Composition and antimicrobial activity of the essential oil of Dicyclophora persica Boiss. from Iran

The chemical composition of the essential oil of Dicyclophora persica Boiss. was identified by GC and GC-MS analysis. The analysis of the oil resulted in the identification of forty-five components constituting 98.6% of the total oil. The main constituents were a-pinene (31.5%), (Z)-beta-ocimene (23.3%), p-cymene (6.7%) and (E)-beta-ocimene (5.4%). The antimicrobial activity of the oil was tested by the disk diffusion method against four Gram-positive (Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis) and three Gram-negative (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) bacteria together with a fungus (Aspergillus niger). The oil showed strong inhibition activity toward all the tested microorganisms except for Pseudomonas aeruginosa.     

Bioengineered chimeric tRNA/pre-miRNAs as prodrugs in cancer therapy

Today, biologic prodrugs have led to targeting specific tumor markers and have increased specificity and selectivity in cancer therapy. Various studies have shown the role of ncRNAs in cancer pathology and tumorigenesis and have suggested that ncRNAs, especially miRNAs, are valuable molecules in understanding cancer biology and therapeutic processes. Most miRNAs-based research and treatment are limited to chemically synthesized miRNAs. Synthetic alterations in these miRNA mimics may affect their folding, safety profile, and even biological activity. However, despite synthetic miRNA mimics produced by automated systems, various carriers could be used to achieve efficient production of bioengineered miRNAs through economical microbial fermentation. These bioengineered miRNAs as biological prodrugs could provide a new approach for safe therapeutic methods and drug production. In this regard, bioengineered chimeric miRNAs could be selectively processed to mature miRNAs in different types of cancer cells by targeting the desired gene and regulating cancer progression. In this article, we aim to review bioengineered miRNAs and their use in cancer therapy, as well as offering advances in this area, including the use of chimeric tRNA/pre-miRNAs.     

The association between adiponectin (+45T/G) and adiponectin receptor-2 (+795G/A) single nucleotide polymorphisms with cirrhosis in Iranian population

Adiponectin which possesses anti-inflammatory and insulin-sensitizing properties is elevated in blood circulation of liver cirrhosis patients. The genetic variations in the adiponectin gene can affect the circulating adiponectin level and stimulation of adiponectin receptor that may affect the activity of adiponectin. We investigated the effect of adiponectin single nucleotide polymorphisms (SNP) 45 T/G and adiponectin receptor-2 gene SNP 795G/A in cirrhotic Iranian population. A total of 97 cirrhotic patients and 128 healthy controls from Iranian population were genotyped for the adiponectin and adiponectin receptor 2 gene (+45T>G and 795G/A) by polymerase chain reaction-restriction fragment length polymorphism. G frequency was 21.1% versus 12.89% (P = 0.001) for SNP45, and G frequency was 75.8% versus 76.2% (P = 0.526) for SNP795G/A in the patients and control group, respectively. Based on our findings, the expression of the G allele at SNP45 is higher in the patient group compared with healthy subjects, suggesting that it may affect liver injury through changes in the plasma adiponectin level.     

Toxicity of depleted uranium on isolated rat kidney mitochondria

Background

Kidney is known as the most sensitive target organ for depleted uranium (DU) toxicity in comparison to other organs. Although the oxidative stress and mitochondrial damage induced by DU has been well investigated, the precise mechanism of DU-induced nephrotoxicity has not been thoroughly recognized yet.

Methods

Kidney mitochondria were obtained using differential centrifugation from Wistar rats and mitochondrial toxicity endpoints were then determined in both in vivo and in vitro uranyl acetate (UA) exposure cases.

Results

Single injection of UA (0, 0.5, 1 and 2 mg/kg, i.p.) caused a significant increase in blood urea nitrogen and creatinine levels. Isolated mitochondria from the UA-treated rat kidney showed a marked elevation in oxidative stress accompanied by mitochondrial membrane potential (MMP) collapse as compared to control group. Incubation of isolated kidney mitochondria with UA (50, 100 and 200 μM) manifested that UA can disrupt the electron transfer chain at complex II and III that leads to induction of reactive oxygen species (ROS) formation, lipid peroxidation, and glutathione oxidation. Disturbances in oxidative phosphorylation were also demonstrated through decreased ATP concentration and ATP/ADP ratio in UA-treated mitochondria. In addition, UA induced a significant damage in mitochondrial outer membrane. Moreover, MMP collapse, mitochondrial swelling and cytochrome c release were observed following the UA treatment in isolated mitochondria.

General significance

Both our in vivo and in vitro results showed that UA-induced nephrotoxicity is linked to the impairment of electron transfer chain especially at complex II and III which leads to subsequent oxidative stress.     

Molecular pathogenesis of a new glycogenosis caused by a glycogenin-1 mutation

Glycogenin-1 initiates the glycogen synthesis in skeletal muscle by the autocatalytic formation of a short oligosaccharide at tyrosine 195. Glycogenin-1 catalyzes both the glucose-O-tyrosine linkage and the α1,4 glucosidic bonds linking the glucose molecules in the oligosaccharide. We recently described a patient with glycogen depletion in skeletal muscle as a result of a non-functional glycogenin-1. The patient carried a Thr83Met substitution in glycogenin-1. In this study we have investigated the importance of threonine 83 for the catalytic activity of glycogenin-1. Non-glucosylated glycogenin-1 constructs, with various amino acid substitutions in position 83 and 195, were expressed in a cell-free expression system and autoglucosylated in vitro. The autoglucosylation was analyzed by gel-shift on western blot, incorporation of radiolabeled UDP-(14)C-glucose and nano-liquid chromatography with tandem mass spectrometry (LC/MS/MS). We demonstrate that glycogenin-1 with the Thr83Met substitution is unable to form the glucose-O-tyrosine linkage at tyrosine 195 unless co-expressed with the catalytically active Tyr195Phe glycogenin-1. Our results explain the glycogen depletion in the patient expressing only Thr83Met glycogenin-1 and why heterozygous carriers without clinical symptoms show a small proportion of unglucosylated glycogenin-1.     

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209–216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.     

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.