Gene note. An alcohol dehydrogenase-like gene is specifically expressed in potato pistils

The reproductive function of the pistil requires the production of compounds essential for pollen tube growth. A cold-plaque screening of a pollinated pistil cDNA library of Solanum tuberosum resulted in the isolation of cDNA clone cp67. Northern blot analyses revealed that cp67 is specifically expressed in potato pistils and in a limited number of plant species. The deduced CP67 protein displays similarity to long-chain zinc-containing alcohol dehydrogenases (ADHs), although at a similarity level much lower than between other plant ADHs.     

Structural Redesign of Lipase B from Candida antarctica by Circular Permutation and Incremental Truncation

Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp283, one of the most active hydrolase variants. Our initial investigation has focused on the role of an extended surface loop, created by linking the native N- and C-termini, on protein integrity. Incremental truncation of the loop partially compensates for observed losses in secondary structure and the permutants' temperature of unfolding. Unexpectedly, the improvements are accompanied by quaternary-structure changes from monomer to dimer. The crystal structures of one truncated variant (cp283Δ7) in the apo-form determined at 1.49 Å resolution and with a bound phosphonate inhibitor at 1.69 Å resolution confirmed the formation of a homodimer by swapping of the enzyme's 35-residue N-terminal region. Separately, the new protein termini at amino acid positions 282/283 convert the narrow access tunnel to the catalytic triad into a broad crevice for accelerated substrate entry and product exit while preserving the native active-site topology for optimal catalytic turnover.     

Immunoprophylaxis against filarial parasite,Brugia malayi: potential of excretory-secretory antigens in inducing immunity

The role of excretory-secretory antigens in inducing immunity in the host againstBrugia malayi microfilariae and infective larvae was studied byin vitro antibody dependent cell-mediated reaction as well asin vivo inoculation of filarial parasites within a microchamber in the host. The immune sera of jirds raised againstBrugia malayi microfilarial and infective larval excretory-secretory antigens(Bm Mf ESA andBm L₃ ESA) promoted the adherence of peritoneal exudate cells toBrugia malayi microfilariae and infective larvaein vitro and induced cytotoxicity to the parasites within 48 h. The antiBm Mf ESA serum was more effective than antiBm L3 ESA serum in inducing cytotoxicity to microfilariae and both antisera had a similar cytotoxic effect on infective larvae. In the microchambers implanted in the immune jirds, host cells could migrate and adhere to the microfilariae and infective larvae and kill them within 48–72 h. Further,Mastomys natalensis immunized againstBm Mf ESA and L₃ ESA generated a high degree of protective response against circulating microfilariae. These results suggest that excretory-secretory antigens are effective in inducing resistance against filarial parasites and thus have potential in immunoprophylaxis.     

Cloning and Molecular Characterization of a SDS‐Activated Tyrosinase from Marinomonas mediterranea

The sequence of the tyrosinase gene cloned from Marinomonas mediterranea is reported. It is the second tyrosinase cloned from a Gram negative bacterium. Its size is higher than that of Gram positive tyrosinases from Streptomyces, and more similar to the eukaryotic enzymes. Its sequence shares the features of copper‐binding sites found in all tyrosinases. Based in the comparison of tyrosinases from all types of organisms, an extension of the characteristic signatures existing at Prosite is proposed. This tyrosinase shares with some plant and amphibian tyrosinases a strong specific activation by submicellar concentrations of SDS. Intrinsic fluorescence and kinetic properties indicate that the activation is caused by an SDS‐dependent conformational change that facilitates the substrate accessibility to the dicopper active site.     

Peculiarities of interaction of synthetic polyribonucleotide poly(rA)-poly(rU) with some intercalators

Abstract

The viral cathepsin from Bombyx mori Nuclear Polyhedrosis Virus (BmNPV-Cath) is a broad-spectrum protease that participates in the horizontal transmission of this virus in silkworm by facilitating solubilization of the integument of infected caterpillars. When a B. mori farm is attacked by BmNPV, there are significant sericultural losses because no drugs or therapies are available. In this work, the structure of viral cathepsin BmNPV-Cath was used as a target for virtual screening simulations, aiming to identify potential molecules that could be used to treat the infection. Virtual screening of the Natural Products library from the Zinc Database selected four molecules. Theoretical calculations of ΔG_binding by the molecular mechanics Poisson–Boltzmann surface analysis (MM-PBSA) method indicated that the molecule Zinc12888007 (Bm5) would have high affinity for the enzyme. The in vivo infection models of B. mori caterpillars with BmNPV showed that treatment with a dose of 100?μg Bm5 dissolved in Pluronic-F127 0.02% was able to reduce the mortality of caterpillars in 22.6%, however, it did not impede the liquefaction of dead bodies. Our results suggest a role of BmNPV-Cath in generating a pool of amino acids necessary for viral replication and indicate a mechanism to be exploited in the search for treatments for grasserie disease of the silkworm.     

Chloroplast DNA in Pinus monticola

Summary Within-species variability of a restriction site in the chloroplast (cp) DNA in Pinus monticola has been surveyed. Frequencies of two variants of the cp genome are significantly different in interior versus coastal populations. Paternal inheritance of the cp genome predominates, though some individuals have both variants of the genome. The presence of heteroplasmic individuals indicates occasional biparental inheritance.     

Expression profiles of glutathione <Emphasis Type="Italic">S</Emphasis>-transferase genes in larval midgut of <Emphasis Type="Italic">Bombyx mori</Emphasis> exposed to insect hormones

Glutathione S-transferases (GSTs) are believed to play a role in the detoxification of xenobiotics, resistance to insect viruses and pesticides, intracellular transport, biosynthesis of hormones and protection against oxidative stress. In this study, we used quantitative real time RT-PCR to examine expression profiles of the silkworm Bombyx mori GST-Sigma (BmGSTS2) and GST-Delta (BmGSTD2) genes in the larval midgut of the silkworm after exposure to 2-hydroxyecdysone (20E) and juvenile hormone analog (JHA). In concentration-course study, 20E at higher concentrations (1.0 and 2.0 μg/μl) caused significant upregulation of BmGSTD2, and all concentrations (0.5–2.0 μg/μl) of 20E caused significant upregulation of BmGSTS2. However, JHA in all concentrations downregulated the expression of BmGSTD2 and BmGSTS2. When exposed to either 20E (2.0 μg/μl) or JHA (2.0 μg/μl) on the third day of the fifth instar, the silkworm had higher BmGSTD2 at later time points: 15, 18, and 24 h for 20E and 24 h for JHA. BmGSTS2 expression was downregulated within 24 h after exposure to JHA and showed a time-dependent response after exposure to 20E. We also did a stage-dependent study, in which JHA downregulated BmGSTD2 expression and upregulated BmGSTS2 expression significantly at both day 1 and day 3 of the fifth instar. 20E upregulated the expression of BmGSTD2 and BmGSTS2 at the two stages. These findings imply that hormones have an important role in the regulation of basal GST expression. However, further validation and field trials should be carried out on the regulatory elements relevant to BmGSTD2 and BmGSTS2 gene expression.     

Characterization of mitochondrial genome of Chinese wild mulberry silkworm, <Emphasis Type="Italic">Bomyx mandarina</Emphasis> (Lepidoptera: Bombycidae)

The complete mitochondrial genome of Chinese Bombyx mandarina (ChBm) was determined. The circular genome is 15682 bp long, and contains a typical gene complement, order, and arrangement identical to that of Bombyx mori (B. mori) and Japanese Bombyx mandarina (JaBm) except for two additional tRNA-like structures: tRNA ^Ser(TGA)-like and tRN ^AIle(TAT)-like. All protein-coding sequences are initiated with a typical ATN codon except for the COI gene, which has a 4-bp TTAG putative initiator codon. Eleven of 13 protein-coding genes (PCGs) have a complete termination codon (all TAA), but the remaining two genes terminate with incomplete codons. All tRNAs have the typical clover-leaf structures of mitochondrial tRNAs, with the exception of tRNA ^Ser(TGA)-like, with a four stem-and-loop structure. The length of the A+T-rich region of ChBm is 484 bp, shorter than those of JaBm (747 bp) and B. mori (494–499 bp). Phylogenetic analysis among B. mori, ChBm, JaBm, and Antheraea pernyi (Anpe) showed that B. mori is more closely related to ChBm than JaBm. The earliest divergence time estimate for B. mori-ChBm and B. mori-JaBm is about 1.08±0.18–1.41±0.24 and 1.53±0.20–2.01±0.26 Mya, respectively. ChBm and JaBm diverged around 1.11±0.16–1.45±0.21 Mya.     

家蚕基质金属蛋白酶基因Bm-MMP的克隆、序列分析及表达研究

基质金属蛋白酶（matrix metalloproteinases, MMPs）家族是一类蛋白水解酶, 能够降解基底膜和细胞外基质中大部分蛋白质。为了研究MMPs对家蚕Bombyx mori基本生理功能的影响, 本文利用RACE和RT-PCR方法, 首次从家蚕蛹中克隆了一个MMP基因的全长cDNA, 命名为Bm-MMP。序列分析表明, Bm-MMP的mRNA存在两个选择性剪切变体, 分别命名为Bm-MMP-V1和Bm-MMP-V2。其中Bm-MMP-V1 cDNA全长为2 257 bp, 包含一个1 686 bp的开放阅读框, 编码561个氨基酸, 预测蛋白质分子量约为62.3 kD; Bm-MMP-V2 cDNA全长为2 188 bp。同源性分析表明, Bm-MMP-V1和Bm-MMP-V2的氨基酸序列与蜡螟Galleria mellonella的Gm1-MMP的氨基酸序列同源性最高, 均为88.8%;与黑腹果蝇Drosophila melanogaster的Dm1-MMP的氨基酸序列同源性, 分别为61.2%和64.3%。将Bm-MMP-V1的编码区连接到表达载体pET28a(+)上, 并在大肠杆菌BL21中进行原核表达, SDS-PAGE和Western blot分析结果表明, 带有6×His标签的融合蛋白被成功表达。半定量RT-PCR分析表明, Bm-MMP-V1和Bm-MMP-V2在4龄眠蚕、熟蚕、吐丝后36及48 h、预蛹中的表达量比5龄中食期与化蛹后的表达量高, 推测该基因与家蚕幼虫蜕皮变态有关;LPS诱导5龄3 d的幼虫, 其Bm-MMP-V1和Bm-MMP-V2在血液中的表达量升高, 推测Bm-MMP可能与免疫相关。本研究为进一步研究Bm-MMP在家蚕体内的作用机制奠定了基础。     

Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L

The peritrophic membrane (PM) is a semi‐permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin‐binding proteins (BmPM‐P43 and BmPM‐P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C‐terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM‐P43 and BmPM‐P41. The chitin‐binding activity analysis showed that the BmPM‐P43 and BmPM‐P41 could bind to chitin strongly. It is concluded that BmPM‐P43 and BmPM‐P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin‐binding protein family. © 2010 Wiley Periodicals, Inc.     

Structural Requirements for Rapid Inactivation and Voltage Dependence in Splice Variants of Lobster Shaker Potassium Channels

We studied the properties of currents generated in Xenopus oocytes by nine splice variants of the spiny lobster Shaker gene. These isoforms differ in their amino termini and in the P-loop region of the pore. Both the voltage dependence and kinetic properties of the currents varied significantly, depending on which amino terminus was present. A cluster of net positive charges at the N-terminus was not necessary for rapid inactivation: negatively charged N-termini also inactivated rapidly. There was no obvious correlation between N-terminus length and inactivation rate. These N-terminal effects were additive with a separate set of voltage and kinetic properties controlled by the two alternative P-loop exons.     

Are turns required for the folding of ribonuclease T1?

Ribonuclease T1 (RNase T1) is a small, globular protein of 104 amino acids for which extensive thermodynamic and structural information is known. To assess the specific influence of variations in amino acid sequence on the mechanism for protein folding, circularly permuted variants of RNase T1 were constructed and characterized in terms of catalytic activity and thermodynamic stability. The disulfide bond connecting Cys-2 and Cys-10 was removed by mutation of these residues to alanine (C2, 10A) to avoid potential steric problems imposed by the circular permutations. The original amino-terminus and carboxyl-terminus of the mutant (C2, 10A) were subsequently joined with a tripeptide linker to accommodate a reverse turn and new termini were introduced throughout the primary sequence in regions of solvent-exposed loops at Ser-35 (cp35S1), Asp-49 (cp49D1), Gly-70 (cp70G1), and Ser-96 (cp96S1). These circularly permuted RNase T1 mutants retained 35-100% of the original catalytic activity for the hydrolysis of guanylyl(3'-->5')cytidine, suggesting that the overall tertiary fold of these mutants is very similar to that of wild-type protein. Chemical denaturation curves indicated thermodynamic stabilities at pH 5.0 of 5.7, 2.9, 2.6, and 4.6 kcal/mol for cp35S1, cp49D1, cp70G1, and cp96S1, respectively, compared to a value of 10.1 kcal/mol for wild-type RNase T1 and 6.4 kcal/mol for (C2, 10A) T1. A fifth set of circularly permuted variants was attempted with new termini positioned in a tight beta-turn between Glu-82 and Gln-85. New termini were inserted at Asn-83 (cp83N1), Asn-84 (cp84N1), and Gln-85 (cp85Q1). No detectable amount of protein was ever produced for any of the mutations in this region, suggesting that this turn may be critical for the proper folding and/or thermodynamic stability of RNase T1.     

Antiviral activity of selected medicinal plants and marine seaweeds on the grasserie infected larvae of silkworm,Bombyx mori

Nuclear polyhedrosis virus (NPV) is the most harmful virus responsible for the manifestation of grasserie disease in the larvae of silkworm, Bombyx mori. It causes a huge economic loss in the sericulture industry. An attempt was made in the present investigation for the screening of antiviral activity using medicinal plants such as Lantana camara, Phyllanthus amarus and marine seaweeds such as Sargassum wightii, Turbinaria ornata against BmNPV. Crude extracts were prepared using different solvents, such as hexane, ethyl acetate, methanol and water. The silkworm feeding bioassay study was carried out with the crude extracts to investigate the presence of anti-BmNPV activity after inoculating fifth instar larvae of silkworm with occlusion bodies (OBs) of BmNPV. Each extract was tested for their anti-BmNPV activity using various concentrations of crude extracts ranging from 200 μg to 1000 μg. Among the crude extracts tested, methanol and aqueous extracts of P. amarus showed significant anti-BmNPV activity.     

家蚕二分浓核病毒ns1基因序列的改造、表达和鉴定

探讨翻译起始区(TIR)部分密码子发生同义突变后,对家蚕二分浓核病毒(BmBDV)ns1基因表达的影响,以及对BmBDV NS1蛋白毒性进行鉴定,设计特异性上游引物,对BmBDV ns1基因中第3、4、9和10个密码子进行同义突变,利用原核表达系统对野生型和改造后的ns1序列进行表达,通过SDS-PAGE电泳对这两种序列的表达产量进行分析。利用Protein Iso~(TM)GST Resin从超声破碎的菌液上清中纯化融合有GST的NS1蛋白,进而对纯化的靶蛋白在细胞水平和家蚕体内进行毒性分析。结果表明:TIR突变后的BmBDV ns1序列,其与野生型序列的表达产量之间没有明显差异;BmBDV NS1蛋白具有抑制细胞增殖和诱导家蚕致死的生化活性。     

Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis

We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment.     

Circularly permuted monomeric red fluorescent proteins with new termini in the ��-sheet

Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein‐based biosensors for Ca²⁺ and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)‐based biosensors by providing a means for varying the critical dipole–dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th β‐strand of mCherry. These new variants may ultimately prove useful for the creation of single FP‐based Ca²⁺ biosensors.