On the specificity of lipid hydroperoxide fragmentation by fatty acid hydroperoxide lyase from Arabidopsis thaliana

Fatty acid hydroperoxide lyase (HPL) is a membrane associated P450 enzyme that cleaves fatty acid hydroperoxides into aldehydes and omega-oxo fatty acids. One of the major products of this reaction is (3Z)-hexenal. It is a constituent of many fresh smelling fruit aromas. For its biotechnological production and because of the lack of structural data on the HPL enzyme family, we investigated the mechanistic reasons for the substrate specificity of HPL by using various structural analogues of HPL substrates. To approach this 13-HPL from Arabidopsis thaliana was cloned and expressed in E. coli utilising a His-Tag expression vector. The fusion protein was purified by affinity chromatography from the E. coli membrane fractions and its pH optimum was detected to be pH 7.2. Then, HPL activity against the respective (9S)- and (13S)-hydroperoxides derived either from linoleic, alpha-linolenic or gamma-linolenic acid, respectively, as well as that against the corresponding methyl esters was analysed. Highest enzyme activity was observed with the (13S)-hydroperoxide of alpha-linolenic acid (13alpha-HPOT) followed by that with its methyl ester. Most interestingly, when the hydroperoxy isomers of gamma-linolenic acid were tested as substrates, 9gamma-HPOT and not 13gamma-HPOT was found to be a better substrate of the enzyme. Taken together from these studies on the substrate specificity it is concluded that At13HPL may not recognise the absolute position of the hydroperoxy group within the substrate, but shows highest activities against substrates with a (1Z4S,5E,7Z)-4-hydroperoxy-1,5,7-triene motif. Thus, At13HPL may not only be used for the production of C6-derived volatiles, but depending on the substrate may be further used for the production of Cg-derived volatiles as well.     

Phosphorylation of sucrose synthase at serine 170: occurrence and possible role as a signal for proteolysis

Sequence analysis identified serine 170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an sucrose non-fermenting 1 (SNF1)-related protein kinase. Phosphorylation of the novel S170 and the known serine 15 (S15) site by these protein kinases was determined in peptide substrates and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein-kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild-type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was threefold lower than that for S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by early nodulin 40 (ENOD40) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: (i) were detected and possessed relatively high phosphorylated-S170 (pS170) stoichiometry; (ii) were spatially coincident with proteasome activity within developing leaves; and (iii) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves.     

Structural evidence for ligand specificity in the binding domain of the human androgen receptor. Implications for pathogenic gene mutations

The crystal structures of the human androgen receptor (hAR) and human progesterone receptor ligand-binding domains in complex with the same ligand metribolone (R1881) have been determined. Both three-dimensional structures show the typical nuclear receptor fold. The change of two residues in the ligand-binding pocket between the human progesterone receptor and hAR is most likely the source for the specificity of R1881 to the hAR. The structural implications of the 14 known mutations in the ligand-binding pocket of the hAR ligand-binding domains associated with either prostate cancer or the partial or complete androgen receptor insensitivity syndrome were analyzed. The effects of most of these mutants could be explained on the basis of the crystal structure.     

Tet repressor induction without Mg2+

We have examined anhydrotetracycline (atc) binding to Tet repressor (TetR) in dependence of the Mg(2+) concentration. Of all tc compounds tested so far, atc has the highest affinity for TetR, with a K(A) of 9.8 x 10(11) M(-1) in the presence of Mg(2+) and 6.5 x 10(7) M(-1) without Mg(2+). Thus, it binds TetR with 500-fold higher affinity than tc under both conditions. The Mg(2+)-free binding of atc to TetR leads to induction in vitro, demonstrating that the metal is not necessary to trigger the associated conformational change. To obtain more detailed information about Mg(2+)-free induction, we constructed and prepared to homogeneity four single-alanine substitution mutants of TetR. Three of them affect residues involved in contacting Mg(2+) (TetR H100A, E147A, and T103A), and one altered residue contacts tc TetR N82A. TetR H100A and E147A are induced by atc, with and without Mg(2+), showing 110-fold and 1000-fold decreased Mg(2+)-dependent and unchanged Mg(2+)-independent atc binding, respectively. Thus, the contacts of these residues to Mg(2+) are not necessary for induction. TetR N82A is not inducible under any of the conditions employed and shows an about 4000-fold decreased atc binding constant. The Mg(2+)-dependent affinity of TetR T103A for atc is only 400-fold reduced, but no induction with atc was observed. Thus, Thr103 must be essential for the conformational change associated with induction in the absence of Mg(2+).     

Decreased neutrophil adhesion to human cytomegalovirus-infected retinal pigment epithelial cells is mediated by virus-induced up-regulation of Fas ligand independent of neutrophil apoptosis

Human CMV (HCMV) retinitis frequently leads to blindness in iatrogenically immunosuppressed patients and in the end stage of AIDS. Despite the general proinflammatory potential of HCMV, virus infection is associated with a rather mild cellular inflammatory response in the retina. To investigate this phenomenon, the influence of HCMV (strains AD169 or Hi91) infection on C-X-C chemokine secretion, ICAM-1 expression, and neutrophil recruitment in cultured human retinal pigment epithelial (RPE) cells was studied. Supernatants from infected cultures contained enhanced levels of IL-8 and melanoma growth-stimulating activity/Gro alpha and induced neutrophil chemotaxis compared with supernatants from uninfected RPE cells. Despite HCMV-induced ICAM-1 expression on RPE cells, binding of activated neutrophils to HCMV-infected RPE cells and subsequent transepithelial penetration were significantly reduced. Reduced neutrophil adhesion to infected RPE cells correlated with HCMV-induced up-regulation of constitutive Fas ligand (FasL) expression. Functional blocking of FasL on RPE cells with the neutralizing mAbs NOK-1 and NOK-2 or of the Fas receptor on neutrophils with mAbB-D29 prevented the HCMV-induced impairment of neutrophil/RPE interactions. Fas-FasL-dependent impairment of neutrophil binding had occurred by 10 min after neutrophil/RPE coculture without apoptotic signs. Neutrophil apoptosis was first detected after 4 h. Treatment of neutrophils with a specific inhibitor of caspase-8 suppressed apoptosis, whereas it did not prevent impaired neutrophil binding to infected RPE. The current results suggest a novel role for FasL in the RPE regulation of neutrophil binding. This may be an important feature of virus escape mechanisms and for sustaining the immune-privileged character of the retina during HCMV ocular infection.     

Molecular genetic analysis for taxonomic determination of fossil skull fragments of Warendorf-Neuwarendorf

The isolation and examination DNA segments from prehistoric and fossil bone samples has become one of the biggest challenges in anthropology within the past years. By using specially developed and/or adapted genetic methods, it is possible under laboratory conditions to amplify portions of DNA from bone remains in states of good preservation by the polymerase chain reaction (PCR). DNA sequence data can provide far more specific answers to palaeanthropological questions than one would expect solely by morphologic comparison. Here we introduce an alternative approach for the classification of total ancient DNA by means of Southern hybridisation techniques.     

Immediate localized CDKN1A (p21) radiation response after damage produced by heavy-ion tracks

Using confocal microscopy on immunofluorescence-stained cells, we have investigated the response of CDKN1A (p21), one of the key proteins involved in the DNA damage response pathway, after irradiation with accelerated lead or chromium ions. Each traversal of an accelerated ion leads to the formation of a single, bright focus of the CDKN1A protein in the nuclei of human fibroblasts within 2 min after irradiation at 4 degrees C. This immediate, localized CDKN1A response is specific for particle irradiation with a high linear energy transfer (LET), whereas X irradiation, after a period of induction, yields a diffusely spread pattern, in line with the differences in the microscopic dose deposition pattern of both radiation types. The particle-induced CDKN1A foci persist for several hours until they become diffuse and vanish. These findings suggest that CDKN1A accumulates at the sites of primary DNA damage, possibly mediated by the interaction with proteins involved in DNA repair. Here, for the first time, an immediate biological response confined to the radial extension of low-energy particle tracks ( approximately 1 micrometer) is directly visualized and correlated to ion traversals. This indicates that particle irradiation represents an ideal tool to study the processing of biological damage induced in defined subnuclear regions.     

Trypanosoma cruzi infection in Didelphis marsupialis in Santa Catarina and Arvoredo Islands, southern Brazil

Between 1984 and 1993 the prevalence of the Trypanosoma cruzi infection in opossums (Didelphis marsupialis) was studied in Santa Catarina and Arvoredo Islands, State of Santa Catarina, Brazil. The association of the triatomine bug Panstrongylus megistus with opossums nests and the infection rate of these triatomines by T. cruzi was also studied. Thirteen different locations were studied in Santa Catarina Island (SCI), in which 137 D. marsupialis were collected. Sixty two opossums were collected at the Arvoredo Island (AI), located 12 miles north from SCI. All captured animals were submitted to parasitological examinations that revealed the presence of T. cruzi in 21.9% of the opossums captured in SCI and 45.2% among opossums captured in the AI. The presence of P. megistus was detected in most of the D. marsupialis nests collected in the SCI, however, in the non-inhabited AI only eight triatomines were collected during the whole study. The presence of T. cruzi-infected D. marsupialis associated with P. megistus in human dwellings in the SCI, and the high infection rate of D. marsupilais by T. cruzi in the absence of a high vector density are discussed.     

Viability and differential function of rainbow trout liver cells in primary culture: Coculture with two permanent fish cells

Summary The study investigates the influence of different culture conditions on attachment, viability and functional status of rainbow trout (Oncorhynchus mykiss) liver cells in primary culture. Cells were isolated by a two-step collagenase perfusion and incubated in serum-free, chemically defined minimal essential medium (MEM), (a) as a monolayer on uncoated PRI-MARIA? dishes, (b) as a monolayer on culture dishes coated with calf collagen type 1, and (c) in coculture with the established fish cell lines RTH-149 or RTG-2. Cell attachment was assessed from DNA and protein concentrations per dish, viability was estimated from cellular lactate dehydrogenase release, and the metabolic status was investigated by measuring activities of the phosphoenolpyruvate carboxykinase and biotransformation enzymes as well as the total cytochrome P450 contents. Seeding of hepatocytes on collagen-coated dishes did not alter cell attachment or detachment from the culture substrate, but had a small, but not significant effect on cell viability and metabolic parameters. Coculture of liver cells and RTG-2 cells reduced hepatocyte detachment from the culture substrate, and it was associated with a significant elevation of 7-ethoxyresorufin-O-deethylase activities in the hepatic cells. Cytochrome P450 contents, however, were not altered. The coculture effect on liver cell physiology clearly depended on the type of cell line, because coculture with RTH-149 cells led to similar, but much weaker effects than obtained in cocultures with RTG-2 cells. Electron microscopical observations revealed the existence of gap junctions and possible exocytosis-like transport between cell lines and hepatocytes. The results point to the potential of coculture systems to improve physiological parameters of trout liver cells in primary culture.