Preventing oxygen free-radical injury in ischemic revascularized bone grafts

Superoxide radicals have been shown to play a role in the cellular injury of reperfused ischemic tissues. We examined the protective effect of superoxide dismutase (SOD), a superoxide radical scavenger, on the reperfusion injury of replanted vascularized bone grafts after 4- and 8-hour periods of ischemia in a rat model. Histologic, fluorochrome, and histomorphometric analyses showed no difference between 4-hour superoxide dismutase-treated and control grafts, with both groups appearing viable. Similar analyses of the 8-hour ischemic grafts revealed both a qualitative and statistically significant quantitative difference (p less than 0.001) between the superoxide dismutase-treated and control grafts in parameters related to viability. Our results indicated that the administration of superoxide dismutase to free vascularized grafts by means of intraarterial perfusion after prolonged periods of warm ischemia significantly enhances the survival of these grafts.     

A Novel Folate-Targeted Nanoliposomal System of Doxorubicin for Cancer Targeting

Targeted drug delivery systems for cancer improves anti-tumor efficacy and reduces systemic toxicity by restricting availability of cytotoxic drugs within tumors. Targeting moieties, such as natural ligands (folic acid, transferrin, and biotin) which are overexpressed on tumors, have been used to enhance liposome-encapsulated drug accumulation within tumors and resulted in better control. In this report, we explored the scope of targeting ligand folic acid, which is incorporated in liposome systems using folic acid-modified cholesterol (CPF), enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in increased cytotoxicity within tumors. Folate-tagged poloxamer-coated liposomes (FDL) were found to have significantly higher cellular uptake than conventional poloxamer-coated liposomes (DL), as confirmed by fluorometric analysis in B16F10 melanoma cells. Biodistribution study of the radiolabeled liposomal system indicated the significantly higher tumor uptake of FDL as compared to DL. Anti-tumor activity of FDL against murine B16F10 melanoma tumor-bearing mice revealed that FDL inhibited tumor growth more efficiently than the DL. Taken together, the results demonstrated the significant potential of the folate-conjugated nanoliposomal system for drug delivery to tumors.     

Lidocaine-loaded fish scale-nanocellulose biopolymer composite microneedles

Microneedle (MN) technology has emerged as an effective drug delivery system, and it has tremendous potential as a patient friendly substitute for conventional methods for transdermal drug delivery (TDD). In this paper, we report on the preparation of lidocaine-loaded biodegradable microneedles, which are manufactured from fish scale-derived collagen. Lidocaine, a common tissue numbing anaesthetic, is loaded in these microneedles with an aim of delivering the drug with controlled skin permeation. Evaluation of lidocaine permeation in porcine skin has been successfully performed using Franz diffusion cell (FDC) which has shown that the drug permeation rate increases from 2.5 to 7.5% w/w after 36 h and pseudo steady state profile is observed from 5.0 to 10.0% w/w lidocaine-loaded microneedle. Swelling experiments have suggested that the microneedles have negligible swellability which implies that the patch would stick to the tissue when inserted. The experiments on MN dissolution have depicted that the lidocaine loaded in the patch is lower than the theoretical loading, which is expected as there can be losses of the drug during initial process manufacture.     

Receptor association and tyrosine phosphorylation of S6 kinases

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB/Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein-protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme complex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR). The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Furthermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localization. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine/s in response to PDGF or serum.     

Growth enhancement of black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis>) by a newly isolated <Emphasis Type="Italic">Bacillus tequilensis</Emphasis> NII-0943

A Gram positive, rod-shaped potential strain was selected from the pool of bacterial isolates obtained from the Western Ghats forest (India) on the basis of zone of P-solubilization activity. Identification based on 16S rRNA gene sequence revealed that the strain is of Bacillus species, sharing highest sequence similarity to Bacillus tequilensis NRRL B-41771^T (99.5%). Strain NII-0943 was able to produce good amount of indole acetic acid (IAA) and was positive for siderophore production. In addition to IAA and siderophore attributes, strain NII-0943 also possessed the characteristics like Ca₃(PO₄)₂ solubilization and growth in nitrogen-free medium. Seed inoculation with the strain NII-0943 resulted in significantly higher root initiation in black pepper cuttings grown under pots. The contents of nitrogen and phosphorus in both soil and plant were also enhanced significantly in treatments inoculated with these bacterial inocula. Hence, based on this evidence it is proposed that strain NII-0943 could be deployed as a plant growth-promoting inoculant to attain the desired results of bacterization.     

Comparative Membrane Channel Size and Activity of Botulinum Neurotoxins A and E

In an effort to compare the molecular basis of differential toxic activity of botulinum neurotoxin A (BoNT/A) and BoNT/E, we have analyzed their membrane channel activity by measuring calcein release from liposomes. Both BoNT/A and /E showed a same level of membrane channel activity that was specifically blocked by IgG specific to the neurotoxins. With the use of fluorescein-labeled dextran, we determined that the size of the channel is at least 24.2 A which is appropriate for the translocation of a protein of 50 kDa (the light chain of BoNT). These findings would suggest that the difference in the toxicity level of the two BoNT serotypes might reflect differences in either endopeptidase activity or their binding to receptor(s).     

Effects of acorn masting on population dynamics of three forest-dwelling rodent species in Hokkaido,Japan

The effects of the abundance of acorns of the oak, Quercus crispula, on the population dynamics of three rodent species (Apodemus speciosus, A. argenteus, and Clethrionomys rufocanus) were analyzed using time series data (1992–2006). The data were obtained in a forest in northern Hokkaido, Japan, by live trapping rodents and directly counting acorns on the ground. Apodemus speciosus generally increased in abundance following acorn masting. However, the clear effect of acorn abundance was not detected for the other two rodent species. Acorns of Q. crispula contain tannins, which potentially have detrimental effects on herbivores. Apodemus speciosus may reduce the damage caused by acorn tannins with tannin-binding salivary proteins and tannase-producing bacteria, whereas such physiological tolerance to tannins is not known in the other two rodent species. The differences in the effects of acorns between the three species may be due to differences in their physiological tolerance to tannins.     

Strategic mining of cyanobacterial patents from the USPTO patent database and analysis of their scope and implications

Patent analysis with the help of the strategic mining of patents from databases is important and useful within the framework of application-oriented research and its commercialization. In the analysis reported here, we have mined cyanobacterial patents from the patent database of the United States Patent and Trademark Office (USPTO). In order to make an assessment of the commercial potentials of cyanobacteria, we conducted the patent search (from 1976 to April 2006) using certain generic terms and the 84 genera of cyanobacteria as keywords. The search was performed in two major ways – searching the abstracts and claims of the patents cumulatively and searching the entire patent documents by the mode of ‘all fields’ in USPTO. In the abstract- and claims-based search, 234 patents were obtained after the removal of overlapping patents among the keywords. An additional 31 patents were added following the ‘all fields’ search; these patents were not covered in the search that was based on abstracts and claims. The entire package of 265 patents, of which 244 were related to cyanobacteria, was then analyzed. Information derived from these patents identified five major areas of cyanobacterial utilization. Cyanobacteria have been patented as a source of a wide spectrum of products, for medical, agriculture and environmental applications, for gene-based products, for methods of cultivation and for methods of control. The chronological development in granting cyanobacterial patents was also traced. This study demonstrates that such strategic mining and analysis of patent data can be used as an index for future development.     

Secretory delivery of heterologous proteins in attenuated <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> for potential use in vaccine design

To synthesize and secrete heterologous proteins in an attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different antigen-delivery systems based on bacterial-originated secretion signal peptides (SPs) were designed and identified in this work. Four SPs were derived from hemolysin of Escherichia coli, RTX protein of V. cholerae, hemolysin of V. anguillarum, zinc-metalloprotease of V. anguillarum, respectively, and their abilities to support secretion of green fluorescent protein (GFP) in an attenuated V. anguillarum strain MVAV6203 were assayed. Immunodetection of GFP showed that the capability of the tested signal leaders to direct secretion of GFP varied greatly. Although all the four signal peptide-fused GFPs could be expressed correctly and trapped intracellularly in recombinant strains, only the EmpA signal peptide could confer efficient secretion to GFP. For the investigation of its potential application in live bacteria carrier vaccines, a heterologous protein EseB of Edwardsiella tarda was fused to the SP(empA) antigen-delivery system and introduced into the strain MVAV6203. Further analysis of EseB demonstrated that the constructed SP(empA) antigen-delivery system could be used to secrete foreign protein in attenuated V. anguillarum and be available for carrier vaccines development.     

The significance of the sub-Antarctic Kerguelen Islands for the assessment of the vulnerability of native communities to climate change,alien insect invasions and plant viruses

The suite of environments and anthropogenic modifications of sub-Antarctic islands provide key opportunities to improve our understanding of the potential consequences of climate change and biological species invasions on terrestrial ecosystems. The profound impact of human introduced invasive species on indigenous biota, and the facilitation of establishment as a result of changing thermal conditions, has been well documented on the French sub-Antarctic Kerguelen Islands (South Indian Ocean). The present study provides an overview of the vulnerability of sub-Antarctic terrestrial communities with respect to two interacting factors, namely climate change and alien insects. We present datasets assimilated by our teams on the Kerguelen Islands since 1974, coupled with a review of the literature, to evaluate the mechanism and impact of biological invasions in this region. First, we consider recent climatic trends of the Antarctic region, and its potential influence on the establishment, distribution and abundance of alien insects, using as examples one fly and one beetle species. Second, we consider to what extent limited gene pools may restrict alien species’ colonisations. Finally, we consider the vulnerability of native communities to aliens using the examples of one beetle, one fly, and five aphid species taking into consideration their additional impact as plant virus vectors. We conclude that the evidence assimilated from the sub-Antarctic islands can be applied to more complex temperate continental systems as well as further developing international guidelines to minimise the impact of alien species.