Gene cloning, heterologous expression and activity identification of latroeggtoxin-Ⅵ / 生物工程学报

One of the distinct characters of Latrodectus tredecimguttatus is that its toxic components exist not only in the venomous glands, but also in the tissues outside the venomous glands and even in the eggs. Investigation on the toxins outside the venomous glands can deepen our understanding of spider toxins and discover new lead molecules with important application prospects. In order to explore the low-abundance proteinaceous toxins in the L. tredecimguttatus eggs, we used bioinformatic strategies to mine a gene sequence encoding a peptide toxin from the transcriptome of L. tredecimguttatus eggs, and then heterologously expressed the gene successfully with a 3'-RACE combined with nest PCR strategy. Biological activity analyses indicated that the expressed peptide toxin, named latroeggtoxin-Ⅵ (LETX-Ⅵ), could inhibit Na⁺ channel currents in ND7/23 cells and promote dopamine release from PC12 cells, without obvious toxicity against Periplaneta americana and bacteria as well as fungi including Staphylococcus aureus and Candida albicans, demonstrating that LETX-Ⅵ is a mammal-specific neurotoxin with a potential application prospect in development of the tool reagents for neurobiological study and the drugs for treating related diseases.

Antitumoral effects of Amblyomma sculptum Berlese saliva in neuroblastoma cell lines involve cytoskeletal deconstruction and cell cycle arrest / Efeito antitumoral da saliva do carrapato Amblyomma sculptum Berlese em células de neuroblastoma envolve desconstrução do citoesqueleto e parada do ciclo celular

Abstract The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (Δψm) was slightly, but significantly (p < 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and Δψm were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.

Resumo As propriedades antitumorais de extratos de glândulas salivares de carrapatos ou proteínas recombinantes foram relatadas recentemente, mas pouco se sabe sobre as propriedades antitumorais dos componentes secretados da saliva. O objetivo deste estudo foi investigar o efeito in vitro da saliva bruta do carrapato duro Amblyomma sculptum sobre as linhagens celulares de neuroblastoma. Células SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32 e CHLA-20 foram suscetíveis à saliva, com redução de 80% na sua viabilidade em comparação com controles não tratados, como demonstrado pelo ensaio de Azul de Metileno. Investigações posteriores utilizando CHLA-20 revelaram apoptose, com aproximadamente 30% de células positivas para anexina-V, e G0/G1 (> 60%) após tratamento com saliva. O potencial de membrana mitocondrial (Δψm) foi reduzido significativamente (p <0,05), e o citoesqueleto de actina foi desestruturado, como indicado pela microscopia de fluorescência. A viabilidade do fibroblasto humano (células HFF-1), usado como controle não tumoral, diminuiu em aproximadamente 40%. No entanto, não foram observadas alterações na progressão do ciclo celular, morfologia e Δψm nestas células. O presente trabalho fornece novas perspectivas para a caracterização das moléculas presentes na saliva e suas propriedades antitumorais.

Mass fingerprinting and electrophysiological analysis of the venom from the scorpion Centruroides hirsutipalpus (Scorpiones: Buthidae)

Centruroides hirsutipalpus, of the family Buthidae, is a scorpion endemic to the Western Pacific region of Mexico. Although medically important, its venom has not yet been studied. Therefore, this communication aims to identify their venom components and possible functions. Methods Fingerprinting mass analysis of the soluble venom from this scorpion was achieved by high-performance liquid chromatography and electrospray mass spectrometry. Furthermore, the soluble venom and its toxic effects were evaluated extensively via electrophysiological assays in HEK cells expressing human voltage-gated Na+ channels (hNav 1.1 to Nav1.6), CHO cells expressing hNav 1.7, potassium channel hERG 1 (Ether-à-go-go-related-gene) and the human K+-channel hKv1.1. Results The separation of soluble venom produced 60 fractions from which 83 distinct components were identified. The molecular mass distribution of these components varies from 340 to 21,120 Da. Most of the peptides have a molecular weight between 7001 and 8000 Da (46% components), a range that usually corresponds to peptides known to affect Na+ channels. Peptides with molecular masses from 3000 to 5000 Da (28% of the components) were identified within the range corresponding to K+-channel blocking toxins. Two peptides were obtained in pure format and completely sequenced: one with 29 amino acids, showing sequence similarity to an "orphan peptide" of C. limpidus, and the other with 65 amino acid residues shown to be an arthropod toxin (lethal to crustaceans and toxic to crickets). The electrophysiological results of the whole soluble venom show a beta type modification of the currents of channels Nav1.1, Nav1.2 and Nav1.6. The main effect observed in channels hERG and hKv 1.1 was a reduction of the currents. Conclusion The venom contains more than 83 distinct components, among which are peptides that affect the function of human Na+-channels and K+-channels. Two new complete amino acid sequences were determined: one an arthropod toxin, the other a peptide of unknown function.(AU)

Processing and Modification of Recombinant Spider Silk Proteins / 生物医学工程学杂志

Due to its special sequence structure, spider silk protein has unique physical and chemical properties, mechanical properties and excellent biological properties. With the expansion of the application value of spider silk in many fields as a functional material, progress has been made in the studies on the expression of recombinant spider silk proteins through many host systems by gene recombinant techniques. Recombinant spider silk proteins can be processed into high performance fibers, and a wide range of nonfibrous morphologies. Moreover, for their excellent biocompatibility and low immune response they are ideal for biomedical applications. Here we review the process and mechanism of preparation in vitro, chemistry and genetic engineering modification on recombinant spider silk protein.

Construction of a vector encoding T-cell epitopes of Dermatophagoides pteronyssinus major allergen group 1 as a vaccine delivered by MHC class II pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway.</p><p><b>METHODS</b>The nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET-28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p 1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay.</p><p><b>RESULTS</b>The recombinant plasmid pET-28a-TAT-IhC-Der p 1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing and the expression of the recombinant protein TAT-IhC-Der p 1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p 1.</p><p><b>CONCLUSION</b>We successfully constructed a recombinant expression vector pET-28a-TAT-IhC-Der p 1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.</p>

Effect of formaldehyde inhalation on allergic rhinitis in mice / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To observe the effect of formaldehyde inhalation on the allergic rhinitis mice model.@*METHOD@#Forty-eight male BALB/C mice in six experimental group were exposure to (A) saline control; (B) Der p1; (C) formaldehyde (3.0 mg/m3); (D) Derp1 + formaldehyde (1.5 mg/m3); (E) Der p1 + formaldehyde (3.0 mg/M3); (F) Der p1+ formaldehyde (6.0 mg/m3). The concentrations of IL-4, IL-10 and IFN-γ in the peripheral serum were measured by enzyme-linked immunosorbent assay(ELISA). Nasal mucosal inflammation was evaluated by HE staining. Result: Formaldehyde exposure could increase the number of allergic rhinitis mice with sneezing and rubbing nose. The levels of IL-4 and IL-10 in group B, D, E and F were higher than that ingroup A (P < 0.05). Compared with the group C, the group D, E and F could effectively increase serum IL-4 and IL-10. The concentration of IL-4 in group E and F was higher than that of group B, while the group C was lower (P < 0.05). The concentration of IL-10 in group D, E and F was higher than that in group B (P < 0.05). The expression of IFN-γ in group B, D, E and F was lower than that in group A. While, the IFN-γ expression in group B was lower than that of group C and higher than that in group F (P < 0.05). Moreover, the concentration of IFN-γ in group D, E and F was lower compared with group C (P < 0.05). The nasal mucosa HE staining showed that the density of EOS increased simultaneously in formaldehyde exposure allergic rhinitis groups.@*CONCLUSION@#The study showed that formaldehyde exposure can promote Th2 cytokines and eosinophil infiltration and then aggravate the allergic rhinitis symptoms.

Distribution characteristics and influencing factors of children with allergic rhinitis in a domestic dust mites allergens content distribution characteristics and influencing factors / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To observe the household environment dust mites allergens content distribution characteristics and influence factors of children with allergic rhinitis to dust mites in Wuhu.@*METHOD@#Collect the surface dust in bedroom and living room floor, mattresses, pillows, sofa of 102 children with allergic rhinitis families. Dust mite allergen components 1 (Der f1) and house dust mites allergens 1 components (Der p1) of the dust samples were determined by enzyme-linked immunosorbent assay (ELISA).@*RESULT@#One hundred and twenty samples were collected . In a domestic dust mites samples, with a median of M (Min and Max) said dust mite allergen levels, Der f1 and Der p1 content was 2.66 (0.03, 26.63), 3.48 (0, 03, 33.68), respectively. Der f1 was significantly less than Der p1, and the difference was statistically significant (P 0.05).@*CONCLUSION@#The household dust mites of children allergic rhinitisin Wuhu area is given priority to with Der p1, and urban dust mites are significantly more than village's and town's. Enhancing health education, controlling dust mites allergens contamination inside the bedroom, especially urban areas, are positive differences for the prevention and treatment of allergic diseases such as allergic rhinitis in children.

Screening and Identification of Antigenic Proteins from the Hard Tick Dermacentor silvarum (Acari: Ixodidae)

In order to explore tick proteins as potential targets for further developing vaccine against ticks, the total proteins of unfed female Dermacentor silvarum were screened with anti-D. silvarum serum produced from rabbits. The results of western blot showed that 3 antigenic proteins of about 100, 68, and 52 kDa were detected by polyclonal antibodies, which means that they probably have immunogenicity. Then, unfed female tick proteins were separated by 12% SDS-PAGE, and target proteins (100, 68, and 52 kDa) were cut and analyzed by LC-MS/MS, respectively. The comparative results of peptide sequences showed that they might be vitellogenin (Vg), heat shock protein 60 (Hsp60), and fructose-1, 6-bisphosphate aldolase (FBA), respectively. These data will lay the foundation for the further validation of antigenic proteins to prevent infestation and diseases transmitted by D. silvarum.

Proteomic Screening of Antigenic Proteins from the Hard Tick, Haemaphysalis longicornis (Acari: Ixodidae)

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.

Proteomic Screening of Antigenic Proteins from the Hard Tick, Haemaphysalis longicornis (Acari: Ixodidae)

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.

The expression of BmK AngM1 in Mut(s) and Mut(+) recombinants of Pichia pastoris / 药学学报

BmK AngM1 is a long-chain scorpion toxin purified from the venom of Buthus martensii Karsch. It has been reported to exhibit evident analgesic effect and low toxicity, and has the potential to be a novel analgesic drug. The BmKAngM1 gene was transformed into Pichiapastoris GS115. Mut+ and Mut(s) recombinant strains were screened by phenotype and Mut+ recombinant strains were used to detect BmK AngMl gene copy number in the real-time PCR. Expression of BmK AngM1 in the Mut+ recombinant strain was compared with that of the Mut(s) recombinant strain with the same single copy of BmK AngM1 gene under the same condition. The results indicated that the transcription level of BmK AngM1 gene in the Mut(s) recombinant strain was 2.7 fold of that in the Mut recombinant strain in the real-time PCR, and the expression of BmK AngM 1 in the Mut(s) recombinant strain was 1.5 fold of that in the Mut+ recombinant strain. Therefore, Mut(s) recombinant strain showed better ability to express BmK AngM1 than Mut+ recombinant strain.

Pharmacophore based approach to design inhibitors in Crustaceans: An insight into the molt inhibition response to the receptor guanylyl cyclase.

The first set of competitive inhibitors of molt inhibiting hormone (MIH) has been developed using the effective approaches such as Hip-Hop, virtual screening and manual alterations. Moreover, the conserved residues at 71 and 72 positions in the molt inhibiting hormone is known to be significant for selective inhibition of ecdysteroidogenesis; thus, the information from mutation and solution structure were used to generate common pharmacophore features. The geometry of the final six-feature pharmacophore was also found to be consistent with the homology-modeled MIH structures from various other decapod crustaceans. The Hypo-1, comprising six features hypothesis was carefully selected as a best pharmacophore model for virtual screening created on the basis of rank score and cluster processes. The hypothesis was validated and the database was virtually screened using this 3D query and the compounds were then manually altered to enhance the fit value. The hits obtained were further filtered for drug-likeness, which is expressed as physicochemical properties that contribute to favorable ADME/Tox profiles to eliminate the molecules exhibit toxicity and poor pharmacokinetics. In conclusion, the higher fit values of CI-1 (4.6), CI-4 (4.9) and CI-7 (4.2) in conjunction with better pharmacokinetic profile made these molecules practically helpful tool to increase production by accelerating molt in crustaceans. The use of feeding sub-therapeutic dosages of these growth enhancers can be very effectively implemented and certainly turn out to be a vital part of emerging nutritional strategies for economically important crustacean livestock.

Physiological and biochemical characterization of egg extract of black widow spiders to uncover molecular basis of egg toxicity

BACKGROUND: Black widow spider (L. tredecimguttatus) has toxic components not only in the venomous glands, but also in other parts of the body and its eggs. It is biologically important to investigate the molecular basis of the egg toxicity. RESULTS: In the present work, an aqueous extract was prepared from the eggs of the spider and characterized using multiple physiological and biochemical strategies. Gel electrophoresis and mass spectrometry demonstrated that the eggs are rich in high-molecular-mass proteins and the peptides below 5 kDa. The lyophilized extract of the eggs had a protein content of 34.22% and was shown to have a strong toxicity towards mammals and insects. When applied at a concentration of 0.25 mg/mL, the extract could completely block the neuromuscular transmission in mouse isolated phrenic nerve-hemidiaphragm preparations within 12.0 ± 1.5 min. Using whole-cell patch-clamp technique, the egg extract was demonstrated to be able to inhibit the voltage-activated Na+, K+and Ca2+ currents in rat DRG neurons. In addition, the extract displayed activities of multiple hydrolases. Finally, the molecular basis of the egg toxicity was discussed. CONCLUSIONS: The eggs of black widow spiders are rich in proteinous compounds particularly the high-molecular-mass proteins with different types of biological activity The neurotoxic and other active compounds in the eggs are believed to play important roles in the eggs' toxic actions.

Recent progress in protein chemistry and proteomics of Latrodectus tredecimguttatus toxins / 生物工程学报

Latrodectus tredecimguttatus (commonly known as black widow spiders) have toxins not only in their venom glands, but also in other parts of their body, in their eggs and even in the newborn spiderlings. The study on the toxins in venom and materials outside the venom glands of the spiders to elucidate their differences and similarities, evolutional relationship and biological functions is of important theoretical and applicable significance. The development of modern protein chemistry and proteomics techniques has provided efficient means for the study of protein and peptide toxins of L. tredecimguttatus. By using such techniques, the molecular base and action mechanism of the toxins can be revealed at the levels of both single purified proteins and omics. Up to now, although protein chemistry and proteomics study on L. tredecimguttatus toxins have achieved a certain progress, the relevant work particularly that on the toxins in the materials outside the venom glands has to be further deepened.

Identification of Scolopendra subspinipes mutilans and its adulterants using DNA barcode / 中国中药杂志

In this study, the COI barcode was used to identify the Scolopendra medicinal materials and its adulterants in order to provide a new method for the identification of Scolopendra. Genomic DNA was extracted from the experimental samples. The COI sequences were amplified and sequenced bi-directionally. Sequence alignment and NJ tree construction was carried out by MEGA6.0 software. The results showed that the COI sequences can be obtained from all experimental samples. The average inter-specific K2P distance of Scolopendra was 0.222 and the minimum inter-specific distance was 0.190. All the Scolopendra subspinipes mutilans medicinal samples clustered into a clade in the NJ tree and can be distinguished from its adulterants. In a conclusion, COI can be used to correctly identify Scolopendra medicinal materials, and it will be a potential DNA barcode for identifying other animal medicinal materials.

Antitumor activity of recombinant antimicrobial peptide penaeidin-2 against kidney cancer cells / 华中科技大学学报（医学）（英德文版）

Penaeidin-2 (Pen-2) is an important antimicrobial peptide derived from the Pacific white shrimp, Penaeus vannamei, and possesses both antibacterial and antifungal activities. Recent studies suggest that recombinant penaeidins show similar activities to the native Pen-2 protein. Previous researches have shown that some antimicrobial peptides (AMPs) exhibit cytotoxic activity against cancer cells. To date, there have been no studies on the antitumor effects of Pen-2. This study evaluated the potential of recombinant pen-2 (rPen-2) in the selective killing of kidney cancer cell lines ACHN and A498, and its action mechanism. MTT assays found the maximal growth inhibition of HK-2, ACHN and A498 cells treated with 100 μg/mL rPen-2 at 48 h was 13.2%, 62.4%, and 70.4%, respectively. DNA-specific fluorescent dye staining showed a high percentage of apoptosis on cancer cells. Flow cytometry revealed that the apoptosis rate of HK-2, ACHN and A498 cells was 15.2%, 55.2%, and 61.5% at 48 h respectively, suggesting that rPen-2 induced higher apoptosis rate in cancer cells than in HK-2 cells. Laser confocal scanning microscopy demonstrated that the plasma membrane was the key site where rPen-2 interacted with and destroyed tumor cells. Scanning electron microscopy showed the morphologic changes of the cell membranes of kidney cancer cells treated with rPen-2. These results suggest that rPen-2 is a novel potential therapeutic agent that may be useful in treating kidney cancers.

Expression of limulus Factor C in silkworm larvae by Bac-to-Bac/BmNPV baculovirus expression system / 生物工程学报

Limulus Factor C, a serine protease zymogen from the amoebocytes of the limulus, has high affinity for endotoxin. When Factor C is activated by endotoxin, it hydrolyses artificial tripeptide substrate and measurable products are released, so it can be used as an alternative reagent for endotoxin analysis. Factor C gene of Tachypleus tridentatus was obtained through RT-PCR and the recombinant protein was expressed by Bac-to-Bac/BmNPV baculovirus expression system in silkworm larvae. The activity of Factor C was detected with diluted serum of silkworm larvae, and the sensitivity of endotoxin detected was 0.2 EU/mL when the serum was diluted at 1:500. The silkworm larvae expressed limulus Factor C could be used to develop a new low-cost endotoxin test reagent.

Gene Cloning, Expression and Immunogenicity of the Protective Antigen Subolesin in Dermacentor silvarum

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl beta-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.

Rhinovirus-Infected Epithelial Cells Produce More IL-8 and RANTES Compared With Other Respiratory Viruses

PURPOSE: The environmental factors human rhinoviruses (HRVs) and house dust mites (HDMs) are the most common causes of acute exacerbations of asthma. The aim of this study was to compare the chemokine production induced by HRVs in airway epithelial cells with that induced by other respiratory viruses, and to investigate synergistic interactions between HRVs and HDMs on the induction of inflammatory chemokines in vitro. METHODS: A549 human airway epithelial cells were infected with either rhinovirus serotype 7, respiratory syncytial virus (RSV)-A2 strain, or adenovirus serotype 3 and analyzed for interleukin (IL)-8 and regulated on activation, normal T-cell expressed and secreted (RANTES) release and mRNA expression. Additionally, activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 were evaluated. The release of IL-8 and RANTES was also measured in cells stimulated simultaneously with a virus and the HDM allergen, Der f1. RESULTS: HRV caused greater IL-8 and RANTES release and mRNA expression compared with either RSV or adenovirus. NF-kappaB and AP-1 were activated in these processes. Cells incubated with a virus and Der f1 showed an increased IL-8 release. However, compared with cells incubated with virus alone as the stimulator, only HRV with Der f1 showed a statistically significant increase. CONCLUSIONS: IL-8 and RANTES were induced to a greater extent by HRV compared with other viruses, and only HRV with Der f1 acted synergistically to induce bronchial epithelial IL-8 release. These findings may correspond with the fact that rhinoviruses are identified more frequently than other viruses in cases of acute exacerbation of asthma.

Cloning, bioinformatics analysis, and expression of the dust mite allergen Der f 5 of Dermatophagoides farinae

Crude extracts of house dust mites are used clinically for diagnosis and immunotherapy of allergic diseases, including bronchial asthma, perennial rhinitis, and atopic dermatitis. However, crude extracts are complexes with non-allergenic antigens and lack effective concentrations of important allergens, resulting in several side effects. Dermatophagoides farinae (Hughes; Acari: Pyroglyphidae) is one of the predominant sources of dust mite allergens, which has more than 30 groups of allergen. The cDNA coding for the group 5 allergen of D. farinae from China was cloned, sequenced and expressed. According to alignment using the VECTOR NTI 9.0 software, there were eight mismatched nucleotides in five cDNA clones resulting in seven incompatible amino acid residues, suggesting that the Der f 5 allergen might have sequence polymorphism. Bioinformatics analysis revealed that the matured Der f 5 allergen has a molecular mass of 13604.03 Da, a theoretical pI of 5.43 and is probably hydrophobic and cytoplasmic. Similarities in amino acid sequences between Der f 5 and allergens of other domestic mite species, viz. Der p 5, Blo t 5, Sui m 5, and Lep d 5, were 79, 48, 53, and 37%, respectively. Phylogenetic analysis indicated that Der f 5 and Der p 5 clustered together. Blo t 5 and Ale o 5 also clustered together, although Blomia tropicalis and Aleuroglyphus ovatus belong to different mite families, viz. Echimyopodidae and Acaridae, respectively.