Regulation of glucose transporters by connective tissue activating peptide-III isoforms.

Connective tissue activating peptide-III (CTAP-III) is a component of platelet alpha-granules which elicits a series of responses in connective tissue cells referred to as activation, including increased glucose consumption and mitogenesis and increased secretion of hyaluronic acid and glycosaminoglycans. As anticipated by a requirement for glucose or glucose precursors in the activation process, an early event following CTAP-III activation of connective tissue cells is an increase in glucose transport. The present study investigates the molecular basis for this increase in glucose transport. Murine 3T3-F442A fibroblasts were found to respond to CTAP-III in a manner similar to human connective tissue cells (synovial cells, chondrocytes, skin fibroblasts). CTAP-III increases the rate of glucose transport to similar extents at 4 and 24 h, and at physiologic (micrograms/ml) concentrations of CTAP-III. A proteolytic cleavage product of recombinant CTAP-III (rCTAP-III-Leu-21 (des-1-15)), also known as neutrophil-activating peptide-2 (NAP-2), was found to be equally effective as CTAP-III, whereas NAP-1/interleukin-8, another member of the CTAP-III super-family, was ineffective in stimulating glucose transport. This contrasts with neutrophil chemotaxis, in which CTAP-III (des-1-15)/NAP-2 acts similarly to NAP-1/interleukin 8 while CTAP-III is ineffective. CTAP-III appears to elicit a different type of glucose transport response than many other growth factors in that its response is sustained (greater than or equal to 24 h) rather than transient (peak approximately 4 h) in confluent as well as in subconfluent cells. Western blot analysis using antibodies to the GLUT-1 glucose transporter revealed an increased level of GLUT-1 protein in response to CTAP-III isoforms that corresponded in magnitude (on a percentage basis) to the increased level of glucose transport. The increased levels of GLUT-1 protein in response to CTAP-III and rCTAP-III-Leu-21 (des-1-15)/NAP-2 were accompanied by an increase in levels of GLUT-1 mRNA of a magnitude sufficient to account for observed increased levels of GLUT-1. These results are consistent with CTAP-III isoforms stimulating glucose transport in connective tissue cells by increasing levels of GLUT-1 mRNA and is one of the few known instances in which increases in levels of GLUT-1 mRNA and protein are sufficient to account for observed increases in glucose transport. They also provide further evidence that CTAP-III (des-1-15)/NAP-2 binds to more than one type of receptor and that CTAP-III acts in a manner different than other well characterized growth factors (e.g. platelet-derived growth factor, transforming growth factor-beta) in that it causes a sustained (greater than or equal to 24 h) elevation in glucose transport in confluent as well as subconfluent cells.     

A possible receptor-binding function for the N-terminus of connective tissue activating peptide III

Connective tissue activating peptide III (CTAP-III) is an 85-residue peptide which has been purified from platelets and shown to possess mitogenic activity toward a variety of fibroblastic cell lines. beta-Thromboglobulin (beta TG) is an 81-residue peptide which is derived from CTAP-III by cleavage of the N-terminal tetrapeptide Asn-Leu-Ala-Lys which results in the loss of mitogenic activity. The near-UV CD spectra for the two proteins indicated that the conformations as well as the electronic environments of the two disulfide bonds, and also of the single aromatic tyrosine residue, were similar in CTAP-III and beta TG. However, differences in the far-UV CD spectra of these proteins indicated a substantial decrease in alpha-helical content for beta TG (29%) as compared to CTAP-III (38%). Structure prediction analysis also suggested that the longer N-terminal segment of CTAP-III may form an alpha-helix. The N-terminal region of beta TG, which lacks this tetrapeptide, was predicted to be in an unordered, or possibly a turn, conformation. This predicted structural difference appears to be due to the high helix-forming potential of the N-terminal tetrapeptide Asn-Leu-Ala-Lys in CTAP-III. These results suggest a possible structural role for the N-terminal region of CTAP-III in the expression of the biologic activities of this protein. On the basis of these studies, a reasonable hypothesis to account for the difference in mitogenic activity between beta TG and CTAP-III is that the N-terminal region must be helical for receptor binding to occur.     

Inhibition of agonist-induced platelet aggregation, Ca2+ mobilization and granule secretion by guanosine 5''-[beta-thio]diphosphate and GDP in intact platelets. Evidence for an inhibitory mechanism unrelated to the inhibition of G-protein-GTP interaction.

(1) We [Muir, Offord & Davies (1986) Biochem. J. 237, 631-637 and Davies, Muir & Offord (1986) Biochem. J. 240, 609-612] have previously identified a major product in the degradation of insulin by insulin proteinase (the N-terminal fragment produced by cleavage between residues LeuA13 and TyrA14, SerB9 and HisB10) together with evidence for a minor cleavage site between HisB10 and LeuB11 or between LeuB11 and ValB12. (2) We now present evidence for minor sites of cleavage between TyrA14 and GlnA15, GluB13 and AlaB14 as well as HisB10 and LeuB11.     

Interaction between bovine trypsin and a synthetic peptide containing 28 residues of the bait region of human alpha 2-macroglobulin.

The time course of the interaction between trypsin and a synthetic peptide corresponding to a segment (residues 676-703) of the bait region (residues 666-706) of human alpha 2-macroglobulin (alpha 2M) was studied by measuring the generation of cleavage products as a function of time by HPLC. Three primary cleavage sites for trypsin were present in the synthetic peptide. The fastest cleavage occurred at the bond corresponding to Arg696-Leu in alpha 2M with an estimated kcat/Km = 1-2 x 10(6) M-1.s-1. This value is of the same magnitude as that characterizing the interaction of alpha 2M and trypsin when taking into account the fact that alpha 2M is a tetramer, kcat/Km = 5 x 10(6) M-1.s-1 [Christensen, U. & Sottrup-Jensen, L. (1984) Biochemistry 23, 6619-6626]. The values of kcat/Km for cleavage at bonds corresponding to Arg681-Val and Arg692-Gly in alpha 2M were 1.5 x 10(5) M-1.s-1 and 1.3 x 10(5) M-1.s-1, respectively. Cleavage of intermediate product peptides was slower, with kcat/Km in the range 13-1.3 x 10(6) M-1.s-1. The value of Km determined for fast cleavage in the synthetic peptide was 8-10 microM. 1H-NMR spectroscopy indicated no ordered structure of the peptide. Hence, the very fast cleavage of the peptide is compatible with a loose structure that readily adopts a conformation favorable for recognition and cleavage by trypsin.     

Reaction pathway of the trans-acting hepatitis delta virus ribozyme: a conformational change accompanies catalysis.

The hepatitis delta virus (HDV), an infectious human pathogen and satellite of hepatitis B virus, leads to intensified disease symptoms, including progression to liver cirrhosis. Both the circular RNA genome of HDV and its complementary antigenome contain the same cis-cleaving catalytic RNA motif that plays a crucial role in virus replication. Previously, the high-resolution crystal structure of the product form of a cis-acting genomic HDV ribozyme has been determined, while a trans-acting version of the ribozyme was used to dissect the cleavage reaction pathway. Using fluorescence resonance energy transfer (FRET) on a synthetic trans-cleaving form of the ribozyme, we are able to directly observe substrate binding (at a rate constant k(on) of 7.8 x 10(6) M(-1) min(-1) at pH 7.5, 11 mM MgCl(2), and 25 degrees C) and dissociation (at 0.34 min(-1)). Steady-state and time-resolved FRET experiments in solution and in nondenaturing gels reveal that the substrate (precursor) complex is slightly more compact (by approximately 3 A) than the free ribozyme, yet becomes significantly extended (by approximately 15 A) upon cleavage and product complex formation. We also find that trans cleavage is characterized by a high transition-state entropy (-26 eu). We propose that the significant global conformational change that we observe between the precursor and product structures occurs on the reaction trajectory into a constrained product complex-like transition state. Our observations may present the structural basis of the recently described utilization of intrinsic substrate binding energy to the overall catalytic rate enhancement by the trans-acting HDV ribozyme.     

Micro RNA, C/D RNA and parental genoming imprinting.

Experimental and computer-assisted approaches have led us to identify hundreds of small non-coding RNA (ncRNAs) whose genes - clustered at two chromosomal domains (the DLK1-GTl2 region/human 15q11q13 and the SNURF-SNRPN/human 14q32 loc) - are subjected to genomic imprinting. Here, we discuss their genomic organization, their expression pattern and their potential functions. A travers des approches in silico et expérimentales, nous avons récemment identifié des centaines de petits ARN non-codants, ARNnc - ARN C/D et microARN - dont les gènes sont soumis à l'empreinte génomique parentale (EGP). Ils sont regroupés au sein de deux loci chromosomiques conservés: le domaine DLK1-GTL2 (locus Callypige, position chromosomique humaine 14q32) et le domaine SNURF-SNRPN (locus Prader-Willi, position chromosomique humaine 15q11q13). Dans cet article, l'organisation génique, le mode d'expression et les fonctions de ces petits ARN sont présentés.     

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1–3)insulin-like growth factor I

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1–3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1–3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1–3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.     

Characterization of ICP6::lacZ insertion mutants of the UL15 gene of herpes simplex virus type 1 reveals the translation of two proteins.

The herpes simplex virus type 1 (HSV-1) UL15 gene is a spliced gene composed of two exons and is predicted to encode an 81-kDa protein of 735 amino acids (aa). Two UL15 gene products with molecular masses of 75 and 35 kDa have been observed (J. Baines, A. Poon, J. Rovnak, and B. Roizman, J. Virol. 68:8118-8124, 1994); however, it is not clear whether the smaller form represents a proteolytic cleavage product of the larger form or whether it is separately translated. In addition, an HSV-1 temperature-sensitive mutant in the UL15 gene (ts66.4) is defective in both cleavage of viral DNA concatemers into unit-length monomers and packaging of viral DNA into capsids (A. Poon and B. Roizman, J. Virol. 67:4497-4503, 1993; J. Baines et al., J. Virol. 68:8118-8124, 1994). In this study, we detected two UL15 gene products of 81 and 30 kDa in HSV-1-infected cells, using a polyclonal antibody raised against a maltose binding protein fusion construct containing UL15 exon 2. In addition, we report the isolation of two HSV-1 insertion mutants, hr81-1 and hr81-2, which contain an ICP6::lacZ insertion in UL15 exon 1 and exon 2 and thus would be predicted to encode C-terminally truncated peptides of 153 and 509 aa long, respectively. hr81-1 and hr81-2 are defective in DNA cleavage and packaging and accumulate only B capsids. However, both mutants are able to undergo wild-type levels of DNA replication and genomic inversion, suggesting that genomic inversion is a result of DNA replication rather than of DNA cleavage and packaging. We also provide evidence that the 81- and 30-kDa proteins are the products of separate in-frame translation events from the UL15 gene and that the 81-kDa full-length UL15 protein is required for DNA cleavage and packaging.     

In vitro stability of growth hormone releasing factor (GRF) analogs in porcine plasma.

The stability of growth-hormone releasing factor (growth regulating factor; GRF) analogs in porcine plasma was examined. GRF analogs were incubated in porcine plasma at 37 degrees C, extracted and subsequently analyzed using high performance liquid chromatography (HPLC). GRF(1-29)-NH2 was rapidly broken down in the plasma with a degradation rate of t1/2 = 13 min. The primary degradation product was identified as GRF(3-29)-NH2. Substitution of Gly15 by Ala15 slightly prolonged the plasma half-life (t1/2 = 17 min) and the major degradative fragment was found to be [Ala15]GRF(3-29)-NH2. The cleavage between the 2 and 3 position of the peptide was not inhibited by trasylol at a concentration of 1,000 KIU/ml but was dramatically reduced by the combined use of diprotin A and trasylol. Absence of the free amino group at the N-terminus and/or substitution of a D-amino acid residue at the penultimate position completely prevented cleavage between the 2 and 3 position in the structural linear GRF analogs. Side-chain to side-chain cyclization between Asp8 and Lys12 amino acid residues significantly improved the stability of GRF in plasma with t1/2 greater than 2 hr. An additional stability was provided by substitution of D-Ala2 for Ala2 in the structural cyclic analog. Cyclization between Lys21 and Asp25 also improved the stability of the GRF peptide in the plasma. Stability was further enhanced by the presence of D-Ala2 or by forming a dicyclic analog through an additional linkage between Asp8 and Lys12.     

Structure and Bioactivity of Recombinant Human CTAP-III and NAP-2

Connective tissue-activating peptide III (CTAP-III) and neutrophil-activating peptide-2 (NAP-2) are both derived from a common precursor, platelet basic protein (PBP), which is stored in the -granules of platelets and released upon their activation. CTAP-III is an 85-residue peptide which is converted to NAP-2 by enzymic removal of the 15 amino-terminal residues. Both peptides play a role in the early stages of wound healing and inflammation through different activities. We have cloned the cDNA for PBP and expressed constructs coding for the CTAP-III and NAP-2 polypeptides in Escherichia coli. We have purified and renatured these recombinant proteins. The integrity of the recombinant proteins has been ascertained by in vitro bioassays. CTAP-III causes 51% histamine release from the basophilic cell lin KU812 at 10^–7 M, whereas NAP-2 only causes 28% release at the same concentration. In assays on human neutrophils, NAP-2 had an EC ₅₀ of 2×10^–8 M in chemotaxis, an EC ₅₀ of 3×10^–8 M for shape change, and could displace IL-8 from neutrophils with a K _d of 7.5×10^–9 M. CTAP-III had no activity in these assays. The disulfide bonds have been identified by peptide mapping and sequence analysis, and are in the positions predicted by homology to interleukin-8 and platelet factor 4. Measurement of the molecular mass at physiologic concentrations by gel permeation chromatography has shown that CTAP-III forms predominantly tetramers and dimers, whereas NAP-2 is only dimetric. SDS/PAGE analysis of samples cross-linked with disuccinimidyl suberate support these topologies. We postulate a mechanism for tetramer formation based on the interaction of the amino-terminal extension in CTAP-III involving a helix–helix interaction that could stabilize the association of two CTAP-III dimers.     

Analysis of the cleavage reaction of a trans-acting human hepatitis delta virus ribozyme.

The cleavage reaction catalyzed by the trans -acting genomic ribozyme of human hepatitis delta virus (HDV) was analyzed with a 13mer substrate (R13) and thio-substituted [SR13(Rp) and SR13(Sp)] substrates under single-turnover conditions. The cleavage of RNA by the trans -acting HDV ribozyme proceeded as a first order reaction. The logarithm of the rate of cleavage (kclv) increased linearly (with a slope of approximately 1) between pH 4.0 and 6.0, an indication that a single deprotonation reaction occurred. This result suggests that kclv reflects the rate of the chemical cleavage step, at least around pH 5. The amount of active complex with the SR13(Sp) substrate was almost as large as with R13 (60-80%), whereas the amount of the corresponding active complex formed with the SR13(Rp) substrate was, at most, 20% of this value (with 0.5-100 mM Mg2+ions) at pH 5.0. Nonetheless, the value of kclv for all substrates was almost the same (0.4-0.5 min-1). Neither a 'thio effect' nor a 'Mn2+rescue effect' were observed. These results suggest that Mg2+ions do not interact with pro-R oxygen directly but are essential to the formation of the active complex of the ribozyme and its substrate.     

Chemical synthesis and expression in yeast of a gene encoding connective tissue activating peptide-III. A novel approach for the facile assembly of a gene encoding a human platelet-derived mitogen

A synthetic gene encoding the platelet-derived factor, connective tissue activating peptide-III (CTAP-III) (Castor, C.W., Miller, J.W., and Walz, D.A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 765-769), has been expressed and secreted from Saccharomyces cerevisiae by using a yeast expression vector and an alpha-factor leader segment. Mitogenic activity reported for naturally derived CTAP-III has been demonstrated here for recombinant CTAP-III. Active CTAP-III has been purified to apparent homogeneity. Structural studies have confirmed its identity. A general approach for the facile synthesis of genes is presented which has permitted the assembly of the entire structural gene and flanking regions (280 base pairs) from 20 oligomers in a single annealing and ligation reaction pool.     

Orientation of the cleavage site of the herpes simplex virus glycoprotein G-2.

During the synthesis of glycoprotein G-2 (gG-2) of herpes simplex virus type 2, the 104,000-Da gG-2 precursor (104K precursor) is cleaved to generate the 72K and the 31K intermediates. The 72K product is processed to generate the mature gG-2 (molecular mass, 108,000 Da), while the 31K product is additionally processed and secreted into the extracellular medium as the 34K component (H. K. Su, R. Eberle, and R. J. Courtney, J. Virol. 61:1735-1737, 1987). In this study, the orientations of the 31K and 72K products on the 104K precursor were determined by using two antipeptide sera produced in rabbits and a monoclonal antibody, 13 alpha C6, directed against gG-2. The sera prepared against synthetic peptides corresponding to the terminal amino acid residues 67 to 78 and an internal peptide at amino acids 247 to 260 of gG-2 recognized the 104K precursor and the 31K cleavage product but not the 72K intermediate. In contrast, 13 alpha C6 detected the 72K cleavage product and the uncleaved precursor but not the 31K cleavage component. The epitope recognized by 13 alpha C6 was mapped within amino acids 486 to 566. These results suggest that the 31K cleavage product is derived from the amino-terminal portion of the 104K precursor molecule and that the 72K intermediate is derived from the carboxyl terminus. In support of our model described above for the synthesis of gG-2, antibodies recognizing either of the cleavage products reacted with the uncleaved precursor but not with the other cleavage product. By using partial endo-beta-N-acetylglucosaminidase H analysis, two N-linked glycosylation sites were found on each of the cleavage products. The distribution of the N-linked glycosylation sites and the reactivities of the antipeptide sera allowed the cleavage region on the precursor to be mapped to within amino acids 260 to 437.     

Nuclear magnetic resonance analysis of [1-13C]dimethylsulfoniopropionate (DMSP) and [1-13C]acrylate metabolism by a DMSP lyase-producing marine isolate of the alpha-subclass of Proteobacteria.

The prominence of the alpha-subclass of Proteobacteria in the marine bacterioplankton community and their role in dimethylsulfide (DMS) production has prompted a detailed examination of dimethylsulfoniopropionate (DMSP) metabolism in a representative isolate of this phylotype, strain LFR. [1-(13)C]DMSP was synthesized, and its metabolism and that of its cleavage product, [1-(13)C]acrylate, were studied using nuclear magnetic resonance (NMR) spectroscopy. [1-(13)C]DMSP additions resulted in the intracellular accumulation and then disappearance of both [1-(13)C]DMSP and [1-(13)C]beta-hydroxypropionate ([1-(13)C]beta-HP), a degradation product. Acrylate, the immediate product of DMSP cleavage, apparently did not accumulate to high enough levels to be detected, suggesting that it was rapidly beta-hydroxylated upon formation. When [1-(13)C]acrylate was added to cell suspensions of strain LFR it was metabolized to [1-(13)C]beta-HP extracellularly, where it first accumulated and was then taken up in the cytosol where it subsequently disappeared, indicating that it was directly decarboxylated. These results were interpreted to mean that DMSP was taken up and metabolized by an intracellular DMSP lyase and acrylase, while added acrylate was beta-hydroxylated on (or near) the cell surface to beta-HP, which accumulated briefly and was then taken up by cells. Growth on acrylate (versus that on glucose) stimulated the rate of acrylate metabolism eightfold, indicating that it acted as an inducer of acrylase activity. DMSP, acrylate, and beta-HP all induced DMSP lyase activity. A putative model is presented that best fits the experimental data regarding the pathway of DMSP and acrylate metabolism in the alpha-proteobacterium, strain LFR.     

A cascade of 24 histatins (histatin 3 fragments) in human saliva. Suggestions for a pre-secretory sequential cleavage pathway

The systematic search by tandem mass spectrometry of human saliva from four different subjects, of 136 possible fragments originated from histatin 3, allowed the detection of 24 different peptides. They include, with the exception of histatin 4, all the known histatin 3 fragments, namely histatins 5-12 and the peptides corresponding to 15-24, 26-32, 29-32 residues, and 13 new fragments corresponding to 1-11, 1-12, 1-13, 5-13, 6-11, 6-13, 7-11, 7-12, 7-13, 14-24, 14-25, 15-25, and 28-32 residues of histatin 3. On the contrary, none of 119 possible fragments of histatin 1, including histatin 2, was detected. The results suggest that the genesis of histatin 3-related peptides, being under the principal action of trypsin-like activities, is probably not a random process but rather follows a sequential fragmentation pathway. Lack of detection of C-terminal fragments, with the exception of 26-32, 28-32, and 29-32 fragments, suggested that arginine 25 should be the first cleavage site, generating histatin 6 and 26-32 fragments. The genesis of 28-32 and 29-32 fragments and histatin 5 should implicate a subsequent exo-protease action. Similarly, lack of detection of fragments having Lys-5 and Arg-6 at the N terminus and Arg-25 at the C terminus strongly suggested that sequences KRKF (11-14 residues) and AKR (4-6 residues) should be the second and the third cleavage sites, respectively. Lys-17 and Arg-22 are not cleaved at all.     

Endogenous cleavage of annexin I generates a truncated protein with a reduced calcium requirement for binding to neutrophil secretory vesicles and plasma membrane

We have earlier shown that an N-terminal truncated annexin I molecule, annexin I(des1-8), is generated in human neutrophils through cleavage by a membrane localized metalloprotease. The truncated protein showed differences in membrane binding among the neutrophil granule populations as compared to full-length annexin I. In this study, we investigated the cleavage capabilities of isolated neutrophil secretory vesicles and plasma membrane, and the binding of full-length annexin I and annexin I(des1-8) to these membrane fractions. Translocations were performed in vitro to secretory vesicles and plasma membrane, respectively, at different Ca(2+) concentrations. We show that the annexin I-cleaving membrane localized metalloprotease is present both in the secretory vesicles and the plasma membrane. The N-terminal truncation of annexin I gives rise to a molecule with a decreased Ca(2+) requirement for binding, both to secretory vesicles and plasma membrane. There was, thus, no difference in binding of either full-length annexin I or annexin I(des1-8) to the secretory vesicles as compared to the plasma membrane.     

A system for shotgun DNA sequencing.

A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.     

Characterization of a C-5,13-Cleaving Enzyme of 13(S)-Hydroperoxide of Linolenic Acid by Soybean Seed

An activity was found in mature soybean seeds (Glycine max L. cv Century) that cleaved 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13S-HPOT) into 13-oxo-9(Z),11(E)-tridecadienoic acid and two isomeric pentenols, 2(Z)-penten-1-ol and 1-penten-3-ol. Isomeric pentene dimers were also produced and were presumably derived from the combination of two pentene radicals. 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid (13S-HPOD) was, by contrast, a poor substrate. Activity with 13S-HPOT increased 24-fold under anaerobic conditions reminiscent of a similar anaerobic promoted reaction of 13S-HPOD catalyzed by lipoxygenase (LOX) in the presence of linoleic acid. However, prior to ion-exchange chromatography, cleavage activity did not require linoleic acid. After separation by gel filtration followed by ion-exchange chromatography, cleavage activity was lost but reappeared in the presence of either linoleic acid or dithiothreitol. Under these conditions cleavage activity was coincident with the activity of types 1 and 2 LOX. LOX inhibitors suppressed the cleavage reaction in a manner similar to inhibition of LOX activity. Heat-generated alkoxyl radicals derived from either 13S-HPOT or 13S-HPOD afforded similar products and yields of 13-oxo-9(Z),11(E)-tridecadienoic acid compared to the enzymic reaction. The product 1-penten-3-ol may be the precursor of the "raw-bean" volatile ethylvinylketone.     

Cyclooxygenase (COX)-2 and COX-1 potentiate beta-amyloid peptide generation through mechanisms that involve gamma-secretase activity

In previous studies we found that overexpression of the inducible form of cyclooxygenase, COX-2, in the brain exacerbated beta-amyloid (A beta) neuropathology in a transgenic mouse model of Alzheimer's disease. To explore the mechanism through which COX may influence A beta amyloidosis, we used an adenoviral gene transfer system to study the effects of human (h)COX-1 and hCOX-2 isoform expression on A beta peptide generation. We found that expression of hCOXs in human amyloid precursor protein (APP)-overexpressing (Chinese hamster ovary (CHO)-APPswe) cells or human neuroglioma (H4-APP751) cells resulting in 10-25 nM prostaglandin (PG)-E2 concentration in the conditioned medium coincided with an approximately 1.8-fold elevation of A beta-(1-40) and A beta-(1-42) peptide generation and an approximately 1.8-fold induction of the C-terminal fragment (CTF)-gamma cleavage product of the APP, an index of gamma-secretase activity. Treatment of APP-overexpressing cells with the non-selective COX inhibitor ibuprofen (1 microM, 48 h) or with the specific gamma-secretase inhibitor L-685,458 significantly attenuated hCOX-1- and hCOX-2-mediated induction of A beta peptide generation and CTF-gamma cleavage product formation. Based on this evidence, we next tested the hypothesis that COX expression might promote A beta peptide generation via a PG-E2-mediated mechanism. We found that exposure of CHO-APPswe or human embryonic kidney (HEK-APPswe) cells to PG-E2 (11-deoxy-PG-E2) at a concentration (10 nM) within the range of PG-E2 found in hCOX-expressing cells similarly promoted (approximately 1.8-fold) the generation of the CTF-gamma cleavage product of APP and commensurate A beta-(1-40) and A beta-(1-42) peptide elevation. The study suggests that expression of COXs may influence A beta peptide generation through mechanisms that involve PG-E2-mediated potentiation of gamma-secretase activity, further supporting a role for COX-2 and COX-1 in Alzheimer's disease neuropathology.