Reports of Biochemistry and Molecular Biology
Volume:4 Issue: 2, Apr 2016

Seeds of the castor bean plant Ricinuscommunis L (CB) contain ricin toxin (RT), one of the most poisonous naturally-occurring substances known. Ricin toxin, a water-soluble glycoprotein that does not partition into the oil extract, is a ribosome-inactivating toxin composed of two chains, labeled A and B. Severity of the toxicity varies depending on the route of exposure to the toxin. Inhalational is the most toxic route, followed by oral ingestion. Orally-ingested RT accumulates in the liver and spleen but other cells are also affected. The main clinical manifestations are also related to the administration route. Oral ingestion of CB or RT results in abdominal pain, vomiting, diarrhea, and various types of gastrointestinal bleeding that leading to volume depletion, hypovolemic shock, and renal failure. Inhalation of the toxin presents with non-cardiogenic pulmonary edema, diffuse necrotizing pneumonia, interstitial and alveolar inflammation, and edema. Local injection of RT induces indurations at the injection site, swelling of regional lymph nodes, hypotension, and death. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect RT in animal tissues and fluids. Ricinine, an alkaloid of CB, can be detected in rat urine within 48 h of RT exposure. Supportive care is the basic treatment and standard biowarfare decontamination protocols are used for RT intoxication. Dexamethasone and difluoromethylornithine might be effective treatments. This review examines the clinical and molecular aspects of ricin toxicity.

LuxI is a component of the quorum sensing signaling pathway in Vibrio fischeri responsible for the inducer synthesis that is essential for bioluminescence.
Homology modeling of LuxI was carried out using Phyre2 and refined with the GalaxyWEB server. Five models were generated and evaluated by ERRAT, ANOLEA, QMEAN6, and Procheck.
Five refined models were generated by the GalaxyWEB server, with Model 4 having the greatest quality based on the QMEAN6 score of 0.732. ERRAT analysis revealed an overall quality of 98.9%, while the overall quality of the initial model was 54%. The mean force potential energy, as analyzed by ANOLEA, were better compared to the initial model. Sterochemical quality estimation by Procheck showed that the refined Model 4 had a reliable structure, and was therefore submitted to the protein model database. Drug Discovery Workbench V.2 was used to screen 2700 experimental compounds from the DrugBank database to identify inhibitors that can bind to the active site between amino acids 24 and 110. Ten compounds with high negative scores were selected as the best in binding.
The model produced, and the predicted acteyltransferase binding site, could be useful in modeling homologous sequences from other microorganisms and the design of new antimicrobials.

The SRY gene (SRY) provides instructions for making a transcription factor called the sex-determining region Y protein. The sex-determining region Y protein causes a fetus to develop as a male. In this study, SRY of 15 spices included of human, chimpanzee, dog, pig, rat, cattle, buffalo, goat, sheep, horse, zebra, frog, urial, dolphin and killer whale were used for determine of bioinformatic differences.
Nucleotide sequences of SRY were retrieved from the NCBI databank. Bioinformatic analysis of SRY is done by CLC Main Workbench version 5.5 and ClustalW (http:/www.ebi.ac.uk/clustalw/) and MEGA6 softwares.
The multiple sequence alignment results indicated that SRY protein sequences from Orcinus orca (killer whale) and Tursiopsaduncus (dolphin) have least genetic distance of 0.33 in these 15 species and are 99.67% identical at the amino acid level. Homosapiens and Pantroglodytes (chimpanzee) have the next lowest genetic distance of 1.35 and are 98.65% identical at the amino acid level.
These findings indicate that the SRY proteins are conserved in the 15 species, and their evolutionary relationships are similar.

Interfering with cell proliferation and survival is a critical role for antineoplastic drugs leading to cell death through induction of apoptosis. Alternative treatments with herbal extracts offer insights into acute myeloid leukemia (AML) therapy. Parthenolide (PTL), an extract from feverfew, induces apoptosis in primary human leukemia stem cells (LSCs) and bulk leukemic cell populations. Osteopontin (OPN) preserves cell viability in response to anticancer agents and its receptors could be utilized for therapeutic targeting of cancer cells.
U937 cells were cultured in RPMI 1640 with concentrations of 2, 4, 6, 8, and 10 μM PTL for 20-24 hours for MTT assays. Apoptosis assays were performed with Annexin V-Alexa Fluor-488/PI as Annexin Vﳲ and Annexin Vﳲ to measure early and late apoptosis, respectively. Quantitative real-time PCR was used to measure OPN gene expression using the 2-∆∆Ct method. The PTL–treated cells were stained with FITC-CD38 antibody for flow cytometry analyses. Data were compared using one-way analysis of variance (ANOVA) by SPSS 19.
Parthenolide inhibited growth of U937 cells with IC25 and IC50 values of 4 and 5.8 µM, respectively. Death induction with PTL was apoptotic. Flow cytometry showed a significant decrease in the percentage of CD38 U937 cells in response to PTL. Osteopontin gene expression decreased in response to PTL.
PTL induced apoptosis and reduced OPN gene expression in U937 cells.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). Bacille Calmette-Guerin (BCG) vaccine, is not effective in adults, therefore, many efforts have been made to produce an effective adult TB vaccine. The aim of this study was to develop a new tuberculosis DNA vaccine candidate encoding a recombinant HspX-PPE44-EsxV fusion antigen of M. tuberculosis.
A fusion DNA segment consisting of HspX, linker, PPE44, linker, and EsxV, after codon optimization, was designed. The fusion DNA was cloned and its sequence confirmed. Then, expression of a recombinant pcDNA3.1 ()/HspX-PPE44-EsxV plasmid in Chinese hamster ovary (CHO) cells was verified by RT-PCR and Western-blot analysis.
A 1968 bp band in RT-PCR and a 68 kDa band on Western-blot analysis confirmed transcription and expression of recombinant hspX-ppe44-esxV in eukaryotic cells.
A recombinant DNA segment encoding the HspX-PPE44-EsxV fusion antigen of M. tuberculosis was constructed and considered to be tested as a new TB DNA vaccine candidate.

Diagnosis of food allergy is difficult in children. Food allergies are diagnosed using several methods that include medical histories, clinical examinations, skin prick and serum-specific immunoglobulin E (IgE) tests, radio-allergosorbent test (RAST), food challenge, and supervised elimination diets. In this study we evaluated allergies to cow's milk, egg, peanut, and fish in children with suspected food allergies with skin prick tests and serum and feces RAST.
Forty-one children with clinical symptoms of food allergies were enrolled in the study. Skin prick tests and serum and fecal RAST were performed and compared with challenge tests.
The most common sites of food allergy symptoms were gastrointestinal (82.9%) and skin (48.8%). 100% of the patients responded to the challenge tests with cow’s milk, egg, peanut, and fish. 65% of the patients tested positive with the skin prick test, 12.1% tested positive with serum RAST, and 29.2% tested positive with fecal RAST.
The skin prick test was more sensitive than serum or fecal RAST, and fecal RAST was more than twice as sensitive as serum RAST.

Urinary organic acids are water-soluble intermediates and end products of the metabolism of amino acids, carbohydrates, lipids, and a number of other metabolic processes. In the hereditary diseases known as organic acidurias, an enzyme or co-factor defect in a metabolic pathway leads to the accumulation and increased excretion of one or more of these acidic metabolites. Gas chromatography is the most commonly-used technology to separate and identify these metabolites. In this report the analytical conditions for the determination of methylmalonic acid using a gas chromatography/flame ionization detector (GC-FID) are studied with the aim to establish a method to analyze organic acids in human urine.
Studies included the GC-FID method development, the conditions of the derivatization (trimethylsilylation) reaction, and the stability of the methylmalonic acid standard solution and trimethylsilyl derivatives during storage. Also, a systematic comparison between GC-FID and gas chromatography/mass spectrometry (GC-MS) was performed.
The highest resolution and sensitivity were obtained at 60 oC with a 30 min reaction time. Standard solutions and derivatized samples were stable for 7 days at 4-8 oC. Relative standard deviations of within-day and day-to-day assay results were less than 5%. Methylmalonic acid was detected in thirty human urine samples by the proposed GC-FID, and the results were compared with gold standard technique GC-MS. The correlation coefficient between GC-MS and GC-FID was obtained with R2= 0.997.
The developed method was applied to the quantitative analysis of methylmalonic acid in urine from hospitalized children with methylmalonic acidemia. With this method we aim to support pediatric clinics in Iran and assist in clinical diagnostics.