Glomalin: an arbuscular mycorrhizal fungal soil protein

Glomalin is abundant in soils and is closely correlated with aggregate water stability. Glomalin contains carbon and, hence, constitutes a non-trivial portion of the terrestrial carbon pool. Possibly far more importantly, however, stabilization of aggregates amplifies the role of glomalin in soils because carbonaceous compounds are protected from degradation inside of aggregates. Increased atmospheric CO₂ can lead to increased production of glomalin because of the symbiotic association that exists between plants and producers of glomalin, arbuscular mycorrhizal fungi (AMF). Glomalin concentrations in soils are influenced by management practices, for example, in agroecosystems, further highlighting the role of this protein in carbon storage. Glomalin is an unusual molecule that has proven difficult to analyze biochemically due to its recalcitrance and complexity. Future research will be directed towards the elucidation of its structure and controls on its production.     

Viral inosine triphosphatase: A mysterious enzyme with typical activity,but an atypical function

Plant viruses typically have highly condensed genomes, yet the plant-pathogenic viruses Cassava brown streak virus, Ugandan cassava brown streak virus, and Euphorbia ringspot virus are unusual in encoding an enzyme not yet found in any other virus, the “house-cleaning” enzyme inosine triphosphatase. Inosine triphosphatases (ITPases) are highly conserved enzymes that occur in all kingdoms of life and perform a house-cleaning function by hydrolysing the noncanonical nucleotide inosine triphosphate to inosine monophosphate. The ITPases encoded by cassava brown streak virus and Ugandan cassava brown streak virus have been characterized biochemically and are shown to have typical ITPase activity. However, their biological role in virus infection has yet to be elucidated. Here we review what is known of viral-encoded ITPases and speculate on potential roles in infection with the aim of generating a greater understanding of cassava brown streak viruses, a group of the world's most devastating viruses.     

IL-33 and M2a alveolar macrophages promote lung defense against the atypical fungal pathogen Pneumocystis murina

We have recently reported that mice deficient in the myeloid Src-family tyrosine kinases Hck, Fgr, and Lyn (Src triple knockout [TKO]) had augmented innate lung clearance of Pneumocystis murina that correlated with a higher ability of alveolar macrophages (AMs) from these mice to kill P. murina. In this article, we show that despite possessing enhanced killing, AMs from naive Src TKO mice did not demonstrate enhanced inflammatory responses to P. murina. We subsequently discovered that both AMs and lungs from P. murina-infected Src TKO mice expressed significantly greater levels of the M2a markers RELM-α and Arg1, and the M2a-associated chemokines CCL17 and CCL22 than did wild-type mice. IL-4 and IL-13, the primary cytokines that promote M2a polarization, were not differentially produced in the lungs between wild-type and Src TKO mice. P. murina infection in Src TKO mice resulted in enhanced lung production of the novel IL-1 family cytokine IL-33. Immunohistochemical analysis of IL-33 in lung tissue revealed localization predominantly in the nucleus of alveolar epithelial cells. We further demonstrate that experimental polarization of naive AMs to M2a resulted in more efficient killing of P. murina compared with untreated AMs, which was further enhanced by the addition of IL-33. Administration of IL-33 to C57BL/6 mice increased lung RELM-α and CCL17 levels, and enhanced clearance of P. murina, despite having no effect on the cellular composition of the lungs. Collectively, these results indicate that M2a AMs are potent effector cells against P. murina. Furthermore, enhancing M2a polarization may be an adjunctive therapy for the treatment of Pneumocystis.     

Ponticulin is an atypical membrane protein

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha- helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).     

Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly

Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV. The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as beta-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity. In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature. The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis. We have identified a P. carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function. The P. carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Delta yeast. Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Delta yeast to maintain expression of the temperature-inducible beta-1-3-glucan synthetase gene, FKS2. PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation. Since glucan machinery is not present in mammals, a better understanding of this pathway in P. carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall.     

Glomalin, an arbuscular-mycorrhizal fungal soil protein, responds to land-use change

Glomalin is a soil proteinaceous substance produced by arbuscular mycorrhizal fungi. Most of the information available concerning this protein has been collected in relation to its role in soil aggregation. In this study, we explored the distribution of glomalin across soil horizons, decomposition of glomalin, and relationship with soil C and N in an agricultural field, a native forest, and an afforested system. Glomalin was present in A, B, and C horizons in decreasing concentrations. Land-use type significantly affected glomalin concentrations (mg cm^–3), with native forest soils having the highest concentrations of the three land-use types in both A and B horizons. In terms of glomalin stocks (Mg ha^–1), calculated based on corrected horizon weights, the agricultural area was significantly lower than both afforested and native forest areas. As measured after a 413 day laboratory soil incubation, glomalin was least persistent in the A horizon of the afforested area.. In agricultural soils and native soils, ca. 50% of glomalin was still remaining after this incubation, indicating that some glomalin may be in the slow or recalcitrant soil C fraction. Comparison of glomalin decomposition with CO₂-C respired during incubation indicates that glomalin makes a large contribution to active soil organic C pools. Soil C and N were highly correlated with glomalin across all soils and within each land-use type, indicating that glomalin may be under similar controls as soil C. Our results show that glomalin may be useful as an indicator of land-use change effects on deciduous forest soils.     

76例恶性肿瘤患者院内深部真菌感染调查

目的探讨深部真菌感染对于恶性肿瘤患者的影响,以及住院期间应注意的问题。方法调查分析2005年1月到2008年12月期间住院的76例恶性肿瘤患者院内深部真菌感染情况,分析患者一般状态、诊治方法的影响。结果76例患者中真菌感染的部位以呼吸道为主（70．9％）,泌尿系次之（17．7％）;病原学检测标本共培养出真菌79株,以白色念珠菌占第1位（60．8％）,热带念珠菌占第2位（21．5％）;老年（≥60岁）、一般状态评分（Ps评分）〉2分、晚期肿瘤差异均有显著性;多数有侵入性操作者（100％）、经过抗生素治疗者（97．4％）、经过激素治疗者（90．8％）和经过放化疗治疗者（82．9％）。结论肿瘤患者的年龄、病情分期、一般状态是本组病例发生院内深部真菌感染的基本病因,抗生素不合理使用及住院期间侵入性操作、既往放化疗及激素治疗等医疗措施是院内深部真菌感染的危险因素。     

Improvement of the fungal enzyme pyranose 2-oxidase using protein engineering

Native pyranose 2-oxidase (P2Ox) was purified from Peniophora sp. and characterized. To improve its catalytic efficiencies and stabilities by protein engineering, we cloned and expressed the P2Ox gene in Escherichia coli and received active, fully flavinylated recombinant P2OxA. Selenomethionine-labeled P2OxA was used for X-ray analysis and the resulting crystal structure enabled the rational design using variant P2OxA1 with the substitution E542K as template. Besides increased thermal and pH stabilities this variant showed improved catalytic efficiencies (k(cat)/K(m)) for the main substrates. A new variant, P2OxA2H, with an additional substitution T158A and a C-terminal His(6)-tag exhibited significantly decreased apparent K(m) values for D-glucose (0.47 mM), l-sorbose (1.79 mM), and D-xylose (1.35 mM). Compared to native P2Ox, the catalytic efficiencies were substantially improved for D-glucose (230-fold), L-sorbose (874-fold), and D-xylose (1751-fold). This P2Ox variant was used for the bioconversion of L-sorbose under O(2)-saturation in a molar scale. The structure-activity relationships of the amino acid substitutions were analyzed by modelling of the mutated P2Ox structures. Molecular docking calculations of various carbohydrates into the crystal structure of P2OxA and the analysis of the protein-ligand interactions in the docked complexes enabled us to explain the substrate specificity of the enzyme by a conserved hydrogen bond pattern which is formed between the protein and all substrates.     

The predominant protein of canine seminal plasma is an enzyme

One protein in canine seminal plasma accounts for over 90% of the total protein and is present at the high concentration of approximately 10 mg/ml. We demonstrate that this predominant protein is a proteolytic enzyme. The enzyme has been purified and migrates as a single symmetrical peak of apparent molecular mass of 29,000 daltons on a column of Sephadex G-75 and as a single band of approximately 30,000 daltons when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, the enzyme dissociates into subunits of 15,000 and 12,000-14,000 daltons. The 15,000-dalton subunit contains the enzyme active site as determined by labeling with [3H]diisopropyl fluorophosphate. The enzyme hydrolyzed the synthetic ester substrates N alpha-benzoyl-L-arginine ethyl ester and N alpha-tosyl-L-arginine methyl ester with maximum specific activities at 25 degrees C of 105 mumol/min/mg and 33 mumol/min/mg, and Km values of 7.4 and 9.1 mM, respectively. The enzyme exhibited a pH optimum of 8.0. The metal ions, Cu2+, Zn2+, Cd2+, and Co2+ were reversible inhibitors and diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride irreversible inhibitors of enzymatic activity. By immunofluorescence, the enzyme can be detected on the tail and postacrosomal regions of washed ejaculated canine sperm, but it is absent from epididymal sperm.     

Isolated DNA repeat region from fcrA76, the Fc-binding protein gene from an M-type 76 strain of group A streptococci, encodes a protein with Fc-binding activity

The DNA repeat region of fcrA76, the gene encoding a group A streptococcal Fc-binding protein, was subcloned in-frame into an Escherichia coli plasmid expression vector. The expressed protein product displayed the same Fc-binding properties as the full-length Fc-binding protein expressed from fcrA76. The affinity-purified, full-length Fc-binding protein was found to compete with staphylococcal protein A or streptococcal protein G for binding to beads coated with human IgG. These results are consistent with earlier studies suggesting that the binding sites on human IgG for protein A, protein G and the type II Fc-binding protein from group A streptococci are located at the interface of the CH2 and CH3 domains of the Fc region.     

Application of a fusion protein, metapyrocatechase/protein A, to an enzyme immunoassay

A fusion protein of metapyrocatechase and protein A was genetically produced for demonstration of effective conjugation of an enzyme with a binding protein employed in enzyme immunoassay. Plasmid pMPRA3, constructed by inserting the protein A gene into a plasmid pMK12 vector derived directly from the structural gene of metapyrocatechase, was expressed in Escherichia coli. The resulting fusion protein was shown to have promising properties for use in enzyme immunoassays due to the specific binding of the protein A moiety to the Fc portion of immunoglobulin G and to the high amplification of enzyme. Bovine serum albumin, a model antigen, was successfully determined in the concentration range from 1 x 10(-3) to 1 x 10(-7) g/ml.     

The atypical PKC-interacting protein p62 is an important mediator of RANK-activated osteoclastogenesis

The atypical PKCs (aPKCs) have been implicated genetically in at least two independent signaling cascades that control NF-kappa B and cell polarity, through the interaction with the adapters p62 and Par-6, respectively. P62 binds TRAF6, which plays an essential role in osteoclastogenesis and bone remodeling. Recently, p62 mutations have been shown to be the cause of the 5q35-linked Paget's disease of bone, a genetic disorder characterized by aberrant osteoclastic activity. Here we show that p62, like TRAF6, is upregulated during RANK-L-induced osteoclastogenesis and that the genetic inactivation of p62 in mice leads to impaired osteoclastogenesis in vitro and in vivo, as well as inhibition of IKK activation and NF-kappa B nuclear translocation. In addition, RANK-L stimulation leads to the inducible formation of a ternary complex involving TRAF6, p62, and the aPKCs. These observations demonstrate that p62 is an important mediator during osteoclastogenesis and induced bone remodeling.     

Oxytocinase-like enzyme in an ovarian dysgerminoma: a placenta-specific protein

Aminopeptidase from dysgerminoma was purified and characterized using L-leucine-beta-naphthylamide as substrate. The enzyme was resistant to puromycin, methionine, amastatin, bastatin, and EDTA, and it was heat labile at 60 degrees C. The enzyme showed the same electrophoretic mobility as pregnant-patient serum oxytocinase CAP1 on polyacrylamide gel electrophoresis. Km value against S-benzylcysteine-p-nitroanilide was 4.2 X 10(-4) M. Oxytocin and vasopressin competitively inhibited the enzyme activity. Molecular weight of the enzyme was estimated to be 80,000 by Sephadex G-200 column chromatography. These results suggest that aminopeptidase from dysgerminoma is an oxytocinase-like enzyme, a placenta-specific protein.     

Helicobacter pylori expresses an autolytic enzyme: gene identification,cloning, and theoretical protein structure

Helicobacter pylori is an important pathogen of the gastric system. The clinical outcome of infection is thought to be correlated with some genetic features of the bacterium. However, due to the extreme genetic variability of this organism, it is hard to draw definitive conclusions concerning its virulence factors. Here we describe a novel H. pylori gene which expresses an autolytic enzyme that is also capable of degrading the cell walls of both gram-positive and gram-negative bacteria. We designated this gene lys. We found this gene and observed its expression in a number of unrelated clinical strains, a fact that suggests that it is well conserved in the species. A comparison of the nucleotide sequences of lys and the hypothetical gene HP0339 from H. pylori strain ATCC 26695 revealed almost total identity, except for the presence of an insertion consisting of 24 nucleotides in the lys sequence. The coding sequences of lys and HP0339 show a high degree of homology with the coding sequence of bacteriophage T4 lysozyme. Because of this similarity, it was possible to model the three-dimensional structures of both the lys and HP0339 products.     

Sulfolobus solfataricus thiol redox puzzle: characterization of an atypical protein disulfide oxidoreductase

Protein disulfide oxidoreductases (PDOs) are proteins involved in disulfide bond formation playing a crucial role in adaptation to extreme environment. This paper reports the functional and structural characterization of Sso1120, a PDO from the hyperthermophilic archaeon Sulfolobus solfataricus. The protein was expressed in Escherichia coli and purified to homogeneity. The functional characterization showed that the enzyme has reductase activity, as tested by insulin assay, but differently from the other PDOs, it does not present isomerase activity. In addition it is able to form a redox couple with the thioredoxin reductase that could be used in undiscovered pathways. The protein revealed a melting point of around 90 °C in CD spectroscopy-monitored thermal denaturation and high denaturant resistance. The X-ray crystallographic structure was solved at 1.80 Å resolution, showing differences with respect to other PDOs and an unexpected similarity with the N-terminal domain of the alkyl hydroperoxide reductase F component from Salmonella typhimurium. On the basis of the reported data and of bioinformatics and phylogenetic analyses, a possible involvement of this atypical PDO in a new antioxidant system of S. solfataricus has been proposed.