Essential role of the metal-ion in the IPM-assisted domain closure of 3-isopropylmalate dehydrogenase

X-ray structures of 3-isopropylmalate dehydrogenase (IPMDH) do not provide sufficient information on the role of the metal-ion in the metal-IPM assisted domain closure. Here solution studies were carried out to test its importance. Small-angle X-ray scattering (SAXS) experiments with the Thermus thermophilus enzyme (complexes with single substrates) have revealed only a very marginal (0-5%) extent of domain closure in the absence of the metal-ion. Only the metal-IPM complex, but neither the metal-ion nor the free IPM itself, is efficient in stabilizing the native protein conformation as confirmed by denaturation experiments with Escherichia coli IPMDH and by studies of the characteristic fluorescence resonance energy transfer (FRET) signal (from Trp to bound NADH) with both IPMDHs. A possible atomic level explanation of the metal-effect is given.     

Expression Levels of the ABCG2 Multidrug Transporter in Human Erythrocytes Correspond to Pharmacologically Relevant Genetic Variations

We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.     

Rhizome-associated bacterial communities of healthy and declining reed stands in Lake Velencei,Hungary

Lake Velencei is a shallow soda lake with extensive reed coverage. In this study, the bacterial communities of reed (Phragmites australis (Cav.) Trin. ex Steudel) rhizomes from healthy and declining stands were compared. Inner and outer rhizome surfaces were sampled. Samples were plated and isolated in September 1998 and June 1999. Phenotypic data of 371 bacterial strains were used for cluster analysis. Identification of phena was based on partial 16S rDNA sequence analysis of representative strains. Healthy reed stand rhizomes in fall 1998 were dominantly colonised by facultatively fermentative organisms, like Erwinia billingiae, Aeromonas sobria, Pantoea agglomerans, and Pseudomonas azotoformans. In the June 1999 sample, mainly Kocuria rosea and various Bacillus spp. dominated. In declining stands of September 1998, a saprotrophic community was found: Acinetobacter spp., Aeromonas hydrophila, Curtobacterium luteum, Agrobacterium vitis, and two further groups representing presumably new taxa. In June 1999, reed rhizomes were colonised by Kocuria rosea, but Dietzia maris and Bacillus cohnii could be isolated as well. Healthy and declining reed stand rhizomes can be distinguished based on the culturable bacterial community. No obligately plant pathogenic bacteria were found, however the possibility of a local, opportunistic bacterial invasion can not be ruled out (e.g. Curtobacterium). The presence of potentially beneficial bacterial species was demonstrated in the healthy reed rhizome rhizosphere (e.g. Pseudomonas azotoformans, Pantoea agglomerans).

     

Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely related 3beta-hydroxyl sterols, and they reacted with human lipoproteins VLDL, LDL, and HDL. They bound, usually with low avidity, to live human or murine lymphocyte and monocyte-macrophage cell lines, which was enhanced substantially by a moderate papain digestion of the cell surface, removing some protruding extracellular protein domains. Cell-bound ACHAs strongly colocalized with markers of cholesterol-rich lipid rafts and caveolae at the cell surface and intracellularly with markers of the endoplasmic reticulum and Golgi complex. These data suggest that these IgG ACHAs may serve as probes of clustered cholesterol (e.g., different lipid rafts) in live cells and thus may also have immunomodulatory potential.     

Secreted key regulators (Fgf1, Bmp4, Gdf3) are expressed by PAC1-immunopositive retinal ganglion cells in the postnatal rat retina

Identified as a member of the secretin/glucagon/VIP superfamily, pituitary adenylate cyclase-activating polypeptide (PACAP1-38) has been recognized as a hormone, neurohormone, transmitter, trophic factor, and known to be involved in diverse and multiple developmental processes. PACAP1-38 was reported to regulate the production of important morphogens (Fgf1, Bmp4, Gdf3) through PAC1-receptor in the newborn rat retina. To follow up, we aimed to reveal the identity of retinal cells responsible for the production and secretion of Fgf1, Bmp4, and Gdf3 in response to PACAP1-38 treatment. Newborn (P1) rats were treated with 100 pmol PACAP1-38 intravitreally. After 24 h, retinas were dissected and processed for immunohistochemistry performed either on flat-mounted retinas or cryosections. Brn3a and PAC1-R double labeling revealed that 90% of retinal ganglion cells (RGCs) expressed PAC1-receptor. We showed that RGCs were Fgf1, Bmp4, and Gdf3- immunopositive and PAC1-R was co-expressed with each protein. To elucidate if RGCs release these secreted regulators, the key components for vesicle release were examined. No labeling was detected for synaptophysin, Exo70, or NESP55 in RGCs but an intense Rab3a-immunoreactivity was detected in their cell bodies. We found that the vast majority of RGCs are responsive to PACAP, which in turn could have a significant impact on their development or/and physiology. Although Fgf1, Bmp4, and Gdf3 were abundantly expressed in PAC1-positive RGCs, the cells lack synaptophysin and Exo70 in the newborn retina thus unable to release these proteins. These proteins could regulate postnatal RGC development acting through intracrine pathways.Key words: PAC1 receptor, Fgf1, Bmp4, Gdf3, retinal ganglion cell     

Measurement of Orbital Volume after Enucleation and Orbital Implantation

Introduction

This article reports experience relating to the measurement of orbital volume by means of cone beam computed tomography (CBCT) and Cranioviewer program software in patients who have undergone enucleation and orbital implantation.

Patients and Methods

CBCT scans were made in 30 cases, 10 of which were later excluded because of various technical problems. The study group therefore consisted of 20 patients (8 men and 12 women). The longest follow-up time was 7 years, and the shortest was 1 year. In all 20 cases, the orbital volume was measured with Cranioviewer orbital program software. Slices were made in the ventrodorsal direction at 4.8 mm intervals in the frontal plane, in both bony orbits (both that containing the orbital implant and the healthy one). Similar measurements were made in 20 patients with various dental problems. CBCT scans were recorded for the facial region of the skull, containing the orbital region. The Cranioviewer program can colour the area of the slices red, and it automatically measures the area in mm.

Results

In 5 of the 20 cases, the first 4 or all 5 slices revealed that the volume of the operated orbit was significantly smaller than that of the healthy orbit, in 12 cases only from 1 to 3 of the slices indicated such a significant difference, and in 3 cases no differences were observed between the orbits. In the control group of patients with various dental problems, there was no significant difference between the two healthy orbits. The accuracy of the volume measurements was assessed statistically by means of the paired samples t-test.

Summary

To date, no appropriate method is avaliable for exact measurement of the bony orbital volume, which would be of particular importance in orbital injury reconstruction. However, the use of CBCT scans and Cranioviewer orbital program software appears to offer a reliable method for the measurement of changes in orbital volume.     

Comparison of the Drought Stress Responses of Tolerant and Sensitive Wheat Cultivars During Grain Filling: Changes in Flag Leaf Photosynthetic Activity,ABA Levels,and Grain Yield

Water status parameters, flag leaf photosynthetic activity, abscisic acid (ABA) levels, grain yield, and storage protein contents were investigated in two drought-tolerant (Triticum aestivum L. cv. MV Emese and cv. Plainsman V) and two drought-sensitive (cvs. GK élet and Cappelle Desprez) wheat genotypes subjected to soil water deficit during grain filling to characterize physiological traits related to yield. The leaf water potential decreased earlier and at a higher rate in the sensitive than in the tolerant cultivars. The net CO₂ assimilation rate (P _N) in flag leaves during water deficit did not display a strict correlation with the drought sensitivity of the genotypes. The photosynthetic activity terminated earliest in the tolerant cv. Emese, and the senescence of flag leaves lasted 7 days longer in the sensitive Cappelle Desprez. Soil drought did not induce characteristic differences between sensitive and tolerant cultivars in chlorophyll a fluorescence parameters of flag leaves during post-anthesis. Changes in the effective quantum yield of PSII (Φ_PSII) and the photochemical quenching (qP) depended on the genotypes and not on the sensitivity of cultivars. In contrast, the levels of ABA in the kernels displayed typical fluctuations in the tolerant and in the sensitive cultivars. Tolerant genotypes exhibited an early maximum in the grain ABA content during drought and the sensitive cultivars maintained high ABA levels in the later stages of grain filling. In contrast with other genotypes, the grain number per ear did not decrease in Plainsman and the gliadin/glutenin ratio was higher than in the control in Emese during drought stress. A possible causal relationship between high ABA levels in the kernels during late stages of grain filling and a decreased grain yield was found in the sensitive cultivars during drought stress.     

RDH10, RALDH2, and CRABP2 are required components of PPARγ-directed ATRA synthesis and signaling in human dendritic cells

All-trans retinoic acid (ATRA) has a key role in dendritic cells (DCs) and affects T cell subtype specification and gut homing. However, the identity of the permissive cell types and the required steps of conversion of vitamin A to biologically active ATRA bringing about retinoic acid receptor-regulated signaling remains elusive. Here we present that only a subset of murine and human DCs express the necessary enzymes, including RDH10, RALDH2, and transporter cellular retinoic acid binding protein (CRABP)2, to produce ATRA and efficient signaling. These permissive cell types include CD103⁺ DCs, granulocyte-macrophage colony-stimulating factor, and interleukin-4-treated bone marrow-derived murine DCs and human monocyte-derived DCs (mo-DCs). Importantly, in addition to RDH10 and RALDH2, CRABP2 also appears to be regulated by the fatty acid-sensing nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) and colocalize in human gut-associated lymphoid tissue DCs. In our model of human mo-DCs, all three proteins (RDH10, RALDH2, and CRABP2) appeared to be required for ATRA production induced by activation of PPARγ and therefore form a linear pathway. This now functionally validated PPARγ-regulated ATRA producing and signaling axis equips the cells with the capacity to convert precursors to active retinoids in response to receptor-activating fatty acids and is potentially amenable to intervention in diseases involving or affecting mucosal immunity.