Hybrid Bacillus (1-3,1-4)-β-glucanases: engineering thermostable enzymes by construction of hybrid genes

Summary Hybrid (1-3,1-4)--glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)--glucanase genes from Bacillus amyloliquefaciens and B. macerans generated by the polymerase chain reaction (PCR). Four hybrid genes were expressed in Escherichia coli cells. The mature hybrid enzymes contain a 16, 36, 78, or 152 amino acid N-terminal sequence derived from B. amyloliquefaciens (1-3,1-4)--glucanase followed by a C-terminal segment derived from B. macerans (1-3,1-4)--glucanase. Biochemical characterization of parental and hybrid enzymes shows a significant increase in thermostability of three of the hybrid enzymes when exposed to an acidic environment thus combining two important enzyme characteristics within the same molecule. At pH 4.1, 85%-95% of the initial activity was retained after 1 h at 65° C in contrast to 5% and 0% for the parental enzymes from B. amyloliquefaciens and B. macerans. After 60 min incubation at 70° C, pH 6.0, the parental enzymes retained 5% or less of the initial activity whilst one of the hybrids still exhibited 90% of the initial activity. Of the parental enzymes B. macerans (1-3,1-4)--glucanase had the lower specific activity while the hybrid enzymes exhibited specific activities that were 1.5- to 3-fold higher. These experimental results demonstrate that exchange of homologous gene segments from different species may be a useful technique for obtaining new and improved versions of biologically active proteins.Abbreviations AMY mature form of Bacillus amyloliquefaciens (1-3,1-4)--glucanase; - MAC mature form of B. macerans (1-3,1-4)--glucanase - SUB mature form of B. subtilis (1-3,1-4)--glucanase - H(A16-M), H(A36-M), H(A78-M), H(A107-M), H(A152-M) mature forms of hybrid enzymes having 16, 36, 78, 107, 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC     

Displacement in two invasive species of leafminer fly in different localities

Liriomyza sativae was displaced by L. trifolii in the USA probably due to the lower insecticide susceptibility of L. trifolii, but L. trifolii was displaced by L. sativae in Japan. To know the possible causes of the species displacement observed in Japan, differences in the insecticide susceptibility, fecundity, effects of natural enemies, and reproductive interference between L. trifolii and L. sativae were evaluated. As a result, the higher fecundity of L. sativae and differential effects of parasitoids on the two Liriomyza species are considered to be contributing factors.     

Polyketide-nonribosomal peptide epothilone antitumor agents: the EpoA,B, C subunits

The epothilones are a family of macrolactone natural products from the myxobacterial species Sorangium cellulosum. Similar to taxol, they are of current clinical interest as anticancer agents. Sequence analysis of the epothilone gene cluster allowed the identification of polyketide synthase and nonribosomal peptide synthetase modules involved in catalyzing epothilone biosynthesis. Given this information, it has been possible to test the predicted functions of several modules to date. EpoA ACP, EpoB, and EpoC have been overproduced in Escherichia coli, allowing in vitro reconstitution of the EpoA/B/C interface and production of the expected epothilone precursor. Further experiments probed the tolerance of EpoB and EpoC for unnatural substrates. These studies of the first three modules of the epothilone biosynthetic cluster suggest that combinatorial biosynthesis may lead to the production of a variety of epothilone analogs that incorporate diversity into the heterocycle starter unit. Additional efforts with the remaining modules, coupled with increased understanding of the macrocyclizing thioesterase domain, may lead to the production of epothilone variants with improved clinical properties.     

Discrimination of macaques by macaques: the case of Sulawesi species

A series of work by the first author have demonstrated that many macaque species show a visual preference for the pictures of their own species when the monkeys actively press a lever to see the pictures. We expanded this study to Sulawesi macaques kept as a pet by local people with slight modification. All seven species of Sulawesi macaques were passively exposed to a variety of colored slides of Sulawesi macaques. The experimenter recorded the duration of visual fixation onto the pictures. Male monkeys of all the seven species clearly watched the pictures of their own species for longer duration than those of the other species. Such visual preference suggested that the seven Sulawesi macaques discriminate each other species and, thus, they may not be integrated into fewer number of species. This visual preference may work to prevent overall intergradation of the Sulawesi macaques who sometimes have hybrid zones only in limited areas. This preference was in general weaker in female monkeys. In one species,Macaca ochreata, females actively avoided to see the pictures of conspecifics. These results may be related to how female monkeys interact with other individuals.     

The role of the microenvironment in tumor growth and invasion

Mathematical modeling and computational analysis are essential for understanding the dynamics of the complex gene networks that control normal development and homeostasis, and can help to understand how circumvention of that control leads to abnormal outcomes such as cancer. Our objectives here are to discuss the different mechanisms by which the local biochemical and mechanical microenvironment, which is comprised of various signaling molecules, cell types and the extracellular matrix (ECM), affects the progression of potentially-cancerous cells, and to present new results on two aspects of these effects. We first deal with the major processes involved in the progression from a normal cell to a cancerous cell at a level accessible to a general scientific readership, and we then outline a number of mathematical and computational issues that arise in cancer modeling. In Section 2 we present results from a model that deals with the effects of the mechanical properties of the environment on tumor growth, and in Section 3 we report results from a model of the signaling pathways and the tumor microenvironment (TME), and how their interactions affect the development of breast cancer. The results emphasize anew the complexities of the interactions within the TME and their effect on tumor growth, and show that tumor progression is not solely determined by the presence of a clone of mutated immortal cells, but rather that it can be ‘community-controlled’.     

Reading the history of a hybrid fish complex from its molecular record

Squalius alburnoides is a widely distributed intergeneric hybrid complex with fish of both sexes, varying ploidy levels and proportions of the parental genomes. Its dispersal routes were here delineated and framed by the reconstruction of the phylogeny and phylogeography of other Squalius with which it hybridizes, based on the available data on the paleohydrographical history of the Iberian Peninsula. Results based on sequences of cytochrome b and beta-actin genes showed that: proto-Squalius pyrenaicus originated at least five species as it dispersed throughout the Iberian Peninsula in the Mio-Pliocene; the S. alburnoides complex likely had a single origin in the bulk of Iberia, in the Upper Tagus/Guadiana area, when hydrographical rearrangements allowed the contact between its ancestors (around 700,000 years ago); interspecific crosses allowed the introgression of mitochondrial and nuclear genes of S. alburnoides in allopatric species/populations of other Squalius and vice-versa; and reconstituted S. alburnoides non-hybrid males may contribute to the replacement of the typical mtDNA of the complex (in the populations where they occur, crosses with females of other Squalius seem to have been especially frequent). A number of dispersal events and colonization routes are proposed.     

Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases

Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes (phaC1 _Pre , phaC2 _Pre ) responsible for the production of intracellular medium-chain-length (mcl-)PHAs. Sequence analysis showed that the putative gene-products of these genes contain a conserved α/β-hydrolase fold in the carboxy-terminal half of the proteins. Hybrid genes pha7 and pha8 were constructed by exchanging the α/β-hydrolase-fold coding portions of phaC1 _Pre and phaC2 _Pre at the 3′ terminal. When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73–75% β-hydroxydecanoate (β-HD) and 25–27% β-hydroxyoctanoate (β-HO). Deletion mutants, Δpha7 and Δpha8, were isolated during the PCR-based construction of pha7 and pha8, respectively. Cells harboring these mutants produced PHAs containing 55–60 mol% β-HD and 40–45 mol% β-HO. These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.     

Differential sensitivity of Ctenocephalides felis and Drosophila melanogaster nicotinic acetylcholine receptor α1 and α2 subunits in recombinant hybrid receptors to nicotinoids and neonicotinoid insecticides

Nicotinic acetylcholine receptors (nAChRs) are the binding sites for nicotinoid drugs, such as nicotine and epibatidine, and are the molecular targets of the selectively insecticidal neonicotinoids. In this study we report the full length cDNA cloning of the three Ctenocephalides (C.) felis (cat flea) nAChR α subunits Cfα1, Cfα2, and Cfα3. When expressed in Xenopus oocytes as hybrid receptors with the Gallus gallus (chicken) β2 (Ggβ2) subunit, these cat flea α subunits formed acetylcholine-responsive ion channels. Acetylcholine-evoked currents of Cfα2/Ggβ2 were resistant to α-bungarotoxin, while those of Cfα1/Ggβ2 were sensitive to this snake toxin. The pharmacological profiles of Cfα1/Ggβ2, Cfα2/Ggβ2 and the chicken neuronal receptor Ggα4/Ggβ2 for acetylcholine, two nicotinoids and 6 insecticidal neonicotinoids were determined and compared. Particularly remarkable was the finding that Cfα1/Ggβ2 was far more sensitive to acetylcholine, nicotine and neonicotinoid agonists than either Cfα2/Ggβ2 or Ggα4/Ggβ2: for the anti flea neonicotinoid market compound imidacloprid the respective EC??s were 0.02 μM, 1.31 μM and 10 μM. These results were confirmed for another insect species, Drosophila melanogaster, where the pharmacological profile of the Dmα1 and Dmα2 subunits as hybrid receptors with Ggβ2 in Xenopus oocyte expressions resulted in a similar sensitivity pattern as those identified for the C. felis orthologs. Our results show that at least in a Ggβ2 hybrid receptor setting, insect α1 subunits confer higher sensitivity to neonicotinoids than α2 subunits, which may contribute in vivo to the insect-selective action of this pesticide class.     

乌鳢和斑鳢微卫星制备及其对自交与杂交F1代的初步鉴定

通过磁珠富集法分离了乌鳢(Channa argus)和斑鳢(C.maculate)的微卫星DNA序列,根据这些微卫星DNA序列的两翼序列,采用Primer 3.0或Primer 5.O软件设计出乌鳢与斑鳢的微卫星引物各31对.用62对微卫星引物对乌鳢和斑鳢自交与杂交F1代的4个组合进行扩增,10对引物无扩增产物,其余5...     

Synthesis and characterization of a photoresponsive doxorubicin/combretastatin A4 hybrid prodrug

To explore the application of photoremovable protecting groups (PPGs) in the field of combination chemotherapy, we designed and synthesized a photoresponsive hybrid prodrug 4 that bearing both doxorubicin (DOX) and combretastatin A4 (CA4). Light triggered drug release investigation found that DOX release was mainly accomplished by 405?nm light while CA4 release was mainly triggered by 365?nm light, i.e., prodrug 4 exhibited a quasi-sequential release behavior when a sequential light irradiation strategy was applied. Cell viability evaluation confirmed the increased cytotoxicity of prodrug 4 compared with individual drugs towards MDA-MB-231cells, indicating that a synergistic effect was achieved.