Abdulrahman M. Alsenaidy

Oxidative stress and antioxidant status in Saudi asthmatic patients.

OBJECTIVES Asthma is a chronic inflammatory airway disorder associated with recruitment of inflammatory cells. This study aims to clarify the role of oxidative stress and antioxidant status in the deterioration accompanied asthma. DESIGN AND METHODS Vitamin E, Vitamin C, superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GSH), total antioxidant status together with the concentrations of lipid peroxides, total nitrates and oxidative DNA damage (8-oxodeoxyguanine) were determined in plasma or whole blood of 47 Saudi asthmatic patients and compared to age-matching control samples. RESULTS The present study showed that asthmatic patients have significantly decreased levels of GSH, α-tocopherol, GPx, total antioxidant status and higher levels of SOD, lipid peroxides, total nitrate and 8-oxo-dG. Vitamin C recorded more or less similar levels in both groups. CONCLUSION Alteration of the selected measured parameters confirms that oxidative stress and defective antioxidant status could represent the primary causative factor in the pathogenesis of asthma.

Receptor-G protein interaction studied by bioluminescence resonance energy transfer: lessons from protease-activated receptor 1.

Since its development, the bioluminescence resonance energy transfer (BRET) approach has been extensively applied to study G protein-coupled receptors (GPCRs) in real-time and in live cells. One of the major aspects of GPCRs investigated in considerable details is their physical coupling to the heterotrimeric G proteins. As a result, new concepts have emerged, but few questions are still a matter of debate illustrating the complexity of GPCR-G protein interactions and coupling. Here, we summarized the recent advances on our understanding of GPCR-G protein coupling based on BRET approaches and supported by other FRET-based studies. We essentially focused on our recent studies in which we addressed the concept of preassembly vs. the agonist-dependent interaction between the protease-activated receptor 1 (PAR1) and its cognate G proteins. We discussed the concept of agonist-induced conformational changes within the preassembled PAR1-G protein complexes as well as the critical question how the multiple coupling of PAR1 with two different G proteins, Gαi1 and Gα12, but also β-arrestin 1, can be regulated.

Co-Circulation of 72bp Duplication Group A and 60bp Duplication Group B Respiratory Syncytial Virus (RSV) Strains in Riyadh, Saudi Arabia during 2014

Respiratory syncytial virus (RSV) is an important viral pathogen of acute respiratory tract infection (ARI). Limited data are available on molecular epidemiology of RSV from Saudi Arabia. A total of 130 nasopharyngeal aspirates were collected from children less than 5 years of age with ARI symptoms attending the Emergency Department at King Khalid University Hospital and King Fahad Medical City, Riyadh, Saudi Arabia between October and December, 2014. RSV was identified in the 26% of the hospitalized children by reverse transcriptase PCR. Group A RSV (77%) predominated during the study as compared to group B RSV (23%). The phylogenetic analysis of 28 study strains clustered group A RSV in NA1 and ON1 genotypes and group B viruses in BA (BA9) genotype. Interestingly, 26% of the positive samples clustered in genotypes with duplication in the G protein gene (ON1 for group A and BA for group B). Both the genotypes showed enhanced O-linked glycosylation in the duplicated region, with 10 and 2 additional sites in ON1 and BA respectively. Selection pressure analysis revealed purifying selection in both the ON1 and BA genotypes. One codon each in the ON1 (position 274) and BA genotypes (position 219) were positively selected and had high entropy values indicating variations at these amino acid positions. This is the first report describing the presence of ON1 genotype and the first report on co-circulation of two different genotypes of RSV with duplication in the G protein gene from Saudi Arabia. The clinical implications of the simultaneous occurrence of genotypes with duplication in G protein gene in a given population especially in the concurrent infections should be investigated in future. Further, the ongoing surveillance of RSV in this region will reveal the evolutionary trajectory of these two genotypes with duplication in G protein gene from largest country in the Middle East.

Remarkable extension of PAI-1 half-life surprisingly brings no changes to its structure.

Plasminogen activator inhibitor type 1 (PAI-1) is a serpin protein, a natural inhibitor of urokinase (uPA) and tissue plasminogen activators (tPA). By inhibiting uPA it can block growth of the cancer tumors by suppressing angiogenesis, while when acting on tPA in the blood it can avert conversion of plasminogen to plasmin preventing lysis of the clot. Furthermore, blocking PAI-1 activity can protect against thrombosis. Thus PAI-1 makes great impact on human homeostasis and is desirable for clinical application. Wild-type PAI-1 (wt-PAI-1) has a short span of activity with a t1/2 of ~2 h, being spontaneously converted into a latent form. An enormous effort has been made to create a more stable molecule with >600 PAI-1 variants constructed to study its structure-function relationship. In the present study, we evaluate the structure of the active recombinant VLHL-PAI-1 (very long half life, active >700 h) which is glycosylated similarly to wt-PAI-1 at N232 and N288, with the extended reactive center loop, intact engineered -S-S-bridge (Q174C, G323C) that precludes latency without affecting structure, and can be controlled by a reducing agent to terminate activity at will. We have already proven its usefulness to control cancer in human cancer cells, as well as preventing clot lysis in human whole blood and plasma and in a mouse model. Our results demonstrate the potential therapeutic applications (topical or systemic) of this protein in the treatment of cancer, for the trauma patients to ward off an excessive blood loss, or for people with the PAI-1 deficiency, especially during surgery.

Differential Effects of Camel Milk on Insulin Receptor Signaling – Toward Understanding the Insulin-Like Properties of Camel Milk

Previous studies on the Arabian camel (Camelus dromedarius) showed beneficial effects of its milk reported in diverse models of human diseases, including a substantial hypoglycemic activity. However, the cellular and molecular mechanisms involved in such effects remain completely unknown. In this study, we hypothesized that camel milk may act at the level of human insulin receptor (hIR) and its related intracellular signaling pathways. Therefore, we examined the effect of camel milk on the activation of hIR transiently expressed in human embryonic kidney 293 (HEK293) cells using bioluminescence resonance energy transfer (BRET) technology. BRET was used to assess, in live cells and real-time, the physical interaction between hIR and insulin receptor signaling proteins (IRS1) and the growth factor receptor-bound protein 2 (Grb2). Our data showed that camel milk did not promote any increase in the BRET signal between hIR and IRS1 or Grb2 in the absence of insulin stimulation. However, it significantly potentiated the maximal insulin-promoted BRET signal between hIR and Grb2 but not IRS1. Interestingly, camel milk appears to differentially impact the downstream signaling since it significantly activated ERK1/2 and potentiated the insulin-induced ERK1/2 but not Akt activation. These observations are to some extent consistent with the BRET data since ERK1/2 and Akt activation are known to reflect the engagement of Grb2 and IRS1 pathways, respectively. The preliminary fractionation of camel milk suggests the peptide/protein nature of the active component in camel milk. Together, our study demonstrates for the first time an allosteric effect of camel milk on insulin receptor conformation and activation with differential effects on its intracellular signaling. These findings should help to shed more light on the hypoglycemic activity of camel milk with potential therapeutic applications.

Optical Analysis of Zinc Oxide Quantum Dots with Bovine Serum Albumin and Bovine Hemoglobin

Quantum dots (QDs) are widely used in medical, industrial, and household applications owing to their excellent biological property. For its wide medical application, the biocompatibility of QDs is an important aspect of research. The aim of the present study was to synthesize zinc oxide quantum dots (ZnO-QDs) and to investigate the interaction with bovine serum albumin (BSA) and bovine hemoglobin (BHb) using fluorescence quenching method and circular dichroism (CD). The study suggests that the electrostatic force of attraction favors the adsorption of BSA onto ZnO-QDs. The fluorescence quenching of BSA and BHb using QD indicates the formation of QDs-BSA complexes. The CD spectra also showed the changes in secondary structure of proteins by interacting with QDs.

Synthetic food additive dye “Tartrazine” triggers amorphous aggregation in cationic myoglobin

Protein aggregation, a characteristic of several neurodegenerative diseases, displays vast conformational diversity from amorphous to amyloid-like aggregates. In this study, we have explored the interaction of tartrazine with myoglobin protein at two different pHs (7.4 and 2.0). We have utilized various spectroscopic techniques (turbidity, Rayleigh light scattering (RLS), intrinsic fluorescence, Congo Red and far-UV CD) along with microscopy techniques i.e. atomic force microscopy (AFM) and transmission electron microscopy (TEM) to characterize the tartrazine-induced aggregation in myoglobin. The results showed that higher concentrations of tartrazine (2.0-10.0mM) induced amorphous aggregation in myoglobin at pH 2.0 via electrostatic interactions. However, tartrazine was not able to induce aggregation in myoglobin at pH 7.4; because of strong electrostatic repulsion between myoglobin and tartrazine at this pH. The tartrazine-induced amorphous aggregation process is kinetically very fast, and aggregation occurred without the formation of a nucleus. These results proposed that the electrostatic interaction is responsible for tartrazine-induced amorphous aggregation. This study may help in the understanding of mechanistic insight of aggregation by tartrazine.

Unveiling the stimulatory effects of tartrazine on human and bovine serum albumin fibrillogenesis: Spectroscopic and microscopic study

Amyloid fibrils are playing key role in the pathogenesis of various neurodegenerative diseases. Generally anionic molecules are known to induce amyloid fibril in several proteins. In this work, we have studied the effect of anionic food additive dye i.e., tartrazine (TZ) on the amyloid fibril formation of human serum albumins (HSA) and bovine serum albumin (BSA) at pHs7.4 and 3.5. We have employed various biophysical methods like, turbidity measurements, Rayleigh Light Scattering (RLS), Dynamic Light Scattering (DLS), intrinsic fluorescence, Congo red assay, far-UV CD, transmission electron microscopy (TEM) and atomic force microscopy (AFM) to decipher the mechanism of TZ-induce amyloid fibril formation in both the serum albumins at pHs7.4 and 3.5. The obtained results suggest that both the albumins forms amyloid-like aggregates in the presence of 1.0 to 15.0mM of TZ at pH3.5, but no amyloid fibril were seen at pH7.4. The possible cause of TZ-induced amyloid fibril formation is electrostatic and hydrophobic interaction because sulfate group of TZ may have interacted electrostatically with positively charged amino acids of the albumins at pH3.5 and increased protein-protein and protein-TZ interactions leading to amyloid fibril formation. The TEM, RLS and DLS results are suggesting that BSA forms bigger size amyloids compared to HSA, may be due to high surface hydrophobicity of BSA.

Optimization of expression and purification of HSPA6 protein from Camelus dromedarius in E. coli.

The HSPA6, one of the members of large family of HSP70, is significantly up-regulated and has been targeted as a biomarker of cellular stress in several studies. Herein, conditions were optimized to increase the yield of recombinant camel HSPA6 protein in its native state, primarily focusing on the optimization of upstream processing parameters that lead to an increase in the specific as well as volumetric yield of the protein. The results showed that the production of cHSPA6 was increased proportionally with increased incubation temperature up to 37 °C. Induction with 10 μM IPTG was sufficient to induce the expression of cHSPA6 which was 100 times less than normally used IPTG concentration. Furthermore, the results indicate that induction during early to late exponential phase produced relatively high levels of cHSPA6 in soluble form. In addition, 5 h of post-induction incubation was found to be optimal to produce folded cHSPA6 with higher specific and volumetric yield. Subsequently, highly pure and homogenous cHSPA6 preparation was obtained using metal affinity and size exclusion chromatography. Taken together, the results showed successful production of electrophoretically pure recombinant HSPA6 protein from Camelus dromedarius in Escherichia coli in milligram quantities from shake flask liquid culture.

Effect of trifluoroethanol on α-crystallin: folding, aggregation, amyloid, and cytotoxicity analysis

α‐Crystallin, a member of small heat shock proteins, is the major structural protein within the eye lens and is believed to play an exceptional role in the stability of lens proteins and its transparency. In the current manuscript, we have investigated the effect of an organic solvent, trifluoroethanol (TFE), on the structure and function of α‐crystallin isolated from camel eye lens. Incubation of this protein with TFE changed the secondary and tertiary structures, which resulted in the aggregation of α‐crystallin as evidenced by intrinsic fluorescence, Rayleighs scattering, Thioflavin T assay, and circular dichroism spectroscopic studies. The treatment with different concentrations of TFE led to increased exposure of hydrophobic domains of α‐crystallin, which was observed by 8‐anilino 1‐napthalene sulfonic acid extrinsic fluorescence assay. These results clearly indicate that TFE induced significant changes in the secondary and tertiary structures of α‐crystallin, leading to aggregation and amyloid formation. Furthermore, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay established the cytotoxicity of the aggregated α‐crystallin towards HepG2 cell lines through reactive oxygen species production. In conclusion, α‐crystallin protein was found to be susceptible to conformational changes by TFE, suggesting that α‐crystallin, although basically acting like a heat shock protein and functionally displaying chaperone‐like activity, might capitulate to change in lens environment induced by diseased conditions or age‐related changes, resulting in cataract formation. Copyright