Development of glycosylated human interleukin-1α, neoglyco IL-1α, coupled with D-galactose monosaccharide: biological activities in vivo

In our previous study, a galactose monosaccharide with C9 spacer was chemically coupled to recombinant human interleukin 1 (rhIL-1) in order to study the effect of glycosylation on its activities, and to develop IL-1 with less deleterious effects. The glycosylated IL-1 exhibited reduced activities in vitro by 10 to 10 000-fold depending upon different aspects of activities addressed. The affinity to type I and II IL-1 receptors were also reduced. In this study we examined a variety of IL-1 activities in vivo, including upregulation of serum levels of IL-6, 1-acid glycoprotein, NOx, corticosterone, downregulation of serum level of glucose, and recovery of peripheral white blood cells (WBCs) from myelosuppression in 5-fluorouracil-treated mice. In contrast to the biological activities in vitro, these activities in vivo were uniformly reduced by only about 10 to 20-fold compared to untreated IL-1.     

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.     

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.     

Natural dental caries in molars of osteogenic disorder Shionogi rats

Osteogenic disorder Shionogi (ODS) rats are genetically defective in ascorbic acid biosynthesis. They exhibit a gait abnormality due to dysfunctional bone formation and display various dental abnormalities. Conditions of the oral cavity and tooth quality both influence the development of dental caries. This study was designed to determine the characteristics of dental caries in ODS/ ShiJclod/od rats. Caries were scored and compared among ODS/ShiJclod/od, ODS/ShiJcl+/+, and Jcl:Wistar retired breeders. Among male rats, the caries scores of the ODS/ShiJclod/od and ODS/ShiJcl+/+ groups were similar to each other but greater than those in Jcl:Wistar rats, whereas among female rats, caries scores in ODS/ShiJclod/od animals were equivalent to or somewhat greater than those in ODS/ShiJcl+/+ rats, whose scores were markedly greater than those of Jcl:Wistar rats. The results suggest that ODS/ShiJcl rats were more susceptible to dental caries than were Jcl:Wistar rats. Under the conditions of the study, caries scores between ODS/ ShiJclod/od and ODS/ShiJcl+/+ rats differed only among parous females.     

Morphological analysis of ghrelin and its receptor distribution in the rat pancreas

Ghrelin, a novel peptide isolated from stomach tissue of rats and humans, has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to its secretion from the stomach, ghrelin is also expressed in the hypothalamic arcuate nucleus, intestine, kidney, placenta, and pancreas. GHS-R mRNA, on the other hand, is expressed in the hypothalamus, pituitary, heart, lung, liver, pancreas, stomach, intestine, and adipose tissue. Ghrelin is considered to have important roles in feeding regulation and energy metabolism as well as in the release of growth hormone (GH). Recent physiological experiments on the pancreas have shown that ghrelin regulates insulin secretion. However, sites of action of ghrelin in the pancreas are yet to be identified. In this study, to gain insight into the role of ghrelin in rat pancreatic islets, we used immunohistochemistry to determine the localization of ghrelin and GHS-R in islet cells. Double fluorescence immunohistochemistry revealed that weak GHS-R-like immunoreactivity was found in B cells containing insulin. GHS-R immunoreactivity overlapped that of glucagon-like immunoreactive cells. Moreover, both ghrelin and GHS-R-like immunoreactivities were detected mostly in the same cells in the periphery of the islets of Langerhans. These observations suggest that ghrelin is synthesized and secreted from A cells, and acts back on A cells in an autocrine and/or paracrine manner. In addition, ghrelin may act on B cells via GHS-R to regulate insulin secretion.     

Rapid construction of Drosophila RNAi transgenes using pRISE, a P-element-mediated transformation vector exploiting an in vitro recombination system

RNAi is a gene-silencing phenomenon mediated by double-stranded RNA (dsRNA) and has become a powerful tool to elucidate gene function. To accomplish rapid construction of transgenes expressing dsRNA in Drosophila, we developed a novel transformation vector, pRISE, which contains an inverted repeat of the attR1-ccdB-attR2 cassette for in vitro recombination and a pentameric GAL4 binding site for conditional expression. These features enabled us to construct RNAi transgenes without a complicated cloning scheme. In cultured cells and transgenic flies, pRISE constructs carrying dsRNA transgenes induced effective RNAi against an EGFP transgene and the endogenous white gene, respectively. These results indicate that pRISE is a convenient transformation vector for studies of multiple Drosophila genes for which functional information is lacking.