Cloning and expression of cyclophilin from Platanus orientalis pollens in Escherichia coli

Background:

Allergy is a clinical disorder affecting the human population with wide geographical distribution. Platanus orientalis (P. orientalis) trees are planted in many countries and their pollen causes allergic reactions.Cyclophilin has recently been identified as one of the most important allergens of P. orientalis pollen. We aimed to clone and purify this allergen in Escherichia coli for further studies and therapeutic and diagnostic purposes for allergy to P. orientalis.

Methods:

RNA was extracted from P. orientalis. A full-length fragment encoding cyclophilin was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from P. orientalis RNA. The cDNA was inserted into the pET32b (+) vector, and the construct transformed into E. coli Top10 and BL21 cells. The expressed protein was purified by the CuSO4 method.

Results:

The cDNA for the cyclophilin of P. orientalis pollen was cloned, and a specific reactivity of recombinant cyclophin was confirmed by immunoblotting using sera from patients allergic to P. orientalis pollen.

Conclusion:

The recombinant cyclophilin has a potential for immunologic assays for evaluation of allergy to P. orientalis pollen.Key Words: Allergy; Recombinant allergen; Cyclophilin, Escherichia coli, Platanus orientalis, Pollen, Cloning     

Investigation of DNA integration into reproductive organs following intramuscular injection of DNA in mice

Background:

DNA immunization with plasmid DNA encoding bacterial, viral, parasitic, and tumor antigens has been reported to trigger protective immunity. The use of plasmid DNA vaccinations against many diseases has produced promising results in animal and human clinical trials; however, safety concerns about the use of DNA vaccines exist, such as the possibility of integration into the host genome, and elicitation of adverse immune responses.

Methods:

In this study, we examined the potential integration and bio-distribution of pcDNA3.1+PA, a new vaccine candidate with GenBank accession # {"type":"entrez-nucleotide","attrs":{"text":"EF550208","term_id":"147743335","term_text":"EF550208"}}EF550208, encoding the PA63 gene, in reproductive organs of mice; ovaries and uterus in female, and testis in male. Animals of both sexes were injected intramuscularly with pcDNA3.1+PA. Host genome integration and tissue distribution were examined using PCR and RT-PCR two times monthly for six months.

Results:

RT-PCR confirmed that pcDNA3.1+PA was not integrated into the host genome and did not enter reproductive organs.

Conclusions:

This finding has important implications for the use of pcDNA3.1+PA plasmid as a vaccine and opens new perspectives in the DNA vaccine area.Key Words: DNA, Intramuscular injection, Integration, Mice, Reproductive organs     

Investigations on possible roles of C-terminal propeptide of a Ca-independent α-amylase from bacillus

Previously, an extracellular α-amylase (BKA) had been purified from the culture of Bacillus sp. KR8104. Subsequently, the crystal structure of the active enzyme revealed a 422 amino acids polypeptide. In this study, the bka was cloned into E. coli, which encoded a polypeptide of 659 amino acids including two additional fragments: one 44 residues N-terminal fragment and another 193 residues C-terminal fragment. In order to investigate the role of the C-terminal fragment, two constructs with and without this region [BKAΔ(N44) and BKAΔ(N44C193)] were designed and expressed in E. coli BL21. The optimum pH, thermal stability, and the end-products of starch hydrolysis were found to be similar in both constructs. The Km and V(max) values for BKAΔ(N44) were lower than BKAΔ(N44C193), using either starch or ethylidene-blocked 4-nitrophenylmaltoheptaoside as a substrate.     

Efficient Room-Temperature O-Silylation of Alcohols Using a SBA-15-Supported Cobalt(II) Nanocatalyst

The O-silylation of OH groups of alcohols and phenols with hexamethyldisilazane (HMDS) was achieved in high-to-excellent yields using catalytic quantities of a SBA-15-supported cobalt(II) nanocatalyst (typically 0.5?mol-%) at room temperature and under solvent-free conditions. Furthermore, the heterogeneous catalyst showed an excellent durability and can be conveniently reused by filtration for at least twelve times without any noticeable loss of activity.     

Evaluation of inhibitory effect and apoptosis induction of Zyzyphus Jujube on tumor cell lines,an in vitro preliminary study

In the present study, water extract of dried fruit of Zyzyphus Jujube was tested for its possible anticancer effect and induction of apoptosis on human tumor cell lines, HEp-2, HeLa and Jurkat cell lines. The inhibitory effect of water extract of this fruit on cell proliferation was assessed by MTT colorimetric assay. The induction of apoptosis of this extract was analyzed by DNA fragmentation analysis. Zyzyphus Jujube extract showed inhibitory effects on mentioned cell lines. Jurkat leukemic line was found the most sensitive cells with IC50 of 0.1 μg mL⁻¹. Our study also showed a typical DNA laddering in this cell line. The present study showed cytotoxic activity of Zyzyphus Jujube on tumor cells. Although Zyzyphus Jujube has useful compounds for medical applications.     

Enhancement of protective humoral immune responses against Herpes simplex virus-2 in DNA-immunized guinea-pigs using protein boosting

Genital Herpes is a common sexually transmitted disease that is caused mostly by Herpes simplex virus type 2 (HSV-2). Its prevalence has increased in developing countries in spite of the availability of valuable antiviral drug therapy. Considering the importance of HSV-2 infections, effective vaccines remain the most likely hope for controlling the spread of HSV diseases. In the present study, the complete HSV-2 glycoprotein D gene was isolated and cloned into different plasmid vectors to construct a DNA vaccine and prepare recombinant subunit vaccines using a baculovirus expression system. The vaccines were tested alone or in combination to evaluate their ability to induce protective immunity in guinea-pigs against genital HSV infections. Immunization elicited humoral responses as measured by neutralization tests and enzyme-linked immunosorbent assay, and immunized animals had less severe genital skin disease as well as reduced replication of the challenging virus in the genital tract during experimental infection. Our results further demonstrate that DNA priming-protein boosting induced a neutralizing antibody titer higher than that obtained with DNA-DNA vaccination. The massive increase of antibody titer following DNA priming-protein boosting might be attributed to a recall of B cell memory.     

Atomic insight into prion disorder: An intricate detail gained by 0.5 μs molecular dynamics simulation of preventive G127V and deleterious D178V mutation in prion protein

In this study we are looking into two contradicting mutations found in prion protein (PrP) viz G127V and D178V, that are reportedly protective and pathogenic, respectively. Despite significant advances in comprehension of the role of pathogenic mutations, the role of protective mutation in amyloid fold inhibition still lacks a substantial basis. To understand the structural basis of protective mutation, molecular dynamics simulation coupled with protein-protein docking and molecular mechanics/Poisson-Boltzmann surface area analysis was used to understand the instant structural variability brought about by these mutations alone and in combination on PrP and prion-prion complex. Atomic-scale investigations successfully revealed that the binding pattern of prion-prion varies differentially in protective and pathogenic mutations with secondary structure showing distinct contrasting patterns, which could supposedly be a critical factor for differential prion behavior in protective and pathogenic mutations. Considering the reported role of an amyloid fold in prion-prion binding, the contrasting pattern has given us a lead in comprehending the role of these mutations and has been used in this study to look for small molecules that can inhibit amyloid fold for prion-prion interaction in pathogenic mutant carrying PrP.     

Hypermethylated RASSF1 and SLC5A8 promoters alongside BRAFV600E mutation as biomarkers for papillary thyroid carcinoma

Circulating cell-free DNA (cfDNA) has been considered as a diagnostic source to track genetic and epigenetic alterations in cancer. We aimed to study mutation in addition to the methylation status in the promoter regions of RASSF1 and SLC5A8 genes in tissues and circulating free DNA samples of patients affected with papillary thyroid carcinoma (PTC) and thyroid nodules as controls. BRAF^V600E mutation was studied by ARMS-scorpion real-time polymerase chain reaction method in 57 PTC and 45 thyroid nodule cases. Methylation status of RASSF1 and SLC5A8 promoter regions was analyzed by methylation-specific high-resolution melting curve analysis. BRAF^V600E mutation was found in 39 (68.4%) out of 57 PTC tissue samples, while in 33 (49.1%) cases of cfDNA, this mutation was detected. The frequency of BRAF^V600E mutation in cfDNA was significantly different between metastatic and nonmetastatic PTC cases (22 of 33 PTC cases vs. 5 of 34 thyroid nodule samples). Methylation levels of three promoter regions of SLC5A8 and proximal promoter region of RASSF1 was significantly different between PTC and thyroid nodule cases in both cfDNA and tissue DNA. In addition, the methylation status of these two genes in tissue DNA was reflected in methylation status observed in cfDNA. This study confirmed that BRAF^V600E mutation is better for discrimination between papillary thyroid carcinoma and thyroid nodules. On the other hand, hypermethylation in the more proximal promoter regions to RASSF1 and SLC5A8 genes showed higher sensitivity and more acceptable specificity for this discrimination.