Oxidation of oxa and thia fatty acids and related compounds catalysed by 5-and 15-lipoxygenase

The modified fatty acids, (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid, (Z,Z,Z)-(octadeca-9,12,15-trienyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid, 3-[(all-Z)-(eicosa-5,8,11,14-tetraenylthio)]propionic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)succinic acid, N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]glycine and N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]aspartic acid, all react with soybean 15-lipoxygenase. The products were treated with triphenylphosphine to give alcohols, which were isolated using HPLC. Analysis of the alcohols using negative ion tandem electrospray mass spectrometry, and by comparison with compounds obtained by autoxidation of arachidonic acid, shows that each enzyme catalysed oxidation occurs at the omega -6 position of the substrate. In a similar fashion, it has been found that (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid, (Z,Z,Z)-(octadeca-9,12,15-trienyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid and N-[(all-Z)-(eicosa-5,8, 11.14-tetraenylthio)]propionic acid each undergoes regioselective oxidation at the carboxyl end of the polyene moiety on treatment with potato 5-lipoxygenase. Neither (all-Z)-(eicosa-5,8,11,14-tetraenylthio)succinic acid nor N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]aspartic acid reacts in the presence of this enzyme, while N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]glycine affords the C11' oxidation product. The alcohol derived from (Z,Z,Z)-(octadeca-6,9, 12-trienyloxy)acetic acid using the 15-lipoxygenase reacts at the C6' position with the 5-lipoxygenase. (C) 2001 Elsevier Science Ltd. All rights reserved.

In vivo protein cyclization promoted by a circularly permuted Synechocystis sp PCC6803 DnaB mini-intein

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH2-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures similar to14 degreesC higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH2 and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (DeltaDeltaG approximate to 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.

A coiled-coil region of an insect immune suppressor protein is involved in binding and uptake by hemocytes

Polydnaviruses are associated with certain parasitoid wasps and are introduced into the body cavity of the host caterpillar during oviposition. Some of the viral genes are expressed in host tissues and corresponding proteins are secreted into the hemocoel causing suppression of the host immune system. The Cotesia rubecula polydnavirus gene product, CrV1, effectively inactivates hemocytes by mediating cytoskeleton break-down. A precondition for the CrV1 function is the incorporation of the extracellular protein by hemocytes. Here, we show that a coiled-coil domain containing a putative leucine zipper is required for CrV1 function, since removal of this domain abolishes binding and uptake of the CrV1 protein by hemocytes. (C) 2002 Elsevier Science Ltd. All rights reserved.

p53 Suppresses c-Myb-induced trans-Activation and Transformation by Recruiting the Corepressor mSin3A

p53 is known to repress transcription of a number of genes, but the mechanism of p53 recruitment to these target genes is unknown. The c-myb proto-oncogene product (c-Myb) positively regulates proliferation of immature hematopoietic cells, whereas p53 blocks cell cycle progression. Here, we demonstrate that p53 inhibits c-Myb-induced transcription and transformation by directly binding to c-Myb. The ability of c-Myb to maintain the undifferentiated state of M1 cells was also suppressed by p53. p53 did not affect the ability of c-Myb to bind to DNA but formed a ternary complex with the corepressor mSin3A and c-Myb. Thus, p53 antagonizes c-Myb by recruiting mSin3A to down-regulate specific Myb target genes.

H-1 NMR spectroscopic studies of the stability of an oxazolidine condensation product

Condensation of (-)-norephedrine with excess formaldehyde under mild conditions leads to formation of the 2:1 condensation product N,N'-methylenebis(4-methyl-5-phenyl)oxazolidine compared with the reaction with 1 mol of formaldehyde, which leads to 4-methyl-5-phenyloxazolidine. H-1 and C-13 NMR spectroscopy was used to monitor the stability of this compound and its decomposition products. The 2:1 condensation product is found to be stable in CDC1(3) but breaks down rapidly in CD3OD to yield a 50:50 mixture of 4-methyl-5-phenyloxazolidine and 3-hydroxymethyl-4-methyl-5-phenyloxazolidine. Upon addition of D2O to this equimolar mixture, the latter compound decomposes to norephedrine and formaldehyde, whereas the former compound is stable. (C) 1997 by John Wiley & Sons, Ltd.

Matrix-product codes over Fq

Codes C-1,...,C-M of length it over F-q and an M x N matrix A over F-q define a matrix-product code C = [C-1 (...) C-M] (.) A consisting of all matrix products [c(1) (...) c(M)] (.) A. This generalizes the (u/u + v)-, (u + v + w/2u + v/u)-, (a + x/b + x/a + b + x)-, (u + v/u - v)- etc. constructions. We study matrix-product codes using Linear Algebra. This provides a basis for a unified analysis of /C/, d(C), the minimum Hamming distance of C, and C-perpendicular to. It also reveals an interesting connection with MDS codes. We determine /C/ when A is non-singular. To underbound d(C), we need A to be 'non-singular by columns (NSC)'. We investigate NSC matrices. We show that Generalized Reed-Muller codes are iterative NSC matrix-product codes, generalizing the construction of Reed-Muller codes, as are the ternary 'Main Sequence codes'. We obtain a simpler proof of the minimum Hamming distance of such families of codes. If A is square and NSC, C-perpendicular to can be described using C-1(perpendicular to),...,C-M(perpendicular to) and a transformation of A. This yields d(C-perpendicular to). Finally we show that an NSC matrix-product code is a generalized concatenated code.

On the direct product of two weak uniquely completable partial latin squares

In this note we show by counter-example that the direct product of two weak uniquely completable partial latin squares is not necessarily a uniquely completable partial latin square. This counter-example rejects a conjecture by Gower (see [3]) on the direct product of two uniquely completable partial latin squares.

Calculation of product state distributions from resonance decay via Lanczos subspace filter diagonalization: Application to HO2

Resonance phenomena associated with the unimolecular dissociation of HO2 have been investigated quantum-mechanically by the Lanczos homogeneous filter diagonalization (LHFD) method. The calculated resonance energies, rates (widths), and product state distributions are compared to results from an autocorrelation function-based filter diagonalization (ACFFD) method. For calculating resonance wave functions via ACFFD, an analytical expression for the expansion coefficients of the modified Chebyshev polynomials is introduced. Both dissociation rates and product state distributions of O-2 show strong fluctuations, indicating the dissociation of HO2 is essentially irregular. (C) 2001 American Institute of Physics.

Epidermal growth factor sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia

Epidermal growth factor (EGF) has been reported to either sensitize or protect cells against ionizing radiation. We report here that EGF increases radiosensitivity in both human fibroblasts and lymphoblasts and downregulates both ATM (mutated in ataxia-telangiectasia (A-T)) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). No further radiosensitization was observed in A-T cells after pretreatment with EGF. The down-regulation of ATM occurs at the transcriptional level. Concomitant with the down-regulation of ATM, the DNA binding activity of the transcription factor Spl decreased. A causal relationship was established between these:observations by demonstrating that upregulation of Spl DNA binding activity by granulocyte/ macrophage colony-stimulating factor rapidly reversed the EGF-induced decrease in ATM protein and restored radiosensitivity to normal levels. Failure to radiosensitize EGF-treated cells to the same extent as observed for A-T cells ban be explained by induction of ATM protein and kinase activity with time post-irradiation, Although ionizing radiation damage to DNA rapidly activates ATM kinase and cell cycle checkpoints, we have provided evidence for the first time that alteration in the amount of ATM protein occurs in response to both EGF and radiation exposure. Taken together these data support complex control of ATM function that has important repercussions for targeting ATM to improve radiotherapeutic benefit.

Transcriptional downregulation of ATM by EGF is defective in ataxia-telangiectasia cells expressing mutant protein

There is evidence that ATM plays a wider role in intracellular signalling in addition to DNA damage recognition and cell cycle control, In this report we show that activation of the EGF receptor is defective in ataxia-telangiectasia (A-T) cells and that sustained stimulation of cells with EGF downregulates ATM protein in control cells but not in A-T cells expressing mutant protein, Concomitant with the downregulation of ATM, DNA-binding activity of the transcription factor Spl decreased in controls after EGF treatment but increased from a lower basal level in A-T cells to that in untreated control cells, Mutation in two Spl consensus sequences in the ATM promoter reduced markedly the capacity of the promoter to support luciferase activity in a reporter assay. Overexpression of anti-sense ATM cDNA in control cells decreased the;basal level of Spl, which in turn was increased by subsequent treatment of cells with EGF, similar to that observed in,A-T cells. On the other hand full-length ATM cDNA increased the basal level of Spl binding in A-T cells, and in response to EGF Spl binding decreased, confirming that this is an ATR I-dependent process. Contrary to that observed in control cells there was no radiation-induced change in ATM protein in EGF-treated A-T cells and likewise no alteration in Spl binding activity. The results demonstrate that EGF-induced downregulation of ATM (mutant) protein in A-T cells is defective and this appears to be due to less efficient EGFR activation and abnormal Spl regulation.

Ataxia-telangiectasia: chronic activation of damage-responsive functions is reduced by alpha-lipoic acid

Cells from patients with the genetic disorder ataxia-telangiectasia (A-T) are hypersensitive to ionizing radiation and radiomimetic agents, both of which generate reactive oxygen species capable of causing oxidative damage to DNA and other macromolecules. We describe in A-T cells constitutive activation of pathways that normally respond to genotoxic stress, Basal levels of p53 and p21(WAF1/CIP1), phosphorylation on serine 15 of p53, and the Tyr15-phosphorylated form of cdc2 are chronically elevated in these cells. Treatment of A-T cells with the antioxidant alpha -lipoic acid significantly reduced the levels of these proteins, pointing to the involvement of reactive oxygen species in their chronic activation. These findings suggest that the absence of functional ATM results in a mild but continuous state of oxidative stress, which could account for several features of the pleiotropic phenotype of A-T.

Photosensitization of the sunscreen octyl p-dimethylaminobenzoate by UVA in human melanocytes but not in keratinocytes

Sunscreens penetrate human epidermis and modify the biology of proliferating cells. This study addressed the question whether the UV response of cultured human cells is affected by direct treatment with nontoxic levels of sunscreens. Cell survival following exposure to UVC or unfiltered UVB was not altered by preincubation with 25 μg/mL of octyl p-dimethylaminobenzoate (o-PABA), 2-ethylhexyl p-methoxycinnamate (EHMC) or oxybenzone. However, UVA or UVB filtered to reproduce the solar UV spectrum penetrating to the basal layer of the epidermis, highly sensitized cells to killing by o-PABA but not by its hydrolysis product, 4-dimethylaminobenzoic acid. Sensitization was found in all cell types tested, except normal keratinocytes, and could be prevented by certain antioxidants particularly pyruvate and the hydroxyl radical scavenger mannitol. o-PABA and EHMC applied without UV reduced the adherence of cells. The results indicate that sunscreens may increase cell mobility and the combination of o-PABA with solar UV may selectively damage melanocytes in the skin.

Adsorption kinetics for the removal of chromium(VI) from aqueous solution by adsorbents derived from used tyres and sawdust

The batch removal of hexavalent chromium (Cr(Vl)) from wastewater under different experimental conditions using economic adsorbents was investigated in this study. These adsorbents were produced from the pyrolysis and activation of the waste tyres (TAC) and from the pyrolysis of sawdust (SPC). The performance of these adsorbents against commercial activated carbon F400 (CAC) has also been carried out. The removal was favoured at low pH, with maximum removal at pH = 2 for all types of carbon. The effects of concentration, temperature and particle size have been reported. All sorbents were found to efficiently remove Cr(VI) from solution. The batch sorption kinetics have been tested for a first-order reversible reaction, a first-order and second-order reaction. The rate constants of adsorption for all these kinetic models have been calculated. The applicability of the Langmuir isotherm for the present system has been tested at different temperatures. The thermodynamic parameters (AGO, K,) obtained indicate the endothermic nature of Cr(Vl) adsorption on TAC, SPC and CAC. (C) 2001 Elsevier Science B.V. All rights reserved.

Improved detection of lacZ reporter gene expression in transgenic epithelia by immunofluorescence microscopy

The bacterial lacZ gene is commonly used as a reporter for the in vivo analysis of gene regulation in transgenic mice. However, several laboratories have reported poor detection of beta-galactosidase (the lacZ gene product) using histochemical techniques, particularly in skin. Here we report the difficulties we encountered in assessing lacZ expression in transgenic keratinocytes using classic X-gal histochemical protocols in tissues shown to express the transgene by mRNA in situ hybridization. We found that lacZ reporter gene expression could be reliably detected in frozen tissue sections by immunofluorescence analysis using a beta-galactosidase-specific antibody. Moreover, we were able to localize both transgene and endogenous gene products simultaneously using double-label immunofluorescence. Our results suggest that antibody detection of beta-galactosidase should be used to verify other assays of lacZ expression, particularly where low expression levels are suspected or patchy expression is observed.

Factorizations of and by powers of complete graphs

Let K-k(d) denote the Cartesian product of d copies of the complete graph K-k. We prove necessary and sufficient conditions for the existence of a K-k(r)-factorization of K-pn(s), where p is prime and k > 1, n, r and s are positive integers. (C) 2002 Elsevier Science B.V. All rights reserved.