Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na⁺ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K⁺ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na⁺ currents verified here, this venom certainly warrant further investigation.     

pLR‐HL: A Novel Amphibian Bowman–Birk‐type Trypsin Inhibitor from the Skin Secretion of the Broad‐folded Frog,Hylarana latouchii

In this study, we report a novel heptadecapeptide (LIGGCWTKSIPPKPCLV) of the pLR/ranacyclin family, named pLR‐HL, whose structure was deduced from its biosynthetic precursor‐encoding cDNA cloned from the skin secretion‐derived cDNA library of the broad‐folded frog, Hylarana latouchii, by employing a ‘shotgun’ cloning technique. It contains a disulphide loop between Cys⁵ and Cys¹⁵ which is consistent with Bowman–Birk‐type protease inhibitors. The primary structure of pLR‐HL deduced from the cDNA sequence was confirmed by fractionating the skin secretion using reverse‐phase HPLC and subsequent analysis using MALDI‐TOF mass spectrometry and LC/MS/MS fragmentation sequencing. On the basis of the establishment of unequivocal amino acid sequence, a synthetic replicate was synthesized by solid‐phase Fmoc chemistry, and it displayed a moderately potent trypsin inhibition with a K_i of 143 nm . The substitution of Lys‐8 by Phe (Phe⁸‐pLR‐HL) resulted in abolition of trypsin inhibition but generation of modest inhibition on chymotrypsin with a K_i of 2.141 μm . Additionally, both the disulphide loops of pLR‐HL and Phe⁸‐pLR‐HL were synthesized and tested. Both of the catalytic loops retained similar inhibitory potencies towards trypsin or chymotrypsin in comparison with the original intact molecules. Thus, the replacement of reactive site residues could alter the specificity of these protease inhibitors, while the canonical reactive loop alone can independently constitute biologically active moiety.     

Patent News

Novelty: The genes encoding Pneumocystis carinii surface antigens have been cloned. These genes could be used to design probes for diagnostic purposes, the preparation of vaccines and the production of targeted therapeutic methods for treating Pneumocystis carinii infections.

Biology: A cDNA expression library derived from rat Pneumocystis carinii was screened with monoclonal antibodies active against the gp116 major surface antigen of rat. A sequence containing a 1197 bp open reading frame was obtained and sequenced. This sequence was cloned into a Vaccinia vector.     

Protective Immunity Induced by DNA-library Immunization against an Intracellular Bacterial Infection

DNA-based immunization has shown to be a viable alternative approach to induce protective immunity against Brucella abortus infection. However, the use of a unique gene may not be sufficient to induce full protection. Therefore, a new strategy based on library immunization has been described to improve the level of protection against different pathogens and to identify new protective genes. In the present study, a B. abortus library was subcloned into the mammalian expression vector pCMV-Ubi. This plasmid was designed to create a fusion between the gene of interest with ubiquitin. The analysis of this Brucella-library showed approximately 72% of clones containing inserts with an average size of 500–2000 bp. Further, homology searches were performed using the BLASTn program, and all sequenced clones showed homology with Brucella genes, as expected. BALB/c mice immunised intramuscularly with the Brucella genomic expression library showed a strong specific total IgG antibody response to a Brucella protein extract, with production of IgG1 and IgG2a isotypes. Regarding cellular immunity, high levels of IFN-γ and no IL-4 were detected in primed mouse splenocytes and partial protection against infection was reached in animals vaccinated with the Brucella library compared to the control group.     

Nine novel precursors of Buthus martensii scorpion α-toxin homologues

The cDNAs encoding nine novel α-toxin homologues were isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). They are rich in AAAA and TTTT elements at the 5′ UTRs. The flanking region of the translation initiation codon ATG is AAAATGAA, which is highly conserved in scorpion Na⁺, K⁺ and Cl⁻ channel toxin genes. These putative scorpion α-toxins shared 45.5–98.4% homology with the characterized BmK α-toxins, and were completely conserved in the positions of all eight cysteines. This showed, together with higher homology at nucleotide level than that at amino acid level, that these toxins may originate from a common ancestor. The discovery of a series of homologues of scorpion α-toxin with a different degree of natural mutation in the primary structure will provide us with a valuable system for studying the structure–function relationship of scorpion toxins.     

RNA-Seq analysis and comparison of the enzymes involved in ionone synthesis of three cultivars of Osmanthus

Abstract

To comprehend the molecular mechanisms that control the differences in the composition of Osmanthus essential oils, the RNA-seq data and differentially expressed genes in different cultivar Osmanthus were studied. cDNA libraries of “jinqiugui,” “baijie,” and “rixianggui” were sequenced using Illumina HiSeq ^TM 2000. All of the enzymes involved in ionone synthesis were verified. DEGs were revealed and their enriched pathways were analyzed. A total of 20 DEGsencoding four enzymes that were potential candidates involved in ionone biosynthesis, as well as ispH, GPPS, ZDS, and CCD. It provided a way for Osmanthus oil monomer material to be synthesized in vitro.     

Genetic insight into cytochrome P450 in Chinese from the Chinese Millionome Database

Genetic variations of cytochrome P450 (CYP) influence the inter‐individual differences in drug response. Here, we collected 8682 variants of 57 CYP genes and cytochrome P450 oxidoreductase (POR) from a large‐scale sequencing project in Chinese, Chinese Millionome Database (CMDB). In addition, 52 294 variants from the Genome Aggregation Database (gnomAD) had been simultaneously identified and analysed. Rare variants with a variant allele frequency (VAF) < 0.01 comprised 41.4% (3594/8682) of identified variations in the CMDB, while 98.1% (51 320/52 294) in the gnomAD were rare. Out of 8682 variants in the CMDB, 66.9% (5808/8682) were in introns and only 4.3% (377/8682) were missense variants. In contrast, 36.2% (18 929/52 294) variants in the gnomAD were missense. The common alleles with a VAF over 0.1 were found in CYP1A2*1C, CYP1A2*1F, CYP2C19*2, CYP2D6*2, CYP2D6*10, CYP3A5*3 and CYP4F2*3, with a VAF of 0.161, 0.6, 0.27, 0.274, 0.678, 0.92 and 0.233, respectively. The growing number of genetic variations in CYP genes as more genomes are sequenced would increase the power to predict drug metabolism and response based on the genotype of the particular individual.     

Isolation of the detoxification enzyme EgP450 from an oil palm EST library

Context: Sequencing of cDNA clones from plant tissue to generate expressed sequence tags (ESTs) is an effective tool for gene discovery. Together with powerful bioinformatics tools, EST sequences allow the prediction of functions of putative bioactive compounds that can later be confirmed.

Objective: To isolate a detoxification enzyme from an EST library from the oil palm (Elaeis guineensis Jacq. Arecaceae).

Methods: In total, 750 clones from an oil palm cDNA library were randomly sequenced and analyzed. A clone homologous to cytochrome P450 monooxygenases (P450) was selected from the list of highly expressed genes. The full-length cDNA of P450 from E. guineensis (EgP450) was generated and transformed into a bacterial host to produce recombinant protein. A 3D model of EgP450 was generated and used in a molecular docking analysis to screen for target herbicide substrates. Finally, the detoxification activity of EgP450 was confirmed by an herbicide tolerance test with rice seedlings.

Results and discussion: The full-length EgP450 has an open reading frame (ORF) of 1515?bp that encodes a protein of 505 amino acids. Docking analysis showed that EgP450 bound to phenylurea-like herbicides such as isoproturon, chlortoluron and fluometuron. The herbicide tolerance test demonstrated that the presence of EgP450 protected the rice seedlings from the killing action of the phytotoxic agent isoproturon.

Conclusions: The gene EgP450 was detected in the roots and stems of oil palm tissues, and its recombinant product was shown to protect rice seedlings from exogenous herbicides of the phenylurea family.     

Differential gene expression triggered by highly cytotoxic α-Emitter-immunoconjugates in gastric cancer cells

Immunoconjugates composed of the α-emitter ²¹³Bi and the monoclonal antibody d9MAb specifically target HSC45-M2 gastric cancer cells expressing mutant d9-E-cadherin. These conjugates efficiently killed tumor cells in a nude mouse peritoneal carcinomatosis model. To elucidate the molecular responses of HSC45-M2 cells to α-emitter irradiation, whole genome gene expression profiling was performed. For that purpose HSC45-M2 cells were incubated with lethal doses of ²¹³Bi-d9MAb. RNA was isolated at 6, 24 and 48 h after irradiation, transcribed into cDNA and hybridized to whole genome microarrays. Results of microarray analysis were validated using RTQ-PCR showing correspondence of approximately 90%. Following incubation with ²¹³Bi-d9MAb, 682-1125 genes showed upregulation and 666-1278 genes showed downregulation at one time point, each. Eight genes appeared upregulated and 12 genes downregulated throughout. Molecular functions and biological processes of differentially expressed genes were categorized according to the PANTHER database. Following ²¹³Bi-d9MAb irradiation also a time-dependent shift in terms of overrepresentation of biological processes was observed. Among the genes showing continuous upregulation, COL4A2, NEDD9 and C3 have not been associated with the cellular response to high LET radiation so far. The same holds true for WWP2, RFX3, HIST4H4 and JADE1 that showed continuous downregulation. According to PANTHER, three of the consistently upregulated (ITM2C, FLJ11000, MSMB) and downregulated (HCG9, GAS2L3, FLJ21439) genes, respectively, have not been associated with any biological process or molecular function so far. Thus, these findings revealed interesting new targets for selective elimination of tumor cells and new insights regarding response of tumor cells to α-emitter exposure.     

Synthesis, characterization and evaluation of thiolated quaternary ammonium-chitosan conjugates for enhanced intestinal drug permeation

In a previous report quaternary ammonium-chitosan conjugates (N⁺-Chs) endowed with intestinal drug permeability-enhancing properties were described. They are characterized by short pendant chains of n adjacent diethyl-dimethylene-ammonium groups substituted onto the primary amino group of the chitosan (Ch) repeating units. In the present work two N⁺-Chs, one having DS (degree of substitution) = 59.2 ± 4.5%, n = 1.7 ± 0.1 (N⁺(60)-Ch), the other one having DS = 40.6 ± 1.3%, n = 3.0 ± 0.2 (N⁺(40)-Ch) were used to synthesize novel multifunctional non-cytotoxic Ch derivatives, each carrying thiol along with quaternary ammonium groups (N⁺-Ch–SH), with increased potential to enhance transepithelial drug transport. They have been obtained by transforming the residual free amino groups of N⁺(60)-Ch and N⁺(40)-Ch into 3-mercaptopropionamide moieties. The former yielded 4.5 ± 0.7% thiol-bearing groups, the latter, 5.2 ± 1.1% of such groups, on a Ch repeating unit basis. The multifunctional derivatives have improved the ability of the parent N⁺-Chs to enhance the permeability of the water-soluble macromolecular fluorescein isothiocyanate dextran, MW 4400 Da (FD4) and that of the lipophilic dexamethasone (DMS) across the excised rat intestinal mucosa and Caco-2 cell monolayer, respectively. The data from the present work altogether point to a synergism of quaternary ammonium and thiol groups to improve the intestinal drug absorption enhancing properties of the multifunctional Ch derivatives.     

Non‐invasive three‐dimensional power Doppler imaging for the assessment of acute cerebral blood flow alteration in a mouse model of subarachnoid haemorrhage

We aimed to evaluate the feasibility of a non‐invasive method of cerebral blood flow (CBF) measurement using high‐frequency power Doppler ultrasound imaging in a mouse model of subarachnoid haemorrhage (SAH). The 3‐dimensionally (3D) reconstructed blood flow signals (%vascularity) within the brain volume of the middle cerebral artery territory correlated well with reference parameters, baseline carotid artery blood flow (r² = 0.52, P < 0.0001) and normalized CBF changes (r² = 0.74 P < 0.0001). These data suggest that the 3D power Doppler analysis may have the potential for reflecting real‐time CBF changes during the acute phase of experimental SAH, which may be applicable to preclinical studies on early brain injury.     

UM171 promotes expansion of autologous peripheral blood hematopoietic stem cells from poorly mobilizing lymphoma patients

BackgroundAutologous hematopoietic stem cell transplantation is an effective therapeutic strategy for lymphoma patients. However, some patients have to give up receiving transplantation because of failing to obtain sufficient CD34⁺ cells yields. Therefore, we ex vivo expanded HSCs of lymphoma patients using UM171 to solve the problem of HSCs deficiency.MethodsMobilized peripheral blood-derived CD34⁺ cells from lymphoma patients were cultured for 10 days with or without UM171. The fold of cell expansion and the immunophenotype of expanded cells were assessed by flow cytometry. RNA-seq experiment was performed to identify the mechanism by which UM171 promoted HSCs expansion.ResultsUM171 treatment increased the proportion of CD34⁺ (68.97 ± 6.91%), CD34⁺ CD38⁻ cells (44.10 ± 9.20%) and CD34⁺CD38⁻CD45RA⁻CD90⁺ LT-HSCs (3.05 ± 2.08%) compared to vehicle treatment (36.08 ± 11.14%, 18.30 ± 9.49% and 0.56 ± 0.45%, respectively). UM171 treatment led to an 85.08-fold increase in LT-HSC numbers relative to initial cells. Importantly, UM171 promoted expansion of LT-HSCs achieved 138.57-fold in patients with poor mobilization. RNA-seq data showed that UM171 upregulated expression of HSC-, mast cell-specific genes and non-canonical Wnt signaling related genes, and inhibited genes expression of erythroid, megakaryocyte and inflammatory mediated chemokine.ConclusionsOur study shows that UM171 can efficiently promote ex vivo expansion of HSCs from lymphoma patients, especially for poorly mobilizing patients. In terms of mechanism, UM171 upregulate HSC-specific genes expression and suppress erythroid and megakaryocytic differentiation, as well as activate non-classical Wnt signaling.     

Purification and pharmacological characterization of BmKK2 (α-KTx 14.2), a novel potassium channel-blocking peptide, from the venom of Asian scorpion Buthus martensi Karsch

BmKK2 (α-KTx 14.2) is one of the novel short-chain peptides found in molecular cloning of a venom gland cDNA library from Asian scorpion Buthus martensi Karsch. Based upon its amino acid sequence, the peptide was proposed to adopt a classical α/β-scaffold for α-KTxs. In the present study, we purified BmKK2 from the venom of B. martensi Karsch, and investigated its action on voltage-dependent K⁺ currents in dissociated hippocampal neurons from neonatal rats. BmKK2 (10–100 μM) selectively inhibited the delayed rectifier K⁺ current, but did not affect the fast transient K⁺ current. The inhibition of BmKK2 on the delayed rectifier K⁺ current was reversible and voltage-independent. The peptide did not affect the steady-state activation of the current, but caused a depolarizing shift (about 9 mV) of its steady-state inactivation curve. The results demonstrate that BmKK2 is a novel K⁺ channel-blocking scorpion peptide.     

Quantitative structure activity relationship (QSAR) studies of a series of 2,5-disubstituted-1,3,4-oxadiazole derivatives as biologically potential compounds

2,5-disubstituted-1,3,4-oxadiazoles and its derivatives are reported to possess wide spectrum of biological activities ranging from antibacterial, antiviral, anti-Parkinson, anti-HIV, and anti-inflammatory activity. Quantitative structure activity relationship (QSAR) analysis is generally carried out to study quantitative relationship between the physicochemical parameters and the biological activity. In the present study, QSAR studies for a set of some disubstituted-1,3,4-oxadiazole derivatives were conducted using TSAR 3.3 software. The in vitro antibacterial activity (MICs) of the compounds against S. aureus and E. coli exhibited good correlation (n = 36, r ² > 0.7, r _cv² > 0.5, s = 0.069) with the prediction made by the model. It was found that the polarizability, thermodynamic, and lipophilicity were major responsible factors for exhibiting the activity.     

Subchronic cadmium exposure upregulates the mRNA level of genes associated to hepatic lipid metabolism in adult female CD1 mice

Cadmium (Cd) is a persistent environmental and occupational contaminant that accumulates in humans and shows adverse effects on health. Accumulating evidence reveals that environmental Cd exposure is associated with hepatic lipid accumulation and metabolic alterations in adult male mice. However, whether Cd exposure induces hepatic lipid accumulation and metabolic alterations in female mice remains poorly understood. In the present study, we aimed to investigate the effects of Cd exposure on insulin resistance, hepatic lipid accumulation and associated metabolic pathways. Female CD1 mice were administrated with CdCl₂ (10 and 100 mg l^–1) by drinking water. We found that Cd exposure did not induce obesity, insulin resistance and hepatic lipid accumulation. By contrary, mice in the Cd‐100 mg l^–1 group presented a significant reduction of the glucose area under the curve during the glucose tolerance test. However, there was a significant elevation in the mRNA level of Fasn and Scd‐1, which were critical genes during hepatic fatty acid synthesis. Moreover, hepatic Fabp1 and Fabp4, two genes for hepatic fatty acid uptake were upregulated in Cd‐treated mice. Of interest, Lpl, a key gene for hepatic lipoprotein lysis, was also upregulated in Cd‐treated mice. Collectively, our results suggest that Cd exposure upregulated mRNA level of genes related to hepatic lipid metabolism although there was no insulin resistance and hepatic lipid accumulation shown in the present study.