The carbohydrate-binding module (CBM)-like sequence is crucial for rice CWA1/BC1 function in proper assembly of secondary cell wall materials

We recently reported that the cwa1 mutation disturbed the deposition and assembly of secondary cell wall materials in the cortical fiber of rice internodes. Genetic analysis revealed that cwa1 is allelic to bc1, which encodes glycosylphosphatidylinositol (GPI)-anchored COBRA-like protein with the highest homology to Arabidopsis COBRA-like 4 (COBL4) and maize Brittle Stalk 2 (Bk2). Our results suggested that CWA1/BC1 plays a role in assembling secondary cell wall materials at appropriate sites, enabling synthesis of highly ordered secondary cell wall structure with solid and flexible internodes in rice. The N-terminal amino acid sequence of CWA1/BC1, as well as its orthologs (COBL4, Bk2) and other BC1-like proteins in rice, shows weak similarity to a family II carbohydrate-binding module (CBM2) of several bacterial cellulases. To investigate the importance of the CBM-like sequence of CWA1/BC1 in the assembly of secondary cell wall materials, Trp residues in the CBM-like sequence, which is important for carbohydrate binding, were substituted for Val residues and introduced into the cwa1 mutant. CWA1/BC1 with the mutated sequence did not complement the abnormal secondary cell walls seen in the cwa1 mutant, indicating that the CBM-like sequence is essential for the proper function of CWA1/BC1, including assembly of secondary cell wall materials.Key words: carbohydrate-binding module, COBRA-LIKE, CWA1/BC1, glycosylphosphatidylinositol-anchored protein, secondary cell wall formationThe main function of carbohydrate-binding modules (CBMs) of microbes and plants is to attach the enzyme to a variety of cell surface glycans and thereby increase the local concentration of substrate, leading to more efficient catalysis.^1–4 Almost all CBMs studied to date contain surface-exposed aromatic rings, which have been shown to be the main sites of interaction with polysaccharides. These residues form face-to-face hydrophobic stacking interactions in which a Trp residue or ring of a Tyr residue interacts with the non-polar face of a sugar ring.^5–9 CBMs have been classified into families based on amino acid sequence similarity. Currently, there are 59 defined families of CBMs and these CBMs display substantial variation in ligand specificity (http://www.cazy.org/Carbohydrate-Binding-Modules.html). Among these CBM families, the large family of CBM2 has been further classified into two subgroups, CBM2a and 2b, which have shown to bind cellulose and xylan, respectively.^10–12 CBM2a characteristically possess three exposed Trp residues,¹³ whereas CBM2b have two Trp residues,¹⁴ which are conserved among the CBM2 members (Fig. 1A).Open in a separate windowFigure 1Sequence alignment of the CBM-like sequence of CWA1/BC1, the BC1L proteins and bacterial CBM2 members. (A) Sequence alignment between bacterial CBM2a, 2b and CWA1/BC1. The three surface-exposed Trp residues of CBM2a members are indicated by asterisks and W. The CBM sequences of CBM2a are: CfiCenA, Cellulomonas fimi endo-1,4-glucanase; CfiCex, C. fimi exo-beta-1,4-glucanase. Those of CBM2b are: CfiXylD1, C. fimi endo-1,4-beta-xylanase D; CfiXylD2, C. fimi endo-1,4-beta-xylanase. CWA1/BC1 shows weak similarity to CBM2, and some Trp residues are conserved with bacterial CBM2 members. (B) Sequence alignment of CWA1/BC1, the BC1L proteins and CWA1/BC1 orthologs, Zea maiz Brittle Stalk 2 (ZmBk2) and Arabidopsis thaliana COBRA-LIKE 4 (AtCOBL4). The CBM-like sequence of CWA1/BC1, especially the Trp residues, is highly conserved among the analyzed sequences. Substituted Trp (W) residues to Val (V) in CWA1/BC1 are indicated by closed triangles. Numbers at the left are the positions of the amino acids in each protein, with gaps (dashes) included to maximize alignments. Identical and similar amino acids are shaded and gray, respectively.Our recent study showed that the defect of the rice CWA1/BC1 (CELL WALL ARCHITECTURE 1/BRITTLE CULM 1) gene induced abnormal secondary cell wall formation with amorphous and bulky structures at the cytoplasm side and CWA1/BC1 encodes one of COBRA-like glycosylphosphatidylinositol (GPI)-anchored proteins, which are specifically found in plants, suggesting that CWA1/BC1 regulates assembly of secondary cell wall materials in rice sclerenchyma. Furthermore, several reports have shown that the N-terminus of rice CWA1/BC1 and other COBRA-like GPI-anchored proteins in Arabidopsis (12 members) and maize Brittle Stalk 2 (Bk2) share weak similarity to a CBM2 in several bacterial cellulases.^15,16 However, the importance of CBM-like sequence in COBRA family members has not been clarified. To investigate the nature of CWA1/BC1, we compared the CBM-like sequence in rice CWA1/BC1 with bacterial CBM2, 10 members of the BC1-like (BC1L) protein in rice and CWA1/BC1 orthologs, Arabidopsis COBL4 and maize Bk2. Furthermore, we constructed three-point mutated CWA1/BC1, in which three conserved Trp residues in CBM-like sequence were substituted for Val residues (CWA1/BC1^W→V), and introduced it into the cwa1 mutant to evaluate the necessity of the CBM-like sequence for proper function of CWA1/BC1. We discuss a putative explanation, based on our results, of the properties and possible functions of CWA1/BC1.     

Bacterial flora in the digestive tract of cattle. I. Comparison of nonselective culture medium and changes in fecal bacterial flora with age.

Studies were made on nonselective culture medium and the method of culture for the investigation of the bacterial flora in the digestive tract of cattle. With their results, further studies were done to clarify changes in the fecal bacterial flora in eight calves less than 6 months of age with the lapse of time. Three roll-tube media were used in the gas jet method. They were modified VL agar (VL medium), rumen fluid glucose cellobiose agar (RGCA medium), and Medium 10 (M 10). Moreover, glucose liver blood agar (BL medium) was used in the anaerobic jar method. In this method the steel wool method was applied after the substitution of carbon dioxide. Of the four media used, VL medium was proved to be the most efficient. It was followed by RGCA medium and M 10. BL medium was much less efficient than any other medium. When the fecal bacterial flora was examined in calves for changes with the advance in age, the total bacterial count and the enteric bacterial count decreased in the second half of the experimental period. The lactobacillary group count remained almost at a constant level of 7 approximately 9 (logarithmic value) per gram in breast-fed calves, but decreased to a level of 5 (logarithmic value) per gram in bottle-fed calves at about 2 months of age or later. The streptococcal group count showed no particular tendency to change. When the organic acid contents of the feces were estimated in calves in every stage of growth, the amount of total organic acids and that of propionic acid were larger in bottle-fed than in breast-fed calves.     

Lysophosphatidylcholine potentiates Ca2+ influx, pore formation and p44/42 MAP kinase phosphorylation mediated by P2X7 receptor activation in mouse microglial cells

The P2X7 receptor (P2X7R) is an ATP-gated ion channel highly expressed in microglia. P2X7R plays important roles in inflammatory responses in the brain. However, little is known about the mechanisms regulating its functions in microglia. Lysophosphatidylcholine (LPC), an inflammatory phospholipid that promotes microglial activation, may have some relevance to P2X7R signaling in terms of microglial function. In this study, we examined its effects on P2X7R signaling in a mouse microglial cell line (MG6) and primary microglia. LPC facilitated the sustained increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) through P2X7R channels activated by ATP or BzATP. The potentiated increase in [Ca(2+)](i) was actually inhibited by P2X7R antagonists, brilliant blue G and oxidized ATP. The potentiating effect of LPC was not observed with P2Y receptor systems, which are also expressed in MG6 cells. G2A, a receptor for LPC, was expressed in MG6 cells, but not involved in the facilitating effect of LPC on the P2X7R-mediated change in [Ca(2+)](i). Furthermore, LPC enhanced the P2X7R-associated formation of membrane pores and the activation of p44/42 mitogen-activated protein kinase. These results suggest that LPC may regulate microglial functions in the brain by enhancing the sensitivity of P2X7R.     

Fatty acid composition indicating diverse habitat use in coral reef fishes in the Malaysian South China Sea

Background

In order to understand feeding ecology and habitat use of coral reef fish, fatty acid composition was examined in five coral reef fishes, Thalassoma lunare, Lutjanus lutjanus, Abudefduf bengalensis, Scarus rivulatus and Scolopsis affinis collected in the Bidong Island of Malaysian South China Sea.

Results

Proportions of saturated fatty acids (SAFA) ranged 57.2% 74.2%, with the highest proportions in fatty acids, the second highest was monounsaturated fatty acids (MUFA) ranged from 21.4% to 39.0% and the proportion of polyunsaturated fatty acids (PUFA) was the lowest ranged from 2.8% to 14.1%. Each fatty acid composition differed among fishes, suggesting diverse feeding ecology, habitat use and migration during the fishes’ life history in the coral reef habitats.

Conclusions

Diets of the coral fish species might vary among species in spite of that each species are living sympatrically. Differences in fatty acid profiles might not just be considered with respect to the diets, but might be based on the habitat and migration.     

Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation

Expression of somatostatin in the brain declines during aging in various mammals including apes and humans. A prominent decrease in this neuropeptide also represents a pathological characteristic of Alzheimer disease. Using in vitro and in vivo paradigms, we show that somatostatin regulates the metabolism of amyloid beta peptide (Abeta), the primary pathogenic agent of Alzheimer disease, in the brain through modulating proteolytic degradation catalyzed by neprilysin. Among various effector candidates, only somatostatin upregulated neprilysin activity in primary cortical neurons. A genetic deficiency of somatostatin altered hippocampal neprilysin activity and localization, and increased the quantity of a hydrophobic 42-mer form of Abeta, Abeta(42), in a manner similar to presenilin gene mutations that cause familial Alzheimer disease. These results indicate that the aging-induced downregulation of somatostatin expression may be a trigger for Abeta accumulation leading to late-onset sporadic Alzheimer disease, and suggest that somatostatin receptors may be pharmacological-target candidates for prevention and treatment of Alzheimer disease.     

Involvement of calpain in osteoclastic bone resorption

There is increasing evidence that calpain contributes to the reorganization of the cytoskeleton in the integrin-mediated signaling pathway. Osteoclastic bone resorption requires cell-matrix contact, an event mediated by integrin alphavbeta3, and subsequent cytoskeletal reorganization to form characteristic membrane domains such as the sealing zone and ruffled border. In this study, therefore, we investigated whether calpain is involved in osteoclastic bone resorption. Membrane-permeable calpain inhibitors suppress the resorption activity of human osteoclasts, but an impermeable inhibitor does not. Upon the attachment of osteoclasts to bone, micro-calpain is translocated from the cytosolic to the cytoskeletal fraction and is autolytically activated. Both the activation of micro-calpain and the formation of actin-rings, the cytoskeletal structures essential for bone resorption, are inhibited by membrane-permeable calpain inhibitors. The activated micro-calpain in osteoclasts selectively cleaves talin, which links the matrix-recognizing integrin to the actin cytoskeleton. These findings suggest that calpain is a regulator of the bone resorption activity of osteoclasts through reorganization of the cytoskeleton related to actin-ring formation.     

A mode of assembly of P0, P1, and P2 proteins at the GTPase-associated center in animal ribosome: in vitro analyses with P0 truncation mutants

Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants CDelta65 and CDelta81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0.P1-P2 binding site. The mutant CDelta107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants NDelta21-NDelta92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants NDelta6, NDelta14, and CDelta18-CDelta81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10.L7/L12 complex and L11. Analysis of incorporation of (32)P-labeled P1/P2 into the 50 S subunits with WT-P0 and CDelta81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but CDelta81 bound only one copy each. The hybrid ribosome with CDelta81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on eukaryotic elongation factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.     

Beta-synuclein regulates Akt activity in neuronal cells. A possible mechanism for neuroprotection in Parkinson's disease

Recent studies have shown that the neurodegenerative process in disorders with Lewy body formation, such as Parkinson's disease and dementia with Lewy bodies, is associated with alpha-synuclein accumulation and that beta-synuclein might protect the central nervous system from the neurotoxic effects of alpha-synuclein. However, the mechanisms are unclear. The main objective of the present study was to investigate the potential involvement of the serine threonine kinase Akt (also known as protein kinase B) signaling pathway in the mechanisms of beta-synuclein neuroprotection. For this purpose, Akt activity and cell survival were analyzed in synuclein-transfected B103 neuroblastoma cells and primary cortical neurons. Beta-synuclein transfection resulted in increased Akt activity and conferred protection from the neurotoxic effects of rotenone. Down-regulation of Akt expression resulted in an increased susceptibility to rotenone toxicity, whereas transfection with a lentiviral vector encoding for beta-synuclein was protective. The effects of beta-synuclein on the Akt pathway appear to be by direct interaction between these molecules and were independent of upstream signaling molecules. Taken together, these results indicate that the mechanisms of beta-synuclein neuroprotection might involve direct interactions between beta-synuclein and Akt and suggest that this signaling pathway could be a potential therapeutic target for neurological conditions associated with parkinsonism and alpha-synuclein aggregation.