Fine-scale genetic structure among genetic individuals of the clone-forming monotypic genus Echinosophora koreensis (Fabaceae)

• Background and Aims For rare endemics or endangered plantspecies that reproduce both sexually and vegetatively it iscritical to understand the extent of clonality because assessmentof clonal extent and distribution has important ecological andevolutionary consequences with conservation implications. Asurvey was undertaken to understand clonal effects on fine-scalegenetic structure (FSGS) in two populations (one from a disturbedand the other from an undisturbed locality) of Echinosophorakoreensis, an endangered small shrub belonging to a monotypicgenus in central Korea that reproduces both sexually and vegetativelyvia rhizomes. • Methods Using inter-simple sequence repeats (ISSRs) asgenetic markers, the spatial distribution of individuals wasevaluated using Ripley's L(d)-statistics and quantified thespatial scale of clonal spread and spatial distribution of ISSRgenotypes using spatial autocorrelation analysis techniques(join-count statistics and kinship coefficient, F_ij) for totalsamples and samples excluding clones. • Key Results A high degree of differentiation betweenpopulations was observed (_ST(g) = 0·184, P < 0·001).Ripley's L(d)-statistics revealed a near random distributionof individuals in a disturbed population, whereas significantaggregation of individuals was found in an undisturbed site.The join-count statistics revealed that most clones significantlyaggregate at 6-m interplant distance. The Sp statistic reflectingpatterns of correlograms revealed a strong pattern of FSGS forall four data sets (Sp = 0·072–0·154), butthese patterns were not significantly different from each other.At small interplant distances (2 m), however, jackknifed 95% CIs revealed that the total samples exhibited significantlyhigher F_ij values than the same samples excluding clones. • Conclusion The strong FSGS from genets is consistentwith two biological and ecological traits of E. koreensis: bee-pollinationand limited seed dispersal. Furthermore, potential clone matesover repeated generations would contribute to the observed highF_ij values among genets at short distance. To ensure long-termex situ genetic variability of the endangered E. koreensis,individuals located at distances of 10–12 m should becollected across entire populations of E. koreensis.     

Binding modes of V(=O)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various synthetic DNAs studied by polarized spectroscopy

The interactions between water-soluble cationic oxovanadyl[meso-tetrakis(4-N-methylpyridiumyl)]porphyrin (VOTMPyP) and various synthetic polynucleotide including poly[d(A-T)2], poly[d(G-C)2], and poly[d(I-C)2] were studied using absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy. When VOTMPyP formed a complex with poly[d(A-T)2] and poly[d(I-C)2], a positive CD signal at low [VOTMPyP]/[DNA] ratios (R ratios) and strong excitonic CD signals at above R > or = 0.15 were induced. The appearance of the CD spectra of the VOTMPyP-poly[d(G-C)2] complex were very different: a small negative CD at low R ratios and very small excitonic CD at high R ratios were observed. Considering the facts that the minor grooves of the former two polynucleotides resemble and the major groove of poly[d(I-C)2] is similar with that of poly[d(G-C)2], it is conclusive that VOTMPyP binds to the minor groove of all DNA at lower R ratios while they stack at the outside of DNA at higher R ratios. The binding geometry of VOTMPyP to all polynucleotides studied by LD seemed to be homogenous, irrespective of the R ratio. It has been found that VOTMPyP can have five- and six-fluxional coordination states. Comparing the absorption spectra of VOTMPyP complexed with poly[d(A-T)2] and poly[d(G-C)2], the distinctive absorptions of the five- and six-coordinated species were observed at lower R ratios which centered at 420-430 nm and 442 nm, respectively. While the six-coordinated VOTMPyP favored the poly[d(A-T)2], the five-coordinated species favored the poly[d(G-C)2] at the low R ratios. As the stacked species increased with an increasing R ratio, the six-coordinated species became the major bound species. These observations lead us to conclude that the guanine base' amino group plays a crucial role not only in determining the binding mode of VOTMPyP but also in the conversion of the six-coordinated species to the five-coordinated species.     

Potential application of antibody-mimicking peptides identified by phage display in immuno-magnetic separation of an antigen

Phage display was performed against human IgG (hIgG) through five rounds of 'biopanning'. Each round consisted of: (1) incubating a library of phage-displayed 12-mer peptides sequences on hIgG-coated magnetic beads, (2) washing the unbound phages, and (3) eluting the bound phages. The eluted phages were either amplified to enrich the pool of positive clones or subjected to the next round without amplification. Through ELISA, four clones (F9, D1, G5, and A10) showing specific binding affinity to hIgG were identified. Among these, F9 had the highest affinity (K(d)=6.2nM), only one order of magnitude lower than the native anti-hIgG antibody (0.66nM). Following the DNA sequences of the selected clones, four 12-mer peptides were chemically synthesized. Among them, D1 peptide showed the highest binding affinity to hIgG via SPR biosensor measurements. This peptide was conjugated to biofunctionalized magnetic beads, and its immuno-binding ability was compared with that of the native antibody immobilized to magnetic beads. The mol-to-mol binding efficacy of the peptide-coated magnetic beads was approximately 1000-fold lower than that of the antibody-coated magnetic beads. Our results suggest a feasibility of using antibody-mimicking peptides identified by phage display technique for immuno-magnetic separation of an antigen.     

Discovery of a series of 2-(1H-pyrazol-1-yl)pyridines as ALK5 inhibitors with potential utility in the prevention of dermal scarring

A series of 2-(1H-pyrazol-1-yl)pyridines are described as inhibitors of ALK5 (TGFβ receptor I kinase). Modeling compounds in the ALK5 kinase domain enabled some optimization of potency via substitutions on the pyrazole core. One of these compounds PF-03671148 gave a dose dependent reduction in TGFβ induced fibrotic gene expression in human fibroblasts. A similar reduction in fibrotic gene expression was observed when PF-03671148 was applied topically in a rat wound repair model. Thus these compounds have potential utility for the prevention of dermal scarring.     

A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.     

Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by Src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr(9) antibodies showed that the level of Tyr(9) phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr(9), distinct from Tyr(373/376), is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr(9) phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.     

Chlorophyll-deficient gene and morphological variations in Korean populations of maize (Zea mays)

We studied cultivated and naturalized Korean maize populations to determine the extent to which the chlorophylldeficient mutation and the phenotypic variations of two morphological characters (i.e., red coleoptiles and epicotyls, and the number of the first root hairs) are maintained. The frequency of the chlorophyll-deficient mutant gene (2.73% on average) was highly variable. Frequencies of red coleoptiles and epicotyls also were higher than expected from a mutation-selection balance. The average number of hairy phenotypes within populations was 1.8, ranging from 0.0 to 4.0. Naturalized populations were closely related to with cultivated communities. Most striking, however, was the more significant difference among populations than within populations with regard to both the frequency of chlorophyll-deficient mutant genes and the phenotypic variations of our two morphological characters. On a per-gene basis, the majority of the phenotypic variation (mean of 73.3%) resided among populations.     

Optimization of culture conditions and continuous production of chitosan by the fungi,Absidia coerulea

The production of chitosan from the mycelia ofAbsidia coerulea was studied to improve cell growth and chitosan productivity. Culture conditions were optimized in batch cultivation (pH 4.5 agitator speed of 250 rpm, and aeration rate of, 2 vvm) and the maximum chitosan concentration achieved was 2.3 g/L under optimized conditions. Continuous culture was carried out successfully by the formation of new growth spots under optimized conditions, with a chitosan productivity of 0.052 gL⁻¹ h⁻¹, which is the highest value to date, and was obtained at a dilution rate of 0.05 h⁻¹. Cell chitosan concentrations reached about 14% in the steady state, which is similar to that achieved in batch culture. This study shows that for the continuous culture ofAbsidia coerulea it is vital to control the medium composition.