Synthesis of chicken major histocompatibility complex class II oligomers using a baculovirus expression system

Chicken major histocompatibility complex (MHC) B21 and B19 haplotypes are associated with resistance and susceptibility to Marek's disease (MD), respectively. T-cell-mediated immune response is crucial in coordinating protection against Marek's disease virus (MDV) infection, but it has been difficult to identify and characterize antigen-specific T-cells. MHC class II tetramers and oligomers have been widely used for characterization of antigen-specific T-cells in the context of infectious and autoimmune diseases. Thus, the objective of this study was to synthesize chicken MHC class II oligomers of B21 and B19 haplotypes for the future identification of antigen-specific T-cells. To achieve this objective, full-length coding sequences of chicken MHC class II B21 and B19 molecules were amplified and the molecules were expressed as fusion proteins, carrying Fos and Jun leucine zipper (LZ), histidine-tag and biotin ligase recognition site sequences, using a baculovirus expression system. Recombinant MHC-II were loaded with self-peptides, which stabilized the heterodimer in SDS-PAGE and allowed the detection of these molecules in Western blots with a conformation-specific anti-chicken MHC class II antibody. Biotinylated MHC molecules were conjugated to streptavidin to form oligomers, which were resolved under the transmission electron microscope through immuno-gold labelling, thus confirming success of oligomerization. In conclusion, chicken MHC class II oligomers may be used in the future to study the antigen-specific CD4+ T-cell compartment.     

Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the L-tyrosine supply for melanogenesis in melanocytes

Epidermal phenylalanine hydroxylase (PAH) produces L-tyrosine from the essential amino acid L-phenylalanine supporting melanogenesis in human melanocytes. Those PAH activities increase linearly in the different skin phototypes I-VI (Fitzpatrick classification) and also increase up to 24h after UVB light with only one minimal erythemal dose. Since UVB generates also H(2)O(2), we here asked the question whether this reactive oxygen species could influence the activity of pure recombinant human PAH. Under saturating conditions with the substrate L-phenylalanine (1x10(-3)M), the V(max) for enzyme activity increased 4-fold by H(2)O(2) (>2.0x10(-3)M). Lineweaver-Burk analysis identified a mixed activation mechanism involving both the regulatory and catalytic domains of PAH. Hyperchem molecular modelling and Deep View analysis support oxidation of the single Trp(120) residue to 5-OH-Trp(120) by H(2)O(2) causing a conformational change in the regulatory domain. PAH was still activated by H(2)O(2) in the presence of the electron donor/cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin despite slow oxidation of this cofactor. In vivo FT-Raman spectroscopy confirmed decreased epidermal phenylalanine in association with increased tyrosine after UVB exposure. Hence, generation of H(2)O(2) by UVB can activate epidermal PAH leading to an increased L-tyrosine pool for melanogenesis.     

Efficient Plant Genomic and Viral DNA Isolation from Mature Leaf Midrib of Banana (Musa spp)

The genomic DNA isolation from mature leaf midrib is a tough job, because of the abundance of polysaccharides and secondary metabolites, which interferes with DNA isolation as well as polymerase chain reaction (PCR) studies. The leaf midrib of 3^rd leaf from 3-moths old, ex-vitro developing banana [AAA, Dwarf Cavendish-Basrai (Sindhri banana)] plants (healthy and BBTV infected) was grinded in liquid N₂. Exact 0.3 g of leaf midrib powder was washed with washing buffer (100 mM Tris-Cl, 5 mM EDTA, 0.35 M sorbitol, 1% 2-mercaptoethanol) then homogenized in 0.8 ml of three different pre-heated (60°C) DNA isolation buffers. Supernatant was extracted through phenol: chloroform:isoamyl alcohol (25:24, v/v), chloroform: isoamyl alcohol (24:1, v/v) and finally with chloroform (100%) one by one. Maximum yields were ranged from 49.33 and 27.73 μg mg ^?1 DNA with impurities 5.67 and 5.87 μg mg^?1 through buffer I, while 45.77 and 25.53 μg mg^?1 DNA with 6.13 and 6.16 μg mg^?1 impurities through buffer III from healthy and infected plants respectively. Best one RAPD was observed in all the DNA samples isolated with different buffers, while viral amplification was good in DNA isolated with buffer I and II, when 10 (RAPD) and 25 ng DNA (C ₁ gene) was used as a template in a reaction of 25 μl. Meanwhile, buffer II is limited for viral DNA isolation while buffer I (1M Tris-Cl, 5M NaCl, 2 % cTAB, 50mM EDTA, 1 % PVP, 0.2 % 2-mercaptoethanol) has dual capacity for plant and virus DNA isolation. This described protocol is economic in terms of times, labor and cost.     

Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.     

Molecular Epidemiology of Influenza A(H1N1)pdm09 Viruses from Pakistan in 2009-2010

Background

In early 2009, a novel influenza A(H1N1) virus that emerged in Mexico and United States rapidly disseminated worldwide. The spread of this virus caused considerable morbidity with over 18000 recorded deaths. The new virus was found to be a reassortant containing gene segments from human, avian and swine influenza viruses.

Methods/Results

The first case of human infection with A(H1N1)pdm09 in Pakistan was detected on 18^th June 2009. Since then, 262 laboratory-confirmed cases have been detected during various outbreaks with 29 deaths (as of 31^st August 2010). The peak of the epidemic was observed in December with over 51% of total respiratory cases positive for influenza. Representative isolates from Pakistan viruses were sequenced and analyzed antigenically. Sequence analysis of genes coding for surface glycoproteins HA and NA showed high degree of high levels of sequence identity with corresponding genes of regional viruses circulating South East Asia. All tested viruses were sensitive to Oseltamivir in the Neuraminidase Inhibition assays.

Conclusions

Influenza A(H1N1)pdm09 viruses from Pakistan form a homogenous group of viruses. Their HA genes belong to clade 7 and show antigenic profile similar to the vaccine strain A/California/07/2009. These isolates do not show any amino acid changes indicative of high pathogenicity and virulence. It is imperative to continue monitoring of these viruses for identification of potential variants of high virulence or drug resistance.     

Comparing glycaemic benefits of Active Versus passive lifestyle Intervention in kidney Allograft Recipients (CAVIAR): study protocol for a randomised controlled trial

BackgroundLifestyle modification is widely recommended to kidney allograft recipients post transplantation due to the cardiometabolic risks associated with immunosuppression including new-onset diabetes, weight gain and cardiovascular events. However, we have no actual evidence that undertaking lifestyle modification protects from any adverse outcomes post transplantation. The aim of this study is to compare whether a more proactive versus passive interventional approach to modify lifestyle is associated with superior outcomes post kidney transplantation.Methods/designWe designed this prospective, single-centre, open-label, randomised controlled study to compare the efficacy of active versus passive lifestyle intervention for kidney allograft recipients early post transplantation. A total of 130 eligible patients, who are stable, nondiabetic and between 3 and 24 months post kidney transplantation, will be recruited. Randomisation is being undertaken by random block permutations into passive (n = 65, leaflet guidance only) versus active lifestyle modification (n = 65, supervised intervention) over a 6-month period. Supervised intervention is being facilitated by two dietitians during the 6-month intervention period to provide continuous lifestyle intervention guidance, support and encouragement. Both dietitians are accredited with behavioural intervention skills and will utilise motivational aids to support study recruits randomised to active intervention. The primary outcome is change in abnormal glucose metabolism parameters after 6 months of comparing active versus passive lifestyle intervention. Secondary outcomes include changes in a wide array of cardiometabolic parameters, kidney allograft function and patient-reported outcome measures. Long-term tracking of patients via data linkage to electronic patient records and national registries will facilitate long-term comparison of outcomes after active versus passive lifestyle intervention beyond the 6-month intervention period.DiscussionThis is the first randomised controlled study to investigate the benefits of active versus passive lifestyle intervention in kidney allograft recipients for the prevention of abnormal cardiometabolic outcomes. In addition, this is the first example of utilising behaviour therapy intervention post kidney transplantation to achieve clinically beneficial outcomes, which has potential implications on many spheres of post-transplant care.

Trial registration

This study was registered with the Clinical Trials Registry on 27 August 2014 (ClinicalTrials.org Identifier: {"type":"clinical-trial","attrs":{"text":"NCT02233491","term_id":"NCT02233491"}}NCT02233491).     

Pharmacological characterization of the N-methyl-d-aspartate (NMDA) receptor-channel in rodent and dog brain and rat spinal cord using [3H]MK-801 binding

The biochemical and pharmacological properties of [³H]MK-801 binding to the N-methyl-d-aspartate (NMDA) receptor-channel in homogenates of mouse, guinea pig and dog brain, dog cerebral cortex and rat spinal cord were determined using radioligand binding techniques. Specific [³H]MK-801 binding increased linearily with increasing tissue concentration and in general represented 80–93% of the total binding at 6–8 nM radioligand concentration. [³H]MK-801 interacted with brain and spinal homogenates with high affinity. The dissociation constants (K _d ) for all tissues studied were similar ranging between 7.9 and 11.9 nM, whereas the maximum number of binding sites (B_max) showed a wide, tissue-dependent range (0.1–6.75 pmol/mg protein). The rank order of tissue enrichment was found to be as follows: mouse brain>>dog cerebral cortex>>dog brain>> guinea pig brain>>rat spinal cord. Specific [³H]MK-801 binding in rodent and dog brain, dog cerebral cortex and rat spinal cord exhibited a similar pharmacological profile 9correlation coefficients=0.93–0.99). The rank order of potency of unlabelled compounds competing for [³H]MK-801 binding was: (+)MK-801>(–)MK-801>phencyclidine>(–)cyclazocine>>(+)cyclazocine ketamine>(+)N-allyl-N-normetazocine>(–)N-allyl-N-normetazocine>(–)pentazocine>(+)pentazocine. NMDA, Kainate, quisqualate and several other compounds failed to inhibit [³H]MK-801 binding at 100 M. In modulation studies conducted on extensively washed dog cortex membranes, Mg²⁺ ions stimulated [³H]MK-801 binding at 10 M-1 mM (EC₅₀=91.5 M) and then inhibited the binding from 1 mM to 10 mM (IC₅₀=3.1 mM). Glycine stimulated [³H]MK-801 binding at 30 nM-1 mM (EC₅₀=256 nM). In contrast, Zn²⁺ ions inhibited the binding of [³H]MK-801 binding site exhibited similar pharmacological and biochemical properties. These data appear to suggest that the pharmacological profile of the NMDA-receptor-channel is species and tissue independent.     

Seroprevalence of Toxocara canis antibodies in humans in northern Jordan.

Sera of 699 individuals, aged between 5-24 years, from the Irbid area, Jordan, were tested for Toxocara canis antibodies using an ELISA-IgG test. Crude prevalence was 10.9% (76 of 699) but age-adjusted prevalence was 14.3%. The highest prevalence was observed in females aged 5-9 years, 23.3% (7 of 30), and males of 15-19 years of age, 19.5% (16 of 82). The lowest prevalence was observed in females aged 20-25 years, 5.2% (8 of 155). Significant differences (P < 0.05) between the prevalences of the toxocaral antibodies in males and females were observed in the age groups 5-9, 15-19 and 20-24 years. The trend of prevalence in relation to age was different according to sex.