Inhibitory effects of bee venom and its components against viruses <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Bee venom (BV) from honey bee (Apis Melifera L.) contains at least 18 pharmacologically active components including melittin (MLT), phospholipase A₂ (PLA₂), and apamin etc. BV is safe for human treatments dose dependently and proven to possess different healing properties including antibacterial and antiparasitidal properties. Nevertheless, antiviral properties of BV have not well investigated. Hence, we identified the potential antiviral properties of BV and its component against a broad panel of viruses. Co-incubation of non-cytotoxic amounts of BV and MLT, the main component of BV, significantly inhibited the replication of enveloped viruses such as Influenza A virus (PR8), Vesicular Stomatitis Virus (VSV), Respiratory Syncytial Virus (RSV), and Herpes Simplex Virus (HSV). Additionally, BV and MLT also inhibited the replication of non-enveloped viruses such as Enterovirus-71 (EV-71) and Coxsackie Virus (H3). Such antiviral properties were mainly explained by virucidal mechanism. Moreover, MLT protected mice which were challenged with lethal doses of pathogenic influenza A H1N1 viruses. Therefore, these results provides the evidence that BV and MLT could be a potential source as a promising antiviral agent, especially to develop as a broad spectrum antiviral agent.     

Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR

Induction of endoplasmic reticulum (ER)‐to‐Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)‐mediated, Golgi‐independent unconventional cell‐surface trafficking of the folding‐deficient ΔF508‐cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation‐dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508‐CFTR, such as induction of ER‐to‐Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C‐terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation‐mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation‐inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER‐retained ΔF508‐CFTR and mediates the ER stress‐induced unconventional secretion pathway.      

cDNA cloning and mRNA expression of LIM protein gene homologue from the silkworm, Bombyx mori

LIM protein cDNA, from Bombyx mori that contains an open reading frame of 622 bp encoding 94 amino acids, was identified and characterized. The B. mori LIM protein homologue is classified into group 2 LIM proteins that contain glycine-rich LIM domain. B. mori LIM protein mRNA is up-regulated at late embryogenesis and detected in the mid-gut of 5th instar larvae.     

Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE)

One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.     

Indoleamine 2,3-dioxygenase-expressing dendritic cells are involved in the generation of CD4+CD25+ regulatory T cells in Peyer's patches in an orally tolerized, collagen-induced arthritis mouse model

Introduction

The present study was devised to understand the role of systemic indoleamine 2,3-dioxygenase (IDO) in the tolerance induction for orally tolerized mice in collagen-induced arthritis (CIA). We examined whether IDO-expressing dendritic cells (DCs) are involved in the generation of CD4⁺CD25⁺ regulatory T cells during the induction of oral tolerance in a murine CIA model.

Methods

Type II collagen was fed six times to DBA/1 mice beginning 2 weeks before immunization, and the effect on arthritis was assessed. To examine the IDO expression, the DCs of messenger RNA and protein were analyzed by RT-PCR and Flow cytometry. In addition, a proliferative response assay was also carried out to determine the suppressive effects of DCs through IDO. The ability of DCs expressing IDO to induce CD4⁺CD25⁺ T regulatory cells was examined.

Results

CD11c⁺ DCs in Peyer's patches from orally tolerized mice expressed a higher level of IDO than DCs from nontolerized CIA mice. IDO-expressing CD11c⁺ DCs were involved in the suppression of type II collagen-specific T-cell proliferation and in the downregulation of proinflammatory T helper 1 cytokine production. The suppressive effect of IDO-expressing CD11c⁺ DCs was mediated by Foxp3⁺CD4⁺CD25⁺ regulatory T cells.

Conclusion

Our data suggest that tolerogenic CD11c⁺ DCs are closely linked with the induction of oral tolerance through an IDO-dependent mechanism and that this pathway may provide a new therapeutic modality to treat autoimmune arthritis.     

Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium

The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10% fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P<0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P<0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P<0.05), and 72 h (70.4% versus 49.1 and 46 4%; P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and calving rate (43.7%; P<0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer.     

A complete library of amino acid alterations at R306 in Streptomyces clavuligerus deacetoxycephalosporin C synthase demonstrates its structural role in the ring-expansion activity

In a previous study, the conserved arginine residue at position 306 of Streptomyces clavuligerus deacetoxycephalsoporin C synthase (scDAOCS), when mutated to leucine, resulted in 191% increase in converting ampicillin to its expanded cephalosporin moiety compared with that of the wild-type enzyme. However, the role of this residue in eliciting the improved enzymatic activity is not well understood. In this study, probing the molecular basis of amino acid substitutions at position 306 has underscored its importance for engineering various improvements in the ring expansion activity. Structural modeling using SwissPdbViewer revealed that R306 is surrounded by a hydrophobic cleft formed by residues Y184, L186, W297, I298, and V303. Hence, the improved activity achieved by the R306L mutation was probably because of better hydrophobic packing in this region. To evaluate the role of amino acids at position 306 of scDAOCS and its influence on the molecular status of the enzyme at this locality, alteration to 18 other amino acids was done by site-directed mutagenesis. The effects of each substitution on the enzyme activity were determined by bioassay using penicillin substrates: ampicillin, penicillin G, phenethicillin, and carbenicillin. Results obtained showed a drastic reduction in enzyme activity when R306 was replaced with charged or polar residues, thus emphasizing the importance of hydrophobic packing around this site. The bioassay results also illustrated that apart from leucine, substitutions to nonpolar residues, isoleucine and methionine, were able to improve the ampicillin conversion activity of scDAOCS by 168 and 113% of the wild-type enzyme activity, respectively. Similar trend of effects from each mutation was also observed for penicillin G, phenethicillin, and carbenicillin conversions. The enhanced enzyme activities were supported by spectrophotometric assay indicating that all these mutants have lower K(m) values (R306L: 1.09 mM; R306I: 2.64 mM; R306M: 5.68 mM) than the wild-type enzyme (8.33 mM), resulting in improvement in the enzyme's substrate binding affinity. Hence, this mutational study of amino acids situated at 306 of scDAOCS has provided a better understanding of the significance of specific amino acid residues at this position which can improve its ring-expansion activity when given a plethora of beta-lactam substrates to generate corresponding, possibly new, cephalosporins.