The crystal structures of human S100B in the zinc- and calcium-loaded state at three pH values reveal zinc ligand swapping

S100B is a homodimeric zinc-, copper-, and calcium-binding protein of the family of EF-hand S100 proteins. Zn²⁺ binding to S100B increases its affinity towards Ca²⁺ as well as towards target peptides and proteins. Cu²⁺ and Zn²⁺ bind presumably to the same site in S100B. We determined the structures of human Zn²⁺- and Ca²⁺-loaded S100B at pH 6.5, pH 9, and pH 10 by X-ray crystallography at 1.5, 1.4, and 1.65 Å resolution, respectively. Two Zn²⁺ ions are coordinated tetrahedrally at the dimer interface by His and Glu residues from both subunits. The crystal structures revealed that ligand swapping occurs for one of the four ligands in the Zn²⁺-binding sites. Whereas at pH 9, the Zn²⁺ ions are coordinated by His15, His25, His 85′, and His 90′, at pH 6.5 and pH 10, His90′ is replaced by Glu89′. The results document that the Zn²⁺-binding sites are flexible to accommodate other metal ions such as Cu²⁺. Moreover, we characterized the structural changes upon Zn²⁺ binding, which might lead to increased affinity towards Ca²⁺ as well as towards target proteins. We observed that in Zn²⁺-Ca²⁺-loaded S100B the C-termini of helix IV adopt a distinct conformation. Zn²⁺ binding induces a repositioning of residues Phe87 and Phe88, which are involved in target protein binding. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Hormonal regulation of DNA synthesis in primary cultures of adult rat hepatocytes : Action of glucagon

Primary cultures of adult rat hepatocytes, grown in modified minimal essential medium (Eagle's) containing 10% calf serum, could be induced into DNA replication by combinations of epidermal growth factor (EGF), insulin and glucagon. The three hormones acted synergistically, and cells began entering DNA synthesis 48 h after hormone addition. The ability of the hormones to stimulate DNA synthesis was enhanced by plating cells at high cell concentrations or by conditioned medium, and was diminished by daily medium change. The contribution of glucagon to DNA synthesis was replaced by cAMP plus 1-methyl, 3-isobutyl xanthine or by adrenergic agents. Evidence is presented which suggests that all three hormones are required on the first day of culture, and that EGF and insulin are also required after the first day. This appears to be a useful system for studies on the hormonal initiation of growth in quiescent cells.     

Blood Glucose Measurement with Dextrostix and New Reflectance Meter

A new instrument, Eyetone, has been produced for use with a Dextrostix reagent strip for estimating blood sugar levels. It differs from the Dextrostix Reflectance Meter in having only one meter scale, a two-point calibration, and a range of measurement limited to 10-400 mg/100 ml. Results with the manufacturers'' original calibration were unsatisfactory, but when recalibrated the performance of the instrument gave a regression equation line close to the ideal. The three Eyetone instruments tested were comparable in their accuracy and functional stability. Packed cell volumes in the blood samples in the range of 30-50% had a negligible effect on the results. The Dextrostix-Eyetone method represents a quick and reliable alternative to conventional laboratory methods for blood sugar estimation. It is especially useful for patients outside hospital.     

Stimulation by glucagon of the incorporation of U-14C-labeled substrates into glucose by isolated hepatocytes from fed rats.

The effect of glucagon on the incorporation of U-14C-labeled lactate, pyruvate or alanine into glucose has been studied using isolated hepatocytes from livers of fed rats. Rates of incorporation into glucose were about the same as observed in perfused liver preparations provided precautions were taken to avoid depletion of certain metabolities by the preparative procedures. With each substrate, stimulation of the incorporation into glucose by a maximally effective concentration of glucagon (10 nM) was associated with about a 75% reduction in the substrate concentration required for a half-maximal rate and with about a 30% increase in maximum rate. Consequently, the hormone caused a substantial (2--4-fold) stimulation when any one of the above substrates was present at a near physiological concentration, but brought about only a relatively small stimulation (1.4-fold) when very high substrate concentrations were used. Provision of cytoplasmic reducing equivalents (by ethanol addition), or of precursor for acetyl-coenzyme A formation (by acetate addition)-stimulated incorporation of labeled alanine into glucose and their effects were additive with that of glucagon. This suggested that provision of either of these intermediates was not a means by which the hormone increased the incorporation of labeled substrate into glucose. NH4+ stimulated the incorporation of 20 mM [U-14C] lactate into glucose 2-fold, probably by promoting glutamate synthesis and thus enhancing the transamination of oxaloacetate to aspartate. Evidence was obtained to support the view that glucagon also increases glutamate production (presumably from endogenous protein). However, the stimulation of incorporation into glucose from 20 mM [U-14C] lactate by NH4+ plus glucagon was synergistic. This suggested that glucagon also stimulated the incorporation of labeled substrate into glucose by additional means. Stimulation of the incorporation of [U-14C] alanine into glucose by beta-hydroxybutyrate plus glucagon was also synergistic. This suggested that another action of glucagon may be to provide more intramitochondrial reducing potential.     

Traversing arbuscular mycorrhizal fungi and Pseudomonas fluorescens for carrot production under salinity

Carrot is a vital supply of dietary fiber, vitamins, and carotenoids, and it is also rich in antioxidants and minerals. Soil salinity significantly reduces the yield and quality of carrots. Mycorrhiza inoculum (AMF) is known to improve morphological and biochemical traits of vegetables even under saline conditions. But the role of AMF in combating soil salinity effect in carrot is not studied in detail. Therefore here, in the first set, carrot seeds are inoculated with microbes in a pot experiment under polyhouse condition. In total, we applied 7 treatments with different combinations of Mycorrhiza inoculum (Glomus mosseae (G_m) and Gigaspora gigantea (G_g)) and phosphate solubilizing bacteria (Pseudomonas fluroscens (P_f)). In pot experiment study the best two treatments were the combination of G_m + P_f + G_G and P_f + G_G. Both of these treatments were selected for validation under the open field conditions. Primarily, there seems to be a promising opportunity for AMF application to carrots under pot culture as well as under field trials because of promising effect towards morphological parameters, especially root weight, and disparities in nutrients and metabolites. Overall, our study highlights mycorrhizal fungi and other microbes' efficacy in achieving a successful carrot production under salinity stress.     

Toward a Healthier Iraq

Health care in Iraq is the sector most influenced by variables such as political, cultural, social, and economic environments. The current status of national security, the deterioration of infrastructure, difficulty in accessing clean water and sewage services in some areas, and the national levels of poverty, malnutrition, and social fragility are all factors that negatively affect the health of the Iraqi people [1].There is a real need for a solid national health policy that is able to match the available resources against the health needs of a growing population, as provided in the Constitution of Iraq and National Development Plan, and oriented to a set of strategies and objectives that can drive development of the Iraqi health sector in the future [2].Establishment of a fair and comprehensive national health system requires sustainable commitment to this policy by all stakeholders and partners in order to focus efforts and achieve synergy in the optimal investment of available resources and to provide health care services and attain maximum effectiveness and efficiency of systemic response to the health needs of the Iraqi people and society.     

Identification of progressors in osteoarthritis by combining biochemical and MRI-based markers

Introduction  

At present, no disease-modifying osteoarthritis drugs (DMOADS) are approved by the FDA (US Food and Drug Administration); possibly partly due to inadequate trial design since efficacy demonstration requires disease progression in the placebo group. We investigated whether combinations of biochemical and magnetic resonance imaging (MRI)-based markers provided effective diagnostic and prognostic tools for identifying subjects with high risk of progression. Specifically, we investigated aggregate cartilage longevity markers combining markers of breakdown, quantity, and quality.