Temporal Changes in Photoreducible Mercury, Photoreduction Rates, and the Role of Dissolved Organic Matter in Freshwater Lakes.

Total photoreducible mercury [Hg(II)RED] and photoreduction rates in the surface waters of four lakes in Kejimkujik National Park, Nova Scotia were measured monthly over a summer. The percent of THg that was photoreducible [%Hg(II)RED] decreased significantly in two of the four lakes from early to late summer: North Cranberry (maximum 42% to minimum 14%) and Big Dam East (maximum 51% to minimum 6%). Hg(II)RED was found to have a linear relationship with THg for all combined site data. THg and Hg(II)RED were found to have positive linear relationships with DOC concentrations (R2 = 0.97; n = 36; p < 0.01 and R2 = 0.75; n = 36; p < 0.01, respectively). A smaller proportion of THg was found to be photoreducible with increasing DOC concentration.

Study of the mode of action of endopolygalacturonase from Fusarium moniliforme.

One endopolygalacturonase from Fusarium moniliforme was purified from the culture broth of a transformed strain of Saccharomyces cerevisiae. Its kinetic parameters and mode of action were studied on galacturonic acid oligomers and homogalacturonan. The dimer was not a substrate for the enzyme. The enzyme was shown to follow Michaelis-Menten behaviour towards the other substrates tested. Affinity and maximum rate of hydrolysis increased with increasing chain length, up to the hexamer or heptamer, for which V(max) was in the same range as with homogalacturonan. The enzyme was demonstrated to have a multi-chain attack mode of action and its active site included five subsites ranging from -3 to +2. The final products of hydrolysis of homogalacturonan were the monomer and the dimer of galacturonic acid.

Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion.

Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants.

Analysis of different de-esterification mechanisms for pectin by enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from A. niger.

A series of pectins with different distribution patterns of methyl ester groups was produced by treatment with either plant (p-PME) or fungal pectin methyl esterases (f-PME) and compared with those obtained by base catalysed de-esterification. The products generated by digestion of these pectins with either endopectin lyase (PL) or endopolygalacturonase II (PG II) from Aspergillus niger were analysed using matrix assisted laser desorption ionisation mass spectrometry (MALDIMS) and high-performance anion-exchange chromatography with pulsed amperometric or UV detection (HPAEC-PAD/UV). Time course analysis using MALDIMS was used to identify the most preferred substrate for each enzyme. For PL, this was shown to be fully methyl esterified HG whereas for PG II, long regions of HG without any methyl esterification, as produced by p-PME was the optimal substrate. The blockwise de-esterification caused by p-PME treatment gave a decrease of partly methylated oligomers in PL fingerprints, which did not effect the relative composition of partly methylated oligomers. PG II fingerprints showed a constant increase of monomers and oligomers without any methyl ester groups with decreasing degree of esterification (DE), but almost no change in the concentration of partly methylated compounds. PL fingerprints of f-PME and chemically treated pectins showed decreasing amounts of partly methyl esterified oligomers with decreasing DE, together with a relative shift towards longer oligomers. PG II fingerprints were characterised by an increase of partly methylated and not methylated oligomers with decreasing DE. But differences were also seen between these two forms of homogenous de-esterification. Introduction of a certain pattern of methyl ester distribution caused by selective removal of certain methyl ester groups by f-PME is the most reasonable explanation for the detected differences.

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation.

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.

Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillus niger.

A method to determine the amount of galacturonic acid in blocksequence (BS) in pectin homogalacturonan (HG) is described. The method is based on a combination of endopolygalacturonase II (endo-PG II) and exopolygalacturonase (exo-PG) digestion followed by quantification of the liberated galacturonic acid monomer. The amount of monomers released is directly related to the amount of non-esterified galacturonic acid units located between two other non-esterified galacturonic acids units on the HG chain. The amount released for exo-PG digestion only corresponds to the BS located at the non-reducing end of the polymer. The difference between total- and exo-BS was calculated to be the amount of endo-BS located either within or on the reducing end of the HG. Three series of model pectins obtained by de-esterification of a high-ester pectin with either plant pectin methyl-esterase (p-PME, P-series), fungal pectin methyl-esterase (f-PME, F-series) and chemical de-esterification using base (B-series) were analysed and compared with a fully de-esterified pectic acid sample obtained from the same raw material. Clear differences for the increase of the amounts of blocksequence could be seen between de-esterification of the P- and F-series samples supporting a blockwise and a homogenous de-esterification mechanism, respectively. f-PME and base treatment showed only minor differences in the increase of galacturonic acid units in BS, despite differences seen in their methyl-esterification pattern. Differences between the amounts of galacturonic acid located in exo- and endo-BS, provided evidence for the need of a certain start side or blocklength for p-PME to de-esterify blockwise.

Uniaxial yield strains for bovine trabecular bone are isotropic and asymmetric.

Although evidence suggests that yield strains for trabecular bone are isotropic, i.e., independent of loading direction, decisive support for this hypothesis has remained elusive. To explicitly test whether yield strains for trabecular bone are isotropic, compressive and tensile yield strains of 51 specimens of bovine tibial trabecular bone (0.41 +/- 0.08 g/cm3 [mean apparent density +/- SD]) were measured without end artifacts in on-axis (along the principal trabecular orientation) and off-axis (30-40 degrees oblique to on-axis) orientations. Yield strains for the on-axis and off-axis orientations were similar in tension (0.80 +/- 0.03% compared with 0.85 +/- 0.04%, p = 0.21) and compression (0.97 +/- 0.05% compared with 0.96 +/- 0.07%, p > 0.99); as expected, modulus and strength depended on loading direction. When considered with an ancillary experiment on bovine tibial trabecular bone that showed yield strains to be similar between on-axis and 90 degrees off-axis bone, these results firmly establish the isotropy of uniaxial yield strains for bovine tibial trabecular bone. This bone is of high density and has a strong, plate-type, anisotropic architecture. Therefore, yield strains for uniaxial loading are expected to be isotropic, or nearly so, for other types of dense trabecular bone, although further work is required to confirm this and to establish this behavior for bone of lower density.

Sequencing of partially methyl-esterified oligogalacturonates by tandem mass spectrometry and its use to determine pectinase specificities.

Complex mixtures of acidic oligosaccharides were produced by enzymatic digestion of partially methyl-esterified pectin with Aspergillus niger pectin lyase, endopolygalacturonase II, and exopolygalacturonase. To determine the specificities of these pectolytic enzymes toward non-esterified and methyl-esterified galacturonic acid residues, we have studied the methyl esterification patterns of selected oligomers in unseparated pectin digests. Collision-induced dissociation in a nanoelectrospray ionization ion trap mass spectrometer was used to locate methyl-esterified galacturonic acid residues in oligomers up to a degree of polymerization of 10. Analysis of the methyl esterification patterns gave insight into the substrate specificities of these pectolytic enzymes. Isomeric fragment ions containing the reducing and nonreducing ends were differentiated by 18O-labeling of the reducing end.

Pectin methyl esterase from orange fruit: characterization and localization by in-situ hybridization and immunohistochemistry.

Pectin methyl esterase (PME) from orange (Citrus sinensis L.) fruit peels has been purified by ammonium sulphate precipitation, and ion-exchange and gel-filtration chromatography. Characterization of the enzyme revealed a 36-kDa protein with an isoelectric point > 9, a pH optimum at 7 and temperature optimum at 50 degrees C. The substrate specificity and kinetic experiments showed that the affinity of PME for pectin was highly dependent on the degree of esterification (DE) of the pectin, with K(m) values of 0.7 mg ml-1 for pectin with a DE of 70% and 17 mg ml-1 for pectin with a DE of 25%. The sequences of the NH2-terminal end of digested peptides from the mature protein were obtained. A DNA fragment of 501 bp was cloned by polymerase chain reaction amplification using degenerate primers and was further used for screening of a cDNA library. Two cDNA clones were isolated encoding PMEs of 584 amino acids and 362 amino acids, respectively, including a putative signal peptide. The deduced amino acid sequence showed full identity to the sequenced peptides. Polyclonal antibodies raised against orange peel PME were used for immunohistochemistry. The main localization of PMEs was in the outer cell layers of the juice vesicles, in the outer cell layers of the lamellae between the segments and in the inner cell layers of the albedo in the peel. In-situ hybridization showed that the mRNA is very abundant in the fruit and was found in the same cell layers as the native enzyme. A very intensive staining for PME mRNA was also seen in the core and in the flavedo close to the oil glands.

Gentacoll hampers epithelialisation and neovascularisation in excisional wounds in hairless mice.

Our aim was to analyse the effect of Gentacoll on the rate of epithelialisation and neovascularisation in wound healing. Standardised circular full thickness dermal wounds 2.25 mm in diameter were created on the dorsum of each ear on 24 hairless homozygous mice (n = 48). The cartilaginous layer was left intact. The wounds were treated in a randomised blinded fashion with bovine collagen implants with gentamicin (Gentacoll) (n = 17); bovine collagen implants without gentamicin (n = 15); and Silicone film (n = 16). Epithelialisation and neovascularisation were measured directly by intravital video-microscopy and computerised planimetry immediately after the wounds had been made and every third day until the wounds closed. Only five of the wounds treated with Gentacoll (n = 17) epithelialised completely; and their mean (SEM) epithelialisation time was 22.8 (1.6) days, significantly longer than controls without gentamicin (n = 15) for which the corresponding figures were 14.5 (0.6) days. In nine wounds treated with Gentacoll the ear cartilage in the wound bed perforated and two wounds developed severe inflammation, which was followed by self-mutilation. Neovascularisation was incomplete in all of the wounds in the Gentacoll group, whereas it was completed by 25.3 (0.7) days in the control group treated with implants without gentamicin. In the silicone treated group (n = 16), epithelialisation was completed by 12.7 (0.7) days and neovascularisation by 25.1 (0.5) days. None of wounds treated with collagen or silicone alone showed reactions similar to the Gentacoll-treated ears. Gentacoll hampers epithelialisation and neovascularisation, and might damage exposed cartilage.

Efficient purification, characterization and partial amino acid sequencing of two alpha-1,4-glucan lyases from fungi.

alpha-1,4-Glucan lyases from the fungi Morchella costata and M. vulgaris were purified by affinity chromatography on beta-cyclodextrin-sepharose, followed by ion exchange and gel filtration. The purified enzymes produced 1,5-anhydro-D-fructose from glucose oligomers and polymers with alpha-1,4-glucosidic linkages, such as maltose, maltosaccharides, amylopectin, and glycogen. The lyases were basically inactive towards glucans linked through alpha-1,1, alpha-1,3 or alpha-1,6 linkages. For both enzymes the molecular mass was around 121,000 Da as determined by matrix-assisted laser desorption mass spectrometry. The pI for the lyases from M. costata and M. vulgaris was 4.5 and 4.4, respectively. The lyases exhibited an optimal pH range of pH 5.5 to pH 7.5 with maximal activity at pH 6.5. Optimal temperature was between 37 degrees C and 48 degrees C for the two lyases, depending on the substrates. The lyases were examined with 12 inhibitors to starch hydrolases and it was found that they were inhibited by the -SH group blocking agent PCMB and the following sugars and their analogues: glucose, maltitol, maltose, 1-deoxynojirimycin and acarbose. Partial amino acid sequences accounting for about 35% of the lyase polypeptides were determined. In the overlapping region of the sequences, the two lyases showed 91% identity. The two lyases also cross-reacted immunologically.

Endogenous TGF-beta 1 and TGF-beta 2 are not essential for epithelialization and neovascularization in the hairless mouse ear wound model.

BACKGROUND AND AIMS: TGF-beta stimulates neovascularization and epithelialization in healing wounds, yet relatively little is known about the mechanisms involved. Using the hairless mouse ear wound model, we studied the effects endogenous TGF-beta 1 and TGF-beta 2 have on epithelialization and neovascularization following the application of neutralizing antibodies. MATERIAL AND METHODS: Forty-three adult male hairless mice had an excisional wound made on the dorsum of each ear. Using vital microscopy, epithelialization and neovascularization were measured every third day until completion. TGF-beta 1 and TGF-beta 2 antibody, control-IgG, or phosphate buffered saline (PBS) were applied to the wounds. RESULTS AND CONCLUSIONS: Excisional wounds treated with anti-TGF-beta 1 and anti-TGF-beta 2, IgGcontrol IgG, and PBS epithelialized in 11.2 +/- 0.5 days (N = 22), 10.9 +/- 0.6 days (N = 17), and 10.6 +/- 0.6 days (N = 15), respectively and neovascularized in 27.9 +/- 0.5 days (N = 17), 27.1 +/- 0.8 days (N = 14), and 26.1 +/- 0.8 days (N = 10), respectively (mean +/- SEM). There were no significant differences in time to epithelialization and neovascularization between the three groups. Furthermore, there were no differences in the average time course of epithelialization and neovascularization between the three groups throughout the healing process. We conclude that endogenous TGF-beta 1 and TGF-beta 2 are not essential for epithelialization and neovascularization in the hairless mouse ear wound model.

Choline kinase II is present only in nodules that synthesize stable peribacteroid membranes.

Host-cell cytoplasm from soybean plants infected with the peribacteroid membrane (PBM)-building Rhizobium japonicum strain 61-A-101 (effective, N(2)-fixing) had much higher choline kinase activity than cytoplasm from either uninfected tissue or tissue infected with the non-PBM-building (ineffective, non-N(2)-fixing) strain 61-A-24. Ion-exchange chromatography showed that both types of nodule and root tissue possessed constitutive choline kinase I activity that had a K(m) for choline of approximately 150 muM. The nodules of the effective symbiosis had another activity, choline kinase II (K(m) = 81 muM). Nondenaturing and NaDodSO(4) electrophoresis revealed no multimeric subunit structure of the two enzyme forms but did show the molecular sizes for choline kinase I, 58-59 kDa, and choline kinase II, 60 kDa. Choline kinase I and II and pI values of 8.1 and 8.5, respectively, and two-dimensional gel electrophoresis of whole cytoplasm from control and infected tissue showed a spot corresponding to choline kinase II only in the case of the effective symbiosis, whereas both tissue types had spots corresponding to choline kinase I. Choline kinase II is presumed to be encoded by the plant as neither free-living nor symbiotic (bacteroid) forms of the prokaryote showed any choline kinase activity.

Enzymes of ureide synthesis in pea and soybean.

Soybean (Glycine max) and pea (Pisum sativum) differ in the transport of fixed nitrogen from nodules to shoots. The dominant nitrogen transport compounds for soybean are ureides, while amides dominate in pea. A possible enzymic basis for this difference was examined.The level of enzymes involved in the formation of the ureides allantoin and allantoic acid from inosine 5'-monophosphate (IMP) was compared in different tissues of pea and soybean. Two enzymes, 5'-nucleotidase and uricase, from soybean nodules were found to be 50- and 25-fold higher, respectively, than the level found in pea nodules. Other purine catabolizing enzymes (purine nucleosidase, xanthine dehydrogenase, and allantoinase) were found to be at the same level in the two species. From comparison of enzyme activities in nodules with those from roots, stems, and leaves, two enzymes were found to be nodule specific, namely uricase and xanthine dehydrogenase. The level of enzymes found in the bacteroids indicated no significant contribution of Rhizobium japonicum purine catabolism in the overall formation of ureides in the soybean nodule. The presence in the nodules of purine nucleosidase and ribokinase activities makes a recirculation of the ribose moiety possible. In concert with phosphoribosylpyrophosphate synthetase, ribose becomes available for a new round of purine de novo synthesis, and thereby ureide formation.