REPORTS OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
Year:2013 | Volume:2 | Issue:1|Papers Count:8

Background: During the last two decades, significant advances have been made in the fields of lactococcal genetics and protein expression. Lactococcus lactis (L. lactis) is an effective vector for protein expression and can be used as an antigen delivery system. Hence, L. lactis is an ideal candidate for mucosal immunotherapy. Profilin (Che a 2), the major allergen in Chenopodium album, is one of the most important causes of allergic diseases in desert and semi-desert areas, especially in Iran, Saudi Arabia, and Kuwait that was cloned and expressed in L. lactis for the first time.Methods: To construct L. lactis that expressed Che a 2, a DNA sequence was cloned and used to transform bacteria. Expression of Che a 2 was analyzed via monitoring of related RNA and protein. Hydrophobicity, adherence to HT-29 cells, antibiotic resistance, resistance to gastrointestinal contents, pH, and bile salt in recombinant and native L. lactis were evaluated.Results: Immunoblot analyses demonstrated that recombinant Che a 2 is expressed as a 32 kDa dimeric protein immunological studies showed it can bind human IgE. Both native and recombinant bacteria were sensitive to low pH and simulated gastric conditions. Bacterial survival was reduced 80-100% after 2 h of exposure to pH 1.5-2. Both native and recombinant bacteria were able to grow in 0.3 and 2% bile salts. After incubation of recombinant L. lactis in simulated gastric and intestinal juices for one and two hours, respectively, cell survival was reduced by 100%. Adhesion capability in both strains was minimal and there were no significant differences in any of our tests between native and recombinant bacteria.Conclusion: Successfully recombinant L. lactis with capability of expression Che a 2 was produced and revealed it is sensitive to gastrointestinal contents.

Background: Although parental obesity is a well-established predisposing factor for the development of obesity, associations between regional body compositions, resting metabolic rates (RMR), and physical activity (PA) of parents and their pre-school children remain unknown. The objective of this study was to investigate parent-child correlations for total and regional body compositions, resting energy expenditures, and physical activity.Methods: Participants were 89 children aged 2-6 years and their parents, consisting of 61 families. Resting metabolic rate was assessed using indirect calorimetry. Total and regional body compositions were measured by both dual energy X-ray absorptiometry (DXA) and deuterium dilution. Physical activity was assessed by an accelerometer.Results: There was a significant parent-offspring regression for total fat free mass (FFM) between children and their mothers (P=0.02), fathers (P=0.02), and mid-parent (average of father and mother value) (P=0.002) when measured by DXA. The same was true for fat mass (FM) between children and mothers (P<0.01), fathers (P=0.02), and midparent (P=0.001). There was no significant association between children and parents for physical activity during the entire week, weekend, weekdays, and different parts of days, except for morning activity, which was positively related to the mothers’ morning activities (P<0.01) and mid-parent (P=0.009). No association was found between RMR of children and parents before and after correction for FFM and FM.Conclusion: These data suggest a familial resemblance for total body composition between children and their parents. Our data showed no familial resemblance for PA and RMR between children and their parents.

Holt-Oram syndrome (HOS) is a developmental disorder inherited in an autosomal-dominant pattern. Affected organs are the heart and forelimbs with upper extremity skeletal defects and congenital heart malformation. In this study we present three cases of HOS in the same family. In one of these three individuals we detected a transition of C to T (CTG-GTT, V205V) in exon 7 of the TBX5 gene. This nucleotide change causes no amino acid change and potential pathologic effects remain unknown.

Background: Candida albicans (C. albicans) is a major cause of candidaemia in people with impaired immunity. Blood culture is a “gold standard” for candidaemia detection but is timeconsuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis.Methods: Five milliliter blood samples from healthy volunteers were spiked with 100-106 C. albicans cells to determine the detection limit of our method. DNA was extracted from whole blood using glass beads and the QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany). DNA from C.albicans isolates were amplified with primers and inserted into Escherichia coli (E. coli) DH5a.1 cells with the TA cloning vector (Invitrogen). The plasmid was used for standardization and optimization. A quantitative PCR assay with the LightCycler amplification and detection system based on fluorescence resonance energy transfer (FRET) with two different specific probes was established. To assess the precision and reproducibility of real-time PCR the intra-assay precision was determined in six consecutive assays.Results: No cross-reactivity of the hybridization probes with the DNA of non-C. albicans species or human genomic DNA was observed, which confirmed its 100% specificity. The minimum limit detected was one C. albicans cell or 100 CFU/ml (10 fg) per PCR reaction. The real-time PCR efficiency rate for Candida was high (E=1.95). Melting curve analysis of C. albicans showed a specific melting peak temperature of 65.76 oC.Conclusion: The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis.

Background: Hepatitis delta virus (HDV) is a subviral human pathogen that exploits host RNA editing activity to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the large form (L-HDAg), which is required for RNA packaging.Methods: In this study, PCR-based site-directed mutagenesis by the overlap extension method was used to create the point mutation converting the small-HDAg (S-HDAg) stop codon to a tryptophan codon through three stages.Results: Sequencing confirmed the desirable mutation and integrity of the L-HDAg open reading frame. The amplicon was ligated into pcDNA3.1 and transfected to Huh7 and HEK 293 cell lines. Western blot analysis using enhanced chemiluminescence confirmed L-HDAg expression. The recombinant L-HDAg localized within the nuclei of cells as determined by immunofluorescence and confocal microscopy.Conclusion: Because L-HDAg requires extensive post-translational modifications, the recombinant protein expressed in a mammalian system might be fully functional and applicable as a tool in HDV molecular studies, as well as in future vaccine research.

Background: Mycobacterium (M.) bovis is the agent of bovine tuberculosis (TB) in a range of animal species, including humans. Recent advances in immunology and the molecular biology of Mycobacterium have allowed identification of a large number of antigens with the potential for the development of a new TB vaccine. The ESAT-6 and CFP-10 proteins of M. bovis are important structural and functional proteins known to be important immunogens.Methods: In the current study, the DNAs encoding these genes were utilized in the construction of pcDNA 3.1+/ESAT-6 and pcDNA3.1+/CFP-10 plasmids. After intramuscular injection of BALB/c mice with these plasmids, ESAT-6 and CFP-10 mRNA expression was assessed by RT-PCR. Mice were inoculated and boosted with the plasmids to evaluate their effects on lymphocyte proliferation.Results: Our results indicate the plasmids are expressed at the RNA level and can induce lymphocyte proliferation.Conclusion: Further study is needed to characterize the effect of these antigens on the immune system and determine whether they are effective vaccine candidates against M. bovis.

Molecular understanding of three-dimensional (3D) peptide: MHC models require both basic knowledge of computational modeling and skilled visual perception, which are not possessed by all students. The present model aims to simulate MHC molecular structure with the hands and make a profound impression on the students.

Background: Hepatitis E virus (HEV) infection is a self-limiting viral infection that can lead to severe complications and death. In different regions the epidemiology of this infection varies. In this study we evaluated the seroepidemiology of hepatitis E infection in Jahrom, a city in southern Iran.Methods: This was a cross-sectional descriptive study of serum samples from 477 subjects, including 30 females and 447 males. HEV immunoglobulin G (IgG) and immunoglobulin M (IgM) were measured by enzyme-linked immunosorbent assays (ELISA). Alanine transaminase (ALT) and aspartate transaminase (AST) levels were also determined. Four hundred forty-seven subjects were male and 30 were female. Subjects were classified by age and sex.Results: One woman (3.3%) and 25 men (5.5%) were positive for HEV antibodies (IgG and/or IgM). There was found an association between serum level of aminotransferases and seropositivity for HEV.Conclusion: The result of this study indicates that HEV is an etiological factor for hepatitis in this area of IRAN. The cost benefit of active immunization in endemic regions should be evaluated because an outbreak could have tragic consequences.