Mapping of functional epitopes of osteopontin by monoclonal antibodies raised against defined internal sequences.

Osteopontin (OPN) is a secreted protein that has been implicated in diverse physiological and pathological processes. OPN can bind to integrins, via GRGDS or SVVYGLR amino acid sequences, and to other cell surface receptors, and many of OPN's functions are likely mediated via cell adhesion and subsequent signaling. Here we developed and characterized a series of five monoclonal antibodies, raised to distinct internal peptide sequences of human OPN, and have used these sequence-specific reagents, along with the previously described anti-OPN monoclonal antibody mAb53, to map functional epitopes of OPN that are important to cell adhesion and migration. All antibodies were reactive with native as well as recombinant human OPN. One antibody (2K1) raised against the peptide VDTYDGRGDSVVYGLRS could inhibit RGD-dependent cell binding to OPN, with an efficacy comparable to that of mAb53. Furthermore, 2K1 could inhibit alpha9 integrin-dependent cell binding to OPN. The epitope recognized by 2K1 was not destroyed by thrombin digestion, whereas mAb53 has been shown to be unable to react with OPN following thrombin cleavage. The two distinct epitopes defined by 2K1 and mAb53 antibodies are closely related to the SVVYGLR cell-binding domain and the GLRSKS containing thrombin cleavage site, respectively, and are involved in cell binding and cell migration.     

Production of recombinant rotavirus VP6 from a suspension culture of transgenic tomato (Lycopersicon esculentum Mill.) cells

Recombinant rotavirus VP6 expressed in transgenic tomato cells was found primarily in the intracellular fraction and had a molecular weight of 44 kDa. In a shake flask, transgenic tomato cells produced 0.33 mg recombinant VP6 l^–1 after 18 days of incubation. In a high aspect rotating-wall vessel designed by NASA to simulate microgravity, the transgenic cells produced up to 0.15 mg recombinant VP6 l^–1.     

Ecological factors of flowering and pollen quality in three willow species

During 1977 to 1980 the course of flowering and pollen quality were investigated inSalix cinerea, S. fragilis, andS. pentandra, the dominant woody species in a wetland area called “Mokré louky” near T?eboň, South Bohemia, Czechoslovakia. Results were compared with basíc climatological data, and decisive factors of flowering were estimated. BothS. cinerea andS. pentandra produced morphologically well developed pollen with a high degree of viability. Comparison in twelve habitats showed that the pollen ofS. fragilis was very sensitive to climatic factors, and that the marsh habitat “Mokré Louky” was hostile to the generative reproduction of this species. Optimal conditions for pollen germination in vitro were defined for all the three willows.     

ESR study of sublevel structure of anionic bis(phthalocyaninato)gadolinium(III)

Sublevel structure of the ⁸S_7/2 electronic ground state of anionic bis(phthalocyaninato)gadolinium(III) has been determined by simulation analysis of an ESR spectrum in frozen solution. The simplex multidimensional minimization algorithm was employed to find the zero-field-splitting parameter set giving the minimum RMS error from the observed spectrum. The parameter set {B₂⁰,B₄⁰,B₆⁰} for the potential of D_4d symmetry has been determined to be ±{(1.54 ± 0.01)× 10⁻² cm⁻¹, (0.9 ± 0.1)× 10⁻⁴ cm⁻¹, (−0.6 ± 0.9)× 10⁻⁶ cm⁻¹}. The energy difference between the lowest and highest sublevels has been found to be about 0.5 cm⁻¹.     

Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to activation of the lectin complement pathway

The complement system plays an important role in innate immunity. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme. It has been suggested that MASP-2 is responsible for the activation of C4. Other serine proteases (MASP-1 and MASP-3) are also associated with MBL or ficolins; however, their functions are still controversial. In this study, a MASP-1- and MASP-3-deficient mouse model (MASP1/3(-/-)) was generated by a gene targeting strategy to investigate the roles of MASP-1 and MASP-3 in the lectin pathway. Serum derived from MASP1/3(-/-) mice showed significantly lower activity of both C4 and C3 deposition on mannan-agarose, and this low activity was restored by the addition of recombinant MASP-1. MASP-1/3-deficient serum showed a significant delay for activation of MASP-2 compared with normal serum. Reconstitution of recombinant MASP-1 in MASP-1/3-deficient serum was able to promote the activation of MASP-2. From these results, we propose that MASP-1 contributes to the activation of the lectin pathway, probably through the activation of MASP-2.     

Atorvastatin attenuates mitochondrial toxin-induced striatal degeneration, with decreasing iNOS/c-Jun levels and activating ERK/Akt pathways

Mitochondrial dysfunction is a major contributor to neurodegeneration, and causes vulnerability to oxidative stress and the activations of downstream cell death pathways. 3-Hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, statins, were originally developed as cholesterol lowering agents, and have cholesterol-independent anti-excitotoxic and anti-oxidative properties. We investigated whether atorvastatin can prevent the neurodegeneration induced by a mitochondrial toxin, 3-nitropropionic acid (3NP), which inhibits succinate dehydrogenase complex II. Male Lewis rats were administered 3NP (63 mg/kg/day) using osmotic pumps for 5 days to induce striatal degeneration, and were also treated with either atorvastatin (1 or 10 mg/kg/day, orally) or vehicle (control) on five consecutive days. Atorvastatin-treated rats showed fewer neurologic deficits than control animals as measured at day 3–5. Atorvastatin-treated animals showed reduced striatal lesion volumes by Nissl staining, and decreased numbers of TUNEL-positive apoptosis and Fluoro-Jade C-positive degenerating neurons at 5 days. Atorvastatin reduced the numbers of c-Jun-positive and p-c-Jun-positive cells, as well as 3-nitrotyrosin-positive cells. In addition, atorvastatin increased p - extracellular signal-regulated kinase and p-Akt levels, and attenuated the up-regulation of inducible nitric oxide synthase by 3NP. When N (omega)-nitro- l -arginine methyl ester hydrochloride was administered concomitantly with the 3NP infusion, atorvastatin failed to further reduce the striatal lesion volume and c-Jun levels compared to the vehicle treatment. In summary, atorvastatin decreased striatal neurodegeneration induced by 3NP, with attenuating inducible nitric oxide synthase and c-Jun levels as well as activating extracellular signal-regulated kinase and Akt.     

Antiangiogenic photodynamic therapy (PDT) by using long-circulating liposomes modified with peptide specific to angiogenic vessels

For the improvement of therapeutic efficacy in photodynamic therapy (PDT) by using a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), we previously prepared polyethylene glycol (PEG)-modified liposomes encapsulating BPD-MA (PEG-Lip BPD-MA). PEGylation of liposomes enhanced the accumulation of BPD-MA in tumor tissue at 3 h after injection of it into Meth-A-sarcoma-bearing mice, but, unexpectedly, decreased the suitability of the drug for PDT when laser irradiation was performed at 3 h after the injection of the liposomal photosensitizer. To improve the bioavailability of PEG-Lip BPD-MA, we endowed the liposomes with active-targeting characteristics by using Ala-Pro-Arg-Pro-Gly (APRPG) pentapeptide, which had earlier been isolated as a peptide specific to angiogenic endothelial cells. APRPG-PEG-modified liposomal BPD-MA (APRPG-PEG-Lip BPD-MA) accumulated in tumor tissue similarly as PEG-Lip BPD-MA and to an approx. 4-fold higher degree than BPD-MA delivered with non-modified liposomes at 3 h after the injection of the drugs into tumor-bearing mice. On the contrary, unlike the treatment with PEG-Lip BPD-MA, APRPG-PEG-Lip BPD-MA treatment strongly suppressed tumor growth after laser irradiation at 3 h after injection. Finally, we observed vasculature damage in the dorsal air sac angiogenesis model by APRPG-PEG-Lip BPD-MA-mediated PDT. The present results suggest that antiangiogenic PDT is an efficient modality for tumor treatment and that tumor neovessel-targeted, long-circulating liposomes are a useful carrier for delivering photosensitizer to angiogenic endothelial cells.