Regulation of bone biology by prostaglandin endoperoxide H synthases (PGHS): A rose by any other name...

It is well established that prostaglandins are essential mediators of bone resorption and formation. In the early 1990s, it was discovered that enzymatic reactions producing prostaglandins were regulated by two cyclooxygenase enzymes, one producing prostaglandins constitutively in tissues like the stomach, prostaglandin endoperoxide H synthase-1 (PGHS-1 or COX-1), and another induced by mitogens or inflammatory mediators (PGHS-2 or COX-2). This neat distinction has not been maintained because both enzymes act in different cell systems to provide physiological signaling, constitutively or by induction under certain conditions. For example, the regulation patterns of PGHS-1 and PGHS-2 are distinct, but the evidence shows that PGHS-2 functions constitutively in the skeleton. PGHS-2 hits quickly been established, therefore, as a key regulator of bone biology, capable of rapid and transient expression in bone cells, and mediating osteoclastogenesis, mechanotransduction, bone formation and fracture repair. The goal of this review is to Summarize the current state of our knowledge of PGHS regulation of bone metabolism and to identify some of the key unresolved challenges and questions that require further study. (c) 2006 Elsevier Ltd. All rights reserved.

Characterisation of three ACC synthase gene family members during post-harvest-induced senescence in broccoli (Brassica oleracea L. var. italica)

We investigated the gene expression profiles of different members of the 1-aminocyclopropane-1-carboxilic acid (ACC) synthase (EC 4.4.1.14) gene family in broccoli (Brassica oleracea L. var. italica) during the post-harvest-induced senescence process. Using RT-PCR, three different cDNAs coding for ACC synthase (BROCACS1, BROCACS2 and BROCACS3) were amplified from floret tissue at the start of the senescence process. The three genes share relatively little homology, but have highly homologous sequences in Arabidopsis thaliana, and could be functionally related to these counterparts. Southern analyses suggest that BROCACS1 and BROCACS3 are present as single copy genes, while there are probably two copies of BROCACS2. All three genes showed different expression patterns: BROCACS1 is likely to be either wound - or mechanical stress-induced showing high transcript levels after harvesting, but no detectable expression afterwards. BROCACS2 shows steady expression throughout senescence, increasing at the latest stages, and BROCACS3 is almost undetectable until the final stages. Our results suggest that BROCACS1 could be required to initiate the senescence process, while BROCACS2 would be the main ACC synthase gene involved throughout the post-harvest-induced senescence. BROCACS3's expression pattern indicates that it is not directly involved in the initial stages of senescence, but in the final remobilization of cellular resources.

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-Angstrom crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 Angstrom, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.

Crystallization of Arabidopsis thaliana acetohydroxyacid synthase in complex with the sulfonylurea herbicide chlorimuron ethyl

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyses the formation of 2-acetolactate and 2-aceto-2-hydroxybutyrate as the first step in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. The enzyme is inhibited by a wide range of substituted sulfonylureas and imidazolinones and many of these compounds are used as commercial herbicides. Here, the crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of Arabidopsis thaliana AHAS in complex with the sulfonylurea herbicide chlorimuron ethyl are reported. This is the first report of the structure of any plant protein in complex with a commercial herbicide. Crystals diffract to 3.0 Angstrom resolution, have unit-cell parameters a = b = 179.92, c = 185.82 Angstrom and belong to space group P6(4)22. Preliminary analysis indicates that there is one monomer in the asymmetric unit and that these are arranged as pairs of dimers in the crystal. The dimers form a very open hexagonal lattice, with a high solvent content of 81%.

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

Vasodilator mechanisms in the dorsal aorta of the giant shovelnose ray, Rhinobatus typus (Rajiformes; Rhinobatidae)

This study investigated the nature of vasodilator mechanisms in the dorsal aorta of the giant shovelnose ray, Rhinobatus typus. Anatomical techniques found no evidence for an endothelial nitric oxide synthase, but neural nitric oxide synthase was found to be present in the perivascular nerve fibres of the dorsal aorta and other arteries and veins using both NADPH-diaphorase staining and immunohistochemistry with a specific neural NOS antibody. Arteries and veins both contained large nNOS-positive nerve trunks from which smaller nNOS-positive bundles branched and formed a plexus in the vessel wall. Single, varicose nNOS-positive nerve fibres were present in both arteries and veins. Within the large bundles of both arteries and veins, groups of nNOS-positive cell bodies forming microganglia were observed. Double-labelling immunohistochemistry using an antibody to tyrosine hydroxylase showed that nearly all the NOS nerves were not sympathetic. Acetylcholine always caused constriction of isolated rings of the dorsal aorta and the nitric oxide donor, sodium nitroprusside, did not mediate any dilation. Addition of nicotine (3 x 10(-4) M) to preconstricted rings caused a vasodilation that was not affected by the nitric oxide synthase inhibitor, L-NNA (10(-4) M), nor the soluble guanylyl cyclase inhibitor, ODQ (10(-5) M). This nicotine-mediated vasodilation was, therefore, not due to the synthesis and release of NO. Disruption of the endothelium significantly reduced or eliminated the nicotine-mediated vasodilation. In addition. indomethacin (10(-5) M), an inhibitor of cyclooxygenases, significantly increased the time period to maximal dilation and reduced, but did not completely inhibit the nicotine-mediated vasodilation. These data support the hypothesis that a prostaglandin is released from the vascular endothelium of a batoid ray, as has been described previously in other groups of fishes. The function of the nitrergic innervation of the blood vessels is not known because nitric oxide does not appear to regulate vascular tone. (C) 2003 Elsevier Inc. All rights reserved.

Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site

Sulfadoxine is predominantly used in combination with pyrimethamine, commonly known as Fansidar, for the treatment of Plasmodium falciparum. This combination is usually less effective against Plasmodium vivax, probably due to the innate refractoriness of parasites to the sulfadoxine component. To investigate this mechanism of resistance by P. vivax to sulfadoxine, we cloned and sequenced the P. vivax dhps (pvdhps) gene. The protein sequence was determined, and three-dimensional homology models of dihydropteroate synthase (DHPS) from P. vivax as well as P. falciparum were created. The docking of sulfadoxine to the two DHPS models allowed us to compare contact residues in the putative sulfadoxine-binding site in both species. The predicted sulfadoxine-binding sites between the species differ by one residue, V585 in P. vivax, equivalent to A613 in P. falciparum. V585 in P. vivax is predicted by energy minimization to cause a reduction in binding of sulfadoxine to DHPS in P. vivax compared to P. falciparum. Sequencing dhps genes from a limited set of geographically different P. vivax isolates revealed that V585 was present in all of the samples, suggesting that V585 may be responsible for innate resistance of P. vivax to sulfadoxine. Additionally, amino acid mutations were observed in some P. vivax isolates in positions known to cause resistance in P. falciparum, suggesting that, as in P. falciparum, these mutations are responsible for acquired increases in resistance of P. vivax to sulfadoxine.

Hybrid 'Sinta' papaya exhibits unique ACC synthase 1 cDNA isoforms

Five ripening-related ACC synthase cDNA isoforms were cloned from 80% ripe papaya cv. 'Sinta' by reverse transcription-PCR using gene-specific primers. Clone 2 had the longest transcript and contained all common exons and three alternative exons. Clones 3 and 4 contained common exons and one alternative exon each, while clone 1, the most common transcript, contained only the common exons. Clone 5 could be due to cloning artifacts and might not be a unique cDNA fragment. Thus, there are only four isoforms of ACC synthase mRNA. Southern blot analysis indicates that all five clones came from only one gene existing as a single copy in the 'Sinta' papaya genome. Multiple sequence alignment indicates that the four isoforms arise from a single gene, possibly through alternative splicing mechanisms. All the putative alternative exons were present at the 5'-end of the gene comprising the N-terminal region of the protein. 'Sinta' ACC synthase cDNAs were of the capacs 1 type and are most closely related to a 1.4 kb capacs 1-type DNA (AJ277160) from Eksotika papaya. No capacs 2-type cDNAs were cloned from 'Sinta' by RT-PCR. This is the first report of possible alternative splicing mechanism in ripening-related ACC synthase genes in hybrid papaya, possibly to modulate or fine-tune gene expression relevant to fruit ripening.

Nitric oxide synthase inhibition in sepsis? Lessons learned from large-animal studies

Nitric Oxide (NO) plays a controversial role in the pathophysiology of sepsis and septic shock. Its vasodilatory effects are well known, but it also has pro- and antiinflammatory properties, assumes crucial importance in antimicrobial host defense, may act as an oxidant as well as an antioxidant, and is said to be a vital poison for the immune and inflammatory network. Large amounts of NO and peroxynitrite are responsible for hypotension, vasoplegia, cellular suffocation, apoptosis, lactic acidosis, and ultimately multiorgan failure. Therefore, NO synthase (NOS) inhibitors were developed to reverse the deleterious effects of NO. Studies using these compounds have not met with uniform success however, and a trial using the nonselective NOS inhibitor N-G-methyl-L-arginine hydrochloride was terminated prematurely because of increased mortality in the treatment arm despite improved shock resolution. Thus, the issue of NOS inhibition in sepsis remains a matter of debate. Several publications have emphasized the differences concerning clinical applicability of data obtained from unresuscitated, hypodynamic rodent models using a pretreatment approach versus resuscitated, hyperdynamic models in high-order species using posttreatment approaches. Therefore, the present review focuses on clinically relevant large-animal studies of endotoxin or living bacteria-induced, hyperdynamic models of sepsis that integrate standard day-today care resuscitative measures.

Elucidating the specificity of binding of sulfonylurea herbicides to acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulforneturon methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 Angstrom), sulforneturon methyl (2.34 Angstrom), and two other sulfonylureas, metsulfuron methyl (2.29 Angstrom) and tribenuron methyl (2.58 Angstrom). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethyl-ThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme.

Suicide inhibition of acetohydroxyacid synthase by hydroxypyruvate

Acetohydroxyacid synthase (Ec 2.2.1.6) catalyses the thiamine diphosphate-dependent reaction between two molecules of pyruvate yielding 2-acetolactacte and CO2. The enzyme will also utilise hydroxypyruvate with a k(cat) value that is 12% of that observed with pyruvate. When hydroxypyruvate is the substrate, the enzyme undergoes progressive inactivation with kinetics that are characteristic of suicide inhibition. It is proposed that the dihydroxyethyl-thiamine diphosphate intermediate can expel a hydroxide ion forming an enol that rearranges to a bound acetyl group.