Honokiol,a small molecular weight natural product,inhibits angiogenesis in vitro and tumor growth in vivo

Natural products comprise a major source of small molecular weight angiogenesis inhibitors. We have used the transformed endothelial cell line SVR as an effective screen of natural product extracts to isolate anti-angiogenesis and anti-tumor compounds. Aqueous extracts of Magnolia grandiflora exhibit potent activity in our SVR proliferation assays. We found that the small molecular weight compound honokiol is the active principle of magnolia extract. Honokiol exhibited potent anti-proliferative activity against SVR cells in vitro. In addition, honokiol demonstrated preferential inhibition of primary human endothelial cells compared with fibroblasts and this inhibition was antagonized by antibodies against TNF alpha-related apoptosis-inducing ligand. In vivo, honokiol was highly effective against angiosarcoma in nude mice. Our preclinical data suggests that honokiol is a systemically available and non-toxic inhibitor of angiogenesis and should be further evaluated as a potential chemotherapeutic agent.     

Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently

We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods.     

Evolution of model proteins on a foldability landscape

We model the evolution of simple lattice proteins as a random walk in a fitness landscape, where the fitness represents the ability of the protein to fold. At higher selective pressure, the evolutionary trajectories are confined to neutral networks where the native structure is conserved and the dynamics are non self-averaging and nonexponential. The optimizability of the corresponding native structure has a strong effect on the size of these neutral networks and thus on the nature of the evolutionary process. Proteins 29:461–466, 1997. © 1997 Wiley-Liss, Inc.     

Endothelin-induced cardiac myocyte hypertrophy: role for focal adhesion kinase

Endothelin-1 (ET) produces neonatal rat ventricular myocyte (NRVM) hypertrophy and activates focal adhesion kinase (FAK) in other cell types. In the present study, we examined whether ET activated FAK in NRVM and whether FAK was necessary and/or sufficient for ET-induced NRVM hypertrophy. Chronic ET-1 stimulation (100 nM, 48 h) increased protein-to-DNA and myosin heavy chain (MHC)-to-DNA ratios and stimulated the assembly of newly synthesized MHC into sarcomeres. ET-1 also induced the assembly of focal adhesions and costameres, as evidenced by increased phosphotyrosine, FAK, and paxillin immunostaining. Acutely, ET treatment rapidly increased tyrosine phosphorylation of FAK and paxillin. FAK was also activated by phorbol 12-myristate 13-acetate (2 microM, 5 min). Pretreatment with chelerythrine (5 microM) or rottlerin (10 microM) completely blocked ET-induced FAK phosphorylation, indicating that protein kinase C activation was upstream of ET-induced FAK activation. In contrast, ET-induced FAK activation was not affected by blocking calcium influx via L-type voltage-gated calcium channels. Adenoviruses (Adv) containing FAK and FAK-related nonkinase (FRNK) were used to specifically define the role of FAK in ET-induced hypertrophy. ET stimulation failed to increase total protein-to-DNA or MHC-to-DNA ratios or to stimulate sarcomeric assembly in myocytes infected with Adv-FRNK. However, Adv-FAK alone did not increase total protein-to-DNA or MHC-to-DNA ratios and failed to increase the number or size of myofibrils as evidenced by double immunofluorescence labeling for MHC and FAK. Thus, although FAK is necessary for ET-induced NRVM hypertrophy, other ET-generated signals are also required to elicit the hypertrophic phenotype.     

Curcumin for malaria therapy

Malaria remains a major global health concern. New, inexpensive, and effective antimalarial agents are urgently needed. Here we show that curcumin, a polyphenolic organic molecule derived from turmeric, inhibits chloroquine-resistant Plasmodium falciparum growth in culture in a dose dependent manner with an IC(50) of approximately 5 microM. Additionally, oral administration of curcumin to mice infected with malaria parasite (Plasmodium berghei) reduces blood parasitemia by 80-90% and enhances their survival significantly. Thus, curcumin may represent a novel treatment for malarial infection.     

Levels of protein kinase C and nitric oxide synthase activity in rats exposed to sub chronic low level lead

Previous studies from our laboratories have linked the protective abilities of IH636 grape seed proanthocyanidin extract (GSPE) with inactivation of anti-apoptotic gene bcl-XL, and modification of several other critical molecular targets such as DNA-damage/DNA-repair, lipid peroxidation and intracellular Ca²⁺ homeostasis. Especially, GSPE provided dramatic protection against acetaminophen (APAP)-induced hepatotoxicity, significantly increased bcl-XL expression in the liver [1], and antagonized both necrotic and apoptotic deaths of liver cells in vivo. However, it was not clear from this study whether anti-apoptogenic and anti-necrotic effects of GSPE were: (i) due to its interference with endonuclease activity, (ii) due to its antioxidant effect, or, (iii) due to its ability to inhibit microsomal drug metabolizing enzyme(s), such as CYP-4502E1. Since CYP-4502E1 primarily metabolizes acetaminophen in mice and rats, this study specifically focused on CYP-4502E1's catalytic activity in vitro. Overall this investigation compared the in vitro aniline hydroxylation patterns of: (i) in vivo GSPE-exposed and unexposed (control) mouse liver microsomes, (ii) induced (1% acetone in drinking water for 3 days) and uninduced rat liver microsomes in the presence and absence of GSPE in vitro, and (iii) control rat liver microsomes in the presence of an anti-APAP agent 4-aminobenzamide (4-AB) in vitro. For the in vivo assessment, male B6C3F1 mice were fed GSPE diet (ADI 100 mg/kg body wt) for 4 weeks, and liver microsomes were isolated from both control and GSPE-fed mice for aniline hydroxylation, a specific marker of CYP-4502E1 activity. Data show that hydroxylation was 40% less in microsomes from GSPE-exposed livers compared to control microsomes. Similarly, when rat liver microsomes were incubated with various concentrations of GSPE in vitro (100 and 250 g/ml), aniline hydroxylation was inhibited to various degrees (uninduced: 40 and 60% and induced: 25 and 50%, respectively with 100 and 250 g/ml). Influence of GSPE on hydroxylation patterns were compared with another hepatoprotective agent 4-aminobenzamide (4-AB), a well-known modulator of nuclear enzyme poly(ADP-ribose) polymerase, and the data shows that 4-AB did not alter aniline hydroxylation at all. Collectively, these results may suggest that GSPE has the ability to inhibit CYP-4502E1, and this is an additional cytoprotective attribute, in conjunction with its novel antioxidant and/or antiendonucleolytic potential.     

A landscape-level assessment of Asian elephant habitat,its population and elephant–human conflict in the Anamalai hill ranges of southern Western Ghats,India

Spatial information at the landscape scale is extremely important for conservation planning, especially in the case of long-ranging vertebrates. The biodiversity-rich Anamalai hill ranges in the Western Ghats of southern India hold a viable population for the long-term conservation of the Asian elephant. Through rapid but extensive field surveys we mapped elephant habitat, corridors, vegetation and land-use patterns, estimated the elephant population density and structure, and assessed elephant–human conflict across this landscape. GIS and remote sensing analyses indicate that elephants are distributed among three blocks over a total area of about 4600 km². Approximately 92% remains contiguous because of four corridors; however, under 4000 km² of this area may be effectively used by elephants. Nine landscape elements were identified, including five natural vegetation types, of which tropical moist deciduous forest is dominant. Population density assessed through the dung count method using line transects covering 275 km of walk across the effective elephant habitat of the landscape yielded a mean density of 1.1 (95% CI = 0.99–1.2) elephant/km². Population structure from direct sighting of elephants showed that adult male elephants constitute just 2.9% and adult females 42.3% of the population with the rest being sub-adults (27.4%), juveniles (16%) and calves (11.4%). Sex ratios show an increasing skew toward females from juvenile (1:1.8) to sub-adult (1:2.4) and adult (1:14.7) indicating higher mortality of sub-adult and adult males that is most likely due to historical poaching for ivory. A rapid questionnaire survey and secondary data on elephant–human conflict from forest department records reveals that villages in and around the forest divisions on the eastern side of landscape experience higher levels of elephant–human conflict than those on the western side; this seems to relate to a greater degree of habitat fragmentation and percentage farmers cultivating annual crops in the east. We provide several recommendations that could help maintain population viability and reduce elephant–human conflict of the Anamalai elephant landscape.