Confirmation of the anomeric structure of galacturonic acid in the galacturonosyl-ceramide of Sphingomonas yanoikuyae

The anomeric structure of glycosphingolipids significantly influences their activity to stimulate natural killer T cells. In this study the chemical structure of the galacturonosyl-ceramide in Sphingomonas yanoikuyae, designated GSL-1'sy, was re-examined to prove the anomeric structure of the Dgalacturonic acid (GalA) in the lipid, which was reported as beta-configuration by Naka et al., but was suggested as alpha-configuration in our preliminary study. GSL-1'sy was purified from the bacterial cells with the same procedure as Naka et al. The 1H-NMR analysis of GSL-1'sy revealed that the coupling constant of the anomeric proton of GalA was 3.0 Hz, indicating that GalA in GSL-1'sy is alpha-anomer, the configuration active for the stimulation of natural killer T cells.     

Characterization of a novel satellite virus and a strain of Himetobi P virus (Dicistroviridae) from the brown planthopper, Nilaparvata lugens

Two distinct spherical virus-like particles were purified from the brown planthopper, Nilaparvata lugens. One was a geographical isolate of Himetobi P virus (Cripavirus, Dicistroviridae). The other was 30 nm in diameter and contained positive-stranded RNA. The RNA was 1647 nucleotides in length and encoded only its own capsid protein, indicating that this particle is a satellite virus. Transmission tests showed that the satellite was transmitted vertically; however, its helper virus was unknown. We named this satellite Nilaparvata lugens commensal X virus (NLCXV).     

Maintenance of higher H(2)O(2) levels, and its mechanism of action to induce growth in breast cancer cells: Important roles of bioactive catalase and PP2A

We assessed the catalase bioactivity and hydrogen peroxide (H(2)O(2)) production rate in human breast cancer (HBC) cell lines and compared these with normal human breast epithelial (HBE) cells. We observed that the bioactivity of catalase was decreased in HBC cells when compared with HBE cells. This was also accompanied by an increase in H(2)O(2) steady-state levels in HBC cells. Silencing the catalase gene led to a further increase in the steady-state level of H(2)O(2) which was also accompanied by an increase in growth rate of HBC cells. Catalase activity was up regulated on treatment with superoxide (O(2)(-)) scavengers such as pegylated SOD (PEG-SOD, indicating inhibition of catalase by the increased O(2)(-) produced by HBC cells. Transfection of either catalase or glutathione peroxidase to HBC cells decreased intracellular H(2)O(2) levels and led to apoptosis of these cells. The H(2)O(2) produced by HBC cells inhibited PP2A activity accompanied by increased phosphorylation of Akt and ERK1/2. The importance of catalase bioactivity in breast cancer was further confirmed as its bioactivity was also decreased in human breast cancer tissues when compared to normal breast tissues. We conclude that inhibition of catalase bioactivity by O(2)(-) leads to an increase in steady-state levels of H(2)O(2) in HBC cells, which in turn inhibits PP2A activity, leading to phosphorylation of ERK 1/2 and Akt and resulting in HBC cell proliferation.     

Site-specific isotope labeling of long RNA for structural and mechanistic studies

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.     

Three new species of Fancy Case caterpillars from threatened forests of Hawaii (Lepidoptera, Cosmopterigidae, Hyposmocoma)

The endemic Hawaiian moth genus Hyposmocoma includes 348 described species and perhaps twice as many that remain undescribed. The genus is unusual within Lepidoptera in that its larvae create distinctive silk cases in which they perambulate while protected and camouflaged. An extraordinary diversity of case types exists, and to date more than ten different types have been identified, each corresponding roughly to a separate evolutionary lineage. In this study, we describe three new species of Hyposmocoma: Hyposmocoma ipohapuusp. n. from Big Island, Hyposmocoma makawaosp. n. from Makawao Forest Reserve in Mauiand Hyposmocoma tantalasp. n. from Mt. Tantalus, Oahu, all of which produce tubular purse cases during their larval stage. We also describe the female of Hyposmocoma inversella Walsingham, which was previously undescribed, and re-describe two closely related species, Hyposmocoma auropurpurea Walsingham and Hyposmocoma nebulifera Walsingham, neither which have been formally described in recent years. We present for the first time, primer sequences for a 705 bp fragment of CAD, designed for Hyposmocoma and relatives. The molecular phylogeny based on mitochondrial and nuclear loci demonstrates that all are distinct species. The discovery of a new, endemic species from Mt. Tantalus, an area with many invasive species, suggests that even relatively degraded areas in Hawaii would be worthy of active conservation efforts.     

Multipotent nestin-expressing stem cells capable of forming neurons are located in the upper,middle and lower part of the vibrissa hair follicle

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells. Nestin-expressing cells were initially identified in the hair follicle bulge area (BA) using a transgenic mouse model in which the nestin promoter drives the green fluorescent protein (ND-GFP). The hair-follicle ND-GFP-expressing cells are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro. Subsequently, we showed that the nestin-expressing stem cells could affect nerve and spinal cord regeneration after injection in mouse models. In the present study, we separated the mouse vibrissa hair follicle into three parts (upper, middle and lower). Each part of the follicle was cultured separately in DMEM-F12 containing B-27 and 1% methylcellulose supplemented with basic FGF. After 2 mo, the nestin-expressing cells from each of the separated parts of the hair follicle proliferated and formed spheres. Upon transfer of the spheres to RPMI 1640 medium containing 10% FBS, the nestin-expressing cells in the spheres differentiated to neurons, as well as glia, keratinocytes, smooth muscle cells and melanocytes. The differentiated cells were produced by spheres which formed from nestin-expressing cells from all segments of the hair follicle. However, the differentiation potential is greatest in the upper part of the follicle. This result is consistent with trafficking of nestin-expressing cells throughout the hair follicle from the bulge area to the dermal papilla that we previously observed. The nestin-expressing cells from the upper part of the follicle produced spheres in very large amounts, which in turn differentiated to neurons and other cell types. The results of the present study demonstrate that multipotent, nestin-expressing stem cells are present throughout the hair follicle and that the upper part of the follicle can produce the stem cells in large amounts that could be used for nerve and spinal cord repair.     

Solution structure of the c-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method

The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform ¹³C and ¹⁵N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the β-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.     

Protocol for the production of viable bimaternal mouse embryos

A reliable nuclear transfer method was first reported in 1983; it provided definite evidence that parthenogenetic embryos are lethal at early postimplantation in mammals. Subsequently, nuclear transfer has been extensively used as an important and versatile tool for investigating embryo and somatic-cell cloning and nucleo-cytoplasmic interactions. Further development of this technique has enabled the generation of bimaternal embryos containing two haploid sets of maternal genomes from female germ cells of different origins. By using a 2-d nuclear transfer system for oocyte reconstruction, viable mice can be produced solely from maternal genomes, without the participation of the paternal genome. This oocyte reconstruction system, as described in this protocol, could provide valuable guidelines for exploring the potential endowments of gametes and for conferring novel properties to them.     

Protein phosphatase 2A regulatory subunit Bbeta promotes MAP kinase-mediated migration of A431 cells.

BACKGROUND/AIMS: Phosphatases are involved in regulation of MAP kinase (MAPK). A431 cells migrate on collagen after EGF stimulation using MAPK. To clarify the involvement of PP2A in this MAPK-dependent migration, the expression of an isoform of the B regulatory subunit was inhibited. METHODS: An antisense sequence corresponding to Bbeta cDNA was transfected into A431 cells. Their migratory activity on collagen was examined using Transwell, and MAPK phosphorylation and phosphatase activity were measured, and the results were compared with those obtained with mock-transfected cells. RESULTS: Antisense-transfected cells showed less Bbeta protein and phosphatase activity than mock-transfected controls. Migration of antisense-transfected cells showed a low response to EGF. The response of MAPK phosphorylation of antisense-transfected cells to EGF stimulation and adhesion to collagen in the presence or absence of EGF were markedly decreased. Phosphatase activity of PP2A-Bbeta also did not respond to EGF, collagen or EGF plus collagen, and remained at low levels. CONCLUSION: These results suggested that PP2A-Bbeta promotes cell migration through the MAPK cascade.