X-ray structure of methanol dehydrogenase from<Emphasis Type="Italic"> Paracoccus denitrificans</Emphasis> and molecular modeling of its interactions with cytochrome<Emphasis Type="Italic"> c</Emphasis>-551i

The X-ray structure of methanol dehydrogenase (MEDH) from Paracoccus denitrificans (MEDH-PD) was determined at 2.5 A resolution using molecular replacement based on the structure of MEDH from Methylophilus methylotrophus W3A1 (MEDH-WA). The overall structures from the two bacteria are similar to each other except that the former has a longer C-terminal tail in each subunit and shows local differences in several insertion regions. The "X-ray sequence" of the segment alphaGly444-alphaLeu452 was established, including one insertion and seven replacements compared with the reported sequence. The primary electron acceptor of MEDH-PD is cytochrome c-551i (Cyt c551i). Based on the crystal structure of MEDH-PD and of the published structure of Cyt c551i, their interactions were investigated by molecular modeling. As a guide and starting point, the covalently attached cytochrome and PQQ domains of the alcohol dehydrogenase from Pseudomonas putida HK5 (ADH2B) were used. In the modeling, two molecules of Cyt c551i could be accommodated in their interaction with the MEDH heterotetramer in accordance with the two-fold molecular symmetry of the latter. Two models are proposed, in both of which electrostatic and hydrogen bonding interactions make major contributions to inter-protein binding. One of these models involves salt bridges from alphaArg99 of MEDH to the heme propionic acids of Cyt c551i and the other involves salt bridges from alphaArg426 of MEDH to Glu112 of Cyt c551i. Both involve salt bridges from alphaLys93 of MEDH to Asp75 of Cyt c551i. The size and nature of the cytochrome/quinoprotein heterodimer interfaces and calculations of electronic coupling and electron transfer rates favor one of these models over the other.     

The discovery of a new class of large-conductance Ca2+-activated K+ channel opener targeted for overactive bladder: synthesis and structure-activity relationships of 2-amino-4-azaindoles

2-Amino-4-azaindoles have been identified as a structurally novel class of BK(Ca) channel openers. Their synthesis from 2-chloro-3-nitropyridine is described together with their in vitro properties assessed by 86Rb(+) efflux and whole-cell patch-clamp assays using HEK293 cells stably transfected with the BK(Ca) alpha subunit. In vitro functional characterization of BK(Ca) channel opening activity was also assessed by measurement of relaxation of smooth muscle tissue strips obtained from Landrace pig bladders. The preliminary SAR data indicate the importance of steric bulk around the 2-amino substituent.     

Objective threshold selection procedure (OTS) for segmentation of scanning laser confocal microscope images

The determination of volumes and interface areas from confocal laser scanning microscopy (CLSM) images requires the identification of component objects by segmentation. An automated method for the determination of segmentation thresholds for CLSM imaging of biofilms was developed. The procedure, named objective threshold selection (OTS), is a three-dimensional development of the approach introduced by the popular robust automatic threshold selection (RATS) method. OTS is based on the statistical properties of local gray-values and gradients in the image. By characterizing the dependence between a volumetric feature and the intensity threshold used for image segmentation, the former can be determined with an arbitrary confidence level, with no need for user intervention. The identification of an objective segmentation procedure renders the possibility for the full automation of volume and interfacial area measurement. Images from two distinct biofilm systems, acquired using different experimental techniques and instrumental setups were segmented by OTS to determine biofilm volume and interfacial area. The reliability of measurements for each case was analyzed to identify optimal procedure for image acquisition. The automated OTS method was shown to reproduce values obtained manually by an experienced operator.     

Telomerase inhibitors

There has been a vast increase in telomerase research over the past several years, with many different pre-clinical approaches being tested for inhibiting the activity of this enzyme as a novel therapeutic modality to treat malignancy. In this review, we will provide some basic background information about telomeres and telomerase and then discuss the pros, cons and challenges of the approaches that are currently under investigation, and what we might expect in the future of this emerging field.     

Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions

Chromatin is composed of nucleosomes, the universally repeating protein-DNA complex in eukaryotic cells. The crystal structure of the nucleosome core particle from Saccharomyces cerevisiae reveals that the structure and function of this fundamental complex is conserved between single-cell organisms and metazoans. Our results show that yeast nucleosomes are likely to be subtly destabilized as compared with nucleosomes from higher eukaryotes, consistent with the idea that much of the yeast genome remains constitutively open during much of its life cycle. Importantly, minor sequence variations lead to dramatic changes in the way in which nucleosomes pack against each other within the crystal lattice. This has important implications for our understanding of the formation of higher order chromatin structure and its modulation by post-translational modifications. Finally, the yeast nucleosome core particle provides a structural context by which to interpret genetic data obtained from yeast. Coordinates have been deposited with the Protein Data Bank under accession number 1ID3.     

Immune system dysfunction and autoimmune disease in mice lacking Emk (Par-1) protein kinase

Emk is a serine/threonine protein kinase implicated in regulating polarity, cell cycle progression, and microtubule dynamics. To delineate the role of Emk in development and adult tissues, mice lacking Emk were generated by targeted gene disruption. Emk(-/-) mice displayed growth retardation and immune cell dysfunction. Although B- and T-cell development were normal, CD4(+)T cells lacking Emk exhibited a marked upregulation of the memory marker CD44/pgp-1 and produced more gamma interferon and interleukin-4 on stimulation through the T-cell receptor in vitro. In addition, B-cell responses to T-cell-dependent and -independent antigen challenge were altered in vivo. As Emk(-/-) animals aged, they developed splenomegaly, lymphadenopathy, membranoproliferative glomerulonephritis, and lymphocytic infiltrates in the lungs, parotid glands and kidneys. Taken together, these results demonstrate that the Emk protein kinase is essential for maintaining immune system homeostasis and that loss of Emk may contribute to autoimmune disease in mammals.     

Association of insulin receptor substrate 1 (IRS-1) y895 with Grb-2 mediates the insulin signaling involved in IRS-1-deficient brown adipocyte mitogenesis

We have recently generated immortalized fetal brown adipocyte cell lines from insulin receptor substrate 1 (IRS-1) knockout mice and demonstrated an impairment in insulin-induced lipid synthesis as compared to wild-type cell lines. In this study, we investigated the consequences of IRS-1 deficiency on mitogenesis in response to insulin. The lack of IRS-1 resulted in the inability of insulin-stimulated IRS-1-deficient brown adipocytes to increase DNA synthesis and enter into S/G2/M phases of the cell cycle. These cells showed a severe impairment in activating mitogen-activated protein kinase kinase (MEK1/2) and p42-p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. IRS-1-deficient cells also lacked tyrosine phosphorylation of SHC and showed no SHC-Grb-2 association in response to insulin. The mitogenic response to insulin could be partially restored by enhancing IRS-2 tyrosine phosphorylation and its association with Grb-2 by inhibition of phosphatidylinositol 3-kinase activity through a feedback mechanism. Reconstitution of IRS-1-deficient brown adipocytes with wild-type IRS-1 restored insulin-induced IRS-1 and SHC tyrosine phosphorylation and IRS-1-Grb-2, IRS-1-SHC, and SHC-Grb-2 associations, leading to the activation of MAPK and enhancement of DNA synthesis. Reconstitution of IRS-1-deficient brown adipocytes with the IRS-1 mutant Tyr895Phe, which lacks IRS-1-Grb-2 binding, restored SHC-IRS-1 association and SHC-Grb-2 association. However, the lack of IRS-1-Grb-2 association impaired MAPK activation and DNA synthesis in insulin-stimulated mutant cells. These data provide strong evidence for an essential role of IRS-1 and its direct association with Grb-2 in the insulin signaling pathway leading to MAPK activation and mitogenesis in brown adipocytes.     

Endothelial dysfunction is induced by proinflammatory oxidant hypochlorous acid

The myeloperoxidase (MPO)-derived oxidant hypochlorous acid (HOCl) plays a role in tissue injury under inflammatory conditions. The present study tests the hypothesis that HOCl decreases nitric oxide (NO) bioavailability in the vasculature of Sprague-Dawley rats. Aortic ring segments were pretreated with HOCl (1-50 microM) followed by extensive washing. Endothelium-dependent relaxation was then assessed by cumulative addition of acetylcholine (ACh) or the calcium ionophore A23187. HOCl treatment significantly impaired both ACh- and A23187-mediated relaxation. In contrast, endothelium-independent relaxation induced by sodium nitroprusside was unaffected. The inhibitory effect of HOCl on ACh-induced relaxation was reversed by exposure of ring segments to L-arginine but not D-arginine. In cellular studies, HOCl did not alter endothelial NO synthase (NOS III) protein or activity, but inhibited formation of the NO metabolites nitrate (NO3(-) and nitrite (NO2(-). The reduction in total NO metabolite production in bovine aortic endothelial cells was also reversed by addition of L-arginine. These data suggest that HOCl induces endothelial dysfunction via modification of L-arginine.     

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.