Impaired L-arginine metabolism marks endothelial dysfunction in CD73-deficient mice.

Changes in the ecto-5'-nucleotidase activity-an extracellular nucleotide catabolic enzyme may lead to the inflammation and endothelial dysfunction. We investigated the effect of CD73 deletion on the endothelial function and L-arginine metabolism in various age groups of mice. 1-,3-,6-, and 12-month-old, male C57BL/6 J wild type (WT) and C57BL/6 J CD73-/- (CD73-/-) mice were used. Blood samples were used for the analysis of adenine nucleotide concentrations. Serum samples were analyzed for the concentration of amino acids, Interleukin 6 (IL-6), Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), and endothelial nitric oxide synthase (eNOS) level. Serum and aortic nitrate/nitrite, as well as aortic arginase and NOS activity in endothelial cells (EC) were evaluated. CD73 deletion led to age-dependent increase in IL-6, ICAM-1, and VCAM-1 concentration compared to WT. All CD73-/- mice age groups were characterized by reduced L-Arginine concentration and eNOS level. Significantly lower NOS activity was noticed in EC isolated from CD73-/- mice lungs in comparison to EC isolated from WT lungs. The L-Arginine/ADMA ratio in the CD73-/- decreased in age-dependent manner in comparison to WT. The nitrate/nitrite ratio was reduced in serum and in aortas of 6-month-old CD73-/- mice as compared to WT. The ornithine/arginine and ornithine/citrulline ratios were increased in CD73-/- compared to controls. Blood (erythrocyte) Adenosine-5'-triphosphate and Adenosine-5'-diphosphate levels were reduced in favor to higher blood Adenosine-5'-monophosphate concentration in CD73-/- mice in comparison to WT. The CD73 deletion leads to the development of age-dependent endothelial dysfunction in mice, associated with impaired L-arginine metabolism. CD73 activity seems to protect endothelium.

Complete remission of Bomirski Ab amelanotic melanoma in hamsters treated with the angiogenesis inhibitor TNP-470.

The growth of solid tumors and their metastasis is dependent on the development of new blood vessels (angiogenesis). In our previous studies it was found that angiogenesis inhibitor TNP-470 acting systematically can decrease the rate of growth of transplantable Bomirski Abmelanoma in hamsters. In this study we applied TNP-470 (30 mg/kg) peritumorally from the day when tumor was palpable over 10 days, once daily. Animals were killed 6 months later and examination by autopsy and histological preparations showed the complete remission of transplanted tumor and the lack of metastasis. Thus, Ab melanoma can be effectively cured with TNP-470 angiogenesis inhibitor when the substance is applied locally.

Synergistic effect of the angiogenesis inhibitor TNP-470 and tumor necrosis factor (TNF) on Bomirski Ab melanoma in hamsters.

Bomirski Ab transplantable melanoma bearing hamsters were treated with angiogenesis inhibitor TNP-470 or with rat tumor necrosis factor (TNF) at doses of 20 micrograms and 40 micrograms or with both TNP-470 and TNF at a dose of 20 micrograms each. The size of the growing tumor was measured everyday from the day when it was palpable. After the death of the animals the final size of their tumors was measured and the number and localisation of metastasis were determined. It was found that the rate of tumor growth was lowest in the group of animals treated with TNF at a dose of 40 micrograms and in the group of animals treated with TNP-470 and TNF at a dose of 20 micrograms. The frequency of metastasis was different in the experimental groups although their location was similar. The longest living animals belonged to the group treated with both TNP-470 and TNF. The results of the investigation indicate that TNP-470 and TNF act in a synergistic way that allows a decrease in the effective dose of TNF thus diminishing its noxious side effects.

Evidence of interactions between Gp27 and Gp28 constituents of the central part of bacteriophage T4 baseplate.

The central part of the bacteriophage T4 baseplate consists of several proteins. However, for a number of the constituents the manner of incorporation are not convincingly established. Recently, we have presented evidence that gp28 is the structural component of the central part of the baseplate, which possesses a hydrophobic region and is membrane bound [Nieradko et al., 1998]. By utilizing extracts prepared from Escherichia coli cells that overexpressed genes 27 and 28 of phage T4, we proved that gp28 forms a complex with an another baseplate structural components: gp27. This complex was located in the membrane fraction. Its affinity to the inner membrane indicates that the identified complex may function as an initiator of the central hub assembly. It was subsequently established that these products interact in the ratio 1:1. We have also demonstrated that the particular components of the complex can be separated by action of SDS and to a lesser extent by Triton X-100.

Cloning, expression, purification of rat tumor necrosis factor and demonstration of its antitumor activity in Bomirski amelanotic melanoma.

In order to analyze the effect of tumor necrosis factor (TNF) on the growth of Bomirski melanomas of hamster, we cloned, expressed and purified to homogeneity the rat tumor necrosis factor. The detailed procedure of chromatographic purification of the cytokine is reported. Polyclonal antibodies generated against the recombinant rat preparation were able to neutralize the rat and hamster TNF. We have found that the preparation of rat TNF is active in vivo and inhibits the growth of a Bomirski (Ab) amelanotic melanoma. Thus, a novel model system has been developed, which should facilitate analysis of antitumor and immunomodulatory function of TNF in cancer hosts.

Characteristics of gene 28 product, the constituent of the central part of bacteriophage T4 baseplate.

The phage TD gene 28 product has been partially characterized and its biological role has been examined. It was found to be a protein with a molecular size of 24 kDa which cosediments with the membrane fraction of the bacterial extracts and could only be washed out by a 0.2% sarcosyl solution. Other observations indicate that gp 28 has a majority of hydrophobic residues on its surface and forms a homotrimeric complex in the absence of other phage proteins. The product was finally identified as a baseplate structural component. Incubation of purified phage preparation in a buffer which contained active protein 28, did not affect the efficiency of the plating. However, incubation of the phage particles with specific antiserum was found to neutralize phage infectivity.