Micromechanical coupling between cell surface receptors and RGD peptides

Contact between an adherent cell and the extracellular matrix (ECM) promotes the recruitment of structural and signaling molecules to the cytoplasmic domain of integrins, which mediate cell adhesion, cell migration, and cell growth. It is unclear whether the intracellular recruitment of these cytoplasmic molecules enhances the affinity between the ECM and the extracellular domain of the cell surface receptors (integrins). Using soft microneedles coated with Arg-Gly-Asp (RGD) peptides, a sequence commonly shared by ECM proteins, we apply a localized ramp shear stress to the surface of a HeLa cell and measure the cell stiffness and the collective (or apparent) unbinding lifetime of its surface receptors to RGD. These measurements demonstrate that both cell stiffness and the collective cell surface receptor-RGD unbinding lifetime increase with the duration of the pre-shear cell-microneedle contact and with the rate of shear applied to the cell membrane. These parameters are also crucially dependent on the integrity of the actin filament network. Our results are consistent with a model of positive feedback signaling where RGD-mediated initial recruitment of cytoskeletal proteins to the cytoplasmic domain of integrins directly enhances the interaction between the extracellular domain of integrins and the RGD sequence of ECM molecules.     

Improvement of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) seed oil quality through introduction of a soybean microsomal omega-3 fatty acid desaturase gene

Microsomal omega-3 fatty acid desaturase is an essential enzyme in the production of the n-3 polyunsaturated fatty acid alpha-linolenic acid during the seed developing stage. We have constructed a chimeric gene consisting of a maize Ubi1-P-int and a soybean GmFAD3 cDNA, which was introduced into rice plants by Agrobacterium-mediated transformation. Ten transformants containing the chimeric gene were established and expression subsequently confirmed by Northern blotting. Furthermore, alpha-linolenic acid content of the T(1) seeds increased dramatically up to tenfold that of the control, and this phenotype was also stably inherited in the T(2) and T(3) progenies. These results demonstrate that the alpha-linolenic acid content of rice seed oil can easily be altered using the combination of a high-activity promoter and a GmFAD3 gene.     

Microsatellite DNA and RAPD fingerprinting,identification and genetic relationships of hybrid poplar (<Emphasis Type="Italic">Populus x canadensis</Emphasis>) cultivars

Microsatellite DNA markers of ten SSR loci and 248 RAPD loci (resolved by 26 RAPD primers) were used for DNA fingerprinting and differentiation of 17 widely grown Populus x canadensis syn. Populus x euramericana (interspecific Populus deltoides x Populus nigra hybrids) cultivars ("Baden 431", "Blanc du Poitou", "Canada Blanc", "Dorskamp 925", "Eugenei", "Gelrica", "Grandis", "Heidemij", "I-55/56", "I-132/56", "I-214", "Jacometti", "Ostia", "Regenerata", "Robusta", "Steckby" and "Zurich 03/3"), and determination of their genetic interrelationships. Informativeness of microsatellite and RAPD markers was also evaluated in comparison with allozyme markers for clone/cultivar identification in P. x canadensis. High microsatellite DNA and RAPD genetic diversity was observed in the sampled cultivars. All of the 17 P. x canadensis cultivars could be differentiated by their multilocus genotypes at four SSR loci, and were heterozygous for their parental species-specific alleles at the PTR6 SSR locus. Except for "Canada Blanc" and "Ostia", which had identical RAPD patterns, all cultivars could also be differentiated by RAPD fingerprints produced by each of the two RAPD primers, OPA07 and OPB15. For microsatellites, the mean number of alleles, polymorphic information content, observed heterozygosity, observed number of genotypes and the number of cultivars with unique genotypes per locus was 5.2, 0.64, 0.67, 5.7 and 2.2, respectively. For RAPD markers, the number of haplotypes per locus, and the number of cultivars with unique RAPD profiles per locus were 1.06 and 0.72, respectively. Overall, microsatellite DNA markers were the most informative for DNA fingerprinting of P. x canadensis cultivars. On the per locus basis, microsatellites were about six-times more informative than RAPD markers and about nine-times more informative than allozyme markers. However, on the per primer basis, RAPD markers were more informative. The UPGMA cluster plots separated the 17 cultivars into two major groups based on their microsatellite genotypic similarities, and into three major groups based on their RAPD fragment similarities. Both the microsatellite and RAPD data suggest that the cultivars "Baden 431", "Heidemij", "Robusta" and "Steckby" are genetically closely related. The inter-cultivar genetic relationships from microsatellite DNA and RAPD markers were consistent with those observed from allozyme markers, and were in general agreement with their speculated origin. Microsatellite DNA and RAPD markers could be used for clone and cultivar identification, varietal control and registration, and stock handling in P. x canadensis.     

Enhanced bioremediation of n-alkane in petroleum sludge using bacterial consortium amended with rhamnolipid and micronutrients

The purpose of the present study was to investigate possible methods to enhance the rate of biodegradation of oil sludge from crude oil tank bottom, thus reducing the time usually required for bioremediation. Enhancement of biodegradation was achieved through bioaugmentation and biostimulation. About 10% and 20% sludge contaminated sterile and non-sterile soil samples were treated with bacterial consortium (BC), rhamnolipid biosurfactant (RL) and nitrogen, phosphorus and potassium (NPK) solution. Maximum n-alkane degradation occurred in the 10% sludge contaminated soil samples. The effects of treatment carried out with the non-sterile soil samples were more pronounced than in the sterile soils. Maximum degradation was achieved after the 56th day of treatment. n-Alkanes in the range of nC8-nC11 were degraded completely followed by nC12-nC21, nC22-nC31 and nC32-nC40 with percentage degradations of 100%, 83-98%, 80-85% and 57-73% respectively. Statistical analysis using analysis of variance and Duncan's multiple range test revealed that the level of amendments, incubation time and combination of amendments significantly influenced bacterial growth, protein concentration and surface tension at a 1% probability level. All tested additives BC, NPK and RL had significant positive effects on the bioremediation of n-alkane in petroleum sludge.     

Conformationally-restricted analogues and partition coefficients of the 5-HT3 serotonin receptor ligands meta-chlorophenylbiguanide (mCPBG) and meta-chlorophenylguanidine (mCPG)

The present investigation examined two features of arylbiguanide and arylguanidine 5-HT(3) ligands: conformation and partition coefficients. Several conformationally-constrained analogues of mCPBG (2) and mCPG (11; K(i)=32 nM) were prepared and of these only 2-amino-5-chloro-3,4-dihydroquinazoline (14; K(i)=34 nM) retained high affinity. The partition coefficient of compound 11 (LogP(app)=-0.64) was less than that of its corresponding arylbiguanide 2 (LogP(app)=-0.38). The quinazoline structure may represent a pharmacologically-active conformation of these agents, and the arylbiguanides were found more lipid soluble than their arylguanidine counterparts at physiological pH.     

Effect of norepinephrine on growth of Salmonella and its enterotoxin production

Salmonella typhimurium was cultured in presence or absence of norepinephrine in conditioned media. Two conditioned media containing bovine and pig serum were prepared. Supplementation of fresh cultures with norepinephrine (5 x 10(-5) M per mL of medium) resulted in ten-fold increase in growth as compared to controls. No significant difference in growth of organisms in media containing bovine and pig serum was observed. Growth was more in culture incubated under shaking condition than in non-shaking condition. Enterotoxin production increased by two to eight-folds in the medium supplemented with norepinephrine.     

Sensitivity of the nested-polymerase chain reaction (PCR) assay for Brugia malayi and significance of 'free' DNA in PCR-based assays

The blood filtration method was used as the gold standard to determine the detection level of simple blood-spot sampling and nested-polymerase chain reaction (PCR) for Brugia malayi. Of 100 samples, 48 were filtration-positive. Of these, 26 had microfilaria counts that were low enough (<1-29 microfilariae/ml) to accurately assess the limit of detection by nested-PCR. Nested-PCR consistently detected B. malayi DNA in samples with > or = 10 microfilariae/ml. Post-filtration, microfilaria-depleted, blood-spots from microfilaria-positive samples were screened by nested-PCR and B. malayi specific 'free' DNA was detected in 51.7% of these samples. There was no evidence for 'free' DNA in microfilaria-negative individuals from this endemic community.