Phylogeny of Saururaceae based on mitochondrial matR gene sequence data

DNA sequences of matR gene from three species of Saururaceae and the selected outgroups, Chloranthus holostegius and Zippelia begoniaefolia, are reported. All DNA sequences of six species in four genera of Saururaceae and the two outgroups are analyzed on PAUP 4.0 8b to reconstruct the phylogeny. A single matR gene tree is generated from parsimony, distance, and likelihood analyses, respectively. The three trees with the same topology are slightly different in bootstrapping support for some clades. The result indicates that Saururaceae is monophyletic. Anemopsis is sister to Houttuynia, and the two genera form the first diverging lineage of the family. The sister group relationship between Saururus and Gymnotheca is also supported by a relatively high bootstrap value. The result is different from all the former phylogenetic opinions on Saururaceae based on morphology, but it is supported by the evolution of flower-bract stalk in Saururaceae. In addition, some characteristics of the matR gene are analyzed. The MatR gene is a relatively better tool to reconstruct the molecular clock because the base substitution bias greatly decreases in the gene.     

Binding potency of peptide fragments of type 1 collagen cross-linked N-telopeptide measured by an enzyme-linked immunosorbant assay

Osteoporosis represents a major healthcare problem affecting elderly person. Urinary level of the crosslinked N-telopeptide of type I collagen is a sensitive marker of bone resorption. Ten overlapping peptides covering the N-telopeptide of alpha-2 type I collagen were synthesized, purified, and assayed for their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibody by using a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). Peptides 1, 2, and 3, containing the N-terminal sequence of N-telopeptide, showed higher binding potency than peptides 4-10, suggesting that these peptides may contain binding sites for anti-NTX antibodies, and can serve as the lead for further preparation of their antibodies in order to develop novel bioassays for monitoring the bone loss in humans.     

Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis

Cleft palate, the most frequent congenital craniofacial birth defects in humans, arises from genetic or environmental perturbations in the multi-step process of palate development. Mutations in the MSX1 homeobox gene are associated with non-syndromic cleft palate and tooth agenesis in humans. We have used Msx1-deficient mice as a model system that exhibits severe craniofacial abnormalities, including cleft secondary palate and lack of teeth, to study the genetic regulation of mammalian palatogenesis. We found that Msx1 expression was restricted to the anterior of the first upper molar site in the palatal mesenchyme and that Msx1 was required for the expression of Bmp4 and Bmp2 in the mesenchyme and Shh in the medial edge epithelium (MEE) in the same region of developing palate. In vivo and in vitro analyses indicated that the cleft palate seen in Msx1 mutants resulted from a defect in cell proliferation in the anterior palatal mesenchyme rather than a failure in palatal fusion. Transgenic expression of human Bmp4 driven by the mouse Msx1 promoter in the Msx1(-/-) palatal mesenchyme rescued the cleft palate phenotype and neonatal lethality. Associated with the rescue of the cleft palate was a restoration of Shh and Bmp2 expression, as well as a return of cell proliferation to the normal levels. Ectopic Bmp4 appears to bypass the requirement for Msx1 and functions upstream of Shh and Bmp2 to support palatal development. Further in vitro assays indicated that Shh (normally expressed in the MEE) activates Bmp2 expression in the palatal mesenchyme which in turn acts as a mitogen to stimulate cell division. Msx1 thus controls a genetic hierarchy involving BMP and Shh signals that regulates the growth of the anterior region of palate during mammalian palatogenesis. Our findings provide insights into the cellular and molecular etiology of the non-syndromic clefting associated with Msx1 mutations.     

Co-operative binding of triplicate carbohydrate-binding modules from a thermophilic xylanase

Family 6 carbohydrate-binding modules were amplified by polymerase chain reaction (PCR) from Clostridium stercorarium strain NCIB11754 genomic DNA as a triplet. Individually, these modules bound to xylooligosaccharides and cellooligosaccharides with affinities varying from approximately 3 x 10(3) M(-1) to approximately 1 x 10(5) M(-1). Tandem and triplet combinations of these modules bound co-operatively to soluble xylan and insoluble cellulose to give approximately 20- to approximately 40-fold increases in affinity relative to the individual modules. This co-operativity was an avidity effect resulting from the modules within the tandems and triplet interacting simultaneously with proximal binding sites on the polysaccharides. This occurred by both intrachain and interchain interactions. The duplication or triplication of modules appears to be linked to the growth temperature of the organism; co-operativity in these multiplets may compensate for the loss of affinity at higher temperatures.     

Functional characterization of an anti-estradiol antibody by site-directed mutagenesis and molecular modelling: modulation of binding properties and prominent role of the V(L) domain in estradiol recognition

The high-affinity monoclonal anti-estradiol antibody 9D3 presents a specificity defect towards estradiol-3-sulphate and 3-glucuronide conjugates incompatible with use in direct immunoassays. The corresponding single-chain variable fragment (scFv), cloned and produced in E. coli, exhibited a 10-fold lower affinity for estradiol (K(a)=1.2 x 10(9) M (-1)) and a slightly increased specificity defect for the 3-position. Site-directed mutagenesis revealed critical residues involved in estradiol recognition and produced mutants exhibiting up to a 3-fold increase of the binding affinity for estradiol and up to a 2-fold decrease of the cross-reactivity with estradiol-3-sulphate. A comparative model of the antibody 9D3-estradiol complex was built in which the estradiol D-ring is buried into the binding pocket while the 3-, 6- and 7-positions are solvent exposed, agreeing with the lack of specificity for these three positions. Two potential alternative orientations of the A-ring, one close to CDR H3 and L2 loops, and the other one close to CDR H2 and L3 loops, have been considered for the docking of estradiol, none of which could be unambiguously privileged taking into account data from cross-reactivity measurements, photolabelling and mutagenesis studies. For both orientations, estradiol is stabilized by hydrogen bonding of the 17beta-OH group with TyrL36, His89 and GlnH35 in the first case, or TyrL36, only, in the second case and by van der Waals contacts from TyrL91 with alpha- or beta-face of estradiol, respectively, and from ValH95 and GlyH97 with the opposite face. To elucidate the molecular basis of antibody 9D3 specificity, as compared with that of another anti-estradiol antibody 15H11, single variable domains (V(H) and V(L)) and scFv hybrids have been constructed. The binding activity of V(L)9D3 as well as the specificity of the V(L)9D3/V(H)15H11 hybrid, both similar to antibody 9D3, revealed a prominent role of V(L) in estradiol recognition. These findings establish premises for antibody engineering to reduce cross-reactivity, especially with estradiol-3-conjugates.     

Determination of andrographolide,deoxyandrographolide and neoandrographolide in the Chinese herb Andrographis paniculata by micellar electrokinetic capillary chromatography

In this paper a micellar electrokinetic capillary chromatographic (MEKC) method has been developed for determining the active components (andrographolide, deoxyandrographolide and neoandrographolide) in water:ethanol extracts of the Chinese crude herb Andrographis paniculata and its preparations (Chuanxinlian and Xiaoyan Lidan tablets). The optimum separation conditions were 15 mM sodium dodecyl sulphate in 30 mM borate buffer (pH 9.5) with UV detection wavelength at 214 nm and a constant voltage of 16 kV. An HPLC method was employed in order to validate the MEKC method with respect to separation efficiency, sensitivity, linearity and repeatability. The two methods are shown to be complementary because of their different selectivity and thus are very suitable for cross-validation studies. The MEKC method is demonstrated to be more appropriate for the analysis of the active compounds in A. paniculata in that it is easier and less expensive to use and does not suffer from contamination of the chromatographic column.     

3-Pyrroline containing arylacetamides: a novel series of remarkably selective kappa-agonists

A series of 2-(substituted phenyl)-N-methyl-N-[(1S)-1-(substituted alkyl)-2-(1-(3-pyrrolinyl))ethyl]acetamides were synthesized and evaluated as highly selective kappa-agonists with K(i) values in low nanomolar range. 3-Pyrroline incorporated into the basic amino functionality in combination with 2-(methylthio)ethyl substituent on the carbon adjacent to the amide nitrogen remarkably enhanced the kappa-selectivity. 3,4-Dichlorophenyl derivative 1e was found the most potent and selective analgesic in this series with ED(50) value of 0.023 mg/kg.