Molecular characterization of a K+ channel blocker in the scorpionButhus martensii Karsch

K⁺ channel blockers of scorpion venoms are of important value in studying pharmacology and physiology of specific K⁺ channel of cells. Based on the amino acid sequences of BmP01 previously characterized as a small-conductance Ca²⁺-activated K⁺ channel blocker, two “back to back” degenarate primers have been designed and synthesized for inverse PCR strategy, its full-length cDNA has been cloned from the venom gland of the Chinese scorpionButhus martensii. The cDNA is composed of 3 parts: 5′ UTR, ORF and 3′ UTR. The flanking sequence of translation initiation codon ATG is AAAATGA, which is highly conserved in scorpion Na⁺ channel toxin and protozoan genes, suggesting that these genes may have followed a common mechanism for translation initiation. The 3′ UTR contains poly(A) signal AATAAA. The open reading frame encodes a precursor of 57 residues with a signal peptide of 28 residues and a mature peptide of 29 residues. The signal peptide is rich in hydrophobic amino acid residues and its length is significantly different from that of the determined scorpion Na⁺ channel toxin. The deduced amino acid sequence of mature peptide is completely consistent with BmP01 previously determined by primary structure analysis.     

Crystallization and preliminary X-ray analysis of an anti-insect toxin BmK 16 fromButhus martensii Karsch scorpion

An excitatory anti-insect toxin from BmK scorpion, named BmK 16, has been purified and crystallized recently. Two crystal forms have been obtained by using ammonium sulfate as precipitant at different pH, one of which is single crystal. Diffraction data have been collected for the single crystal on a Mar Research 345 Image Plate. The analyses of the data show that the crystal can diffract to 0.28 nm resolution and belongs to the space group C2 with unit cell parameters a = 4.65 nm,b = 8.52 nm,c = 3.26 nm, α = γ = 90°, β = 110.5°. There are 2 molecules per asymmetric unit according to its unit cell volume and molecular weight.     

Baculovirus-mediated expression of a Chinese scorpion neurotoxin improves insecticidal efficacy

An Buthus martensii Karsch Insect Toxin （BmK IT ） gene was inserted into the genome of Autographa californica multicapsid nucleopolyhedrovirus （AcMNPV） to construct a recombinant baculovirus, AcMNPV-BmK IT. The expression of BmK IT was confirmed using RT-PCR, dot blot and SDS-PAGE analysis. Dose-lethal time responses to Spodoptera exigua larvae were compared between wild-type baculovirus AcMNPV and recombinant virus AcMNPV-BmK IT. At the concentration of 1 × 10^7 PIBs/mL, the median lethal time of recombinant baculovirus （LT50 = 73.6 h） on third instar S. exigua larvae showed an improvement of 13.2% over the efficacy of wild type virus （LT50 = 84.8 h） during a 192 h infection.     

JAK3 mediatesc-myc gene expression induced by interleukin-2

C myc gene expression can be rapidly induced by IL 2 through intracellular signal transduction which is triggered by the interaction between IL 2 and its receptor (IL 2R). JAK3 which associates to the intracellular domain of IL_2R γ may play a critical role in this process. To reveal the action of JAK3 in c myc induction, a chimeric receptor gene IL_2R α/γ/Δ NJAK3 is constructed which consists of extracellular domain derived from IL 2R α subunit (IL_2R α), the transmembrane sequence derived from IL_2R γ and the cytoplasmic domain derived from the catalytic domain of JAK3 (Δ NJAK3), and then transfected this chimeric gene into mouse fibroblast cell line L929 β which had been transfected with IL_2R β gene and stably expressed IL_2R β in high level. In the transfectants coexpressing IL_2R β and α/γ/Δ NJAK3, the stimulation of IL_2 could intensively induce c myc gene expression. Because the whole cytoplasmic domain of IL_2R γ which could recruit signaling molecules was replaced by JAK3 and the c myc could be still induced by IL_2 in this situation, the results here gave the direct evidence demonstrating that JAK3 plays an important role in c myc gene expression induced by IL_2.     

Identification of integrin-like in guard cells ofVicia faba

We addressed the existence and localization of integrin-like in guard cells ofVicia faba by using a probe of polyclonal antibody against the human integrin (α_vβ₃/β₅). Western blot results showed that three integrins-like of about 47.3, 43.7 and 41.1 ku were detected from the preparation of membrane fragments of purified guard cell protoplasts. Further research with immunofluorescent scanning microscopy indicated that those integrins-like were localized on plasma membrane of guard cells, most nearing the dorsal wall, which is consistent with the reception of signals from epidermal cells to guard cells. Thus our results indicate, for the first time, that integrins-like are present at guard cell plasma membrane ofVicia faba.     

Identification of a novel DNA molecule associated with Tobacco leaf curl virus

A circular DNA molecule, designated as DNAβ, was identified in tobacco plants infected with Tobacco leaf curl virus (TLCV) isolates Y5 and Y8 by PCR using primers based on the conserved region of the two reported DNAβ sequences of whitefly-transmitted geminiviruses (WTGs). The complete nucleotide sequences of DNAβ of Y5 and Y8 (TLCV DNAβ) were determined. Y5 DNAβ comprises 1333 nucleotides encoding 8 predicted ORFs with 4 ORFs in virion-sense DNA and 4 ORFs in complementary-sense DNA; Y8 DNAβ consists of 1338 nucleotides encoding 7 predicted ORFs with 4 ORFs in virion-sense DNA and 3 ORFs in complementary-sense DNA. TLCV DNAβ has little sequence homology to DNA-A of TLCV., except that it shares conserved TAATATTAC loop sequence with TLCV DNA-A. Sequence comparison showed that Y5 DNAβ shared 85% sequence homology with Y8 DNAβ, and both Y5 DNAβ and Y8 DNAβ had relatively low sequence identity (51%–65%) with the reported DNAβ molecules associated with Ageratum yellow vein virus and Cotton leaf curl virus. The immunotrapping PCR and whitefly transmission tests showed that DNAβ molecule could be encapsidated in virus particle and transmitted by Bemisia tabaci. This is the first report of DNAβ associated with WTGs in China.     

Comparison study of chitosan/β-cyclodextrin and carboxymethyl chitosan/β-cyclodextrin microspheres

To compare two microspheres of chitosan/β-cyclodextrin and carboxymethyl chitosan/β-cyclodextrin loaded theophylline as pulmonary sustained drug delivery carriers, the characteristics and ciliotoxicity were studied. The drug loadings of chitosan/β-cyclodextrin and carboxymethyl chitosan/β-cyclodextrin microspheres were 21.09% and 21.42%, and the encapsulation efficiencies were 91.40% and 92.80%. The distributions of 50% (d _0.5) of chitosan/β-cyclodextrin and carboxymethyl chitosan/β-cyclodextrin microspheres were 4.89 and 5.83 μm, respectively. Both microspheres showed spherical shape with smooth or wrinkled surfaces. FT-IR of chitosan/β-cyclodextrin microspheres demonstrated that theophylline had formed hydrogen bonds with chitosan and β-cyclodextrin, while for the carboxymethyl chitosan/β-cyclodextrin microspheres theophylline had interaction with carboxymethyl chitosan. The moisture absorption showed that an equilibrium was reached within 24 h. The two microspheres possessed better adaptability. In vitro release of theophylline from chitosan/β-cyclodextrin microspheres was slower than that from carboxymethyl chitosan/β-cyclodextrin microspheres at pH 6.8. Biography: LI Ruobao(1968–), male, Associate professor, research direction: foundation research of respiratory system.     

Down-regulation of αGal epitopes by co-transfection of α1,3-galactosidase gene and α1,2–fucosyltransferase gene

The polycarbohydrate structure of Galα1- 3Ga1β1-4GluNAc-R （known as αGal epitopes of xenoantigen）, produced by α1-3-galactosyltransferase （α1,3-GT） in the course of animal development, is the major xenoantigen on the cell surface of porcine which causes hyperacute rejection in pig-to-human xenotransplantation. Alpha-1,3-galactosidase （AGL）, a hydrolytic enzyme, can remove the terminal α1,3-galactosyi from the Galα1-3Galβ1-4GluNAc-R structure resulting in cleaning αGai epitopes from the porcine cells. Aipha-1,2-fucosyitransferase （HT） can modify the surface carbohydrate phenotype of porcine cells, bringing about reduction of αGai epitopes expression. In this study, human AGL and HT gene were co-transfected to porcine fetal fibroblast （PFFb） in equimolar concentration to reduce the xenoantigen. Gene and protein of hAGL and HT were both detected to express at high level by RT-PCR and Western blot, respectively. There was an 84% reduction in αGai xenoantigen and an 82% increase in H antigen as assayed by flow cytometry in the AGL and HT gene co-transfected PFFb. The number and morphology of transgenic PFFb chromosome were normal. Findings indicate that Galα1-3Gal epitopes of PFFb could be down regulated by AGL and HT co-transfection without deleterious effects on the chromosomal profile of the transgenic ceil.     

Crystal structures of catalytic and regulatory subunits of rat protein kinase CK2

Protein kinase CK2 consists of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). Here, we report the crystal structures of rat CK2α mutant (rCK2α-△C, 1—335) and CK2β (rCK2β). The overall topology of rCK2α-△C and rCK2β are very similar to the human enzyme, although large structural differences could be observed in the N-terminal domain of rCK2α-△C. Our reported structure of rCK2α-△C is in the close conformation state while the counterpart hCK2α is in the open conformation state, indi- cating ...     

Molecular evolution of scorpion a-toxins----Accelerated substitutions and functional divergence

Scorpion a-toxins are a family of toxic proteins with similar scaffold, but possess divergent pharmacological properties. Analysis of cDNA sequences reveals that the numbers of nucleotide substitutions per site ( K ) for 5' and 3' UTRs are smaller than those per synonymous site ( Ks) for the mature peptide-coding sequences, whereas the numbers of nucleotide substitutions per nonsynonymous site (-Ka) are close to or larger than Ks values for relevant pairs of cDNAs. These results, together with phylogenetic analysis, indicate that scorpion a-toxins have evolved by accelerated substitutions in the mature toxin regions. In addition, the 15 amino acids, absolutely conserved in all the scorpion a-toxins described so far. are mostly located in molecular interior, which may be involved in structural constraints for stabilizing the CSa(3 fold in evolution of these molecules. Four hot spot mutation sites in the molecular surface are found to distribute in the putative functional regions of a-toxins. suggesting that positive Darwinian selection drives the accelerated evolution of scorpion a-toxins. These findings reasonably explain the relationship between three-dimensional structure conservation and functional divergence of scorpion a-toxins and are of important value in guiding us in our engineering experiments to obtain higher affinity ligands to Na+ channels.     

Cloning,sequencing and structural analysis of a pea cDNA encoding EF-1α

A cDNA library with the capacity of 2.0 × 10⁶ was constructed using mRNA extracted, from gibberellic acid (GA)-treated G2 pea seedlings and a 1.6 kb cDNA with 100 nucleotides of 5′ non-coding region and 223 nucleotides of 3′ non-coding region was obtained by random screening. The DNA fragment contains an open reading frame of 1 344 nucleotides and encodes 447 amino acids. Sequence analysis of DNA and deduced polypeptide demonstrated that it encoded for a pea elongation factor 1-alpha (EF-1α). The functional domains of EF-1α is well conserved and EF-1α can be a good candidate for studying molecular evolution.     

A new class of harmonic univalent functions by the generalized Salagean operator

A complex-valued harmonic functions that are univalent and sense preserving in the unit disk U can be written in the form f = h ＋ g^-, where h and g are analytic in U. We define and investigate a new class SHPλ（α,β）by generalized Salagean operator of harmonic univalent functions. We give sufficient coefficient conditions for normalized harmonic functions in the class SHPλ（α,β） These conditions are also shown to be necessary when the coefficients are negative. This leads to distortion bounds and extreme points.     

Discovery of low-mature hydrocarbon in manganese nodules and ooze from the Central Pacific deep sea floor

Extracts from manganese nodules and ooze from the Central Pacific deep sea floor were analyzed using the chromatogram-mass spectrum, and it was found that most of the biomarker molecules are of the low-mature type (some have characteristics of mature): the ratio of "A"/C is high between 11.4%-19.75%; CPI is 1.22-1.23; C31-22S/ (22S+22R) hopane is 0.59-0.60, Tm/Ts is 0.99-1.99; βα moretane/(αβ+βα) hopane is 0.12-0.14; C29 sterane 20S/(20S+20R) is 0.35-0.41; ββ/(ββ+αα) is 0.38-0.45; arene TA(I)/TA(I+II) is 0.16-0.21; methyl-phenanthrene index (MPI1) is 0.35-0.67. According to the geological settings of the sampling area and its organic geochemical characteristics, it is considered that the hydrothermal activities on the ocean floor facilitate the decomposition of organic matter in the sediment, which leads to the generation and migration of hydrocarbon into manganese nodules and ooze. This discovery is important for understanding the mechanisms of hydrocarbon generation in the ocean floor and for expanding the potential of oil and gas exploration in the ocean.     

Estimating divergence times among subfamilies in Nymphalidae

The mitochondrial cytochrome oxidase subunit Ⅰ （COⅠ） gene and the nuclear elongation factor 1α （EF-1α） gene were sequenced from 13 species of Nymphalidae. Phylogenetic trees of Nymphalidae, which is the largest family in butterflies, were constructed based on the sequences determined from 13 species sequenced in our laboratory and an additional 43 species obtained from GenBank using the maximum likelihood （ML） and Bayesian methods. Relative-rate tests between lineages in these phylogenetic trees were performed. On the basis of the results of the relative-rate tests and fossil information of Satyrinae, Nymphalinae and Biblidinae, the average divergence times among the subfamilies are estimated as 44.2-87.1 million years ago （Ma）. These results will be helpful for better understanding of the origin and evolution of this family, as well as the divergence time of butterflies and other complex taxa.     

Seasonal variation of snow microbial community structure in the East Rongbuk glacier, Mt. Everest

Mt. Everest, the zenith of the world, plays an impor- tant role in the global climate change. During the lastdecade, some studies on glacier and snow have been undertaken in this region. The climate and environment changes in the past hundred years have b…     

Microstructural evolution of titanium matrix composite coatings reinforced by <Emphasis Type="Italic">in situ</Emphasis> synthesized TiB and TiC by laser cladding

Titanium-based composite coatings reinforced by in situ synthesized TiB and TiC particles were successfully fabricated on Ti6Al4V by laser cladding using Ti-B₄C-Al or Ti-B₄C-C-Al powders as the precursor materials. The microstructural and metallographic analyses were made by X-ray diffraction (XRD), optical microscope (OM), scanning electron microscopy (SEM), and electron probe microanalysis (EPMA). The results show that the coatings are mainly composed of α-Ti cellular dendrites and a eutectic transformation product in which a large number of coarse and fine needle-shaped TiB and a few equiaxial TiC particles are homogeneously embedded. A thin dilution zone with a thickness of about 100 μm is present at the interface, and it consists of a few TiB and TiC reinforcements and a large number of lamella grains growing parallel to the heat flux direction in which a thin needle-shaped microstructure exists due to the martensitic transformation. The microstructural evolution can be divided into four stages: precipitation and growth of primary β-Ti phase, formation of the binary eutecticum β-Ti+TiB, formation of the ternary eutecticum β-Ti+TiB+TiC, and solid transformation from β-Ti to α-Ti.