How Well Do We Know the Neutron-Matter Equation of State at the Densities Inside Neutron Stars? A Bayesian Approach with Correlated Uncertainties.

We introduce a new framework for quantifying correlated uncertainties of the infinite-matter equation of state derived from chiral effective field theory (χEFT). Bayesian machine learning via Gaussian processes with physics-based hyperparameters allows us to efficiently quantify and propagate theoretical uncertainties of the equation of state, such as χEFT truncation errors, to derived quantities. We apply this framework to state-of-the-art many-body perturbation theory calculations with nucleon-nucleon and three-nucleon interactions up to fourth order in the χEFT expansion. This produces the first statistically robust uncertainty estimates for key quantities of neutron stars. We give results up to twice nuclear saturation density for the energy per particle, pressure, and speed of sound of neutron matter, as well as for the nuclear symmetry energy and its derivative. At nuclear saturation density, the predicted symmetry energy and its slope are consistent with experimental constraints.

Power counting of contact-range currents in effective field theory.

We analyze the power counting of two-body currents in nuclear effective field theories (EFTs). We find that the existence of nonperturbative physics at low energies, which is manifest in the existence of the deuteron and the ^{1}S_{0} NN virtual bound state, combined with the appearance of singular potentials in versions of nuclear EFT that incorporate chiral symmetry, modifies the renormalization-group flow of the couplings associated with contact operators that involve nucleon-nucleon pairs and external fields. The order of these couplings is thereby enhanced with respect to the naive-dimensional-analysis estimate. Consequently, short-range currents enter at a lower order in the chiral EFT than has been appreciated up until now, and their impact on low-energy observables is concomitantly larger. We illustrate the changes in the power counting with a few low-energy processes involving external probes and few-nucleon systems, including electron-deuteron elastic scattering and radiative neutron capture by protons.

Measurement of Compton scattering from the deuteron and an improved extraction of the neutron electromagnetic polarizabilities.

The electromagnetic polarizabilities of the nucleon are fundamental properties that describe its response to external electric and magnetic fields. They can be extracted from Compton-scattering data-and have been, with good accuracy, in the case of the proton. In contradistinction, information for the neutron requires the use of Compton scattering from nuclear targets. Here, we report a new measurement of elastic photon scattering from deuterium using quasimonoenergetic tagged photons at the MAX IV Laboratory in Lund, Sweden. These first new data in more than a decade effectively double the world data set. Their energy range overlaps with previous experiments and extends it by 20 MeV to higher energies. An analysis using chiral effective field theory with dynamical Δ(1232) degrees of freedom shows the data are consistent with and within the world data set. After demonstrating that the fit is consistent with the Baldin sum rule, extracting values for the isoscalar nucleon polarizabilities, and combining them with a recent result for the proton, we obtain the neutron polarizabilities as αn=[11.55±1.25(stat)±0.2(BSR)±0.8(th)]×10(-4) fm(3) and ßn=[3.65∓1.25(stat)±0.2(BSR)∓0.8(th)]×10(-4) fm(3), with χ(2)=45.2 for 44 degrees of freedom.

Integrin tyrosine phosphorylation in platelet signaling.

The beta 3 integrin cytoplasmic tyrosine (ICY) motif of alpha IIb beta 3 becomes tyrosine phosphorylated during platelet aggregation, causing Shc and myosin to interact with the beta-integrin cytoplasmic domain. Platelets from mice lacking beta 3 ICY motif tyrosines formed defective aggregates and poorly retracted clots, establishing integrin tyrosine phosphorylation as a key mediator of beta 3-integrin signals.

Identification of membrane proteins mediating the interaction of human platelets.

Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.

Actin filament content and organization in unstimulated platelets.

The extent of actin polymerization in unstimulated, discoid platelets was measured by DNase I inhibition assay in Triton X-100 lysates of platelets washed at 37 degrees C by gel filtration, or in Triton X-100 lysates of platelets washed at ambient temperatures by centrifugation in the presence of prostacyclin. About 40% of the actin in the discoid platelets obtained by either method existed as filaments. These filaments could be visualized by electron microscopy of thin sections. Similar results were obtained when the actin filament content of discoid platelets was measured by sedimentation of filaments from Triton X-100 lysates at high g forces (145,000 g for 45 min). However, few of these filaments sedimented at the lower g forces often used to isolate networks of actin filaments from cell extracts. These results indicate that actin filaments in discoid cells are not highly crosslinked. Platelets isolated by centrifugation in the absence of prostacyclin were not discoid, but were instead irregular with one or more pseudopodia. These platelets also contained approximately 40-50% of their actin in a filamentous form; many of these filaments sedimented at low g forces, however, indicating that they were organized into networks. The discoid shape of these centrifuged platelets could be restored by incubating them for 1-3 h at 37 degrees C, which resulted in the reversal of filament organization. High g forces were then required for the sedimentation of the actin. Approximately 80-90% of the actin in platelets washed at 4 degrees C was filamentous; this high actin filament content could be attributed to actin polymerization during the preparation of the platelets at low temperatures. These studies show that platelet activation involves mechanisms for the structural reorganization of existing filaments, in addition to those previously described for mediating actin polymerization.

Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage.

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.

Adhesive properties of the beta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells.

Glycoprotein IIb-IIIa (alpha IIb beta 3) and the vitronectin receptor (alpha v beta 3), two integrins that share the common beta 3 subunit, have been reported to function as promiscuous receptors for the RGD-containing adhesive proteins fibrinogen, vitronectin, fibronectin, von Willebrand factor, and thrombospondin. The present study was designed to establish a cell system for the expression of either GP IIb-IIIa or the vitronectin receptor in an otherwise identical cellular environment and to compare the adhesive properties of these two integrins with those of native GP IIb-IIIa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP IIb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and von Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP IIb into M21-L cells, a variant of M21 cells (Cheresh, D.A., and R.C. Spiro. 1987. J. Biol. Chem. 262:17703-17711), which lack the expression of functional alpha v and are therefore unable to attach to vitronectin, fibrinogen, and von Willebrand factor. Transfectants expressing GP IIb were isolated by immunomagnetic beads and surface expression of the GP IIb-IIIa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21-L/GP IIb cells. Comparative functional studies demonstrated that GP IIb-IIIa expressed in M21-L/GPIIb cells as well as native GP IIb-IIIa constitutively expressed in HEL-5J20 cells (an HEL variant lacking alpha v beta 3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von Willebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing alpha v beta 3) mediated cell attachment to fibrinogen, vitronectin, and von Willebrand factor. Similarly, PGl2-treated resting platelets attached to immobilized fibrinogen but not to vitronectin or von Willebrand factor, and this attachment could be inhibited by mAb A2A9 (directed against a functional site on the GP IIb-IIIa complex). However, in contrast to platelets, which adhered to vitronectin and von Willebrand factor after stimulation by thrombin or PMA, activation of the protein kinase C pathway in M21-L/GP IIb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor.(ABSTRACT TRUNCATED AT 400 WORDS)

The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering.

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.

Thrombin-induced platelet aggregation is mediated by a platelet plasma membrane-bound lectin.

Throbin-activated human platelets cause agglutination of trypsinized, formalinized bovine erythrocytes. This lectin activity of stimulated platelets was blocked by galactosamine, glucosamine, mannosamine, lysine, and arginine, but not by N-acetylated sugars, other neutral sugars, or other amino acids. Inhibitors of the thrombin-induced lectin activity also blocked thrombin-induced platelet aggregation. It appears that a membrane surface component that has lectin activity mediates platelet aggregation.

First UK report of successful treatment of Mycobacterium simiae and immune reconstitution inflammatory syndrome in an HIV-seropositive patient.

Non-tuberculous mycobacteria (NTM) such as Mycobacterium avium intracellulare are commonly encountered by HIV physicians and management strategies are well established. Experience of other NTM is, however, limited. As HIV epidemiology in the United Kingdom changes, we may expect the emergence of these lesser known mycobacterial infections. We present the first UK report of an AIDS patient who has survived infection with disseminated Mycobacterium simiae despite cerebrospinal fluid involvement, an extremely high level of baseline HIV viraemia and treatment complicated by severe immune reconstitution inflammatory syndrome.

Platelet membrane defects in Glanzmann's thrombasthenia. Evidence for decreased amounts of two major glycoproteins.

Platelets from patients with Glanzmann's thrombasthenia have a distinct molecular alteration of the plasma membrane surface, namely decreased amounts of a major glycoprotein designated as IIb (apparent mol wt 142,000). To identify other possible surface defects of thrombasthenic platelets, we labeled the membrane polypeptides of normal and thrombasthenic platelets by two different techniques: lactoperoxidase-catalyzed iodination and galactose oxidase oxidation, followed by reduction with tritiated sodium borohydride. Labeling patterns were determined after the polypeptides were separated by two-dimensional polyacrylamide gel electrophoresis. Before the second dimension was run, platelet samples were incubated with a reducing agent, beta-mercapto-ethanol, to cleave the disulfide bonds of certain glycoproteins; the resulting changes in electrophoretic mobility permitted better resolution of individual molecules. Comparison of the labeled polypeptides of normal and thrombasthenic samples after reduction indicated decreased labeling of two major glycoproteins in thrombasthenic platelets: IIb and III (apparent mol wt 114,000). The relative proportions of radioactivity incorporated by these polypeptides were about 60 and 80% less than control values, respectively. With either Coomassie Blue or periodic acid-Schiff's reagent, glycoprotein III stained much less intensely in thrombasthenic compared to normal samples, indicating that the observed labeling deficit was caused by a decreased concentration of the molecule rather than steric inaccessibility on the membrane surface. Analysis of normal plasma membranes by affinity chromatography showed that glycoprotein IIb has receptors for lectin from Lens culinaris, the common lentil, whereas III does not. We conclude that a characteristic feature of Glanzmann's thrombasthenia is a decreased concentration of two discrete glycoproteins in the platelet plasma membrane.

Synthesis and expression of the fibroblast fibronectin receptor in human monocytes.

Human monocytes adhere to fibronectin, but the receptor (or receptors) mediating this interaction has not been clearly identified. To examine the nature of this receptor, human monocytes were obtained by counter-current elutriation and were found to adhere to immobilized fibronectin but not to vitronectin or von Willebrand factor. Antibodies and peptides were used to determine whether monocyte adherence to immobilized fibronectin was mediated by a receptor similar to that which mediates the adherence of fibroblasts to fibronectin. Antibodies against both the alpha and beta subunits of the fibroblast fibronectin receptor (VLA-5) inhibited monocyte adherence to fibronectin. These antibodies immunoprecipitated two surface proteins from monocytes that migrated at 140 and 120 kD under nonreducing conditions. Surface proteins with identical apparent molecular weights were immunoprecipitated from surface-labeled MG-63 cells, a fibroblast-like cell line. A synthetic peptide containing the Arg-Gly-Asp-Ser (RGDS) sequence also inhibited monocyte adherence to fibronectin. cDNA hybridization experiments revealed the presence of mRNA for both the alpha and beta subunits of the fibroblast fibronectin receptor in human monocytes. We conclude that circulating monocytes express a fibronectin receptor that is structurally and functionally very similar, if not identical, to the fibronectin receptor in adherent fibroblasts, and that this receptor mediates monocyte adherence to immobilized fibronectin.

Platelet membrane glycoproteins implicated in ristocetin-induced aggregation. Studies of the proteins on platelets from patients with Bernard-Soulier syndrome and von Willebrand's disease.

The antibiotic ristocetin only aggregates platelets in the presence of plasma von Willebrand factor. Platelets from patients with Bernard-Soulier syndrome do not aggregate upon addition of ristocetin although, in contrast to von Willebrand's disease, plasma levels of factor VIII complex (factor VIII clotting activity, von Willebrand factor activity, and von Willebrand antigen) are normal. The membrane surface of normal platelets was modified and compared to the surface of platelets from a patient with Bernard-Soulier syndrome in an attempt to identify the receptor involved in von Willebrand factor-ristocetin-induced aggregation. After the incubation of washed normal platelets with a preparation of ristocetin previously shown to contain a proteolytic contaminant, the aggregation response is significantly decreased on addition or normal plasma. Analaysis by gel electrophoresis of such platelets when stained for carbohydrate revealed a decrease in the relative amounts of membrane glycopro-eins. Chymotrypsin-treated normal platelets had less membrane glycoproteins in addition to giving a reduced aggregation response in ristocetin-induced aggregation. Staining of gels for protein and carbohydrate indicated that there was an extensive change in the surface of Bernard-Soulier platelets, whereas those from patients with von Willebrand's disease appeared the same as normal. Platelets from patients were labeled by the lactoperoxidase iodination technique. Not only was the relative intensity of staining of platelet-specific proteins and glycoproteins changed in Bernard-Soulier platelets, but the iodination of the glycoproteins on the membrane surface relative to other membrane constituents was lower. In contrast, platelets from patients with von Willebrand's disease showed a normal exposure of membrane components. These data suggest therefore that membrane glycoproteins may play a functional role in ristocetin-induced aggregation.

Biochemical studies of two patients with the gray platelet syndrome. Selective deficiency of platelet alpha granules.

The biochemistry of platelets from two unrelated patients with the gray platelet syndrome, a deficiency of platelet alpha-granules, has been evaluated. Ultrastructural studies of their platelets revealed the number of alpha-granules to be less than 15% of normal, whereas the number of dense bodies was within normal limits. Platelets from both patients had severe deficiencies of platelet factor 4 and beta-thromboglobulin (less than 10% of normal). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a marked deficiency of thrombin-sensitive protein in both patients. Analysis of the platelet-derived growth factor in one patient showed it was also markedly reduced. Levels of lysosomal enzymes, adenine nucleotides, serotonin, and catalase, and conversion of arachidonic acid by the lipoxygenase and cyclo-oxygenase enzymes, were within normal limits. The results provide important evidence to define the contents of alpha-granules and to differentiate these contents from the contents of lysosomal granules, dense bodies, and peroxisomes. Functional studies of these platelets showed deficiencies in ADP, thrombin, and collagen aggregation. The results suggest that alpha-granules or their contents make a contribution to normal platelet aggregation.

Formaldehyde activation of mitoxantrone yields CpG and CpA specific DNA adducts.

Recently we have found that mitoxantrone, like Adria-mycin, can be activated by formaldehyde and subsequently form adducts which stabilise double-stranded DNA in vitro. This activation by formaldehyde may be biologically relevant since formaldehyde levels are elevated in those tumours in which mitoxan-trone is most cytotoxic. In vitro transcription analysis revealed that these adducts block the progression of RNA polymerase during transcription and cause truncated RNA transcripts. There was an absolute requirement for both mitoxantrone and formaldehyde in transcriptional blockage formation and the activated complex was found to exhibit site specificity, with blockage occurring prior to CpG and CpA sites in the DNA (non-template strand). The stability of the adduct at 37 degrees C was site dependent. The half-lives ranged from 45 min to approximately 5 h and this was dependent on both the central 2 bp blockage site as well as flanking sequences. The CpG specificity of mitoxantrone adduct sites was also confirmed independently by a lambda exonuclease digestion assay.

Interstrand cross-linking by adriamycin in nuclear and mitochondrial DNA of MCF-7 cells.

Activation of Adriamycin by formaldehyde leads to the formation of drug-DNA adducts in vitro and these adducts stabilise the DNA to such a degree that they function as virtual interstrand cross-links. The formation of these virtual interstrand cross-links by Adriamycin was investigated in MCF-7 cells using a gene-specific interstrand cross-linking assay. Cross-linking was measured in both the nuclear-encoded DHFR gene and in mitochondrial DNA (mtDNA). Cross-link formation increased linearly with Adriamycin concentration following a 4 h exposure to the drug. The rate of formation of Adriamycin cross-links in each of the genomes was similar, reaching maximal levels of 0.55 and 0.4 cross-links/10 kb in the DHFR gene and mtDNA respectively, following exposure to 20 micro M Adriamycin for 8 h. The interstrand cross-link was short lived in both DNA compartments, with a half-life of 4.5 and 3.3 h in the DHFR gene and mtDNA respectively. The kinetics of total Adriamycin adduct formation, detected using [(14)C]Adriamycin, was similar to that of cross-link formation. Maximal adduct levels (30 lesions/10 kb) were observed following incubation at 20 micro M drug for 8 h. The formation of such high levels of adducts and cross-links could therefore be expected to contribute to the mechanism of action of Adriamycin.

Molecular basis for the synergistic interaction of adriamycin with the formaldehyde-releasing prodrug pivaloyloxymethyl butyrate (AN-9).

The interaction of Adriamycin and pivaloyloxymethyl butyrate (AN-9) was investigated in IMR-32 neuroblastoma and MCF-7 breast adenocarcinoma cells. Adriamycin is a widely used anticancer drug, whereas AN-9 is an anticancer agent presently undergoing Phase II clinical trials. The anticancer activity of AN-9 has been attributed to its ability to act as a butyric acid prodrug, although it also releases formaldehyde and pivalic acid. Adriamycin and AN-9 in combination display synergy when exposed simultaneously to cells or when AN-9 treatment is up to 18 h after Adriamycin administration. However, the reverse order of addition results in antagonism. These interactions have been established using cell viability assays and classical isobologram analysis. To understand the molecular basis of this synergy, the relative levels of Adriamycin-DNA adducts were determined using various treatment combinations. Levels of Adriamycin-DNA adducts were enhanced when treatment combinations known to be synergistic were used and were diminished using those treatments known to be antagonistic. The relative timing of the addition of Adriamycin and AN-9 was critical, with a 20-fold enhancement of Adriamycin-DNA adducts occurring when AN-9 was administered 2 h after the exposure of cells to Adriamycin. The enhanced levels of these adducts and the accompanying decreased cell viability were directly related to the esterase-dependent release of formaldehyde from AN-9, providing evidence for the formaldehyde-mediated activation of Adriamycin.

The enzymic cleavage of linoleic acid to C9 carbonyl fragments in extracts of cucumber (Cucumis sativus) fruit and the possible role of lipoxygenase.

1. Homogenates and acetone powders of cucumber fruits catalyse the enzymic conversion of linoleic acid to aldehyde and oxoacid fragments in high yield, up to 60% with acetone powder extracts. 2. The major products are trans2-nonenal--a major component of the characteristic odour of cucumber--and 9-oxononanoic acid. 3. The cleavage reaction is a heat-labile, aerobic process, optimal at pH 6 (approx.). 4. Substrate specificity studies indicate that a lipoxygenase-type of reaction is involved in the cleavage process. 5. The acetone powder extracts have lipoxygenase activity and the proportion of linoleic acid hydroperoxide to carbonyl fragments depends upon incubation conditions. 6. Linoleic acid hydroperoxide isomers are also converted to carbonyl fragments by acetone powder extracts; the 9-hydroperoxide is cleaved at the 9-10 position whereas 12-13 cleavage is predominant with the 13-hydroperoxide isomer.

The formation of cis-3-nonenal, trans-2-nonenal and hexanal from linoleic acid hydroperoxide isomers by a hydroperoxide cleavage enzyme system in cucumber (Cucumis sativus) fruits.

1. A particulate enzyme fraction and an acetone powder preparation from cucumber fruits cleaved 9- and 13-hydroperoxyoctadecadienoic acids to form volatile aldehydes and oxoacid fragments. 2. From the 9-hydroperoxide, the major volatile fragments were cis-3-nonenal and trans-2-nonenal using particulate enzyme and acetone powder preparations, respectively. 3. Hexanal was the only significant volatile fragment from the 13-hydroperoxide. 4. The particulate enzyme system was equally effective on both 9- and 13-hydroperoxide isomers and was fully active under anaerobic conditions and at pH 6.4. 5. An enzymic pathway for the biogenesis of hexanal, cis-3- and trans-2-nonenal (components of the characteristic flavour volatiles of cucumber) from linoleic acid is proposed. This involves the sequential activity of lipoxygenase, hydroperoxide cleavage and cis-3-: trans-2-enal isomerase enzymes.