Levava Roiz

A recombinant human RNASET2 glycoprotein with antitumorigenic and antiangiogenic characteristics: expression, purification, and characterization.

Human RNASET2 is a T2‐RNase glycoprotein encoded by the RNASET2 gene, which is located on chromosome 6 (6q27). Deletion in 6q27 is associated with several human malignancies.

ACTIBIND, an actin-binding fungal T2-RNase with antiangiogenic and anticarcinogenic characteristics.

ACTIBIND is an Aspergillus niger extracellular ribonuclease (T2‐ribonuclease [RNase]) that possesses actin‐binding activity. In plants, ACTIBIND inhibits the elongation and alters the orientation of pollen tubes by interfering with the intracellular actin network. The question rose whether ACTIBIND can also affect mammalian cancer development.

ACTIBIND, a T2 RNase, competes with angiogenin and inhibits human melanoma growth, angiogenesis, and metastasis.

Melanoma is a very aggressive and highly angiogenic tumor in which standard treatments have had only limited success. Patients with advanced disease have a 5-year survival rate of 5%. In search for alternatives, we identified a natural product extracted from the fungus Aspergillus niger, termed ACTIBIND, that inhibits tumor growth and metastasis of melanoma in vivo. ACTIBIND, a T2 RNase, exerts antitumorigenic and antiangiogenic activities by competing with the angiogenic factor angiogenin (itself an RNase homologue). Thus, there was decreased expression and activity of the matrix metalloproteinase 2 in melanoma and vascular endothelial cells, decreased vascularization, and increased tumor cell apoptosis in vivo. ACTIBIND significantly inhibited angiogenesis in an in vivo angiogenesis assay with sponges containing angiogenin. In vitro, ACTIBIND was internalized by both melanoma and human umbilical vein endothelial cells, reached the cell nuclei, and inhibited the activity of angiogenin response elements in a dose-dependent manner. Collectively, our data indicate that ACTIBIND should be tested for its potential as a new antiangiogenic modality for the treatment of melanoma.

Growth modulation of transgenic potato plants by heterologous expression of bacterial carbohydrate-binding module

Transgenic potato plants (Solanum tuberosum cv. Desiree) expressing the bacterial carbohydrate-binding module (CBM) family III, which is part of the Clostridium cellulovorans CBPA, under control of the CaMV 35S promoter were employed to investigate the influence of this protein on plant development. Eleven independent transgenic plants were found to express the cbm gene, at levels varying from one to four copies. Relative to non-transgenic controls, CBM-expressing plants were characterized by significantly more rapid elongation of the main stem. In addition, under both greenhouse and field conditions, the emergence rate of these plants was higher than in the controls, and their leaf area at early stages of development was larger, resulting in faster accumulation of fresh and dry weight than in control plants. Determination of cell size indicated that epidermal cells in young tissue were significantly larger in CBM-expressing than in control potato plants. These findings suggest that the CBM influence at the cellular level my cause significant alterations in plant growth both in tissue culture and in vivo under field conditions.

Cellulose binding domain increases cellulose synthase activity in Acetobacter xylinum, and biomass of transgenic plants.

Recombinant cellulose-binding domain (CBD) was found to modulate the elongation of different plant cells in-vitro. At low concentrations (0.01–1 µg ml−1), CBD enhanced elongation of Arabidopsis thaliana L. roots. At high concentrations (100–500 µg ml−1), CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 µg ml−1, whereas root elongation was inhibited at that concentration. Using Acetobacter xylinum L. as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to fivefold as compared with the control. Electron microscopy examination of the cellulose ribbons produced by A.xylinum, showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits as compared with a thin and uniform ribbon in the control. Expression of cbd modulated the growth of transgenic plants. Biomass production was significantly higher in selected clones as compared with the control.

Stigmatic RNase in Self-Compatible Peach (Prunus persica)

In peach, which is self-compatible, the stigma was found to actively secrete RNase. This stigmatic RNase was developmentally regulated, reaching maximum activity at the beginning of anthesis. A major 21-kD stigmatic RNase was detected by SDS-PAGE. Both this 21-kD stigmatic RNase and pancreatic RNaseA significantly inhibited pollen germination and pollen tube length in vitro. Pollen tube length was fully recovered in the presence of RNA as a competitive inhibitor of the 21-kD stigmatic RNase. These in vitro studies appear to indicate that in peach, the 21-kD RNase plays a role in the pollination process.

Binding assay and preliminary X-ray crystallographic analysis of ACTIBIND, a protein with anticarcinogenic and antiangiogenic activities

ACTIBIND is a T2 RNase extracellular glycoprotein produced by the mould Aspergillus niger B1 (CMI CC 324626) that possesses anticarcinogenic and antiangiogenic activities. ACTIBIND was found to be an actin-binding protein that interacts with rabbit muscle actin in a 1:2 molar ratio (ACTIBIND:actin) with a binding constant of 16.17 x 10(4) M(-1). Autoclave-treated ACTIBIND (EI-ACTIBIND) lost its RNase activity, but its actin-binding ability was conserved. ACTIBIND crystals were grown using 20% PEG 3350, 0.2 M ammonium dihydrogen phosphate solution at room temperature (293 K). One to four single crystals appeared in each droplet within a few days and grew to approximate dimensions of 0.5 x 0.5 x 0.5 mm after about two weeks. Diffraction studies of these crystals at low temperature (100 K) indicated that they belong to the P3(1)21 space group, with unit-cell parameters a = 78, b = 78, c = 104 A.