent with lipid metabolism, venoruton, as vessel tightening drug, may play a protective role in relation to endothelia in hyperlipidemia.

Administration, Cutaneous↗

Protein phosphatase in neuroblastoma cells: [3H]cantharidin binding site in relation to cytotoxicity.

Protein phosphatase 2A (PP2A) plays a central role in essential phosphorylation-dependent signal transduction pathways. It is also a principal target for many natural toxicants (cantharidin, microcystins, diarrhetic shellfish poisons) and a synthetic herbicide (endothall). This study develops a cellular model to explore the toxicology of PP2A inhibitors by use of a [3H]cantharidic acid ([3H]CA) ligand binding assay to quantify interactions at the toxicant site and cell viability to evaluate in vivo toxicity. Mouse neuroblastoma (N1E-115) cells are similar to mouse brain with respect to the affinity (12-15 nM), number (B(max), 9-22 pmol/mg protein) and ligand specificity of this binding site. In addition, the competitive potency of ten analogs of CA (including endothall) and two potent diarrhetic shellfish poisons (okadaic acid and calyculin A) is correlated (r2 = .9) with and therefore predictive of their cytotoxicity. The only exception is microcystin LR which is a potent inhibitor at the binding site but is not cytotoxic, possibly reflecting a lack of cellular uptake. ATP and several other phosphorus-containing bifunctional acids inhibit [3H]CA binding by phosphorylation-independent pathways; pyrophosphate apparently acts as a competitive inhibitor. Mn++ and five other divalent cations are also inhibitors with a unique action of Mn++ at 25 to 50 microM in increasing [3H]CA binding, which suggests a specific role in PP2A function. Neuroblastoma cells are therefore suitable to study the mechanisms by which the toxicant, ATP and Mn++ binding sites regulate PP2A activity and cell physiology.

Animals↗

The effects of cantharidin analogues on xanthine oxidase.

Norcantharidin[3], the demethylated product of cantharidin[1] has been used for the treatment of hepatoma, carcinomas of esophagus and gastric cardia, leukopenia and hepatitis. Since the enzyme xanthine oxidase is involved in the diseases mentioned above, and the reactive oxygen species produced by the enzyme induces DNA damage and oxidative damage of tissues, fourteen cantharidin analogues and cantharidimide derivatives were tested for their effects on xanthine oxidase. The results showed that these compounds, listed in Figure 1, displayed very weak inhibitory effects on xanthine oxidase. Contrary to expectation, disodium cantharidate [2], Norcantharidin [3], dehydronorcantharidin [4], disodium dehydronorcantharidate [5], N-(2-pyridyl) cantharidimide [12], N-(3pyridyl) cantharidimide [13] and N-(4-pyridyl) cantharidimide [14] showed a slight stimulating effect on xanthine oxidase at several concentrations.

Allopurinol↗