Electron Transfer Precedes ATP Hydrolysis during Nitrogenase Catalysis

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein

Negative Cooperativity in the Nitrogenase Fe Protein Electron Delivery Cycle

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the α2β2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αβ active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves

Comment on "Correlation between Compact Radio Lout Quasars and Ultrahigh Energy Cosmic Rays"

In a recent paper, Farrar and Biermann argue that there is a strong correlation between the direction of the five highest-energy cosmic-ray events and compact, radio-loud quasars. This Comment shows that this analysis contains several inconsistencies and errors so that the significance of any such correlation is certainly greatly overestimated and perhaps nonexistent.Comment: 2 pages, REVTE

R375-87, and Pediatric Subunit United States Public Health Service (Bethesda, Maryland) grant MO1-RR0063

Full-field electroretinograms (ERGs) were obtained from very-low-birth-weight (VLBW) neonates to determine whether omega-3 (a>-3) fatty acids are essential for normal human retinal development. Eighty-one infants born at 30.4 (standard deviation, ±1.5) wk gestation were, within 10 d of birth, either enrolled to receive mother's milk (naturally containing both o>-6 and a>-3 essential fatty acids) or randomized to receive one of the infant formulas. Corn oil-based Formula A contained mainly linoleic acid (18:2 co-6) and was low in all co-3 fatty acids. Soy oil-based Formula B contained ample a-linolenic acid (18:3 a>-3) but no long-chain a>-3. Formula C, supplemented with both a-linolenic acid and marine oils, was comparable to human milk in long-chain co-3. Full-field ERGs were obtained in the special care nursery from infants aged 36 and 57 wk postconception. Ten healthy preterm infants born at 35 wk gestation were tested at 36 wk postconception. Significant differences were found among groups in rod ERG function. Post hoc comparisons showed that infants fed Formula A had significantly higher rod thresholds than infants receiving long-chain co-3 (human milk, Formula C, and intrauterine). Infants receiving Formula B had intermediate thresholds that were significantly higher than those of infants receiving intrauterine nutrition. Analysis of the leading edge of the a-wave showed that b-wave differences originated at the photoreceptor level. Differences were not present in infants at 57 wk postconception. No significant differences among groups were found in cone b-waves at 36 or 57 wk postconception. Oscillatory potentials had significantly longer implicit times at 57 wk postconception in infants fed Formula A than in infants receiving human milk. These findings suggest that retinal function varies with the dietary supply of co-3 fatty acids in VLBW infants. Invest Ophthalmol Vis Sci 33: [2365][2366][2367][2368][2369][2370][2371][2372][2373][2374][2375][2376]1992 Linoleic acid (18:2 00-6) and a-linolenic acid (18:3 a>-3) are considered essential fatty acids (EFAs) for humans because of our inability to synthesize them and the resulting deficiency syndromes when they are removed from the diet

R375-87, and Pediatric Subunit United States Public Health Service (Bethesda, Maryland) grant MO1-RR0063

Full-field electroretinograms (ERGs) were obtained from very-low-birth-weight (VLBW) neonates to determine whether omega-3 (a>-3) fatty acids are essential for normal human retinal development. Eighty-one infants born at 30.4 (standard deviation, ±1.5) wk gestation were, within 10 d of birth, either enrolled to receive mother's milk (naturally containing both o>-6 and a>-3 essential fatty acids) or randomized to receive one of the infant formulas. Corn oil-based Formula A contained mainly linoleic acid (18:2 co-6) and was low in all co-3 fatty acids. Soy oil-based Formula B contained ample a-linolenic acid (18:3 a>-3) but no long-chain a>-3. Formula C, supplemented with both a-linolenic acid and marine oils, was comparable to human milk in long-chain co-3. Full-field ERGs were obtained in the special care nursery from infants aged 36 and 57 wk postconception. Ten healthy preterm infants born at 35 wk gestation were tested at 36 wk postconception. Significant differences were found among groups in rod ERG function. Post hoc comparisons showed that infants fed Formula A had significantly higher rod thresholds than infants receiving long-chain co-3 (human milk, Formula C, and intrauterine). Infants receiving Formula B had intermediate thresholds that were significantly higher than those of infants receiving intrauterine nutrition. Analysis of the leading edge of the a-wave showed that b-wave differences originated at the photoreceptor level. Differences were not present in infants at 57 wk postconception. No significant differences among groups were found in cone b-waves at 36 or 57 wk postconception. Oscillatory potentials had significantly longer implicit times at 57 wk postconception in infants fed Formula A than in infants receiving human milk. These findings suggest that retinal function varies with the dietary supply of co-3 fatty acids in VLBW infants. Invest Ophthalmol Vis Sci 33: [2365][2366][2367][2368][2369][2370][2371][2372][2373][2374][2375][2376]1992 Linoleic acid (18:2 00-6) and a-linolenic acid (18:3 a>-3) are considered essential fatty acids (EFAs) for humans because of our inability to synthesize them and the resulting deficiency syndromes when they are removed from the diet

Spectrum of Mutations in the RPGR Gene That Are Identified in 20% of Families with X-Linked Retinitis Pigmentosa

SummaryThe RPGR (retinitis pigmentosa GTPase regulator) gene for RP3, the most frequent genetic subtype of X-linked retinitis pigmentosa (XLRP), has been shown to be mutated in 10%–15% of European XLRP patients. We have examined the RPGR gene for mutations in a cohort of 80 affected males from apparently unrelated XLRP families, by direct sequencing of the PCR-amplified products from the genomic DNA. Fifteen different putative disease-causing mutations were identified in 17 of the 80 families; these include four nonsense mutations, one missense mutation, six microdeletions, and four intronic-sequence substitutions resulting in splice defects. Most of the mutations were detected in the conserved N-terminal region of the RPGR protein, containing tandem repeats homologous to those present in the RCC-1 protein (a guanine nucleotide-exchange factor for Ran-GTPase). Our results indicate that mutations either in as yet uncharacterized sequences of the RPGR gene or in another gene located in its vicinity may be a more frequent cause of XLRP. The reported studies will be beneficial in establishing genotype-phenotype correlations and should lead to further investigations seeking to understand the mechanism of disease pathogenesis

Spectrum of Mutations in the RPGR Gene That Are Identified in 20% of Families with X-Linked Retinitis Pigmentosa

SummaryThe RPGR (retinitis pigmentosa GTPase regulator) gene for RP3, the most frequent genetic subtype of X-linked retinitis pigmentosa (XLRP), has been shown to be mutated in 10%–15% of European XLRP patients. We have examined the RPGR gene for mutations in a cohort of 80 affected males from apparently unrelated XLRP families, by direct sequencing of the PCR-amplified products from the genomic DNA. Fifteen different putative disease-causing mutations were identified in 17 of the 80 families; these include four nonsense mutations, one missense mutation, six microdeletions, and four intronic-sequence substitutions resulting in splice defects. Most of the mutations were detected in the conserved N-terminal region of the RPGR protein, containing tandem repeats homologous to those present in the RCC-1 protein (a guanine nucleotide-exchange factor for Ran-GTPase). Our results indicate that mutations either in as yet uncharacterized sequences of the RPGR gene or in another gene located in its vicinity may be a more frequent cause of XLRP. The reported studies will be beneficial in establishing genotype-phenotype correlations and should lead to further investigations seeking to understand the mechanism of disease pathogenesis

Safety Assessment of Docosahexaenoic Acid in X-Linked Retinitis Pigmentosa: The 4-Year DHAX Trial

Citation: Hughbanks-Wheaton DK, Birch DG, Fish GE, et al. Safety assessment of docosahexaenoic acid in X-linked retinitis pigmentosa: the 4-year DHAX trial. Invest Ophthalmol Vis Sci. 2014;55:4958-4966. DOI: 10.1167/iovs.14-14437 PURPOSE. Docosahexaenoic acid (DHA) continues to be evaluated and recommended as treatment and prophylaxis for various diseases. We recently assessed efficacy of high-dose DHA supplementation to slow vision loss in patients with X-linked retinitis pigmentosa (XLRP) in a randomized clinical trial. Because DHA is a highly unsaturated fatty acid, it could serve as a target for free-radical induced oxidation, resulting in increased oxidative stress. Biosafety was monitored during the 4-year trial to determine whether DHA supplementation was associated with identifiable risks. METHODS. Males (n ¼ 78; 7-31 years) meeting entry criteria were enrolled. The modified intent-to-treat cohort (DHA ¼ 33; placebo ¼ 27) adhered to the protocol ‡ 1 year. Participants were randomized to an oral dose of 30 mg/kg/d DHA or placebo plus a daily multivitamin. Comprehensive metabolic analyses were assessed for group differences. Treatment-emergent adverse events including blood chemistry metabolites were recorded. RESULTS. By year 4, supplementation elevated plasma and red blood cell-DHA 4.4-and 3.6-fold, respectively, compared with the placebo group (P < 0.00001). Over the trial duration, no significant differences between DHA and placebo groups were found for vitamin A, vitamin E, platelet aggregation, antioxidant activity, lipoprotein cholesterol, or oxidized LDL levels (all P > 0.14). Adverse events were transient and not considered severe (e.g., gastrointestinal [GI] irritability, blood chemistry alterations). One participant was unable to tolerate persistent GI discomfort. CONCLUSIONS. Long-term, high-dose DHA supplementation to patients with XLRP was associated with limited safety risks in this 4-year trial. Nevertheless, GI symptoms should be monitored in all patients taking high dose DHA especially those with personal or family history of GI disturbances. (ClinicalTrials.gov number, NCT00100230.) Keywords: biosafety, fatty acids, adverse events R etinitis pigmentosa (RP) is a retinal degenerative disease characterized by night blindness and visual field constriction 1 with four underlying inheritance patterns. The X-linked form of RP (XLRP) is among the most severe with night blindness often detectable by age 5 years 2-4 and legal blindness by the second or third decade. Gene defects are known to cause retinal degeneration, yet factors such as environment, diet, stress, and/or metabolism may modify disease severity. Many patients with RP have lower plasma and red blood cell (RBC) levels of the n3 polyunsaturated fatty acid docosahexaenoic acid (DHA; 22:6n3) than normally-sighted controls. 5 Blood DHA was significantly correlated with age-adjusted ERG responses in XLRP such that patients with lower RBC-DHA tended to have lower ERG amplitudes. 6 These findings were similarly documented in approximately 70% of female XLRP carriers (Hoffman DR, et al. IOVS 1998;39:ARVO Abstract 725). A reduction in DHA biosynthesis was demonstrated in XLRP using stable isotopes to assess in vivo metabolism 7 suggesting that downregulation of hepatic D 5 desaturase may contribute to subnormal blood DHA levels. Thus, daily supplementation with DHA may bypass any decrease in DHA biosynthesis. Docosahexaenoic acid comprises 1% to 5% of membrane fatty acids in most human tissues; however, it is the most abundant fatty acid in the retina. 8 This n3 fatty acid can increase membrane fluidity and modify the mobility of vital proteins and activities of retinal enzymes, 9,10 promote photoreceptor differentiation, 11 and antiapoptotic activity. 12 The highly unsaturated nature of DHA makes it a potential target for free radical oxidative damage. Increased polyunsaturated fatty acid (PUFA) intake, particularly long-chain PUFAs (LCPUFAs; >18 carbons), may lead to elevated oxidative stress and subsequent membrane damage. 15 Numerous n3-supplementation studies report elevations in low-and highdensity lipoprotein (LDL and HDL)-cholesterol

A Seriation Approach for Visualization-Driven Discovery of Co-Expression Patterns in Serial Analysis of Gene Expression (SAGE) Data

Background: Serial Analysis of Gene Expression (SAGE) is a DNA sequencing-based method for large-scale gene expression profiling that provides an alternative to microarray analysis. Most analyses of SAGE data aimed at identifying co-expressed genes have been accomplished using various versions of clustering approaches that often result in a number of false positives. Principal Findings: Here we explore the use of seriation, a statistical approach for ordering sets of objects based on their similarity, for large-scale expression pattern discovery in SAGE data. For this specific task we implement a seriation heuristic we term ‘progressive construction of contigs ’ that constructs local chains of related elements by sequentially rearranging margins of the correlation matrix. We apply the heuristic to the analysis of simulated and experimental SAGE data and compare our results to those obtained with a clustering algorithm developed specifically for SAGE data. We show using simulations that the performance of seriation compares favorably to that of the clustering algorithm on noisy SAGE data. Conclusions: We explore the use of a seriation approach for visualization-based pattern discovery in SAGE data. Using both simulations and experimental data, we demonstrate that seriation is able to identify groups of co-expressed genes more accurately than a clustering algorithm developed specifically for SAGE data. Our results suggest that seriation is a usefu