Heparin-induced cis- and trans-dimerization modes of the thrombospondin-1 N-terminal domain

Through its interactions with proteins and proteoglycans, thrombospondin-1 (TSP-1) functions at the interface of the cell membrane and the extracellular matrix to regulate matrix structure and cellular phenotype. We have previously determined the structure of the high affinity heparin-binding domain of TSP-1, designated TSPN-1, in association with the synthetic heparin, Arixtra. To establish that the binding of TSPN-1 to Arixtra is representative of the association with naturally occurring heparins, we have determined the structures of TSPN-1 in complex with heparin oligosaccharides containing eight (dp8) and ten (dp10) subunits, by x-ray crystallography. We have found that dp8 and dp10 bind to TSPN-1 in a manner similar to Arixtra and that dp8 and dp10 induce the formation of trans and cis TSPN-1 dimers, respectively. In silico docking calculations partnered with our crystal structures support the importance of arginine residues in positions 29, 42, and 77 in binding sulfate groups of the dp8 and dp10 forms of heparin. The ability of several TSPN-1 domains to bind to glycosaminoglycans simultaneously probably increases the affinity of binding through multivalent interactions. The formation of cis and trans dimers of the TSPN-1 domain with relatively short segments of heparin further enhances the ability of TSP-1 to participate in high affinity binding to glycosaminoglycans. Dimer formation may also involve TSPN-1 domains from two separate TSP-1 molecules. This association would enable glycosaminoglycans to cluster TSP-1.     

Protein binding studies of luminescent rhenium(I) diimine complexes

Interaction of four luminescent rhenium(I) diimine complexes, [Re(CO)₃(N-N)L]PF₆ ((N-N = 2,2-bipyridine, L = py-3-COOH) 1a, (N-N = 2,2-bipyridine, L = py-3-CONH₂) 1b, (N-N = 1,10-phenanthroline, L = py-3-COOH) 2a, (N-N = 1,10-phenanthroline, L = py-3-CONH₂) 2b with bovine serum albumin (BSA) at physiological pH has been examined using UV-Vis absorption and luminescence spectroscopy, excited state lifetime measurement and circular dichroism (CD). In the presence of BSA, the luminescence of Re(I) complexes is quenched due to the locking-in of the probe into the protein environment. Interestingly the probe is released from the protein environment in the presence of sodium dodecyl sulfate (SDS) resulting in the restoration of the original luminescence along with a red shift in the emission maximum. These observations are explained in terms of binding constants (K_a) of probe with protein and surfactant and the nature of the binding has been investigated from Scatchard plot and Hill’s coefficient (n) value. These studies point out that the interaction between Re(I) complexes and BSA is cooperative in nature.     

Mass Production Potential of the Bacto-Helminthic Biocontrol Complex Heterorhabditis indica - Photorhabdus luminescens

Heterorhabditis indica is a potential agent for the biological control of grubs in sugarcane fields in India. The type strain LN 2 was transferred to monoxenic cultures on its symbiont Photorhabdus luminescens and successfully produced on solid media. In liquid cultures, a mean dauer juvenile yield of 457 000 was obtained with a maximum of 648 000 per ml. Comparatively high yields have not been reported before. Therefore, costs related to the liquid culture production of H. indica will be lower than for other entomopathogenic nematodes currently used in biocontrol. Different bacterial clones had no significant influence on the dauer juvenile yields in liquid media. The exit from the dauer juvenile stage (recovery) after inoculation and the number of hermaphrodites significantly decreased when culture temperature was increased from 25-30 ° C; the dauer juvenile yields were not affected. The cell density of P. luminescens in batch cultures was higher at 25 and 30 ° C than at growth temperatures of 35 and 37 ° C. In continuous culture, the bacterial growth was inhibited when the growth temperature reached 38 ° C. After approximately 60 h, the bacteria adapted to higher temperature and the growth rate increased again. When the temperature was further increased to 40 ° C, the bacterial growth was inhibited.     

Constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.     

Is the lactose-utilization ability ofRhizobium ofSesbania procumbens a vestigeal function?

Rhizobium SBS-R100, isolated from the stem nodules ofSesbania procumbens, synthesized -galactosidase constitutively. Transposon mutagenesis by Tn9 induced mutants defective in lactose utilization; the mutations did not interfere with growth, nodulation or N₂ fixation. Mouse monoclonal antibody raised against -galactosidase ofEscherichia coli reacted with soluble proteins of wild typeRhizobium SBS-R100. Anin vivo constructed recombinant plasmid pSBS-4 complemented aRhizobium mutant defective in lactose utilization.     

Interaction of ferredoxin with carbon monoxide dehydrogenase from Clostridium thermoaceticum.

Acetogenic bacteria, as determined with Clostridium thermoaceticum, synthesize acetate by the acetyl-CoA pathway which involves the reduction of CO2 to a methyl group and then combination of the methyl with CoA and a carbonyl group formed from CO or CO2 (Wood, H.G., Ragsdale, S.W., and Pezacka, E. (1986) Trends Biochem. Sci. 11, 14-18). Carbon monoxide dehydrogenase (CODH), the key enzyme in this pathway not only catalyzes the oxidation of CO to CO2 but also the final step, the synthesis of acetyl-CoA from a methyl group, CO, and CoA. Previously, it has been shown that ferredoxin can stimulate exchange of CO with CH3 14COSCoA (Ragsdale, S.W., and Wood, H.G. (1985) J. Biol. Chem. 260, 3970-3977). In the present study, it has been observed that ferredoxin and CODH can form an electrostatically stabilized complex. In order to identify the ferredoxin binding region on CODH, the ferredoxin and CODH were cross-linked by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The cross-linked CODH-ferredoxin adduct was enzymatically as active as the uncross-linked complex. The native CODH and cross-linked CODH-ferredoxin complex were subjected to cyanogen bromide cleavage. By comparison of the high-performance liquid chromatography peptide profiles, it was observed that the mobility of at least one peptide is altered in the CODH-ferredoxin cross-linked complex. The peptide was identified with residues 229-239 of the alpha-subunit of CODH.     

Preparation of cyanobacterial peptide toxin as a biopesticide against cotton pests

Glycine-rich peptide toxin of cyanobacterium Scytonema MKU 106 was purified. UV spectral analysis showed an absorption maximum at 228 nm and the molecular mass was less than 12 kDa. The mortality rate of American boll worms (Helicoverpa armigera) was about 80% and 40% 84 h after treatment with 0.001% crude and purified peptide toxins respectively; 100% mortality was observed after 108 h treatment with both purified and crude peptide toxins. The LC₅₀ (lethal concentration to 50% of the population) for Heliothis larvae after 96 h was 8.3 μg/ml purified peptide toxin and 6.2 μg/ml crude peptide toxin. Observations also show that the peptide toxin at 0.01% concentration acts as a biopesticide and at high (0.1%) concentrations it will act as an anti-feeding compound for Stylepta derogata (leaf-roller) larvae of the cotton crop. Received: 22 May 1996 / Accepted: 8 July 1996