In vitro activation of soluble guanylyl cyclase and nitric oxide release: a comparison of NO donors and NO mimetics

Nitric oxide (NO) performs a central role in biological systems, binding to the heme site of soluble guanylyl cyclase (sGC), leading to enzyme activation and elevation of intracellular levels of cGMP. Organic nitrates, in particular, nitroglycerin (GTN), are clinically important nitrovasodilators that function as NO-mimetics in biological systems. Comparison of sGC activation data with electrochemically measured rates of NO release for genuine NO donors, NONOates and nitrosothiols, yields an excellent correlation between the EC(50) for sGC activation and the rate constant for NO release, k(NO). However, activation of sGC by GTN and the nitrates has very different characteristics, including the requirement for specific added thiols, for example, cysteine. The reaction of GTN with cysteine in anaerobic solution yields NO slowly, and NO release, measured by chemiluminescence detection, is quenched by added metal ion chelator. The generation of NO under aerobic conditions is 100-fold slower than the anaerobic reaction. Furthermore, NO release from the reaction of GTN with cysteine in phosphate buffer is too slow to account for sGC activation by GTN/cysteine. The slow rate of the chemical reaction to release NO suggests that nitrates can activate sGC by an NO-independent mechanism. In contrast to the genuine NO donors, GTN behaves as a partial agonist with respect to sGC activation, but in the presence of the allosteric sGC activator, YC-1, GTN exhibits full agonist activity.     

Effects of nitroglycerin on soluble guanylate cyclase: implications for nitrate tolerance

Soluble guanylate cyclase (sGC) is a heterodimeric hemoprotein that catalyzes the conversion of GTP to cGMP. Upon binding NO to its heme cofactor, purified sGC was activated 300-fold. sGC was only activated 67-fold by nitroglycerin (GTN) and Cys; and in the absence of Cys, GTN did not activate sGC. Electronic absorption spectroscopy studies showed that upon NO binding, the Soret of ferrous sGC shifted from 431 to 399 nm. The data also revealed that activation of sGC by GTN/Cys was not via the expected ferrous heme-NO species as indicated by the absence of the 399 nm heme Soret. Furthermore, EPR studies of the reaction of GTN/Cys with sGC confirmed that no ferrous heme-NO species was formed but that there was heme oxidation. Potassium ferricyanide is known to oxidize ferrous sGC to the ferric oxidation state. Spectroscopic and activity data for the reactions of sGC with GTN alone or with K(3)Fe(CN)(6) were indistinguishable. These data suggest the following: 1) GTN/Cys do not activate sGC via GTN biotransformation to NO in vitro, and 2) in the absence of added thiol, GTN oxidizes sGC.     

Rampant horizontal transfer and duplication of rubisco genes in eubacteria and plastids

Previous work has shown that molecular phylogenies of plastids, cyanobacteria, and proteobacteria based on the rubisco (ribulose-1,5- bisphosphate carboxylase/oxygenase) genes rbcL and rbcS are incongruent with molecular phylogenies based on other genes and are also incompatible with structural and biochemical information. Although it has been much speculated that this is the consequence of a single horizontal gene transfer (of a proteobacterial or mitochondrial rubisco operon into plastids of rhodophytic and chromophytic algae), neither this hypothesis nor the alternative hypothesis of ancient gene duplication have been examined in detail. We have conducted phylogenetic analyses of all available bacterial rbcL sequences, and representative plastid sequences, in order to explore these alternative hypothesis and fully examine the complexity of rubisco gene evolution. The rbcL phylogeny reveals a surprising number of gene relationships that are fundamentally incongruent with organismal relationships as inferred from multiple lines of other molecular evidence. On the order of six horizontal gene transfers are implied by the form I (L8S8) rbcL phylogeny, two between cyanobacteria and proteobacteria, one between proteobacteria and plastids, and three within proteobacteria. Alternatively, a single ancient duplication of the form I rubisco operon, followed by repeated and pervasive differential loss of one operon or the other, would account for much of this incongruity. In all probability, the rubisco operon has undergone multiple events of both horizontal gene transfer and gene duplication in different lineages.      

The hisD-hisC gene border of the Salmonella typhimurium histidine operon

Summary We have sequenced the hisD-hisC gene border of the Salmonella typhimurium histidine operon. The translation termination codon of the hisD gene overlaps with the translation initiation codon of the hisC gene in the manner . The Shine-Dalgarno sequence of the hisC gene is contained entirely within hisD and there is no intercistronic space since all of the bases are utilized in coding. Two mutations that alter the hisD-hisC gene border are analyzed. Both mutations simultaneously abolish the termination codon of hisD and modify the initiation codon of hisC. One of the mutations changes the hisC initiation codon from AUG to AUU. The AUU codon is 10 to 20% as efficient as AUG for initiation of translation of the hisC gene. The mutant hisC ribosome binding site is compared to the ribosome binding site of the Escherichia coli infC gene which has been reported to contain an AUU initiation codon. The role of overlapping termination/initiation codons in regulating translation of polycistronic mRNAs in bacterial operons is discussed.     

Performance of Apis mellifera, Bombus impatiens, and Peponapis pruinosa (Hymenoptera: Apidae) as pollinators of pumpkin

Pollination services of pumpkin, Cucurbita pepo L., provided by the European honey bee, Apis mellifera L., were compared with two native bee species, the common eastern bumble bee, Bombus impatiens (Cresson), and Peponapis pruinosa Say, in New York from 2008 to 2010. Performance of each species was determined by comparing single-visit pollen deposition, percentage of visits that contacted the stigma, flower-handling time, fruit and seed set, and fruit weight per number of visits. Fruit yield from small fields (0.6 ha) supplemented with commercial B. impatiens colonies was compared with yield from those not supplemented. A. mellifera spent nearly 2 and 3 times longer foraging on each pistillate flower compared with B. impatiens and P. pruinosa, respectively. A. mellifera also visited pistillate flowers 10-20 times more frequently than B. impatiens and P. pruinosa, respectively. Yet, B. impatiens deposited 3 times more pollen grains per stigma and contacted stigmas significantly more often than either A. mellifera or P. pruinosa. Fruit set and weight from flowers visited four to eight times by B. impatiens were similar to those from open-pollinated flowers, whereas flowers pollinated by A. mellifera and P. pruinosa produced fewer fruit and smaller fruit compared with those from open-pollinated flowers. Fields supplemented with B. impatiens produced significantly more pumpkins per plant than nonsupplemented fields. B. impatiens was a better pollinator of pumpkin than P. pruinosa and should be considered as a promising alternative to A. mellifera for pollinating this crop.