Human chromosome 21q22.2-qter carries a gene(s) responsible for downregulation of mlc2a and PEBP in Down syndrome model mice

Congenital heart disease (CHD) is a major clinical manifestation of Down syndrome (DS). We recently showed that chimeric mice containing a human chromosome 21 (Chr 21) exhibited phenotypic traits of DS, including CHD. Our previous study showed that myosin light chain-2a (mlc2a) expression was reduced in the hearts of chimeric mice and DS patients. We found that phosphatidylethanolamine binding protein (PEBP) was also downregulated in Chr 21 chimeras in this study. As mlc2a is involved in heart morphogenesis, and PEBP controls the proliferation and differentiation of different cell types, these genes are candidates for involvement in DS-CHD. The DS-CHD candidate region has been suggested to span between PFKL and D21S3, which is the STS marker near the ETS2 loci. To identify gene(s) or a gene cluster on Chr 21 responsible for the downregulation of mlc2a and PEBP, we fragmented Chr 21 at the EST2 loci, by telomere-directed chromosome truncation in homologous recombination-proficient chicken DT40 cells. The modified Chr 21 was transferred to mouse ES cells by microcell-mediated chromosome transfer (MMCT), via CHO cells. We used ES cell lines retaining the Chr 21 truncated at the ETS2 locus (Chr 21E) to produce chimeric mice and compared overall protein expression patterns in hearts of the chimeras containing the intact and the fragmented Chr 21 by two-dimensional electrophoresis. While mouse mlc2a and PEBP expression was downregulated in the chimeras containing the intact Chr 21, the expression was not affected in the Chr 21E chimeras. Therefore, we suggest that Chr 21 gene(s) distal from the ETS2 locus reduce mouse mlc2a and PEBP expression in DS model mice and DS. Thus, this chromosome engineering technology is a useful tool for identification or mapping of genes that contribute to the DS phenotypes.     

Reproductive development according to elevation in a seasonally breeding male songbird

Seasonal temperate zone breeders respond to increasing day length to anticipate the approach of spring breeding conditions. Other (supplementary) environmental cues, such as temperature and precipitation, were historically thought to play unimportant roles in reproductive timing. We demonstrate variation in reproductive timing across small geographic distances by examining the vernal testicular recrudescence of adult song sparrows (Melospiza melodia morphna) breeding in coastal (0–10 m elevation) and montane (280–1220 m elevation) habitats. Each year, these birds experienced the same photoperiod, but were exposed to different supplementary cues that varied with altitude. Coastal birds experienced warmer and more stable temperatures during late winter and early spring than did montane birds. We measured bud opening, emergence of new green shoots, and arthropod biomass to monitor the pace of springs approach. New spring shoots emerged 2 months earlier on the coast than in the mountains and buds on flowering trees and shrubs also tended to open earlier at the coast. Arthropod biomass was similar in both the mountains and the coast during early spring, and began to increase in early summer. Reproductive morphology (i.e. testis volume and cloacal protuberance length) developed up to 2 months earlier on the coast than in the mountains. Testicular recrudescence occurred earlier on the coast in most years and proceeded at a faster rate in 1 year. Circulating levels of luteinizing hormone, follicle stimulating hormone and prolactin increased through the season, but did not correlate with differences between sites. Both populations responded similarly when exposed to identical photoperiodic cues in the laboratory. Therefore, we suggest that an integrated response to cues characteristic of location and elevation account for differences in patterns measured in the field.     

Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable isotope tagging

Protein post-translational modifications (PTMs), such as glycosylation and phosphorylation, are crucial for various signaling and regulatory events, and are therefore an important objective of proteomics research. We describe here a protocol for isotope-coded glycosylation site-specific tagging (IGOT), a method for the large-scale identification of N-linked glycoproteins from complex biological samples. The steps of this approach are: (1) lectin column-mediated affinity capture of glycopeptides generated by protease digestion of protein mixtures; (2) purification of the enriched glycopeptides by hydrophilic interaction chromatography (HIC); (3) peptide-N-glycanase-mediated incorporation of a stable isotope tag, 18O18O, specifically at the N-glycosylation site; and (4) identification of 18O-tagged peptides by liquid chromatography-coupled mass spectrometry (LC/MS)-based proteomics technology. The application of this protocol to the characterization of N-linked glycoproteins from crude extracts of the nematode Caenorhabditis elegans or mouse liver provides a list of hundreds to a thousand glycoproteins and their sites of glycosylation within a week.     

Processing of amyloid beta-peptides by neutral cysteine protease bleomycin hydrolase

We studied the processing of amyloid beta-peptides (Abetas) including Abeta(1-40), Abeta(1-42) and pAbeta(3-42) by rat neutral cysteine protease bleomycin hydrolase (BH) according to the methods of SDS-PAGE, HPLC and matrix-assisted laser desorption/inonization time-of-flight mass spectrometry (MALDI-TOF MS). BH significantly processed them by novel features of its diverse activities. It initially cleaved at two sites, His(14)-Gln(15) and Phe(19)-Phe(20) degraded to short intermediates then to amino acids by aminopeptidase and/or carboxypeptidase activities. Also, full-length Abetas were clipped at the carboxyl(C)-terminal region. On the other hand, BH cleaved at only the His(14)-Gln(15) bond in pbetaA(3-42) within a short period of the reaction by endopeptidase activity, and processed the intermediates in order by carboxypeptidase activity. On processing by BH, it found that both fibrillar Abeta(1-40) and Abeta(1-42) were more resistant than non-fibrillar peptides. These results indicate that the processing specificity of BH depends upon the structure and sequence of Abetas.     

A Function of Polar Flagellum and Anisotropic Growth in Vibrio alginolyticus Early-Phase Colonies

We continuously observed growth of Vibrio alginolyticus early-phase colonies on agar plates by phase-contrast microscopy. Two mutants defective in motility on solid surfaces were used in this study: one (YM4) can swim in liquid environments using its polar flagellum, and the other (NMB198) cannot swim because it lacks any flagella. We found that isolated colonies of YM4 were generally more circular than those of NMB198. This observation suggests that YM4 cells moved slightly within a colony by the function of their polar flagella. For clustered colonies, where the distance between the colonies was short (<50 μm), the colonies of YM4 grew rapidly along the line between them, but they grew slowly in the lateral directions. Some colonies of NMB198 grew toward neighboring colonies. These observations indicate colony-to-colony interaction.     

14-3-3 Mediates phosphorylation-dependent inhibition of the interaction between the ubiquitin E3 ligase Nedd4-2 and epithelial Na+ channels

Although recent studies show that the 14-3-3 protein is a negative regulator of ubiquitin E3 protein ligases, the molecular mechanism remains largely unknown. We previously demonstrated that 14-3-3 specifically binds one of the E3 enzymes, Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2), which can be phosphorylated by serum glucocorticoid-inducible protein kinase 1 (SGK1); this binding protects the phosphorylated/inactive hNedd4-2 from phosphatase-catalyzed dephosphorylation [Ichimura, T., et al. (2005) J. Biol. Chem. 280, 13187-13194]. Here we report an additional mechanism of 14-3-3-mediated regulation of hNedd4-2. Using surface plasmon resonance spectrometry, we show that 14-3-3 inhibits the interaction between the WW domains of hNedd4-2 and the PY motif of the epithelial Na(+) channel, ENaC. The inhibition was dose-dependent and was dependent on SGK1-catalyzed phosphorylation of Ser468 located between the WW domains. Importantly, a mutant of hNedd4-2, which can be phosphorylated by SGK1 but cannot bind 14-3-3, reduced SGK1-mediated stimulation of the ENaC-induced current in Xenopus laevis oocytes. In addition, 14-3-3 had similar effects on hNedd4-2 that had been phosphorylated by cAMP-dependent protein kinase (PKA). Our results, together with the recent finding on 14-3-3/parkin interactions [Sato, S., et al. (2006) EMBO J. 25, 211-221], suggest that 14-3-3 suppresses ubiquitin E3 ligase activities by inhibiting the formation of the enzyme/substrate complex.     

Efficient synthesis of [2-15N]guanosine and 2'-deoxy[2'-15N]guanosine derivatives using N-(tert-butyldimethylsilyl)[15N]phthalimide as a 15N-labeling reagent

Nucleophilic aromatic substitution of 9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-6-chloro-2-fluoro-9H-purine with N-(tert-butyldimethylsilyl) [15N]phthalimide in the presence of a catalytic amount of CsF at room temperature in DMF efficiently afforded the 6-chloro-2-[15N]phthalimidopurine derivative, which was subsequently converted to the [2-15N]guanosine derivative. The 2'-deoxy[2'-15N]guanosine derivative was also efficiently synthesized through a similar procedure.     

Estimation of cat medial gastrocnemius fascicle lengths during dynamic contractions

In typical muscle models, it is often assumed that the contractile element (fascicle) length depends exclusively on the instantaneous muscle-tendon length and the instantaneous muscle force. In order to test whether the instantaneous fascicle length during dynamic contractions can be predicted from muscle-tendon length and force, fascicle lengths, muscle-tendon lengths, and muscle forces were directly measured in cat medial gastrocnemii during isometric and dynamic contractions. Two theoretical muscle models were developed: model A was based on force-time data obtained during the activation phase and model D on force-time data obtained during the deactivation phase of isometric contractions. To test the models, instantaneous fascicle lengths were predicted from muscle-tendon lengths and forces during dynamic contractions that simulated cat locomotion for speeds ranging from 0.4 to 1.6m/s. The theoretically predicted fascicle lengths were compared with the experimentally measured fascicle lengths. It was found that fascicle lengths were not uniquely associated with muscle-tendon lengths and forces; that is, for a given muscle-tendon length and force, fascicle lengths varied depending on the contractile history. Consequently, models A and D differed in fascicle length predictions; model D (maximum average error=8.5%) was considerably better than model A (maximum average error=22.3%). We conclude from this study that it is not possible to predict the exact fascicle lengths from muscle-tendon lengths and forces alone, however, adequate predictions seem possible based on such a model. The relationship between fascicle length and muscle force and muscle-tendon length is complex and highly non-linear, thus, it appears unlikely that accurate fascicle length predictions can be made without some reference contractions in which fascicle length, muscle-tendon length, and force are measured simultaneously.     

Sunitinib induces apoptosis in pheochromocytoma tumor cells by inhibiting VEGFR2/Akt/mTOR/S6K1 pathways through modulation of Bcl-2 and BAD

Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors (VEGFRs). Very recently, sunitinib has been shown to be an active agent for the treatment of malignant pheochromocytomas. However, it is unclear whether sunitinib acts only through an antiangiogenic mechanism or whether it may also directly target tumor cells. Sunitinib markedly induced apoptosis of PC12 cells in a dose-dependent and time-dependent manner. Furthermore, in support of these findings, we found that sunitinib induced a reduction in the expression of the antiapoptotic molecule Bcl-2 as well as dephosphorylation of the proapoptotic molecule BAD, which results in the activation of BAD in these cells. Consistent with these apoptotic effects, our results showed that sunitinib inhibited phosphorylation of Akt and mTOR and was followed by a reduction of S6K1, which is a well-known target of mTOR. Knockdown of VEGFR-2 attenuated the sunitinib-induced effects, including apoptosis and inhibition of signaling pathways such as the phosphorylation of Akt as well as mTOR, and Bcl-2, which confirmed that these effects could be mediated by VEGFR-2. In addition, silencing of S6K1 induced apoptosis accompanied by a decrease in the phosphorylation of BAD and Bcl-2, similar to that observed with sunitinib treatment. Thus, these results together suggest that sunitinib initially exerts its apoptotic effect through the inhibition of VEGFR-2, which, when followed by reduction of its downstream effectors, including Akt/mTOR/S6K1, may lead to inhibition of the antiapoptotic molecule Bcl-2 and activation of the proapoptotic molecule BAD in PC12 cells. However, PC12 cells do not precisely reflect the pathogenesis of malignant cells. Therefore, we confirmed the key findings by replicating these experiments in human neuroblastoma SK-N-SH cells.