A mutation in HOXA2 is responsible for autosomal-recessive microtia in an Iranian family

Microtia, a congenital deformity manifesting as an abnormally shaped or absent external ear, occurs in one out of 8,000-10,000 births. We ascertained a consanguineous Iranian family segregating with autosomal-recessive bilateral microtia, mixed symmetrical severe to profound hearing impairment, and partial cleft palate. Genome-wide linkage analysis localized the responsible gene to chromosome 7p14.3-p15.3 with a maximum multi-point LOD score of 4.17. In this region, homeobox genes from the HOXA cluster were the most interesting candidates. Subsequent DNA sequence analysis of the HOXA1 and HOXA2 homeobox genes from the candidate region identified an interesting HOXA2 homeodomain variant: a change in a highly conserved amino acid (p.Q186K). The variant was not found in 231 Iranian and 109 Belgian control samples. The critical contribution of HoxA2 for auditory-system development has already been shown in mouse models. We built a homology model to predict the effect of this mutation on the structure and DNA-binding activity of the homeodomain by using the program Modeler 8v2. In the model of the mutant homeodomain, the position of the mutant lysine side chain is consistently farther away from a nearby phosphate group; this altered position results in the loss of a hydrogen bond and affects the DNA-binding activity.     

Genome-wide linkage analysis of a Parkinsonian-pyramidal syndrome pedigree by 500 K SNP arrays

Robust SNP genotyping technologies and data analysis programs have encouraged researchers in recent years to use SNPs for linkage studies. Platforms used to date have been 10 K chip arrays, but the possible value of interrogating SNPs at higher densities has been considered. Here, we present a genome-wide linkage analysis by means of a 500 K SNP platform. The analysis was done on a large pedigree affected with Parkinsonian-pyramidal syndrome (PPS), and the results showed linkage to chromosome 22. Sequencing of candidate genes revealed a disease-associated homozygous variation (R378G) in FBXO7. FBXO7 codes for a member of the F-box family of proteins, all of which may have a role in the ubiquitin-proteosome protein-degradation pathway. This pathway has been implicated in various neurodegenerative diseases, and identification of FBXO7 as the causative gene of PPS is expected to shed new light on its role. The performance of the array was assessed and systematic analysis of effects of SNP density reduction was performed with the real experimental data. Our results suggest that linkage in our pedigree may have been missed had we used chips containing less than 100,000 SNPs across the genome.     

Mechanism of sulfite cytotoxicity in isolated rat hepatocytes

Sulfite (SO(3)(2-)) has been widely used as preservative and antimicrobial in preventing browning of foods and beverages. SO(2), a common air pollutant, also is capable of producing sulfite and bisulfite depending on the pH of solutions. A molybdenum-dependent mitochondrial enzyme, sulfite oxidase, oxidizes sulfite to inorganic sulfate and prevents its toxic effects. In the present study, sulfite toxicity towards isolated rat hepatocytes was markedly increased by partial inhibition of cytochrome a/a(3) by cyanide or by putting rats on a high-tungsten/low-molybdenum diet, which result in inactivation of sulfite oxidase. Sulfite cytotoxicity was accompanied by a rapid disappearance of GSSG followed by a slow depletion of reduced glutathione (GSH). Depleting hepatocyte GSH beforehand increased cytotoxicity of sulfite. On the other hand, dithiothreitol (DTT), a thiol reductant, added even 1h after the addition of sulfite to hepatocytes, prevented cell death and restored hepatocyte GSH levels. Sulfite cytotoxicity was also accompanied by an increase of oxygen uptake, reactive oxygen species (ROS) formation and lipid peroxidation. Cytochrome P450 inhibitors, metyrapone and piperonyl butoxide also prevented sulfite-induced cytotoxicity and lipid peroxidation. Desferroxamine and antioxidants also protected the cells against sulfite toxicity. These findings suggest that cytotoxicity of sulfite is mediated by free radicals as ROS formation increases by sulfite and antioxidants prevent its toxicity. Reaction of sulfite or its free radical metabolite with disulfide bonds of GSSG and GSH results in the compromise of GSH/GSSG antioxidant system leaving the cell susceptible to oxidative stress. Restoring GSH content of the cell or protein-SH groups by DTT can prevent sulfite cytotoxicity.     

WRR4 encodes a TIR-NB-LRR protein that confers broad-spectrum white rust resistance in Arabidopsis thaliana to four physiological races of Albugo candida

White blister rust in the Brassicaceae is emerging as a superb model for exploring how plant biodiversity has channeled speciation of biotrophic parasites. The causal agents of white rust across a wide breadth of cruciferous hosts currently are named as variants of a single oomycete species, Albugo candida. The most notable examples include a major group of physiological races that each are economically destructive in a different vegetable or oilseed crop of Brassica juncea (A. candida race 2), B. rapa (race 7), or B. oleracea (race 9); or parasitic on wild crucifers such as Capsella bursa-pastoris (race 4). Arabidopsis thaliana is innately immune to these races of A. candida under natural conditions; however, it commonly hosts its own molecularly distinct subspecies of A. candida (A. candida subsp. arabidopsis). In the laboratory, we have identified several accessions of Arabidopsis thaliana (e.g.,. Ws-3) that can permit varying degrees of rust development following inoculation with A. candida races 2, 4, and 7, whereas race 9 is universally incompatible in Arabidopsis thaliana and nonrusting resistance is the most prevalent outcome of interactions with the other races. Subtle variation in resistance phenotypes is evident, observed initially with an isolate of A. candida race 4, indicating additional genetic variation. Therefore, we used the race 4 isolate for map-based cloning of the first of many expected white rust resistance (WRR) genes. This gene was designated WRR4 and encodes a cytoplasmic toll-interleukin receptor-like nucleotide-binding leucine-rich repeat receptor-like protein that confers a dominant, broad-spectrum white rust resistance in the Arabidopsis thaliana accession Columbia to representative isolates of A. candida races 2, 4, 7, and 9, as verified by transgenic expression of the Columbia allele in Ws-3. The WRR4 protein requires functional expression of the lipase-like protein EDS1 but not the paralogous protein PAD4, and confers full immunity that masks an underlying nonhypersensitive incompatibility in Columbia to A. candida race 4. This residual incompatibility is independent of functional EDS1.     

Direct and rapid electrochemical biosensing of the human interleukin-2 DNA in unpurified polymerase chain reaction (PCR)-amplified real samples

Electrochemical detection of polymerase chain reaction (PCR)-amplified human interleukin-2 (IL-2) coding DNA sample (399bp size) without any purification and pre-treatment is described. To achieve this goal, a sensor was made by immobilization of a 20-mer oligonucleotide (chIL-2) as the probe on the pencil graphite electrode (PGE). This probe is related to the antisense strand of human interleukin-2 gene. The results showed that the electrode could effectively sense the PCR product of human interleukin-2 DNA by anodic differential pulse voltammetry (ADPV) based on guanine oxidation signal. In order to inhibit PCR components interfering effects and improve biosensing performance, various factors were investigated. We found that the desorption of non-specifically adsorbed components of the unpurified PCR samples from PGE surface is easily achieved by washing of the electrode in washing solution for about 300s. The effectiveness of this procedure was confirmed using purified PCR samples. The selectivity of the sensor was assessed with negative control PCR sample and seven different non-complementary PCR products corresponding to 16S rDNA (bigger than 1500bp) of various bacterial genuses. Diagnostic performance of the biosensor is described and the detection limit is found to be 69pM. The reliability of the electrochemical biosensing results was verified by electrophoresis of the PCR products.     

Simple procedures for purification and stabilization of human serum paraoxonase-1

Human paraoxonases-1 is one of the most important detoxifying enzymes. In this study using simple chromatographic procedures human paraoxonases-1 was purified from human pooled plasma. The enzyme was purified using DEAE Sephadex an anion exchanger and G-200 a gel filtration chromatographic media. Results showed a single band of approximately 43 KD proteins in SDS–PAGE, corresponding to the human PON1. Using paraoxon as the substrate the activity was related to the concentration of calcium and sodium ions (K_m = 1.2 ± 0.2 mM). Phenyl acetate hydrolyzing activity was independent of sodium and calcium ions (K_m = 0.78 ± 0.08 mM). Keeping at 25 °C for 20 days 75% of the enzyme original activity was restored in 20% (v/v) glycerol. EDTA and zinc chloride both inhibited the enzyme activity. In conclusion the applied procedures can be used for large scale purification. It would greatly facilitate their structural and functional characterization and permit examination of their weak, yet potentially most biologically relevant activities, in the complete absence of other serum proteins.     

An efficient, simple and expedition synthesis of 1-amidoalkyl-2-naphthols as 'drug like' molecules for biological screening

An efficient and direct protocol for the preparation of amidoalkyl naphthols employing a multi-component, one-pot condensation reaction of beta-naphthol, aromatic aldehydes and acetamide in the presence of ferric hydrogensulfate under solvent, solvent-free and microwave conditions is described. The thermal solvent-free and microwave green procedures offer advantages such as shorter reaction times, simple work-up, excellent yield, recovery and reusability of catalyst. It is noteworthy that 1-amidomethyl-2-naphthols can be converted into important biological 'drug like' active 1-aminomethyl-2-naphthols derivatives by amide hydrolysis.     

Predictors and frequency of conduction disturbances after open-heart surgery

Introduction

The risk of developing conduction disturbances after coronary bypass grafting (CABG) or valvular surgery has been well established in previous studies, leading to permanent pacemaker implantation in about 2% to 3% of patients, and in 10% of patients undergoing repeat cardiac surgery. We sought to determine the incidence, features and predictors of conduction disorders in the immediate post-operative period of patients subjected to open-heart surgery, and the need for permanent pacemaker implantation.

Material and Method

We prospectively studied 374 consecutive patients who underwent open-heart surgery in our institution: coronary artery bypass (CABG) (n=128), Mitral valve replacement(MVR)(n=18), aortic valve replacement(AVR) (n=21), MVR and AVR(n=56), repair of ventricular septal defect (VSD) (n=51), repair of tetralogy of Fallot (TOF) (n=57),CABG and valvular surgery (n=6), others (n=37).

Results

Among 374 patients included in our study (mean age 34.46±25.68; 146 males), 192 developed new conduction disorders: symptomatic sinus bradycardia in 8%, atrial fibrillation with slow ventricular response (AF) in 4.5%, first-degree atrioventricular block (AVB)in 6.4%, second-degree AVB in 0.3%, third-degree AVB in 7%, new right bundle branch block (RBBB) in 33%, and new left bundle branch block (LBBB) in 2.1%. In 5.6% patients, a permanent pacemaker was implanted, 47.6% of them underwent valvular surgery. In 44.1% of patients the conduction defects occurred in the first 48 hr. after surgery. In CABG group, 29.7% of patients developed new conduction disturbances; the most common of them was symptomatic sinus bradycardia. After valvular surgery 44.2% of patients developed conduction disturbances, of those the most common was atrial fibrillation with slow ventricular response . After VSD and TOF repair, the most common conduction disturbance was new RBBB. Perioperative myocardial infarction (MI) occurred in 1.9% of patients. The occurrence conduction disturbance was compared with patient age, sex, occurrence of perioperative MI, ejection fraction (EF), postoperative use of ß-adernergic receptor blocking agents and digitalis and type of cardiac surgery. By regression analysis there was a correlation between type of surgery and new conduction defects, being significant for CABG and TOF repair. Only the occurrence of perioperative MI was related to PPM implantation.

Conclusion

Irreversible AVB requiring a PPM is an uncommon complication after open-heart surgery. Peri-operative MI is a risk factor.     

Patterns of co-association of C-reactive protein and nitric oxide in malaria in endemic areas of Iran

In addition to numerous immune factors, C-reactive protein (CRP) and nitric oxide (NO) are believed to be molecules of malaria immunopathology. The objective of this study was to detect CRP and NO inductions by agglutination latex test and Griess microassay respectively in both control and malaria groups from endemic areas of Iran, including Southeastern (SE) (Sistan & Balouchestan, Hormozgan, Kerman) and Northwestern (NW) provinces (Ardabil). The results indicated that CRP and NO are produced in all malaria endemic areas of Iran. In addition, more CRP and NO positive cases were observed amongst malaria patients in comparison with those in control group. A variable co-association of CRP/NO production were detected between control and malaria groups, which depended upon the malaria endemic areas and the type of plasmodia infection. The percentage of CRP/NO positive cases was observed to be lower in NW compare to SE region, which may be due to the different type of plasmodium in the NW (Plasmodium vivax) with SE area (P. vivax, Plasmodium falciparum, mixed infection). The fluctuations in CRP/NO induction may be consistent with genetic background of patients. Although, CRP/NO may play important role in malaria, their actual function and interaction in clinical forms of disease remains unclear.     

Structural, biochemical, and physiological characterization of photosynthesis in two C4 subspecies of Tecticornia indica and the C3 species Tecticornia pergranulata (Chenopodiaceae)

Among dicotyledon families, Chenopodiaceae has the most C(4) species and the greatest diversity in structural forms of C(4). In subfamily Salicornioideae, C(4) photosynthesis has, so far, only been found in the genus Halosarcia which is now included in the broadly circumscribed Tecticornia. Comparative anatomical, cytochemical, and physiological studies on these taxa, which have near-aphyllous photosynthetic shoots, show that T. pergranulata is C(3), and that two subspecies of T. indica (bidens and indica) are C(4) (Kranz-tecticornoid type). In T. pergranulata, the stems have two layers of chlorenchyma cells surrounding the centrally located water storage tissue. The two subspecies of T. indica have Kranz anatomy in reduced leaves and in the fleshy stem cortex. They are NAD-malic enzyme-type C(4) species, with mesophyll chloroplasts having reduced grana, characteristic of this subtype. The Kranz-tecticornoid-type anatomy is unique among C(4) types in the family in having groups of chlorenchymatous cells separated by a network of large colourless cells (which may provide mechanical support or optimize the distribution of radiation in the tissue), and in having peripheral vascular bundles with the phloem side facing the bundle sheath cells. Also, the bundle sheath cells have chloroplasts in a centrifugal position, which is atypical for C(4) dicots. Fluorescence analyses in fresh sections indicate that all non-lignified cell walls have ferulic acid, a cell wall cross-linker. Structural-functional relationships of C(4) photosynthesis in T. indica are discussed. Recent molecular studies show that the C(4) taxa in Tecticornia form a monophyletic group, with incorporation of the Australian endemic genera of Salicornioideae, including Halosarcia, Pachycornia, Sclerostegia, and Tegicornia, into Tecticornia.