Role of soluble guanylyl cyclase in renal afferent and efferent arterioles.

Renal arteriolar tone depends considerably on the dilatory action of nitric oxide (NO) via activation of soluble guanylyl cyclase (sGC) and cGMP action. NO deficiency and hypoxia/reoxygenation are important pathophysiological factors in the development of acute kidney injury. It was hypothesized that the NO-sGC-cGMP system functions differently in renal afferent arterioles (AA) compared with efferent arterioles (EA) and that the sGC activator cinaciguat differentially dilates these arterioles. Experiments were performed in isolated, perfused mouse glomerular arterioles. Hypoxia (0.1% oxygen) was achieved by using a hypoxia chamber. Phosphodiesterase 5 (PDE5) and sGC subunits were considerably expressed on the mRNA level in AA. PDE5 inhibition with sildenafil, which blocks cGMP degradation, diminished the responses to ANG II bolus application in AA, but not significantly in EA. Vasodilation induced by sildenafil in ANG II-preconstricted vessels was stronger in EA than AA. Cinaciguat, an NO- and heme-independent sGC activator, dilated EA more strongly than AA after NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) treatment and preconstriction with ANG II. Cinaciguat-induced dilatation of l-NAME-pretreated and ANG II-preconstricted arterioles was similar to controls without l-NAME treatment. Cinaciguat also induced dilatation in iodinated contrast medium treated AA. Furthermore, it dilated EA, but not AA, after hypoxia/reoxygenation. The results reveal an important role of the NO-sGC-cGMP system for renal dilatation and that EA have a more potent sGC activated dilatory system. Furthermore, AA seem to be more sensitive to hypoxia/reoxygenation than EA under these experimental conditions.

[Ab interno trabeculectomy with the Nd:YLF picosecond laser]. / Ab-interno-Trabekulotomie mit dem Nd:YLF-Pikosekundenlaser.

BACKGROUND: The outcome of glaucoma surgery is limited by scar formation. As an alternative to current techniques, the opening of the trabecular meshwork ab interno may lead to increased outflow. This study examined the Nd:YLF picosecond laser for ab interno ablation of the trabecular meshwork without opening of the eye. PATIENTS AND METHODS: A Nd:YLF picosecond laser was used which allows tissue to be ablated even through fluids. We performed ablation with different spot sizes and energies in postmortem eyes. Afterwards the effect of the laser treatment was examined by electron microscopy. RESULTS: The ablation threshold for the cornea was found to be 20 J/cm2 for a pulse width of 30 ps. At a lower level of energy (10 J/cm2) ablation of the trabecular meshwork was possible. The size of the focus which was limited by the optics of the slit-lamp was about 50 microns. CONCLUSION: These initial results with a Nd:YLF picosecond laser confirm the possibility of ablating tissue in the trabecular meshwork ab interno without the need to open the eye.

Enzymatic mutation detection: enrichment of heteroduplexes from hybrid DNA mixtures by cleavage-deficient GST-tagged endonuclease VII.

A method for the enrichment of heteroduplex DNAs from hybrid DNA mixtures by endonuclease VII is reported. The procedure is based on the ability of a GST-fused cleavage-deficient mutant endonuclease VII (EVII-N62D(GST)) to bind to mismatching nucleotides in heteroduplex DNAs identical to the wild-type enzyme. The GST tag was used for stable immobilisation of the protein to Glutathione Sepharose 4B. This enables the material to withstand the repeated rounds of binding steps required for enrichment of heteroduplex molecules from appropriate samples.

X-ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain-swapped dimer architecture.

Phage T4 endonuclease VII (Endo VII), the first enzyme shown to resolve Holliday junctions, recognizes a broad spectrum of DNA substrates ranging from branched DNAs to single base mismatches. We have determined the crystal structures of the Ca2+-bound wild-type and the inactive N62D mutant enzymes at 2.4 and 2.1 A, respectively. The Endo VII monomers form an elongated, highly intertwined molecular dimer exhibiting extreme domain swapping. The major dimerization elements are two pairs of antiparallel helices forming a novel 'four-helix cross' motif. The unique monomer fold, almost completely lacking beta-sheet structure and containing a zinc ion tetrahedrally coordinated to four cysteines, does not resemble any of the known junction-resolving enzymes, including the Escherichia coli RuvC and lambda integrase-type recombinases. The S-shaped dimer has two 'binding bays' separated by approximately 25 A which are lined by positively charged residues and contain near their base residues known to be essential for activity. These include Asp40 and Asn62, which function as ligands for the bound calcium ions. A pronounced bipolar charge distribution suggests that branched DNA substrates bind to the positively charged face with the scissile phosphates located near the divalent cations. A model for the complex with a four-way DNA junction is presented.

Association of holliday-structure resolving endonuclease VII with gp20 from the packaging machine of phage T4.

Endonuclease VII (endo VII) is the product of gene 49 (gp49) of bacteriophage T4. It is a Holliday-structure resolvase (X-solvase) responsible for clearing branched replicative DNA prior to packaging. Consequently, mutations in gene 49 are unable to fill heads to completion because unresolved branches stop translocation of DNA. A likely association of gp49 with heads or proheads, however, could not be shown in the past. We have investigated whether gp49 could be part of the transiently assembled packaging machine (the "packasome") located at the base of proheads. Using purified proteins gpl6, gpl7 and gp20, which are constituents of the packasome, we found that gp49 binds tightly to gp20 and does not bind to gpl6 or gpl7. Quantification revealed that one dimer of gp49 binds one monomer of gp20. Notably, dimerisation of gp49 was an essential prerequisite for complex formation with gp20, and the dimerisation-deficient point mutation His-EVII-W87R showed only residual affinity to gp20. Furthermore, truncated peptides of gp49 deficient in dimer formation to various degrees were found to be impaired in binding to gp20. In contrast, the cleavage-deficient mutation EVII-N62D bound normally to gp20. The cruciform DNA (cf-DNA) resolving activity typical of endo VII is maintained in gp20-gp49 complexes. Furthermore, the complexes bind cf-DNA in the absence of Mg2+ as demonstrated by electromobility shift assays. The binding of the complexes to cf-DNA occurs via gp49, since gp20 alone does not bind cf-DNA. In conclusion, these findings are consistent with a model in which gp49 is an integral part of the packaging machine of phage T4.

Enzymatic mutation detection. Phosphate ions increase incision efficiency of endonuclease VII at a variety of damage sites in DNA.

The ability of endonuclease VII (endo VII) to cleave at mispairings in double-stranded DNA has recently been used for enzymatic mutation detection (EMD) [R. Youil, B.W. Kemper, R.G.H. Cotton, Proc. Natl. Acad. Sci. USA 92 (1995) 87-91]. The method is based on mapping cleavages in heteroduplex DNAs obtained from mutant and wildtype sequences. Despite the capability of endo VII to cleave at all possible mispairings, relative cleavage efficiencies vary considerably for individual mismatches and may escape detection if located in an unfavorable sequence surrounding. We report here improved reaction conditions which can increase the selectivity of the enzyme for mismatches up to 500-fold, as demonstrated with a mutation in a 247 nt long fragment from exon 7 of human gene p53. The new conditions involve replacement of Tris/HCl buffer by phosphate buffer and change from pH 8.0 to 6.5. Various concentrations of phosphate ions should be tried in the assay to meet individual requirements of the substrate.

Enzymatic mutation detection. Procedure for screening and mapping of mutations by immobilised endonuclease VII.

Endonuclease VII (endo VII) binds to non-pairing nucleotides in DNA. This served as the basis for the development of a mutation detection assay involving immobilised endo VII and heteroduplex DNAs made by hybridisation of mutant and wild type DNA. The use of microtiter plates allows screening of large numbers of samples. Localisation of mutations in positive samples can be done in the same assay in a second optional step.

Identification of amino acids of endonuclease VII essential for binding and cleavage of cruciform DNA.

Endonuclease VII is a Holliday-structure-resolving enzyme of bacteriophage T4. The active protein is a homodimer with 157 amino acids/monomer. An amber mutation (amE727 in codon 151) inactivates the nuclease completely, indicating the importance of the seven C-terminal amino acids for nucleolytic activity. The influence of these amino acids on cruciform-DNA binding and cleavage was investigated through functional analysis of C-terminal-truncated proteins derived from deletion constructs. It was found that the three C-terminal amino acids are not necessary for binding and cleavage. A transition from active to inactive protein occurs gradually with truncations of the next four amino acids. Reduction of DNA-binding ability, as measured by electrophoretic mobility shift assays, was determined to be the primary defect in the cleavage-deficient proteins. This was further concluded by the finding that EVII-(1-150)-peptide(amber), a protein with fairly low affinity to cruciform DNA, contributes cleavage activity to reactions of wild-type EVII with cruciform DNA. [Asp62]EVII-(1-156)-peptide lacking one C-terminal amino acid, contains a point mutation in codon 62 that eliminates the nucleolytic activity of the protein while retaining its DNA-binding proficiency. By mixing binding-deficient and cleavage-deficient mutants in the same assay, cleavage of cruciform DNA resumed. Evidence is presented that complementation occurs by heterodimer formation. Our results show that the zinc-binding motif of EVII is not sufficient for cruciform-DNA binding.

Inhibition of Holliday structure resolving endonuclease VII of bacteriophage T4 by recombination enzymes UvsX and UvsY.

Proteins UvsX, UvsY and Endonuclease VII (Endo VII) of bacteriophage T4 are required for DNA recombination, replication and repair. Endo VII is the product of gene 49 (gp49) and essential for resolution of branches from newly made DNA, prior to packaging into preformed heads. The ability of Endo VII to resolve Holliday structures in vitro suggested an in vivo function for the resolution of recombination intermediates, generated by UvsX and UvsY during the early infection cycle. Here we report results which contrast with this hypothesis. It is shown that the potent endonucleolytic activity of Endo VII with branched DNAs is inhibited in strand transfer reactions by the strand transferase UvsX, and more strongly by the accessory protein UvsY in vitro. The inhibitory effect of UvsX or UvsY is also seen in reactions with Endo VII using two synthetic cruciform DNAs and a C/C-mismatch containing substrate. Low concentrations of UvsY protein (12 ng or 0,76 pmol) were sufficient to reduce the cleavage efficiency of 30 units of Endo VII (about 16 fmol) to 50%. The inhibition is due to a direct protein-protein interaction between Endo VII, UvsX and UvsY as suggested by electrophoretic mobility shift assays (EMSAs). These results were confirmed through affinity chromatography, where UvsX and UvsY bound to Endo VII, immobilized on a NHS-activated Sepharose matrix. This is the first identification of phage-encoded proteins which modulate the potent endonucleolytic activity of gp49 in vitro.

Improved large-scale preparation of phage T4 endonuclease VII overexpressed in E. coli.

Using PCR, we cloned T4 gene 49, which encodes the endonuclease VII, and the inactive mutant gene 49 amE727 into vector pET-11a. In combination with Escherichia coli host strain BL21 (DE3), this system provided excellent repression of the expression of the highly toxic protein before induction with IPTG. After induction, the proteins were made in high quantities while remaining soluble. Dilution of the crude lysate at 1:10,000 continued to show a highly specific activity in the case of the wild-type enzyme. The protein was purified to homogeneity with a recovery of 33% using two chromatography steps. The yield was 20 times higher and the specific activity 500 times higher than that obtained by using the previously published protocol.

Left ventricular hypertrophy in persons age 90 years and older.

Clinical, electrocardiographic and echocardiographic findings of 32 patients age 90 years or older were analyzed to assess the prevalence, characteristics and correlates of left ventricular (LV) hypertrophy. All patients (mean age 92 years, range 90 to 98; 21 women and 11 men) were referred to the echocardiography laboratory with a definite or suspected cardiovascular diagnosis. LV hypertrophy, echocardiographically diagnosed by high LV mass index, was present in 28 patients. The LV mass index ranged from 105 to 215 g/m2 in men and 140 to 262 g/m2 in women. Electrocardiographic evaluation showed LV hypertrophy in only 5 patients. Five patients had low voltage on the electrocardiogram. There was no correlation between the LV mass index and presence of electrocardiographic LV hypertrophy or presence of low voltage on the electrocardiogram. LV hypertrophy was concentric in 19 and eccentric in 9. There was no correlation between types of LV hypertrophy and underlying cardiovascular disease or presence of electrocardiographic LV hypertrophy. It is concluded that LV hypertrophy is frequently present and has a wide range and heterogeneous character in very elderly patients with cardiovascular disease. In the tenth decade of life, echocardiography is a sensitive method for detecting, characterizing and classifying LV hypertrophy, whereas electrocardiography lacks sensitivity in detecting it.

Effects of noradrenaline on the activation and the stability of brain adenylate cyclase.

At constant 1 mM-ATP, the Mg2+-saturation curves for adenylate cyclase (EC 4.6.1.1) particulate preparations obtained from corpus striatum and cortex tissues of rat brain show that addition of 0.1 mM-noradrenaline increases the apparent Vmax. for Mg2+ by 300% in corpus striatum particles, and by 280% in cortex particles. At 10 mM-MgCl2, the addition of 0.1 mM-noradrenaline increased by 800% the adenylate cyclase activity of corpus striatum particles. At all Mg2+ concentrations, the addition of 0.3 mM-CaCl2 suppressed the noradrenaline-induced stimulation of adenylate cyclase of corpus striatum particles, and even resulted in a strong inhibition of the activating effect of Mg2+ itself on adenylate cyclase of corpus striatum particles, and even resulted in a strong inhibition of the activating effect of Mg2+ itself on adenylate cyclase activity of cortex particles. The addition of noradrenaline during a 3 h preincubation of particle preparations of brain cortex at 38 degrees C decreased by more than 4-fold the half-life of the decay of adenylate cyclase activity. The addition of MgATP protected against noradrenaline-induced inactivation.