A tryptophan neutral radical in the oxidized state of versatile peroxidase from Pleurotus eryngii: a combined multifrequency EPR and density functional theory study

Versatile peroxidases are heme enzymes that combine catalytic properties of lignin peroxidases and manganese peroxidases, being able to oxidize Mn(2+) as well as phenolic and non-phenolic aromatic compounds in the absence of mediators. The catalytic process (initiated by hydrogen peroxide) is the same as in classical peroxidases, with the involvement of 2 oxidizing equivalents and the formation of the so-called Compound I. This latter state contains an oxoferryl center and an organic cation radical that can be located on either the porphyrin ring or a protein residue. In this study, a radical intermediate in the reaction of versatile peroxidase from the ligninolytic fungus Pleurotus eryngii with H(2)O(2) has been characterized by multifrequency (9.4 and 94 GHz) EPR and assigned to a tryptophan residue. Comparison of experimental data and density functional theory theoretical results strongly suggests the assignment to a tryptophan neutral radical, excluding the assignment to a tryptophan cation radical or a histidine radical. Based on the experimentally determined side chain orientation and comparison with a high resolution crystal structure, the tryptophan neutral radical can be assigned to Trp(164) as the site involved in long-range electron transfer for aromatic substrate oxidation.     

Yeast nuclear envelope subdomains with distinct abilities to resist membrane expansion

Little is known about what dictates the round shape of the yeast Saccharomyces cerevisiae nucleus. In spo7Delta mutants, the nucleus is misshapen, exhibiting a single protrusion. The Spo7 protein is part of a phosphatase complex that represses phospholipid biosynthesis. Here, we report that the nuclear protrusion of spo7Delta mutants colocalizes with the nucleolus, whereas the nuclear compartment containing the bulk of the DNA is unaffected. Using strains in which the nucleolus is not intimately associated with the nuclear envelope, we show that the single nuclear protrusion of spo7Delta mutants is not a result of nucleolar expansion, but rather a property of the nuclear membrane. We found that in spo7Delta mutants the peripheral endoplasmic reticulum (ER) membrane was also expanded. Because the nuclear membrane and the ER are contiguous, this finding indicates that in spo7Delta mutants all ER membranes, with the exception of the membrane surrounding the bulk of the DNA, undergo expansion. Our results suggest that the nuclear envelope has distinct domains that differ in their ability to resist membrane expansion in response to increased phospholipid biosynthesis. We further propose that in budding yeast there is a mechanism, or structure, that restricts nuclear membrane expansion around the bulk of the DNA.     

Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments

Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.     

Helicobacter pylori-induced downregulation of the secretory leukocyte protease inhibitor (SLPI) in gastric epithelial cell lines and its functional relevance for H. pylori-mediated diseases

The secretory leukocyte protease inhibitor (SLPI) exerts antiproteolytic activity towards serine proteases, as well as anti-microbial and anti-inflammatory effects. To investigate its role in H. pylori-mediated diseases, SLPI expression was analyzed by RT-PCR, ELISA and immunohistochemistry in clinical samples and gastric tumor cell lines. Determination of the mucosal SLPI levels in 126 patients confirmed the previously reported downregulation of SLPI in H. pylori-infected patients. The lower SLPI levels in antral biopsies of H. pylori-positive subjects were associated with a 30-fold increase (p<0.01) in neutrophil elastase activity, and a significant negative correlation was demonstrated for both parameters (R=-0.63, p=0.0002). Eradication of the bacterium in a long-term study (5-7 years) led to a recovery of mucosal SLPI expression. In vitro experiments using four gastric tumor cell lines (AGS, MKN-28, MKN-45, NCI-N87) generally confirmed the clinical findings. While the co-incubation of these cell lines with H. pylori resulted in lower or unchanged SLPI protein levels, the corresponding SLPI mRNA amounts were upregulated by up to five-fold (p=0.006) in all cell lines. Taken together, these results indicate that the reduction in antral SLPI levels in H. pylori-infected subjects has a functional relevance for gastric mucosa and the H. pylori-induced decrease in SLPI is primarily regulated at the posttranslational level.     

Variable responses within epiphytic and benthic microalgal communities to nutrient enrichment

We evaluated how changes in nutrient supply altered the composition of epiphytic and benthic microalgal communities in a Thalassia testudinum (turtle grass) bed in Florida Bay. We established study plots at four sites in the bay and added nitrogen (N) and phosphorus (P) to the sediments in a factorial design. After 18, 24, and 30 months of fertilization we measured the pigment concentrations in the epiphytic and benthic microalgal assemblages using high performance liquid chromatography. Overall, the epiphytic assemblage was P-limited in the eastern portion of the bay, but each phototrophic group displayed unique spatial and temporal responses to N and P addition. Epiphytic chlorophyll a, an indicator of total microalgal load, and epiphytic fucoxanthin, an indicator of diatoms, increased in response to P addition at one eastern bay site, decreased at another eastern bay site, and were not affected by P or N addition at two western bay sites. Epiphytic zeaxanthin, an indicator of the cyanobacterial/coralline red algae complex, and epiphytic chlorophyll b, an indicator of green algae, generally increased in response to P addition at both eastern bay sites but did not respond to P or N addition in the western bay. Benthic chlorophyll a, chlorophyll b, fucoxanthin, and zeaxanthin showed complex responses to N and P addition in the eastern bay, suggesting that the benthic assemblage is limited by both N and P. Benthic assemblages in the western bay were variable over time and displayed few responses to N or P addition. The contrasting nutrient limitation patterns between the epiphytic and benthic communities in the eastern bay suggest that altering nutrient input to the bay, as might occur during Everglades restoration, can shift microalgal community structure, which may subsequently alter food web support for upper trophic levels.     

Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny,Physiology, and Niche Adaptation on Human Skin

Malassezia is a unique lipophilic genus in class Malasseziomycetes in Ustilaginomycotina, (Basidiomycota, fungi) that otherwise consists almost exclusively of plant pathogens. Malassezia are typically isolated from warm-blooded animals, are dominant members of the human skin mycobiome and are associated with common skin disorders. To characterize the genetic basis of the unique phenotypes of Malassezia spp., we sequenced the genomes of all 14 accepted species and used comparative genomics against a broad panel of fungal genomes to comprehensively identify distinct features that define the Malassezia gene repertoire: gene gain and loss; selection signatures; and lineage-specific gene family expansions. Our analysis revealed key gene gain events (64) with a single gene conserved across all Malassezia but absent in all other sequenced Basidiomycota. These likely horizontally transferred genes provide intriguing gain-of-function events and prime candidates to explain the emergence of Malassezia. A larger set of genes (741) were lost, with enrichment for glycosyl hydrolases and carbohydrate metabolism, concordant with adaptation to skin’s carbohydrate-deficient environment. Gene family analysis revealed extensive turnover and underlined the importance of secretory lipases, phospholipases, aspartyl proteases, and other peptidases. Combining genomic analysis with a re-evaluation of culture characteristics, we establish the likely lipid-dependence of all Malassezia. Our phylogenetic analysis sheds new light on the relationship between Malassezia and other members of Ustilaginomycotina, as well as phylogenetic lineages within the genus. Overall, our study provides a unique genomic resource for understanding Malassezia niche-specificity and potential virulence, as well as their abundance and distribution in the environment and on human skin.     

Resurrecting embryos of the tuatara,Sphenodon punctatus,to resolve vertebrate phallus evolution

The breadth of anatomical and functional diversity among amniote external genitalia has led to uncertainty about the evolutionary origins of the phallus. In several lineages, including the tuatara, Sphenodon punctatus, adults lack an intromittent phallus, raising the possibility that the amniote ancestor lacked external genitalia and reproduced using cloacal apposition. Accordingly, a phallus may have evolved multiple times in amniotes. However, similarities in development across amniote external genitalia suggest that the phallus may have a single evolutionary origin. To resolve the evolutionary history of amniote genitalia, we performed three-dimensional reconstruction of Victorian era tuatara embryos to look for embryological evidence of external genital initiation. Despite the absence of an intromittent phallus in adult tuataras, our observations show that tuatara embryos develop genital anlagen. This illustrates that there is a conserved developmental stage of external genital development among all amniotes and suggests a single evolutionary origin of amniote external genitalia.     

Evidence for carrier-mediated,energy-dependent uptake of urea in some bacteria

Evidence for the existence of an energy-dependent urea permease was found for Alcaligenes eutrophus H16 and Klebsiella pneumoniae M5a1 by studying uptake of ¹⁴C-urea. Since intracellular urea was metabolized immediately, uptake did not result in formation of an urea pool. Evidence is based on observations that the in vivo urea uptake and in vitro urease activity differ significantly with respect to kinetic parameters, temperature optimum, pH optimum, response towards inhibitors and regulation. The K _m for urea uptake was 15–20 times lower (38 M and 13 M urea for A. eutrophus and K. pneumoniae, respectively) than the K _m of urease for urea (650 M and 280 M urea), the activity optimum for A. eutrophus was at pH 6.0 and 35°C for the uptake and pH 9.0 and 65°C for urease. Uptake but not urease activity in both organisms strongly decreased upon addition of inhibitors of energy metabolism, while in K. pneumoniae, potent inhibitors of urease (thiourea and hydroxyurea) did not affect the uptake process. Significant differences in the uptake rates were observed during growth with different nitrogen sources (ammonia, nitrate, urea) or in the absence of a nitrogen source; this suggested that a carrier is involved which is subject to nitrogen control. Some evidence for the presence of an energy-dependent uptake of urea was also obtained in Pseudomonas aeruginosa DSM 50071 and Providencia rettgeri DSM 1131, but not in Proteus vulgaris DSM 30118 and Bacillus pasteurii DSM 33.Non-standard abbreviations CCCP Carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide - DNP 2,4-dinitrophenole     

Reciprocal control of pyruvate dehydrogenase kinase and phosphatase by inositol phosphoglycans. Dynamic state set by "push-pull" system

Reversible phosphorylation of proteins regulates numerous aspects of cell function, and abnormal phosphorylation is causal in many diseases. Pyruvate dehydrogenase complex (PDC) is central to the regulation of glucose homeostasis. PDC exists in a dynamic equilibrium between de-phospho-(active) and phosphorylated (inactive) forms controlled by pyruvate dehydrogenase phosphatases (PDP1,2) and pyruvate dehydrogenase kinases (PDK1-4). In contrast to the reciprocal regulation of the phospho-/de-phospho cycle of PDC and at the level of expression of the isoforms of PDK and PDP regulated by hormones and diet, there is scant evidence for regulatory factors acting in vivo as reciprocal "on-off" switches. Here we show that the putative insulin mediator inositol phosphoglycan P-type (IPG-P) has a sigmoidal inhibitory action on PDK in addition to its known linear stimulation of PDP. Thus, at critical levels of IPG-P, this sigmoidal/linear model markedly enhances the switchover from the inactive to the active form of PDC, a "push-pull" system that, combined with the developmental and hormonal control of IPG-P, indicates their powerful regulatory function. The release of IPGs from cell membranes by insulin is significant in relation to diabetes. The chelation of IPGs with Mn2+ and Zn2+ suggests a role as "catalytic chelators" coordinating the traffic of metal ions in cells. Synthetic inositol hexosamine analogues are shown here to have a similar linear/sigmoidal reciprocal action on PDC exerting push-pull effects, suggesting their potential for treatment of metabolic disorders, including diabetes.     

Application of electron transfer dissociation (ETD) for the analysis of posttranslational modifications

Despite major advantages in the field of proteomics, the analysis of PTMs still poses a major challenge; thus far, preventing insights into the role and regulation of protein networks. Additionally, top-down sequencing of proteins is another powerful approach to reveal comprehensive information for biological function. A commonly used fragmentation technique in MS-based peptide sequencing is CID. As CID often fails in PTM-analysis and performs best on doubly-charged, short and middle-sized peptides, confident peptide identification may be hampered. A newly developed fragmentation technique, namely electron transfer dissociation (ETD), supports both, PTM- and top-down analysis, and generally results in more confident identification of long, highly charged or modified peptides. The following review presents the theoretical background of ETD and its technical implementation in mass analyzers. Furthermore, current improvements of ETD and approaches for the PTM-analysis and top-down sequencing are introduced. Alternating both fragmentation techniques, ETD and CID, increases the amount of information derived from peptide fragmentation, thereby enhancing both, peptide sequence coverage and the confidence of peptide and protein identification.