Mineralization of calcium phosphate crystals in starch template inducing a brushite kidney stone biomimetic composite

Dicalcium phosphate dihydrate (brushite) and octacalcium phosphate (OCP) crystals are precursors of hydroxyapatite (HAp) for tooth enamel, dentine, and bones formation in living organisms. Here, we introduce a new method for biomimicking brushite and OCP in starch using single and double diffusion techniques. Brushite and OCP crystals were grown by precipitation in starch after gelation. The obtained materials were analyzed by infrared spectroscopy (IR), scanning electron microscopy (SEM), X-ray diffraction (XRD), and confocal laser scanning microscopy (CLSM). IR spectra demonstrate starch inclusion by peak shifts in the 2900–3500 cm–1 region. SEM showed two different morphologies: plate-shaped and needle-like crystals. Calcium phosphate/starch aggregates bear strong resemblance to prismatic brushite kidney stones. This may open up a clue to understand the mechanism of kidney stone formation.

Association of folate receptor (FOLR1, FOLR2, FOLR3) and reduced folate carrier (SLC19A1) genes with meningomyelocele.

BACKGROUND: Meningomyelocele (MM) results from lack of closure of the neural tube during embryologic development. Periconceptional folic acid supplementation is a modifier of MM risk in humans, leading toan interest in the folate transport genes as potential candidates for association to MM. METHODS: This study used the SNPlex Genotyping (ABI, Foster City, CA) platform to genotype 20 single polymorphic variants across the folate receptor genes (FOLR1, FOLR2, FOLR3) and the folate carrier gene (SLC19A1) to assess their association to MM. The study population included 329 trio and 281 duo families. Only cases with MM were included. Genetic association was assessed using the transmission disequilibrium test in PLINK. RESULTS: A variant in the FOLR2 gene (rs13908), three linked variants in the FOLR3 gene (rs7925545, rs7926875, rs7926987), and two variants in the SLC19A1 gene (rs1888530 and rs3788200) were statistically significant for association to MM in our population. CONCLUSION: This study involved the analyses of selected single nucleotide polymorphisms across the folate receptor genes and the folate carrier gene in a large population sample. It provided evidence that the rare alleles of specific single nucleotide polymorphisms within these genes appear to be statistically significant for association to MM in the patient population that was tested.

Role of acetyl-phosphate in activation of the Rrp2-RpoN-RpoS pathway in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ(54)-σ(S) sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis.

Regulation of pyridoxal $5\prime$-phosphate biosynthesis in {\it Escherichia coli\/} K-12

Vitamin B$\sb6$ (or pyridoxal 5$\sp\prime$-phosphate, PLP) is an essential, ubiquitous coenzyme that affects many aspects of amino acid and cellular metabolism in all organisms. The goal of this thesis is to examine the regulation of PLP biosynthesis in Escherichia coli K-12. First, PdxH oxidase is a PLP biosynthetic enzyme, which uses molecular oxygen as an electron acceptor under aerobic assay conditions. To test if facultative anaerobic E. coli uses another enzyme to replace the function of PdxH oxidase anaerobically, suppressors of a pdxH null mutant were isolated anaerobically after 2-aminopurine or spontaneous mutagenesis. Only one specific bypass mutation in another PLP biosynthetic gene pdxJ was found, suggesting that PdxH oxidase is able to function anaerobically and PdxT utilizes D-1-deoxyxyulose as a substrate. Second, regulation of the serC (pdxF)-aroA operon, which is involved the biosynthesis of L-serine, PLP and aromatic compounds was examined. A serC (pdxF) single gene transcript and a serC (pdXf)-aroA cotranscript initiated at P$\sb{serC\ (pdxF)}$ upstream of serC (pdxF) were detected. The expression of the operon is activated by leucine responsive regulatory protein (LRP) and repressed by cAMP receptor protein-cAMP complex (CRP$\cdot$cAMP) at the transcriptional level. LRP activates the operon by directly binding to the upstream consensus box. Binding of CRP$\cdot$cAMP to the upstream CRP box diminishes the activation effect of LRP. However, deletion of the CRP box did not affect the repression of CRP$\cdot$cAMP, suggesting that CRP$\cdot$cAMP may repress the operon indirectly by stimulating the activity or level of an unidentified repressor. The overall effect of this regulation is to maximize the expression of the operon when the cells are growing in minimal-glucose medium. In addition, the binding and the transcription of P$\sb{serC\ (pdxF)}$ by RNA polymerase require a supercoiled circular DNA, indicating that DNA supercoiling affects the transcription of the operon. Third, regulation of another PLP biosynthetic gene gapB was also examined. gapB is activated by CRP$\cdot$cAMP and repressed by catabolic repressor activator protein (CRA). However, the activation of CRP$\cdot$cAMP is epistatic to the repression of CRA. Due to the CRA repression, gapB was expressed at a low level in all the media tested, suggesting that it may be the rate-limiting step of PLP biosynthesis. In summary, unlike genes in many biosynthetic pathways, PLP biosynthetic genes are regulated by global regulators that are important for carbon and amino acid metabolism, instead of the end product(s) of the pathway. ^

The auxin influx carrier is essential for correct leaf positioning

Auxin is of vital importance in virtually every aspect of plant growth and development, yet, even after almost a century of intense study, major gaps in our knowledge of its synthesis, distribution, perception, and signal transduction remain. One unique property of auxin is its polar transport, which in many well-documented cases is a critical part of its mode of action. Auxin is actively transported through the action of both influx and efflux carriers. Inhibition of polar transport by the efflux inhibitor N-1-naphthylphthalamic acid (NPA) causes a complete cessation of leaf initiation, a defect that can be reversed by local application of the auxin, indole-3-acetic acid (IAA), to the responsive zone of the shoot apical meristem. In this study, we address the role of the auxin influx carrier in the positioning and outgrowth of leaf primordia at the shoot apical meristem of tomato. By using a combination of transport inhibitors and synthetic auxins, we demonstrate that interference with auxin influx has little effect on organ formation as such, but prevents proper localization of leaf primordia. These results suggest the existence of functional auxin concentration gradients in the shoot apical meristem that are actively set up and maintained by the action of efflux and influx carriers. We propose a model in which efflux carriers control auxin delivery to the shoot apical meristem, whereas influx and efflux carriers regulate auxin distribution within the meristem.

High-resolution dynamic computer simulation analysis of the behavior of sample components with pI values outside the pH gradient established by carrier ampholyte CIEF

The behavior of sample components whose pI values are outside the pH gradient established by 101 hypothetical biprotic carrier ampholytes covering a pH 6-8 range was investigated by computer simulation under constant current conditions with concomitant constant electroosmosis toward the cathode. Data obtained with the sample being applied between zones of carrier ampholytes and on the anodic side of the carrier ampholytes were studied and found to evolve into zone structures comprising three regions between anolyte and catholyte. The focusing region with the pH gradient is bracketed by two isotachopheretic zone structures comprising selected sample and carrier components as isotachophoretic zones. The isotachophoretic structures electrophoretically migrate in opposite direction and their lengths increase with time due to the gradual isotachophoretic decay at the pH gradient edges. Due to electroosmosis, however, the overall pattern is being transported toward the cathode. Sample components whose pI values are outside the established pH gradient are demonstrated to form isotachophoretic zones behind the leading cation of the catholyte (components with pI values larger than 8) and the leading anion of the anolyte (components with pI values smaller than 6). Amphoteric compounds with appropriate pI values or nonamphoteric components can act as isotachophoretic spacer compounds between sample compounds or between the leader and the sample with the highest mobility. The simulation data obtained provide for the first time insight into the dynamics of amphoteric sample components that do not focus within the established pH gradient.

Characterization of choline uptake in Trypanosoma brucei and involvement of a mitochondrial carrier in resistance to choline analogs

Choline is an essential nutrient for eukaryotic cells, where it is used as precursor for the synthesis of choline-­containing phospholipids, such as phosphatidylcholine (PC). Our experiments showed – for the first time – that Trypanosoma brucei, the causative agent of human African sleeping sickness, is able to take up choline from the culture medium to use for PC synthesis, indicating that trypanosomes express a transporter for choline at the plasma membrane. Further characterization in procyclic and bloodstream forms revealed that choline uptake is saturable and can be inhibited by HC-3, a known inhibitor of choline uptake in mammalian cells. To obtain additional insights on choline uptake and metabolism, we investigated the effects of choline-analogs that were previously shown to be toxic for T. brucei parasites in culture. Interestingly, we found that all analogs tested effectively inhibited choline uptake into both bloodstream and procyclic form parasites. Subsequently, selected compounds were used to search for possible candidate genes encoding choline transporters in T. brucei, using an RNAi library in bloodstream forms. We identified a protein belonging to the mitochondrial carrier family, previously annotated as TbMCP14, as prime candidate. Down‐regulation of TbMCP14 by RNAi prevented drug-­induced loss of mitochondrial membrane potential and conferred 8­‐fold resistance of T. brucei bloodstream forms to choline analogs. Conversely, over‐expression of the carrier increased parasite susceptibility more than 13-­fold. However, subsequent experiments demonstrated that TbMCP14 was not involved in metabolism of choline. Instead, growth curves in glucose‐depleted medium using RNAi or knock‐out parasites suggested that TbMCP14 is involved in metabolism of amino acids for energy production. Together, our data demonstrate that the identified member of the mitochondrial carrier family is involved in drug uptake into the mitochondrion and has a vital function in energy production in T. brucei.

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Poly(ethylene oxide)-b-poly(3-sulfopropyl methacrylate) block copolymers for calcium phosphate mineralization and biofilm inhibition.

Poly(ethylene oxide) (PEO) has long been used as an additive in toothpaste, partly because it reduces biofilm formation on teeth. It does not, however, reduce the formation of dental calculus or support the remineralization of dental enamel or dentine. The present article describes the synthesis of new block copolymers on the basis of PEO and poly(3-sulfopropyl methacrylate) blocks using atom transfer radical polymerization. The polymers have very large molecular weights (over 10(6) g/mol) and are highly water-soluble. They delay the precipitation of calcium phosphate from aqueous solution but, upon precipitation, lead to relatively monodisperse hydroxyapatite (HAP) spheres. Moreover, the polymers inhibit the bacterial colonization of human enamel by Streptococcus gordonii, a pioneer bacterium in oral biofilm formation, in vitro. The formation of well-defined HAP spheres suggests that a polymer-induced liquid precursor phase could be involved in the precipitation process. Moreover, the inhibition of bacterial adhesion suggests that the polymers could be utilized in caries prevention.

Anesthesia in patients with glucose-6-phosphate dehydrogenase deficiency: case report and perioperative anesthesiologic management

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a frequent congenital human enzyme defect, is the most frequent cause of hemolytic anemia triggered by drugs or infectious diseases. Drugs which induce acute hemolysis in patients with G6PD deficiency are often used in anesthesia and perioperative pain therapy. Considering the fact that patients from geographic regions with a high prevalence of the disease are often treated in European hospitals, special attention should be paid to this problem. We report a case of a 30-year-old female patient with favism and review the disease and anesthesia-related implications.

Phosphate disorders: hyperphosphatemia or pseudohyperphosphatemia?

We report the case of a 79 year old woman presenting with progressive confusion and drowsiness. Renal insufficiency with hyperkalemia as well as hypercalcemia and severe hyperphosphatemia were diagnosed. Renal insufficiency improved with treatment. However, hyperphosphatemia persisted without apparent explanation. We discuss possible causes of hyper- and pseudohyperphosphatemia. Specifically, phosphate analysis may be disturbed by the paraproteins in patients with multiple myeloma, resulting in pseudohyperphosphatemia. We review the standard laboratory phosphate measurement and the mechanisms of interference with paraproteins.