MyD88 expression by CNS-resident cells is pivotal for eliciting protective immunity in brain abscesses

MyD88 KO (knockout) mice are exquisitely sensitive to CNS (central nervous system) infection with Staphylococcus aureus, a common aetiological agent of brain abscess, exhibiting global defects in innate immunity and exacerbated tissue damage. However, since brain abscesses are typified by the involvement of both activated CNS-resident and infiltrating immune cells, in our previous studies it has been impossible to determine the relative contribution of MyD88-dependent signalling in the CNS compared with the peripheral immune cell compartments. In the present study we addressed this by examining the course of S. aureus infection in MyD88 bone marrow chimaera mice. Interestingly, chimaeras where MyD88 was present in the CNS, but not bone marrow-derived cells, mounted pro-inflammatory mediator expression profiles and neutrophil recruitment equivalent to or exceeding that detected in WT (wild-type) mice. These results implicate CNS MyD88 as essential in eliciting the initial wave of inflammation during the acute response to parenchymal infection. Microarray analysis of infected MyD88 KO compared with WT mice revealed a preponderance of differentially regulated genes involved in apoptotic pathways, suggesting that the extensive tissue damage characteristic of brain abscesses from MyD88 KO mice could result from dysregulated apoptosis. Collectively, the findings of the present study highlight a novel mechanism for CNS-resident cells in initiating a protective innate immune response in the infected brain and, in the absence of MyD88 in this compartment, immunity is compromised.     

Furostanol glycoside 26-O-beta-glucosidase from the leaves of Solanum torvum

A beta-glucosidase (torvosidase) was purified to homogeneity from the young leaves of Solanum torvum. The enzyme was highly specific for cleavage of the glucose unit attached to the C-26 hydroxyl of furostanol glycosides from the same plant, namely torvosides A and H. Purified torvosidase is a monomeric glycoprotein, with a native molecular weight of 87 kDa by gel filtration and a pI of 8.8 by native agarose IEF. Optimum pH of the enzyme for p-nitrophenyl-beta-glucoside and torvoside H was 5.0. Kinetic studies showed that Km values for torvoside A (0.06 3mM) and torvoside H (0.068 mM) were much lower than those for synthetic substrates, pNP-beta-glucoside (1.03 mM) and 4-methylumbelliferyl-beta-glucoside (0.78 mM). The enzyme showed strict specificity for the beta-d-glucosyl bond when tested for glycone specificity. Torvosidase hydrolyses only torvosides and dalcochinin-8'-beta-glucoside, which is the natural substrate of Thai rosewood beta-glucosidase, but does not hydrolyse other natural substrates of the GH1 beta-glucosidases or of the GH3 beta-glucosidase families. Torvosidase also hydrolyses C5-C10 alkyl-beta-glucosides, with a rate of hydrolysis increasing with longer alkyl chain length. The internal peptide sequence of Solanum beta-glucosidase shows high similarity to the sequences of family GH3 glycosyl hydrolases.     

A phytochemical investigation of Citrus halimii

Essential leaf oils, isoenzymes, protein and polyphenol oxidase-catalyzed browning patterns of Citrus halimii (Rutaceae) are investigated and compared with those of other species of Citrus in order to support the species standing of the recently described C. halimii.     

An immunodominant site of γ-zein1 is in the region of tandem hexapeptide repeats

The immunochemical data from studies with polyclonal antisera to γ-zein₁, the 27 kD component of the maize prolamin, indicated that the region containing 8 tandem repeats of the sequence PPPVHL is an immunodominant site. In one case, the entire antibody repertoire of an antiserum recognized epitope(s) within this region. Three 17-mer oligopeptides corresponding to the predicted antigenic epitopes of γ-zein₁ were synthesized and reacted with three different anti-γ-zein₁ sera in order to map antigenic sites in the intact protein. These antisera yielded positive reactions with a 17-mer peptide (peptide 37), which was not in a hydrophilic maximum but derived from the repeat region. The same antisera gave little or no reaction with other peptides (peptides 38 and 39), both of which were in a hydrophilic maximum. In addition, an antiserum to peptide 37 reacted strongly with both the homologous antigen and the intact γ-zein₁. Peptide 37 also blocked the binding of antisera to γ-zein₁ in competition assays. Subsequently, the shorter 6-mer (peptide 82) and 12-mer (peptide 80) versions of peptide 37 were synthesized, and both reacted with anti-peptide 37 serum and also with each of the three anti-γ-zein₁ sera. In these reactions and in competition assays, the reactivity and the blocking ability increased in proportion to the length of the peptide. Based on these data, it was concluded that the repeat region of γ-zein₁ is the site of one or more continuous immunodominant epitopes. The data also suggest that the repeat region is exposed on the surface of the folded protein and probably occur as a mobile, random coil.     

Expression of soluble and catalytically active plant (monocot) beta-glucosidases in E. coli

Complementary DNAs encoding mature beta-glucosidase proteins Glu1 and Glu2 of maize were amplified by the polymerase chain reaction (PCR) and cloned into the expression vector pET21a. Both Glu1 and Glu2 isozymes were expressed in high yield ( approximately 3.8% of the total soluble protein and 32% of the total expressed protein) in E. coli. Recombinant enzymes were active on a variety of artificial and natural substrates at levels similar to those of their native counterparts isolated from maize seedlings. Western blot analysis confirmed that both recombinant isozymes were immunoreactive with maize anti-beta-glucosidase sera and their molecular sizes were identical to those of the native maize Glu1 and Glu2 isozymes. Zymogram assays in native gels revealed that recombinant enzymes had the same electrophoretic mobility and substrate specificity as their native counterparts.     

Structural determinants of substrate specificity in family 1 beta-glucosidases: novel insights from the crystal structure of sorghum dhurrinase-1, a plant beta-glucosidase with strict specificity, in complex with its natural substrate

Plant beta-glucosidases play a crucial role in defense against pests. They cleave, with variable specificity, beta-glucosides to release toxic aglycone moieties. The Sorghum bicolor beta-glucosidase isoenzyme Dhr1 has a strict specificity for its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-D-glucoside), whereas its close homolog, the maize beta-glucosidase isoenzyme Glu1, which shares 72% sequence identity, hydrolyzes a broad spectrum of substrates in addition to its natural substrate 2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one. Structural data from enzyme.substrate complexes of Dhr1 show that the mode of aglycone binding differs from that previously observed in the homologous maize enzyme. Specifically, the data suggest that Asn(259), Phe(261), and Ser(462), located in the aglycone-binding site of S. bicolor Dhr1, are crucial for aglycone recognition and binding. The tight binding of the aglycone moiety of dhurrin promotes the stabilization of the reaction intermediate in which the glycone moiety is in a deformed (1)S(3) conformation within the glycone-binding site, ready for nucleophilic attack to occur. Compared with the broad specificity maize beta-glucosidase, this different binding mode explains the narrow specificity of sorghum dhurrinase-1.     

The aglycone specificity-determining sites are different in 2, 4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)-glucosidase (Maize beta -glucosidase) and dhurrinase (Sorghum beta -glucosidase)

The maize beta-glucosidase isozyme Glu1 hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOAGlc (2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-on e), whereas the sorghum beta-glucosidase isozyme Dhr1 hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-d-glucose). To study the mechanism of substrate specificity further, eight chimeric beta-glucosidases were constructed by replacing peptide sequences within the C-terminal region of Glu1 with the homologous peptide sequences of Dhr1 or vice versa, where the two enzymes differ by 4 to 22 amino acid substitutions, depending on the length of the swapped regions. Five Glu1/Dhr1 chimeras hydrolyzed substrates that are hydrolyzed by both parental enzymes, including dhurrin, which is not hydrolyzed by Glu1. In contrast, three Dhr1/Glu1 chimeras hydrolyzed only dhurrin but with lower catalytic efficiency than Dhr1. Additional domain-swapping within the C-terminal domain of Glu1 showed that replacing the peptide (466)FAGFTERY(473) of Glu1 with the homologous peptide (462)SSGYTERF(469) of Dhr1 or replacing the peptide (481)NNNCTRYMKE(490) in Glu1 with the homologous peptide (477)ENGCERTMKR(486) of Dhr1 was sufficient to confer to Glu1 the ability to hydrolyze dhurrin. Data from various reciprocal chimeras, sequence comparisons, and homology modeling suggest that the Dhr1-specific Ser-462-Ser-463 and Phe-469 play a key role in dhurrin hydrolysis. Similar data suggest that DIMBOAGlc hydrolysis determinants are not located within the extreme 47-amino acid-long C-terminal domain of Glu1.     

WNT7B Promotes Bone Formation in part through mTORC1

WNT signaling has been implicated in both embryonic and postnatal bone formation. However, the pertinent WNT ligands and their downstream signaling mechanisms are not well understood. To investigate the osteogenic capacity of WNT7B and WNT5A, both normally expressed in the developing bone, we engineered mouse strains to express either protein in a Cre-dependent manner. Targeted induction of WNT7B, but not WNT5A, in the osteoblast lineage dramatically enhanced bone mass due to increased osteoblast number and activity; this phenotype began in the late-stage embryo and intensified postnatally. Similarly, postnatal induction of WNT7B in Runx2-lineage cells greatly stimulated bone formation. WNT7B activated mTORC1 through PI3K-AKT signaling. Genetic disruption of mTORC1 signaling by deleting Raptor in the osteoblast lineage alleviated the WNT7B-induced high-bone-mass phenotype. Thus, WNT7B promotes bone formation in part through mTORC1 activation.