Molecular cloning and characterization of a beta-L-Arabinobiosidase in Bifidobacterium longum that belongs to a novel glycoside hydrolase family

Extensin is a glycoprotein that is rich in hydroxyprolines linked to β-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel β-L-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38-98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated L-arabinofuranose (Araf)-β1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(3)-Hyp) but not Araf-α1,3-Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(4)-Hyp) or Araf-β1,2-Araf-β-Hyp (Ara(2)-Hyp), which indicated that it was specific for unmodified Ara(3)-Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyp-linked β-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.     

Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.     

Functional activation of Src family kinase yes protein is essential for the enhanced malignant properties of human melanoma cells expressing ganglioside GD3

The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3- cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3- cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3- cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3- cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.     

Spectral tuning in sensory rhodopsin I from Salinibacter ruber

Organisms utilize light as energy sources and as signals. Rhodopsins, which have seven transmembrane α-helices with retinal covalently linked to a conserved Lys residue, are found in various organisms as distant in evolution as bacteria, archaea, and eukarya. One of the most notable properties of rhodopsin molecules is the large variation in their absorption spectrum. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) function as photosensors and have similar properties (retinal composition, photocycle, structure, and function) except for their λ(max) (SRI, ～560 nm; SRII, ～500 nm). An expression system utilizing Escherichia coli and the high protein stability of a newly found SRI-like protein, SrSRI, enables studies of mutant proteins. To determine the residue contributing to the spectral shift from SRI to SRII, we constructed various SRI mutants, in which individual residues were substituted with the corresponding residues of SRII. Three such mutants of SrSRI showed a large spectral blue-shift (>14 nm) without a large alteration of their retinal composition. Two of them, A136Y and A200T, are newly discovered color tuning residues. In the triple mutant, the λ(max) was 525 nm. The inverse mutation of SRII (F134H/Y139A/T204A) generated a spectral-shifted SRII toward longer wavelengths, although the effect is smaller than in the case of SRI, which is probably due to the lack of anion binding in the SRII mutant. Thus, half of the spectral shift from SRI to SRII could be explained by only those three residues taking into account the effect of Cl(-) binding.     

Activation of TLR2 enhances tight junction barrier in epidermal keratinocytes

The epidermis has developed physical and immunological barriers that prevent infiltration of deleterious chemicals and pathogens. As a first step to understanding the relationship between these barriers, we investigated whether TLR2 activation functionally alters tight junctions (TJs) in cultured human keratinocytes. Stimulation with peptidoglycan, a ligand for TLR2, elevated the TJ-associated barrier in the space of 3 h. The increase in TJ-associated barrier function due to peptidoglycan stimulation was suppressed by the knockdown of TLR adaptor MyD88 or the pretreatment with TLR2-neutralizing Ab, indicating that TLR2 activation enhanced TJ-associated barrier. One and 3 h after peptidoglycan stimulation, expression levels of the TJ proteins occludin, claudin-1, claudin-4, and ZO-1 were unchanged. However, immunoprecipitation studies demonstrated that the association of phospho-atypical protein kinase Cζ/ι, crucial for TJ biogenesis, with occludin was increased. Significantly, inhibition of atypical protein kinase Cζ/ι activity completely blocked the immediate elevation of the TJ-associated barrier. Finally, peptidoglycan was applied to the stratum corneum surface of a human skin equivalent, and the TJ barrier was evaluated. In the space of 3 h after the stimulation, the amount of intercellular tracer in the stratum corneum incubated from the dermal side was reduced, indicating that the TJ barrier is strengthened via TLR2 activation. Taken together, our findings indicated that infiltration of pathogens into the epidermis immediately enhanced TJ function via TLR2 signaling. Furthermore, the dynamically controlled TJs in skin are considered fundamental in preventing further invasion of pathogens and maintaining cutaneous barrier homeostasis.     

Analysis of pelvic movement in the elderly during walking using a posture monitoring system equipped with a triaxial accelerometer and a gyroscope

The incidence of falls in the elderly is increasing with the aging of society and is becoming a major public health issue. From the viewpoint of prevention of falls, it is important to evaluate the stability of the gait in the elderly people. The pelvic movement, which is a critical factor for walking stability, was analyzed using a posture monitoring system equipped with a triaxial accelerometer and a gyroscope. The subjects were 95 elderly people over 60 years of age. The criteria for instability were open-eye standing on one leg for 15s or less, and 11s or more on 3m timed up and go test. Forty subjects who did not meet both of these criteria comprised the stable group, and the remaining 55 subjects comprised the unstable group. Pelvic movement during walking was compared between the two groups. The angle, angular velocity, and acceleration were analyzed based on the wave shape derived from the device worn around the second sacral. The results indicated that pelvic movement was lower in all three directions in the unstable group compared to the stable group, and the changes in the pelvic movement during walking in unstable elderly people were also reduced. This report is the first to evaluate pelvic movement by both a triaxial accelerometer and a triaxial gyroscope simultaneously. The characteristics of pelvic movement during walking can be applied in screening to identify elderly people with instability, which is the main risk factor associated with falls.     

Structure and mutation analysis of archaeal geranylgeranyl reductase

The crystal structure of geranylgeranyl reductase (GGR) from Sulfolobus acidocaldarius was determined in order to elucidate the molecular mechanism of the catalytic reaction. The enzyme is a flavoprotein and is involved in saturation of the double bonds on the isoprenoid moiety of archaeal membranes. The structure determined in this study belongs to the p-hydroxybenzoate hydroxylase family in the glutathione reductase superfamily. GGR functions as a monomer and is divided into the FAD-binding, catalytic and C-terminal domains. The catalytic domain has a large cavity surrounded by a characteristic YxWxFPx_7-8GxG motif and by the isoalloxazine ring of an FAD molecule. The cavity holds a lipid molecule, which is probably derived from Escherichia coli cells used for over-expression. One of the two forms of the structure clarifies the presence of an anion pocket holding a pyrophosphate molecule, which might anchor the phosphate head of the natural ligands. Mutational analysis supports the suggestion that the three aromatic residues of the YxWxFPx_7-8GxG motif hold the ligand in the appropriate position for reduction. Cys47, which is widely conserved in GGRs, is located at the si-side of the isoalloxazine ring of FAD and is shown by mutational analysis to be involved in catalysis. The catalytic cycle, including the FAD reducing factor binding site, is proposed on the basis of the detailed analysis of the structure.     

The tomato terpene synthase gene family

Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far.