Dysregulated expression of MIC-1/PDF in human prostate tumor cells

As a part of the study to identify genes associated with hormone-refractory stage of human prostate cancer, we have recently identified several genetic and epigenetic changes that seem to be associated with the progression of androgen-sensitive to androgen-independent prostate tumor cells. In the present study, we report a novel gene, macrophage inhibitory cytokine-1 (MIC-1) also known as prostate derived factor (PDF), that was highly expressed in androgen-independent LNCaP-C81 cells and its metastatic variant LNCaP-Ln3 compared to androgen-sensitive LNCaP-C33 cells. The MIC-1/PDF expression was dysregulated (very low to non-detectable) in the androgen-independent PC3 and DU145 cells. Interestingly, serum factors demonstrated a differential regulation of MIC-1/PDF in the androgen-sensitive and the androgen-independent cells of LNCaP cells. Immunohistochemical analysis on 15 prostatic adenocarcinomas showed a weak staining in the benign prostatic glandular area (intensity score 2.38+/-0.25; n=13), while the immunoreactivity was significantly stronger (p<0.05) in areas of adenocarcinoma (score 7.33+/-0.88; n=15). Altogether, these data suggest that the serum factors (including androgens and cytokines) might contribute to the regulation of the MIC-1/PDF gene that seems to be associated with the progression of prostate cancer.     

The cytotoxic activity of ribosome-inactivating protein saporin-6 is attributed to its rRNA N-glycosidase and internucleosomal DNA fragmentation activities

Saporin-6 produced by the plant Saponaria officinalis belongs to the family of single chain ribosome-inactivating proteins. It potently inhibits protein synthesis in eukaryotic cells, by cleaving the N-glycosidic bond of a specific adenine in 28 S rRNA, which results in the cell death. Saporin-6 has also been shown to be active on DNA and induces apoptosis. In the current study, we have investigated the roles of rRNA depurination and the activity of saporin-6 on genomic DNA in its cytotoxic activity. The role of putative active site residues, Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208), and two invariant residues, Tyr(16) and Arg(24), proposed to be important for structural stability of saporin-6, has been investigated in its catalytic and cytotoxic activity. These residues were mutated to alanine to generate seven mutants, Y16A, R24A, Y72A, Y120A, E176A, R179A, and W208A. We show that for the RNA N-glycosidase activity of saporin-6, residues Tyr(16), Tyr(72), and Arg(179) are absolutely critical; Tyr(120) and Glu(176) can be partially dispensed with, whereas Trp(208) and Arg(24) do not appear to be involved in this activity. The residues Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208) were found to be essential for the genomic DNA fragmentation activity, whereas residues Tyr(16) and Arg(24) do not appear to be required for the DNA fragmentation. The study shows that saporin-6 possesses two catalytic activities, namely RNA N-glycosidase and genomic DNA fragmentation activity, and for its complete cytotoxic activity both activities are required.     

Solid-phase synthesis and bioevaluation of lupeol-based libraries as antimalarial agents

The use of the triterpenoid lupeol as a scaffold for the synthesis of lupeol-based libraries is described. Lupeol was anchored to a solid support (Rink amide/Sieber Amide) through aliphatic dicarboxylic acid moieties, which also served as a site for introducing diversity. The resulting polymer linked 3beta-O (resin-alkanoyl)-lup-20(29)-ene 3 was used to generate key intermediates 3beta-O (resin-alkanoyl)-30-bromo-lup-20(29)-ene 4 and 3beta-O (resin-alkanoyl)-30-amino-lup-20(29)-ene 6 for the generation of libraries based on disubstituted lupeol derivatives. A 96-member library was screened for its in-vitro antimalarial activity against Plasmodium falciparum.     

Agrobacterium-mediated transient GUS gene expression in buffel grass (Cenchrus ciliaris L.)

The study was conducted to standardize a protocol for Agrobacterium-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.). Embryogenic calli, produced from one-year-old mature seeds of buffel grass, were used as target cells for Agrobacterium-mediated transformation. A. tumefaciens strain LBA4404, harbouring pCAMBIA-1301 or pCAMBIA-2301, was used for co-cultivation with embryogenic calli from three genotypes (IG-3108, IG-9757 and IG-97101). Co-culturing of calli with Agrobacterium for 30 minutes, followed by co-cultivation with 0.1 mM acetosyringone for 3 days was found to be optimum for maximum transformation efficiency. Presence of acetosyringone during co-cultivation was found to be necessary for transformation. Transient GUS (beta-glucuronidase) gene expression was used to monitor T-DNA delivery into the target cells. Significant genotypic variations in response to transformation were observed among the tested genotypes. A very high frequency (63.3%) of GUS gene expression was obtained following Agrobacterium-mediated gene transfer into embryogenic calli. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of buffel grass with genes of agronomic importance.     

7,8-Benzoflavone: a phytotoxin from root exudates of invasive Russian knapweed

Root exudates from Acroptilon repens (Russian knapweed) were found to be phytotoxic and the phytotoxin in the exudate was identified as 7,8-benzoflavone (alpha-naphthoflavone), (1), not previously known as a natural product. In tests on growing seedlings both 1 and its isomer 5,6-benzoflavone (2) were phytotoxic. Flavone, a structural analog of 1 and a known granular leaf and stem exudate of other plant species, was also phytotoxic and more potent than 1 or 2.     

Divalent forms of CC49 single-chain antibody constructs in Pichia pastoris: expression, purification, and characterization

Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. In the present study, we engineered and expressed divalent non-covalent [(scFv)(2)-His(6)] and covalent [sc(Fv)(2)-His(6)] scFvs of a tumor-associated monoclonal antibody (MAb) CC49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDS-PAGE, HPLC, and competitive ELISA. The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10(7), 2.75 x 10(7), and 1.14 x 10(8) M(-1) for (scFv)(2)-His(6), sc(Fv)(2)-His(6), and CC49 IgG, respectively. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Biodistribution studies in athymic mice bearing LS-174T human colon carcinoma xenografts showed equivalent tumor-targeting of CC49 dimers generated in yeast (scFv)(2)-His(6) and bacteria (scFv)(2) with 12.52% injected dose/gram (%ID/g) and 11. 42%ID/g, respectively, at 6 h post-injection. Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1 >/= p >/= 0.05). In conclusion, improved yields of divalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFvs suggest possible therapeutic applications.     

Simulated brain tumor growth dynamics using a three-dimensional cellular automaton

We have developed a novel and versatile three-dimensional cellular automaton model of brain tumor growth. We show that macroscopic tumor behavior can be realistically modeled using microscopic parameters. Using only four parameters, this model simulates Gompertzian growth for a tumor growing over nearly three orders of magnitude in radius. It also predicts the composition and dynamics of the tumor at selected time points in agreement with medical literature. We also demonstrate the flexibility of the model by showing the emergence, and eventual dominance, of a second tumor clone with a different genotype. The model incorporates several important and novel features, both in the rules governing the model and in the underlying structure of the model. Among these are a new definition of how to model proliferative and non-proliferative cells, an isotropic lattice, and an adaptive grid lattice.     

Enhanced neointimal growth in cultured rabbit aorta following in vivo balloon angioplasty

Summary We have used in vivo balloon catheterization in combination with in vitro organ culture to develop a model system for vascular neointima formation. A Fogarty balloon catheter was used to deendothelialize and rupture the internal elastic lamina of aortae in adult rabbits. After three d of recovery, aortae were harvested, divided into segments, and placed into organ culture. We obtained a daily index of cell proliferation in cultured vessels using [³H]thymidine incorporation into DNA. Also, segments were collected and processed for routine histology or immunohistochemistry. Aortic segments that had undergone ballooning 3 d before harvest and then cultured exhibited diffuse neointimal growth after several d in vitro, whereas those from sham-operated (nonballooned) rabbits showed generally only a single endothelial cell layer that is characteristic of normal intima. Aortae that were harvested, balloon-damaged in vitro, and then cultured exhibited no neointimal growth. The neointima that developed in cultured segments from in vivo ballooned rabbits was primarily of smooth muscle cell origin as determined by positive immunostaining for α-smooth muscle actin. The intima:media thickness ratios were significantly higher in aortic segments from ballooned rabbits at harvest and after 4 or 7 d in culture compared with those from nonballooned rabbits. Also, the [³H]thymidine index was higher in the in vivo ballooned aorta compared to non-ballooned or in vitro ballooned vessel. We conclude that ballooning in vivo followed by exposure to blood-borne elements produces an enhanced proliferative response in cultured vessels that is distinct from other in vitro models of neointimal growth.     

Genetic Diversity in Lens Species Revealed by EST and Genomic Simple Sequence Repeat Analysis

Low productivity of pilosae type lentils grown in South Asia is attributed to narrow genetic base of the released cultivars which results in susceptibility to biotic and abiotic stresses. For enhancement of productivity and production, broadening of genetic base is essentially required. The genetic base of released cultivars can be broadened by using diverse types including bold seeded and early maturing lentils from Mediterranean region and related wild species. Genetic diversity in eighty six accessions of three species of genus Lens was assessed based on twelve genomic and thirty one EST-SSR markers. The evaluated set of genotypes included diverse lentil varieties and advanced breeding lines from Indian programme, two early maturing ICARDA lines and five related wild subspecies/species endemic to the Mediterranean region. Genomic SSRs exhibited higher polymorphism in comparison to EST SSRs. GLLC 598 produced 5 alleles with highest gene diversity value of 0.80. Among the studied subspecies/species 43 SSRs detected maximum number of alleles in L. orientalis. Based on Nei’s genetic distance cultivated lentil L. culinaris subsp. culinaris was found to be close to its wild progenitor L. culinaris subsp. orientalis. The Prichard’s structure of 86 genotypes distinguished different subspecies/species. Higher variability was recorded among individuals within population than among populations.