Seasonal incidence of airborne fungal allergens in Riyadh,Saudi Arabia

Aerobiological studies to evaluate allergenic fungal spores in the atmosphere of Riyadh were conducted. Burkard personal volumetric sampler were operated as volumetric viable spore traps at two different sites (Al-Batha, a more developed area in the south and Al-Ulia, a less developed area in the north) in Riyadh. Twice a week samplings were carried out over a period of 12 month. The seasonal fluctuations of the most frequent fungi were plotted as major components. The dominant species at the two sites were members of the genera Alternaria, Aspergillus, Cladosporium, Penicillium and Ulocladium. Drechslera, Fusarium, Rhizopus and Stachybotrytis species were minor components or sporadic. Seasonal variations of the total colonies were significantly (p < 0.05) different. They showed higher concentrations in the winter season and the lowest in summer. The Al-Batha site was always higher in spore concentrations than the Al-Ulia site. The results provide valuable information for the diagnosis and prophylaxis of allergic diseases due to airborne fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli

The production of correctly folded protein in Escherichia coli is often challenging because of aggregation of the overexpressed protein into inclusion bodies. Although a number of general and protein-specific techniques are available, their effectiveness varies widely. We report a novel method for enhancing the solubility of overexpressed proteins. Presence of a dipeptide, glycylglycine, in the range of 100 mM to 1 M in the medium was found to significantly enhance the solubility (up to 170-fold) of the expressed proteins. The method has been validated using mycobacterial proteins, resulting in improved solubilization, which were otherwise difficult to express as soluble proteins in E. coli. This method can also be used to enhance the solubility of other heterologous recombinant proteins expressed in a bacterial system.     

Biochemical and crystallographic studies of the Met144Ala, Asp92Asn and His254Phe mutants of the nitrite reductase from Alcaligenes xylosoxidans provide insight into the enzyme mechanism

Dissimilatory nitrite reductase catalyses the reduction of nitrite (NO(2)(-)) to nitric oxide (NO). Copper-containing nitrite reductases contain both type 1 and type 2 Cu sites. Electron transfer from redox partners is presumed to be mediated via the type 1 Cu site and used at the catalytic type 2 Cu centre along with the substrate nitrite. At the type 2 Cu site, Asp92 has been identified as a key residue in substrate utilisation, since it hydrogen bonds to the water molecule at the nitrite binding site. We have also suggested that protons enter the catalytic site via Asp92, through a water network that is mediated by His254. The role of these residues has been investigated in the blue copper nitrite reductase from Alcaligenes xylosoxidans (NCIMB 11015) by a combination of point mutation, enzymatic activity measurement and structure determination.In addition, it has been suggested that the enzyme operates via an ordered mechanism where an electron is transferred to the type 2 Cu site largely when the second substrate nitrite is bound and that this is controlled via the lowering of the redox potential of the type 2 site when it is loaded with nitrite. Thus, a small perturbation of the type 1 Cu site should result in a significant effect on the activity of the enzyme. For this reason a mutation of Met144, which is the weakest ligand of the type 1 Cu, is investigated. The structures of H254F, D92N and M144A have been determined to 1.85 A, 1.9 A and 2.2 A resolution, respectively. The D92N and H254F mutants have negligible or no activity, while the M144A mutant has 30 % activity of the native enzyme. Structural and spectroscopic data show that the loss of activity in H254F is due to the catalytic site being occupied by Zn while the loss/reduction of activity in D92N/M144A are due to structural reasons. The D92N mutation results in the loss of the Asp92 hydrogen bond to the Cu-ligated water. Therefore, the ligand is no longer able to perform proton abstraction. Even though the loss of activity in H254F is due to lack of catalytic Cu, the mutation does cause the disruption of the water network, confirming its key role in proton channel. The structure of the H254F mutant is the first case where full occupancy Zn at the type 2 Cu site is observed, but despite the previously noted similarity of this site to the carbonic anhydrase catalytic site, no carbonic anhydrase activity is observed. The H254F and D92N mutant structures provide, for the first time, observation of surface Zn sites which may act as a Zn sink and prevent binding of Zn at the catalytic Cu site in the native enzyme.     

The N-terminal extension of rusticyanin is not responsible for its acid stability

The N-terminal extension of rusticyanin is a unique structural feature of this protein in the cupredoxin family and has been speculated to be responsible for the extreme acid stability of the protein. We have removed the 35 residues from the N-terminus and show that the resulting -35 mutant is insoluble in aqueous media above pH 5.0 and exists primarily in a hexameric form at lower pHs. Synchrotron radiation circular dichroism (SRCD) and solution X-ray scattering data indicate that much of the beta-sheet structure is retained in acidic solution and indeed there is a small but significant increase in the beta-sheet contribution. We suggest this to be a result of beta-sheet formation between the monomer interfaces. The mutant does not bind copper. These results provide evidence that the unique N-terminus of rusticyanin is not responsible for the acid stability of the hydrophobic beta-barrel core of the protein.     

Phosphorylation of serine 51 in initiation factor 2 alpha (eIF2 alpha) promotes complex formation between eIF2 alpha(P) and eIF2B and causes inhibition in the guanine nucleotide exchange activity of eIF2B

Phosphorylation of serine 51 residue on the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) inhibits the guanine nucleotide exchange (GNE) activity of eIF2B, presumably, by forming a tight complex with eIF2B. Inhibition of the GNE activity of eIF2B leads to impairment in eIF2 recycling and protein synthesis. We have partially purified the wild-type (wt) and mutants of eIF2alpha in which the serine 51 residue was replaced with alanine (51A mutant) or aspartic acid (51D mutant) in the baculovirus system. Analysis of these mutants has provided novel insight into the role of 51 serine in the interaction between eIF2 and eIF2B. Neither mutant was phosphorylated in vitro. Both mutants decreased eIF2alpha phosphorylation occurring in hemin and poly(IC)-treated reticulocyte lysates due to the activation of double-stranded RNA-dependent protein kinase (PKR). However, addition of 51D, but not 51A mutant eIF2alpha protein promoted inhibition of the GNE activity of eIF2B in hemin-supplemented rabbit reticulocyte lysates in which relatively little or no endogenous eIF2alpha phosphorylation occurred. The 51D mutant enhanced the inhibition in GNE activity of eIF2B that occurred in hemin and poly(IC)-treated reticulocyte lysates where PKR is active. Our results show that the increased interaction between eIF2 and eIF2B protein, occurring in reticulocyte lysates due to increased eIF2alpha phosphorylation, is decreased significantly by the addition of mutant 51A protein but not 51D. Consistent with the idea that mutant 51D protein behaves like a phosphorylated eIF2alpha, addition of this partially purified recombinant subunit, but not 51A or wt eIF2alpha, increases the interaction between eIF2 and 2B proteins in actively translating hemin-supplemented lysates. These findings support the idea that phosphorylation of the serine 51 residue in eIF2alpha promotes complex formation between eIF2alpha(P) and eIF2B and thereby inhibits the GNE activity of eIF2B.     

Effects of Porosity and Mixed Convection on MHD Two Phase Fluid Flow in an Inclined Channel

The present study deals with the flow and heat transfer analysis of two immiscible fluids in an inclined channel embedded in a porous medium. The channel is divided in two phases such that a third grade fluid occupies the phase I and a viscous fluid occupies the phase II. Both viscous and third grade fluids are electrically conducting. A constant magnetic field is imposed perpendicular to the channel walls. The mathematical model is developed by using Darcy''s and modified Darcy''s laws for viscous and third grade fluids respectively. The transformed ordinary differential equations are solved numerically using a shooting method. The obtained results are presented graphically and influence of emerging parameters is discussed in detail.     

Identifying Elements of Patient-Centered Care in Underserved Populations: A Qualitative Study of Patient Perspectives

Patient-centered care is an important goal in the delivery of healthcare. However, many patients do not engage in preventive medical care. In this pilot study, we conducted twenty in depth, semi-structured qualitative interviews at the University of Illinois at Chicago Health Sciences campus in a four month time frame. Many patients were underserved and underinsured, and we wanted to understand their experiences in the healthcare system. Using content analysis, several themes emerged from the interview data. Participants discussed the need for empathy and rapport with their providers. They identified provider behaviors that fostered a positive clinical relationship, including step-by step explanations of procedures, attention to body language and clinic atmosphere, and appropriate time management. Participants identified cost as the most common barrier to engaging in preventive care and discussed children and social support as motivating factors. A long-term relationship with a provider was an important motivator for preventive care, suggesting that the therapeutic alliance was essential to many patients. Conversely, many participants discussed a sense of dehumanization in the healthcare system, reporting that their life circumstances were overlooked, or that they were judged based on insurance status or ethnicity. We discuss implications for provider training and healthcare delivery, including the importance of patient-centered medical homes.     

Association of non-heterocystous cyanobacteria with crop plants

Cyanobacteria have the ability to form associations with organisms from all domains of life, notably with plants, which they provide with fixed nitrogen, among other substances. This study was aimed at developing artificial associations between non-heterocystous cyanobacteria and selected crop plants. We isolated several non-heterocystous cyanobacteria from various rice fields. The cultures were tested for their capacity to produce the plant hormone indole-3-acetic acid (IAA), and the possible role of IAA in the association of cyanobacteria with seedling roots was evaluated. Axenic cultures were co-inoculated with 10-day-old plant seedlings of Triticum aestivum, Vigna radiata and Pisum sativum and incubated for 1 week. Cyanobacterial association with the roots of these seedlings was quantified by measuring chlorophyll-a. Cyanobacterial association with the roots was observed by light microscopy as well as by confocal laser scanning microscopy (CLSM). Based on sequence analysis of the 16S rRNA gene, the isolates were identified as Synechocystis sp., Chroococcidiopsis sp., Leptolyngbya sp., and Phormidium sp. CLSM observations revealed the intimate association of cyanobacteria with the seedling roots as well as invasion of the roots and root cells. Strains producing IAA were more efficient in the colonization of the roots than those that lacked this ability. IAA-producing cyanobacteria possess a tryptophan-dependent pathway, and these cyanobacteria showed IAA synthesis activity in the presence of roots in media lacking tryptophan. Based on the results of this study, we conclude that non-heterocystous cyanobacteria also have the potential for use in agriculture to improve the growth and yield of crop plants that do not naturally form associations with cyanobacteria.     

Chromium-resistant bacteria and cyanobacteria: impact on Cr(VI) reduction potential and plant growth

Two chromium-resistant bacterial strains, Bacillus cereus S-6 and Ochrobactrum intermedium CrT-1, and two cyanobacterial strains, Oscillatoria sp. and Synechocystis sp., were used in this study. At initial chromate concentrations of 300 and 600 μg K₂CrO₄ mL⁻¹, and an inoculum size of 9.6×10⁷ cells mL⁻¹, B. cereus S-6 completely reduced Cr(VI), while O. intermedium CrT-1 reduced Cr(VI) by 98% and 70%, respectively after 96 h. At 100 μg K₂CrO₄ mL⁻¹, Synechocystis sp. MK(S) and Oscillatoria sp. BJ2 reduced 62.1% and 39.9% of Cr(VI), respectively, at 30°C and pH 8. Application of hexavalent chromate salts adversely affected wheat seedling growth and anatomical characters. However, bacterial inoculation alleviated the toxic effects, as reflected by significant improvements in growth as well as anatomical parameters. Cyanobacterial strains also led to some enhancement of various growth parameters in wheat seedlings.

     

Glucan, water dikinase activity stimulates breakdown of starch granules by plastidial beta-amylases

Glucan phosphorylating enzymes are required for normal mobilization of starch in leaves of Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), but mechanisms underlying this dependency are unknown. Using two different activity assays, we aimed to identify starch degrading enzymes from Arabidopsis, whose activity is affected by glucan phosphorylation. Breakdown of granular starch by a protein fraction purified from leaf extracts increased approximately 2-fold if the granules were simultaneously phosphorylated by recombinant potato glucan, water dikinase (GWD). Using matrix-assisted laser-desorption ionization mass spectrometry several putative starch-related enzymes were identified in this fraction, among them beta-AMYLASE1 (BAM1; At3g23920) and ISOAMYLASE3 (ISA3; At4g09020). Experiments using purified recombinant enzymes showed that BAM1 activity with granules similarly increased under conditions of simultaneous starch phosphorylation. Purified recombinant potato ISA3 (StISA3) did not attack the granular starch significantly with or without glucan phosphorylation. However, starch breakdown by a mixture of BAM1 and StISA3 was 2 times higher than that by BAM1 alone and was further enhanced in the presence of GWD and ATP. Similar to BAM1, maltose release from granular starch by purified recombinant BAM3 (At4g17090), another plastid-localized beta-amylase isoform, increased 2- to 3-fold if the granules were simultaneously phosphorylated by GWD. BAM activity in turn strongly stimulated the GWD-catalyzed phosphorylation. The interdependence between the activities of GWD and BAMs offers an explanation for the severe starch excess phenotype of GWD-deficient mutants.