The monomeric GIY-YIG homing endonuclease I-BmoI uses a molecular anchor and a flexible tether to sequentially nick DNA

The GIY-YIG nuclease domain is found within protein scaffolds that participate in diverse cellular pathways and contains a single active site that hydrolyzes DNA by a one-metal ion mechanism. GIY-YIG homing endonucleases (GIY-HEs) are two-domain proteins with N-terminal GIY-YIG nuclease domains connected to C-terminal DNA-binding and they are thought to function as monomers. Using I-BmoI as a model GIY-HE, we test mechanisms by which the single active site is used to generate a double-strand break. We show that I-BmoI is partially disordered in the absence of substrate, and that the GIY-YIG domain alone has weak affinity for DNA. Significantly, we show that I-BmoI functions as a monomer at all steps of the reaction pathway and does not transiently dimerize or use sequential transesterification reactions to cleave substrate. Our results are consistent with the I-BmoI DNA-binding domain acting as a molecular anchor to tether the GIY-YIG domain to substrate, permitting rotation of the GIY-YIG domain to sequentially nick each DNA strand. These data highlight the mechanistic differences between monomeric GIY-HEs and dimeric or tetrameric GIY-YIG restriction enzymes, and they have implications for the use of the GIY-YIG domain in genome-editing applications.     

WISE 2005: responses of women to sublingual nitroglycerin before and after 56 days of 6° head-down bed rest

This study tested the hypothesis that cardiovascular effects of sublingual nitroglycerin (NG) would be exaggerated after 56 days of 6° head-down bed rest (HDBR) in women, and that an aerobic and resistive exercise countermeasure (EX, n = 8) would reduce the effect compared with HDBR without exercise (CON, n = 7). Middle cerebral artery maximal blood flow velocity (CBFV), cardiac stroke volume (SV), and superficial femoral artery blood flow (Doppler ultrasound) were recorded at baseline rest and for 5 min following 0.3 mg sublingual NG. Post-HDBR, NG caused greater increases in heart rate (HR) in CON compared with EX (+24.9 ± 7.7 and +18.8 ± 6.6 beats/min, respectively, P < 0.0001). The increase in HR combined with reductions in SV to maintain cardiac output. Systolic, mean, and pulse pressures were reduced 5-10 mmHg by NG, but total peripheral resistance was only slightly reduced at 3 min after NG. Reductions in CBFV of -12.5 ± 3.8 cm/s were seen after NG, but a reduction in the Doppler resistance index suggested dilation of the middle cerebral artery with no differences after HDBR. The femoral artery dilated with NG and blood flow was reduced ～50% with the appearance of large negative waves suggesting a marked increase in downstream resistance, but there were no effects of HDBR. In general, responses of women to NG were not altered by HDBR; the greater increase in HR in CON but not EX was probably a consequence of cardiovascular deconditioning. These results contrast with the hypothesis and a previous investigation of men after HDBR by revealing no change in cardiovascular responses to exogenous nitric oxide.     

Arabidopsis mutants in short- and medium-chain acyl-CoA oxidase activities accumulate acyl-CoAs and reveal that fatty acid beta-oxidation is essential for embryo development

The short-chain acyl-CoA oxidase (ACX4) is one of a family of ACX genes that together catalyze the first step of peroxisomal fatty acid beta-oxidation during early, postgerminative growth in oilseed species. Here we have isolated and characterized an Arabidopsis thaliana mutant containing a T-DNA insert in ACX4. In acx4 seedlings, short-chain acyl-CoA oxidase activity was reduced by greater than 98%, whereas medium-chain activity was unchanged from wild type levels. Despite the almost complete loss of short-chain activity, lipid catabolism and seedling growth and establishment were unaltered in the acx4 mutant. However, the acx4 seedlings accumulated high levels (31 mol %) of short-chain acyl-CoAs and showed resistance to 2,4-dichlorophenoxybutyric acid, which is converted to the herbicide and auxin analogue 2,4-dichlorophenoxyacetic acid by beta-oxidation. A mutant in medium-chain length acyl-CoA activity (acx3) (1) shows a similar phenotype to acx4, and we show here that acx3 seedlings accumulate medium-chain length acyl-CoAs (16.4 mol %). The acx3 and acx4 mutants were crossed together, and remarkably, the acx3acx4 double mutants aborted during the first phase of embryo development. We propose that acx3acx4 double mutants are nonviable because they have a complete block in short-chain acyl-CoA oxidase activity. This is the first demonstration of the effects of eliminating (short-chain) beta-oxidation capacity in plants and shows that a functional beta-oxidation cycle is essential in the early stages of embryo development.     

Nonideality and protein thermal denaturation

We studied the thermal denaturation of eglin c by using CD spectropolarimetry and differential scanning calorimetry (DSC). At low protein concentrations, denaturation is consistent with the classical two-state model. At concentrations greater than several hundred microM, however, the calorimetric enthalpy and the midpoint transition temperature increase with increasing protein concentration. These observations suggested the presence of intermediates and/or native state aggregation. However, the transitions are symmetric, suggesting that intermediates are absent, the DSC data do not fit models that include aggregation, and analytical ultracentrifugation (AUC) data show that native eglin c is monomeric. Instead, the AUC data show that eglin c solutions are nonideal. Analysis of the AUC data gives a second virial coefficient that is close to values calculated from theory and the DSC data are consistent with the behavior expected for nonideal solutions. We conclude that the concentration dependence is caused by differential nonideality of the native and denatured states. The nondeality arises from the high charge of the protein at acid pH and is exacerbated by low buffer concentrations. Our conclusion may explain differences between van't Hoff and calorimetric denaturation enthalpies observed for other proteins whose behavior is otherwise consistent with the classical two-state model.     

Perpetuating the homing endonuclease life cycle: identification of mutations that modulate and change I-TevI cleavage preference

Homing endonucleases are sequence-tolerant DNA endonucleases that act as mobile genetic elements. The ability of homing endonucleases to cleave substrates with multiple nucleotide substitutions suggests a high degree of adaptability in that changing or modulating cleavage preference would require relatively few amino acid substitutions. Here, using directed evolution experiments with the GIY-YIG homing endonuclease I-TevI that targets the thymidylate synthase gene of phage T4, we readily isolated variants that dramatically broadened I-TevI cleavage preference, as well as variants that fine-tuned cleavage preference. By combining substitutions, we observed an ∼10 000-fold improvement in cleavage on some substrates not cleaved by the wild-type enzyme, correlating with a decrease in readout of information content at the cleavage site. Strikingly, we were able to change the cleavage preference of I-TevI to that of the isoschizomer I-BmoI which targets a different cleavage site in the thymidylate synthase gene, recapitulating the evolution of cleavage preference in this family of homing endonucleases. Our results define a strategy to isolate GIY-YIG nuclease domains with distinct cleavage preferences, and provide insight into how homing endonucleases may escape a dead-end life cycle in a population of saturated target sites by promoting transposition to different target sites.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Studies on the interactions of Ca2+ and pyruvate in the regulation of rat heart pyruvate dehydrogenase activity. Effects of starvation and diabetes.

1. Previous studies showed that the activation of pyruvate dehydrogenase within intact rat heart mitochondria of pyruvate is much diminished in mitochondria from starved or diabetic animals [see Kerbey, Randle, Cooper, Whitehouse, Pask & Denton (1976) Biochem. J. 154, 327-348]. In the present study, diminished responses to added Ca2+ and ADP were also found in these mitochondria. 2. Starvation or diabetes did not affect the mitochondrial respiratory control ratio of the ATP content. Moreover, starvation and diabetes did not alter the response of the intramitochondrial Ca2+-sensitive enzyme, 2-oxoglutarate dehydrogenase, to changes in the extramitochondrial concentration of Ca2+ and 2-oxoglutarate, thus indicating that there were no appreciable changes in the distribution of Ca2+ and H+ across the mitochondrial inner membrane. 3. Pyruvate, Ca2+ and ADP were found to have synergistic effects on pyruvate dehydrogenase activity, particularly in mitochondria from starved and diabetic rats. 4. The results suggest that the effects of diabetes and starvation on pyruvate dehydrogenase are not brought about by changes in the distribution of these effectors across the mitochondrial inner membrane or by changes in the intrinsic sensitivity of the kinase or phosphatase of the pyruvate dehydrogenase system to pyruvate, Ca2+ or ADP; rather it is probably that there is an increase in the maximum activity of kinase relative to that of the phosphatase. 6. The results also lend further support to the hypothesis that adrenaline may bring about the activation of pyruvate dehydrogenase in the rat heart by an increase in the intramitochondrial concentration of Ca2+.     

High-resolution preparative gel electrophoresis: separation and recovery of functional messenger RNA species

Certain open-chain polyols were shown to interfere with the determination of phosphorus of the Lowry-Lopez method by forming a complex with Mo₇O₂₄^6?. The ability to interfere with the assay increased with increasing chain length of the polyols: Ethylene glycol and glycerol did not react at all; i-erythritol reacted to a small extent, but hexitols and perseitol formed stronger complexes. Depending on the polyol, interference occurred even at 0.2 mm (hexitols) or 2 mm (xylitol) concentrations. At these concentrations the polyols interfered only to a small extent with the phosphorus assays based on the use of Triton X-100 and molybdate. The complex formation was exploited in the development of a colorimetric polyol assay.