The relation of localized myocardial lesion size to the QRS complex of vectorcardiographic leads

Effects of localized destructive myocardial lesions on the QRS complex of vectorcardiographic leads was studied by graphic derivation of vectors from the detailed activation sequence of the human heart. Lesions of three sizes were postulated at each of 16 cardiac locations and differences between derived vectors in the presence and absence of lesions determined. At each location there was a near linear relation between lesion size and vector differences. Over different locations, however, there were marked variations in the effects of lesions of a particular size. Variations were due to varied activation patterns within each left ventricular wall since the contribution of each wall (anterior, lateral, posterior and septal) to QRS waveform was nearly identical. Findings suggest that the size of extensive destructive lesions will be well represented by the degree of QRS complex change in vectorcardiographic leads but that vectocardiographic estimates of the size of small lesions or of change of size of either small or large lesions are subject to substantial error.     

WNK3 bypasses the tonicity requirement for K-Cl cotransporter activation via a phosphatase-dependent pathway

SLC12A cation/Cl- cotransporters are mutated in human disease, are targets of diuretics, and are collectively involved in the regulation of cell volume, neuronal excitability, and blood pressure. This gene family has two major branches with different physiological functions and inverse regulation: K-Cl cotransporters (KCC1-KCC4) mediate cellular Cl- efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-Cl cotransporters (NCC and NKCC1/2) mediate cellular Cl- influx and are activated by phosphorylation. A single kinase/phosphatase pathway is thought to coordinate the activities of these cotransporters in a given cell; however, the mechanisms involved are as yet unknown. We previously demonstrated that WNK3, a paralog of serine-threonine kinases mutated in hereditary hypertension, is coexpressed with several cation/Cl- cotransporters and regulates their activity. Here, we show that WNK3 completely prevents the cell swelling-induced activation of KCC1-KCC4 in Xenopus oocytes. In contrast, catalytically inactive WNK3 abolishes the cell shrinkage-induced inhibition of KCC1-KCC4, resulting in a >100-fold stimulation of K-Cl cotransport during conditions in which transport is normally inactive. This activation is completely abolished by calyculin A and cyclosporine A, inhibitors of protein phosphatase 1 and 2B, respectively. Wild-type WNK3 activates Na-(K)-Cl cotransporters by increasing their phosphorylation, and catalytically inactive kinase inhibits Na-(K)-Cl cotransporters by decreasing their phosphorylation, such that our data suggest that WNK3 is a crucial component of the kinase/phosphatase signaling pathway that coordinately regulates the Cl- influx and efflux branches of the SLC12A cotransporter family.     

EDEM1 reveals a quality control vesicular transport pathway out of the endoplasmic reticulum not involving the COPII exit sites

Immature and nonnative proteins are retained in the endoplasmic reticulum (ER) by the quality control machinery. Folding-incompetent glycoproteins are eventually targeted for ER-associated protein degradation (ERAD). EDEM1 (ER degradation-enhancing alpha-mannosidase-like protein 1), a putative mannose-binding protein, targets misfolded glycoproteins for ERAD. We report that endogenous EDEM1 exists mainly as a soluble glycoprotein. By high-resolution immunolabeling and serial section analysis, we find that endogenous EDEM1 is sequestered in buds that form along cisternae of the rough ER at regions outside of the transitional ER. They give rise to approximately 150-nm vesicles scattered throughout the cytoplasm that are lacking a recognizable COPII coat. About 87% of the immunogold labeling was over the vesicles and approximately 11% over the ER lumen. Some of the EDEM1 vesicles also contain Derlin-2 and the misfolded Hong Kong variant of alpha-1-antitrypsin, a substrate for EDEM1 and ERAD. Our results demonstrate the existence of a vesicle budding transport pathway out of the rough ER that does not involve the canonical transitional ER exit sites and therefore represents a previously unrecognized passageway to remove potentially harmful misfolded luminal glycoproteins from the ER.     

Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance

The glyoxalase pathway involving glyoxalase I (gly I) and glyoxalase II (gly II) enzymes is required for glutathione-based detoxification of methylglyoxal. We had earlier indicated the potential of gly I as a probable candidate gene in conferring salinity tolerance. We report here that overexpression of gly I+II together confers improved salinity tolerance, thus offering another effective strategy for manipulating stress tolerance in crop plants. We have overexpressed the gly II gene either alone in untransformed plants or with gly I transgenic background. Both types of these transgenic plants stably expressed the foreign protein, and the enzyme activity was also higher. Compared with nontransformants, several independent gly II transgenic lines showed improved capability for tolerating exposure to high methylglyoxal and NaCl concentration and were able to grow, flower, and set normal viable seeds under continuous salinity stress conditions. Importantly, the double transgenic lines always showed a better response than either of the single gene-transformed lines and WT plants under salinity stress. Ionic measurements revealed higher accumulation of Na+ and K+ in old leaves and negligible accumulation of Na+ in seeds of transgenic lines as compared with the WT plants. Comparison of various growth parameters and seed production demonstrated that there is hardly any yield penalty in the double transgenics under nonstress conditions and that these plants suffered only 5% loss in total productivity when grown in 200 mM NaCl. These findings establish the potential of manipulation of the glyoxalase pathway for increased salinity tolerance without affecting yield in crop plants.     

Lipid requirements for reconstitution of the proton-translocating complex of clathrin-coated vesicles.

We have recently reported the reconstitution of the clathrin-coated vesicle proton-translocating complex with liposomes prepared from ethanol-extracted crude bovine brain lipids. Reconstitution of proton pumping by the isolated proton ATPase has now been achieved with liposomes prepared from pure lipids. Optimal proton pumping, as assessed by ATP-generated acridine orange quenching and 32Pi-ATP exchange, is achieved with liposomes prepared from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol at a weight ratio of 40:26.5:7.5:26, and a lipid-to-protein weight ratio of 200:1. Under such conditions, the extent of decrease of ATP-generated acridine orange quenching is 150-fold greater than that of native clathrin-coated vesicles. Cholesterol is required to stabilize the proteoliposome. Phosphatidylserine, which is most effective in activating ATP-hydrolytic activity of the solubilized enzyme, is not obligatory for reconstitution of proton pumping.     

The structure of the GPIb-filamin A complex

Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIbalpha binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIbalpha cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein-cytoskeleton connection. GPIbalpha binds in a groove formed between the C and D beta strands of FLNa domain 17. The interaction is strikingly similar to that between the beta7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.     

Phylogeny of protein-folding trajectories reveals a unique pathway to native structure

To scrutinize how a protein folds at atomic resolution, we performed 200 molecular dynamics simulations (each of 50 ns) of the miniprotein Trp-cage on the computational grid. Within the trajectories, 58 folding and 31 unfolding events were identified and subjected to extensive comparison and classification. Based on an analogy with biological sequences, the folding and unfolding trajectories (arrays of sequential snapshots of structures) were aligned by dynamic programming allowing gaps. A phylogenetic tree derived from the alignments revealed four distinct groups of the trajectories, characterized by the Trp side-chain motions and the main-chain motions. It was found that only one group attained the native structure and that the other three led to pseudonative structures having the correct main-chain trace but different nonnative Trp side-chain rotamers, indicating that those four folded structures were each attained through a unique folding pathway.     

Prefusion structure of syntaxin-1A suggests pathway for folding into neuronal trans-SNARE complex fusion intermediate

The assembly of the three neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins synaptobrevin 2, syntaxin-1A, and SNAP-25 is the key step that leads to exocytotic fusion of synaptic vesicles. In the fully assembled SNARE complex, these three proteins form a coiled-coil four-helix bundle structure by interaction of their respective SNARE motifs. Although biochemical and mutational analyses strongly suggest that the heptad-repeat SNARE motifs zipper into the final structure, little is known about the prefusion state of individual membrane-bound SNAREs and how they change conformation from the unzippered prefusion to the zippered postfusion state in a membrane environment. We have solved the solution NMR structure of micelle-bound syntaxin-1A in its prefusion conformation. In addition to the transmembrane helix, the SNARE motif consists of two well-ordered, membrane-bound helices separated by the “0-layer” residue Gln226. This unexpected structural order of the N- and C-terminal halves of the uncomplexed SNARE motif suggests the formation of partially zippered SNARE complex intermediates, with the 0-layer serving as a proofreading site for correct SNARE assembly. Interferometric fluorescence measurements in lipid bilayers confirm that the open SNARE motif helices of syntaxin interact with lipid bilayers and that association with the other target-membrane SNARE SNAP-25 lifts the SNARE motif off the membrane as a critical prerequisite for SNARE complex assembly and membrane fusion.Neurotransmitter release in eukaryotic cells requires the fusion of synaptic vesicles with the presynaptic plasma membrane. This fast and precise process is mediated by many proteins (1–3). The minimal neuronal exocytotic fusion machinery includes three soluble N-ethylmaleimide-sensitive (NSF) factor attachment protein receptor (SNARE) proteins: synaptobrevin-2 (syb) in the vesicle membrane and syntaxin-1A (syx) and SNAP-25 (SN25) in the plasma membrane (4). These proteins possess either one or two conserved SNARE motifs of 60–70 amino acids, which on SNARE assembly are capable of forming a four-stranded coiled-coil helical bundle (5). Folding, assumed to occur by N- to C-terminal zippering of these helices, is thought to be sufficient to merge the two opposing membranes. However, little is known about how the individual SNARE proteins change their conformations from the unzippered prefusion to the zippered postfusion states in membranes. Whereas SN25 and syb mostly consist of the SNARE motifs and, in the case of syb, a transmembrane (TM) domain, syx possesses two regulatory domains, a three-helix bundle (Habc) domain (6, 7) and a short N-terminal peptide (N-peptide) (8), in addition to its SNARE motif (H3 domain) and TM domain. Although neither of these additional domains are required in vitro for fast fusion per se (9, 10), the interaction between these domains and the SNARE motif in the presence of Munc18-1, Munc13, and other regulatory proteins is required upstream to “precondition” the SNAREs for efficient assembly and subsequent fusion. The Habc and H3 domains of syx are in dynamic equilibrium between “closed” and “open” conformations, which is regulated by Munc18-1 and Munc13, and which thereby controls synaptic vesicle fusion (11–14). Only the open, not the closed, conformation promotes formation of the SNARE complex, and thus membrane fusion.Although the three-helix Habc domain is well-ordered, the H3 domain is flexible in a soluble syx construct without its TM domain (15). Two different modes of binding of Munc18-1 to soluble syx have been observed. In a crystal structure of the syx-Munc18 complex, Munc18 locks syx in a “closed” conformation. The N-terminal half of the H3 domain forms an interrupted helix that is stabilized by interaction with Habc and Munc18 (16). Alternatively, Munc18 can just bind to the N-terminal peptide of syx (13, 17). Interestingly, the Munc18-regulated open/closed equilibrium of syx is shifted toward being more open in the membrane-bound and more closed in the soluble form of syx (18). In the soluble and the detergent-bound postfusion SNARE complex, the SNARE motifs form uninterrupted helices (19, 20). Moreover, the SNARE helices continue through the juxtamembrane linker regions to the TM domains in the latter structure (20). Because some structures and conformational equilibria are different for membrane-bound than for soluble SNAREs (18, 21), and as all previously reported high-resolution structures of prefusion syx have been obtained for their soluble portions only (6, 15, 16), it is of great interest to elucidate the structure of prefusion syx in a membrane-mimetic environment and to demonstrate its relevance in lipid bilayers. Here, we used solution NMR to study the structure and dynamics of syx in lipid micelles and fluorescence interference contrast (FLIC) microscopy to validate its conformation and activity in lipid bilayers.     

Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di-myo-inositol-phosphate

Di-myo-inositol 1,1'-phosphate (DIP) is a major osmoprotecting metabolite in a number of hyperthermophilic species of archaea and bacteria. Although the DIP biosynthesis pathway was previously proposed, genes encoding only two of the four required enzymes, inositol-1-phosphate synthase and inositol monophosphatase, were identified. In this study we used a comparative genomic analysis to predict two additional genes of this pathway (termed dipA and dipB) that remained missing. In Thermotoga maritima both candidate genes (in an originally misannotated locus TM1418) form an operon with the inositol-1-phosphate synthase encoding gene (TM1419). A predicted inositol-mono-phosphate cytidylyltransferase activity was directly confirmed for the purified product of T. maritima gene dipA cloned and expressed in Escherichia coli. The entire DIP pathway was reconstituted in E. coli by cloning of the TM1418-TM1419 operon in pBAD expression vector and confirmed to function in the crude lysate. (31)P NMR and MS analysis revealed that DIP synthesis proceeds via a phosphorylated DIP intermediate, P-DIP, which is generated by the dipB-encoded enzyme, now termed P-DIP synthase. This previously unknown intermediate is apparently converted to the final product, DIP, by an inositol monophosphatase-like phosphatase. These findings allowed us to revise the previously proposed DIP pathway. The genomic survey confirmed its presence in the species known to use DIP for osmoprotection. Among several newly identified species with a postulated DIP pathway, Aeropyrum pernix was directly proven to produce this osmolyte.     

The identification of a 9-cis retinol dehydrogenase in the mouse embryo reveals a pathway for synthesis of 9-cis retinoic acid

The ligand-controlled retinoic acid (RA) receptors and retinoid X receptors are important for several physiological processes, including normal embryonic development, but little is known about how their ligands, all-trans and 9-cis RA, are generated. Here we report the identification of a stereo-specific 9-cis retinol dehydrogenase, which is abundantly expressed in embryonic tissues known to be targets in the retinoid signaling pathway. The membrane-bound enzyme is a member of the short-chain alcohol dehydrogenase/reductase superfamily, able to oxidize 9-cis retinol into 9-cis retinaldehyde, an intermediate in 9-cis RA biosynthesis. Analysis by nonradioactive in situ hybridization in mouse embryos shows that expression of the enzyme is temporally and spatially well controlled during embryogenesis with prominent expression in parts of the developing central nervous system, sensory organs, somites and myotomes, and several tissues of endodermal origin. The identification of this enzyme reveals a pathway in RA biosynthesis, where 9-cis retinol is generated for subsequent oxidation to 9-cis RA.     

The structure and function of the Rh antigen complex

The Rh system is one of the most important and complex blood group systems because of the large number of antigens and the serious complications for the fetus of a woman sensitized by transfusion or pregnancy. Major advances in our understanding of the Rh system have occurred with the cloning of the genes and with functional evidence that the Rh blood group proteins belong to an ancient family of membrane proteins involved in ammonia transport. The arrangement and configuration of the genes at the RH locus promotes genetic exchange, generating new antigens. Importantly, RH genetic testing can now be applied to clinical transfusion medicine and prenatal practice. This includes testing for RHD zygosity, confirmation or resolution of D antigen status, and detection of altered RHD and RHCE genes in individuals at risk for producing antibodies to high-incidence Rh antigens, particularly sickle cell disease (SCD) patients. The Rh proteins form a core complex that is critical to the structure of the erythrocyte membrane, and they may play a physiologic role in the sequestration of blood ammonia. The Rh family of proteins now includes non-erythroid homologs present in many other tissues, and comparative genomics reveal Rh homologs in all domains of life.     

Orthogonal leads for the measurement of QT dispersion: a comparison with conventional leads

To determine the characteristics of QT interval dispersion using orthogonal ECG leads with high paper speed (100 mm/s) and high voltage gain (10 cm/mV) as compared to conventional 12-lead ECG, we measured the QT dispersion in 57 patients at rest and directly after exercise using these two techniques. The measurements were repeated by the same observer and by an independent observer in 29 patients to assess reproducibility. QT dispersion was found to be significantly lower in orthogonal leads than standard lead tracings (24+/-20 ms versus 44+/-17 ms at rest, P<0.001; 29+/-21 ms versus 53+/-27 ms after exercise, P<0.001, respectively). The intrasubject and interobserver reproducibility was better for the orthogonal lead tracings, making this technique a potentially useful tool for future research.     

The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.