Cellobiose Dehydrogenase, an Active Agent in Cellulose Depolymerization

The ability of cellobiose dehydrogenase purified from Phanerochaete chrysosporium to modify a Douglas fir kraft pulp was assessed. Although the addition of cellobiose dehydrogenase alone had little effect, supplementation with cellobiose and iron resulted in a substantial reduction in the degree of polymerization of the pulp cellulose. When the reaction was monitored over time, a progressive depolymerization of the cellulose was apparent with the concomitant production of cellobiono-1,5-lactone. Analysis of the reaction filtrates indicated that glucose and arabinose were the only neutral sugars generated. These sugars are derived from the degradation of the cellobiose rather than resulting from modifications of the pulp. These results suggest that the action of cellobiose dehydrogenase results in the generation of hydroxyl radicals via Fenton's chemistry which subsequently results in the depolymerization of cellulose. This appears to be the mechanism whereby a substantial reduction in the degree of polymerization of the cellulose can be achieved without a significant release of sugar.     

The hypersensitive reaction, membrane damage and accumulation of autofluorescent phenolics in lettuce cells challenged by Bremia lactucae

The expression of resistance to Bremia lactucae determined by the resistance genes Dm5/8 and Dm7 in lettuce was examined; incompatibility involved the hypersensitive reaction (HR) which occurred only within penetrated cells at early and late stages of fungal development, respectively. Autofluorescence observed under UV and blue light excitation in cells undergoing the HR was associated with the accumulation of ester-linked syringaldehyde and caffeic acid on plant cell walls. Two phases of phenolic deposition were identified. The first was highly localized around penetration points and occurred during incompatible and compatible interactions. The second and major phase was only activated after the occurrence of irreversible membrane damage in the penetrated cell and was reduced by inhibitors of mRNA synthesis. Fungal structures, primary and secondary vesicles, intercellular hyphae and haustoria also became autofluorescent during incompatible interactions. Changes in the fluorescence due to preformed phenolics located in the plant cell vacuole were found just before plasma membrane damage became irreversible during the HR. In addition to localized deposition of phenolics, increases in the concentrations of the major free phenolic esters identified as dicaffeoyl tartaric and chlorogenic acids also occurred during incompatible interactions. The results suggest that membrane damage in penetrated cells occurs at different rates in resistance controlled by Dm5/8 and Dm7 and indicate an important role for irreversible membrane damage in lettuce as a key signalling event leading to widespread activation of defence responses in surrounding cells.     

Comparisons of the molecular evolutionary process at rbcL and ndhF in the grass family (Poaceae)

We examine rate heterogeneity among evolutionary lineages of the grass family at two plasmid loci, ndhF and rbcL, and we introduce a method to determine whether patterns of rate heterogeneity are correlated between loci. We show both that rates of synonymous evolution are heterogeneous among grass lineages and that are heterogeneity is correlated between loci at synonymous sites. At nonsynonymous sites, the pattern of rate heterogeneity is not correlated between loci, primarily due to an aberrant pattern of rate heterogeneity at nonsynonymous sites of rbcL. We compare patterns of synonymous rate heterogeneity to predictors based on the generation time effect and the speciation rate hypotheses. Although there is some evidence for generation time effects, neither generation time effects nor speciation rates appear to be sufficient to explain patterns of rate heterogeneity in the grass plastid sequences.      

Abrupt shortening of bird W chromosomes in ancestral Neognathae

As a result of suppressed recombination, heterogametic sex chromosomes (either Y or W) are usually assumed to gradually shorten over evolutionary time as a way to remove accumulated mutations. However, suppressed recombination removes the most obvious mechanism for excising portions of sex chromosomes. We examined ratios of W/Z chromosome size across 224 bird species from 146 genera. Much of the data were obtained from a previous study (Rutkowska et al. 2012. Biology Letters 8 : 636–638), who, similar to ourselves, found no gradual decrease in W chromosome length over evolutionary time. However, we show an abrupt decrease in W chromosome length at or just after the phylogenetic split between the two extant bird superorders, Paleognathae and Neognathae, indicating that the key to understanding sex chromosome evolution may have little to do with gradual suppression of recombination.     

Population Differences in Postural Response Strategy Associated with Exposure to a Novel Continuous Perturbation Stimuli: Would Dancers Have Better Balance on a Boat?

Central or postural set theory suggests that the central nervous system uses short term, trial to trial adaptation associated with repeated exposure to a perturbation in order to improve postural responses and stability. It is not known if longer-term prior experiences requiring challenging balance control carryover as long-term adaptations that influence ability to react in response to novel stimuli. The purpose of this study was to determine if individuals who had long-term exposure to balance instability, such as those who train on specific skills that demand balance control, will have improved ability to adapt to complex continuous multidirectional perturbations. Healthy adults from three groups: 1) experienced maritime workers (n = 14), 2) novice individuals with no experience working in maritime environments (n = 12) and 3) individuals with training in dance (n = 13) participated in the study. All participants performed a stationary standing task while being exposed to five 6 degree of freedom motions designed to mimic the motions of a ship at sea. The balance reactions (change-in-support (CS) event occurrences and characteristics) were compared between groups. Results indicate dancers demonstrated significantly fewer CS events than novices during the first trial, but did not perform as well as those with offshore experience. Linear trend analyses revealed that short-term adaptation across all five trials was dependent on the nature of participant experience, with dancers achieving postural stability earlier than novices, but later than those with offshore experience. These results suggest that long term previous experiences also have a significant influence on the neural control of posture and balance in the development of compensatory responses.     

Synthesis of the low molecular weight heat shock proteins in plants

Heat shock of living tissue induces the synthesis of a unique group of proteins, the heat shock proteins. In plants, the major group of heat shock proteins has a molecular mass of 15 to 25 kilodaltons. Accumulation of these proteins to stainable levels has been reported in only a few species. To examine accumulation of the low molecular weight heat shock proteins in a broader range of species, two-dimensional electrophoresis was used to resolve total protein from the following species: soybean (Glycine max L. Merr., var Wayne), pea (Pisum sativum L., var Early Alaska), sunflower (Helianthus annuus L.), wheat (Triticum aestivum L.), rice (Oryza sativa L., cv IR-36), maize (Zea mays L.), pearl millet (Pennisetum americanum L. Leeke, line 23DB), and Panicum miliaceum L. When identified by both silver staining and incorporation of radiolabel, a diverse array of low molecular weight heat shock proteins was synthesized in each of these species. These proteins accumulated to significant levels after three hours of heat shock but exhibited considerable heterogeneity in isoelectric point, molecular weight, stainability, and radiolabel incorporation. Although most appeared to be synthesized only during heat shock, some were detectable at low levels in control tissue. Compared to the monocots, a higher proportion of low molecular weight heat shock proteins was detectable in control tissues from dicots.     

Studies of the mechanism of action of fusicoccin,the fungal toxin that induces wilting,and its interaction with abscisic acid

Summary Effects of fusicoccin alone and together with abscisic acid were observed on the stomatal complex of Commelina communis. The experimental material consisted of isolated epidermal strips incubated in a medium containing the ions required for stomatal opening. Fusicoccin stimulated opening and this was accompanied by potassium entry into the guard cells, and hydrolysis of the starch in their chloroplasts. Abscisic acid alone inhibited potassium entry and starch hydrolysis, but these effects could be almost entirely overcome by fusicoccin.Attempts were made to measure the solute potential of the guard cells under the various treatments. Abscisic acid clearly increased their solute potential, but no absolute measurements could be made in the presence of fusicoccin owing to a failure of plasmolysis even with mannitol solutions of solute potential as low as —35 bars. Experiments using isotopically labelled mannitol indicated a massive uptake into the epidermis in the presence of fusicoccin.The mechanism of stimulation of stomatal opening by fusicoccin probably depends in part on a stimulation of the normal processes associated with opening in the guard cells, but may also involve release of pressure due to destruction of the surrounding cells. The effectiveness of this toxin under natural conditions may depend on its ability to counteract effects of abscisic acid, the stress hormone that induces stomatal closure.     

Genetics of resistance to the African trypanosomes. III. Variant-specific antibody responses of H-2-compatible resistant and susceptible mice

Genetically based differences in variant-specific immunity to the African trypanosomes were examined. H-2-compatible inbred mouse strains that differed in relative resistance were infected with Trypanosoma rhodesiense clone LouTat 1. Antibody responses to exposed epitopes of the LouTat 1 variant-specific surface glycoprotein (VSG) were measured. Relatively resistant B10.BR mice (H-2k) made predictable IgM antibody responses to the VSG of LouTat 1 which were associated with clearance of the LouTat 1 variant antigenic type from blood; IgG responses to LouTat 1 surface antigen appeared after clearance occurred, and were lower than peak titers of IgM. Intermediately susceptible CBA mice (H-2k) also made predictable IgM and IgG responses which followed the same pattern as the more resistant strain. Peak titers were lower for both Ig classes, however, and a delayed appearance of antibody was correlated with delayed clearance of LouTat 1. In contrast to B10.BR and CBA mice, the susceptible C3H mice (H-2k) failed to make detectable antibodies to LouTat 1 surface antigen and also failed to control the first peak of parasitemia. The absence of immunity in infected C3H mice was selective for antibody to exposed epitopes of LouTat 1 VSG because antibody was detectable to invariant VSG or internal trypanosome antigens. Also, the C3H strain was shown not to be a genetic nonresponder to LouTat 1 surface antigen because VSG-specific antibodies appeared within 1 wk after trypanocidal chemotherapy. Finally, we demonstrated that the susceptibility of C3H mice was not associated with an inability of the mononuclear phagocyte system to clear the parasites because drug cure, passive transfer of immune serum, or sensitization of trypanosomes with antibody all led to trypanosome clearance from blood by the liver. In summary, we show for the first time that major differences in variant-specific immunity occur in MHC-compatible animals after infection with the African trypanosomes.     

Cytochrome distribution across chloroplast thylakoid membranes. Controlled proteolysis of inside-out and right-side-out vesicles

The transverse distribution of chloroplast cytochromes b-559 (high and low potentials), b-563 and f in pea thylakoid membranes was studied by the effects of trypsin and pronase on inside-out and right-side-out thylakoid vesicles. The high potential (HP) form of cytochrome b-559 was degraded to a low potential (LP) form most rapidly in right-side-out vesicles. In either type of vesicle there was no overall loss of the cytochrome from the membrane. This suggests that the haem group is buried in the membrane but that the cytochrome environment is most labile at the outer surface. Cytochrome b-563 was unaffected by trypsin and only slightly degraded by pronase in inverted vesicles. However, pronase caused the loss of an M_r 1000, non-haem fraction from the cytochrome f polypeptide in inside-out vesices only. The total cytochrome f content (measured spectrophotometrically and by staining polyacrylamide gels for haem associated peroxidase activity) decayed only slightly in either type of vesicle. These observations suggest that cytochrome f is, in part, exposed to the intrathylakoid lumen, whilst its haem group is retained in a more hydrophobic region.     

Localization of components of the oxidative cross-linking of glycoproteins and of callose synthesis in papillae formed during the interaction between non-pathogenic strains of Xanthomonas campestris and French bean mesophyll cells

Immunogold labelling was used to probe the responses of mesophyll cells in French bean ( Phaseolus vulgaris L.) to an hrpA mutant of Xanthomonas campestris pv. vesicatoria and a saprophytic strain of X.c. The non-pathogenic strains both caused localized alterations to the plant cell wall and formation of large papillae in adjacent cells. Immunocytochemistry showed the co-localization, in the cell wall and paramural deposits, of an M _r 42 000 proline-rich glycoprotein with chitin-binding activity (CBPRP) and the enzyme responsible for its immobilization, an M _r 46 000 peroxidase. The CBPRP appeared to lose antigenicity after cross-linking, and, unlike the peroxidase, was not detected consistently in the extracellular matrix that encapsulated bacteria onto the plant cell wall. The peroxidase may have a dual function in both the generation and utilization of H₂O₂ for cross-linking of proteins and phenolics during the construction of papillae. A burst of H₂O₂ was detected 1–5 h after inoculation at reaction sites by histochemical staining with cerium chloride. Progressive expansion of papillae and cell-wall alterations was, however, not associated with the maintenance of high levels of H₂O₂. Co-localization of callose and an M _r 65 000 polypeptide component of callose synthase was also demonstrated. Synthesis of callose appeared so rapid that the enzyme became embedded in the polysaccharide so that both were detected as integral to the developing papilla. Localized alterations to the cell wall and deposition of papillae were found to involve co-ordinated synthetic and oxidative activities at microsites within responding cells, without activation of the hypersensitive reaction.