Expression and intracellular localization of TBC1D9, a Rab GTPase-accelerating protein,in mouse testes

Membrane trafficking in male germ cells contributes to their development via cell morphological changes and acrosome formation. TBC family proteins work as Rab GTPase accelerating proteins (GAPs), which negatively regulate Rab proteins, to mediate membrane trafficking. In this study, we analyzed the expression of a Rab GAP, TBC1D9, in mouse organs and the intracellular localization of the gene products. Tbc1d9 showed abundant expression in adult mice testis. We found that the Tbc1d9 mRNA was expressed in primary and secondary spermatocytes, and that the TBC1D9 protein was expressed in spermatocytes and round spermatids. In 293T cells, TBC1D9-GFP proteins were localized in the endosome and Golgi apparatus. Compartments that were positive for the constitutive active mutants of Rab7 and Rab9 were also positive for TBC1D9 isoform 1. In addition, TBC1D9 proteins were associated with Rab7 and Rab9, respectively. These results indicate that TBC1D9 is expressed mainly in spermatocytes, and suggest that TBC1D9 regulates membrane trafficking pathways related to Rab9- or Rab7-positive vesicles.     

Two guard cell mitogen‐activated protein kinases,MPK9 and MPK12, function in methyl jasmonate‐induced stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell‐preferential mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9‐1 and mpk12‐1 single mutants as well as wild‐type plants, but not in mpk9‐1 mpk12‐1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA‐induced stomatal closure in wild‐type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9‐1, mpk12‐1 and mpk9‐1 mpk12‐1 mutants, as well in wild‐type plants. Furthermore, MeJA triggered elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in the mpk9‐1 mpk12‐1 double mutant as well as wild‐type plants. Activation of S‐type anion channels by MeJA was impaired in mpk9‐1 mpk12‐1. Together, these results indicate that MPK9 and MPK12 function upstream of S‐type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca²⁺]_cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.     

Hair follicle stem cell progeny heal blisters while pausing skin development

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that HF development can be sacrificed for the sake of epidermal wound regeneration. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.     

Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, we analyzed the function of the Delta (Gb'Delta) and Notch (Gb'Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb'Delta and Gb'Notch were expressed in developing legs, and that RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. Our results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, we found that Gb'Delta was expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries.     

Neuroendocrine ductal carcinoma in situ of the breast: cytological features in 32 cases

T. Kawasaki, S. Nakamura, G. Sakamoto, T. Kondo, H. Tsunoda‐Shimizu, Y. Ishii, T. Nakazawa, K. Mochizuki, T. Yamane, M. Inoue, S. Inoue and R. Katoh
Neuroendocrine ductal carcinoma in situ of the breast: cytological features in 32 cases Objective: The purpose of this study was to clarify the cytological features of neuroendocrine ductal carcinoma in situ (NE‐DCIS) of the breast. Methods: We analysed the cytopathological findings in 22 fine needle aspiration (FNA) smears and 17 nipple discharge smears obtained from 32 Japanese patients with NE‐DCIS. Results: The background of the FNA smears was clear (59%), mucoid (23%), haemorrhagic (14%) or necrotic (5%). Most of the FNA smears (95%) showed high cellularity. Characteristically, NE‐DCIS cells were loosely arranged in three‐dimensional solid clusters or singly dispersed. Well‐developed vascular cores with or without malignant cells were occasionally recognized. The tumour cells were polygonal or spindle‐shaped with a fine granular, abundant cytoplasm. Nuclei with finely granular chromatin were round or oval and often eccentrically located (plasmacytoid appearance). Mitotic figures were infrequent. Nuclear grade was estimated to be low in 86%. Most nipple discharge smears had fairly low cellularity with poorly preserved cell clusters in a markedly haemorrhagic background, although two (12%) were extremely cellular with cytological characteristics similar to those of the FNA smears. Pre‐operative cytological malignant diagnoses were made in 42% of FNA smears and 0% of nipple discharge smears. Immunohistochemistry for neuroendocrine markers (chromogranin A and synaptophysin) confirmed the neuroendocrine nature of this tumour in adequate cytological specimens. Conclusions: NE‐DCIS has distinctive cytological features and can therefore be diagnosed as a neuroendocrine tumour in most FNAs and some nipple discharge smears by cytological examination employing immunohistochemical techniques. We emphasize that a breast lesion with these features may be in situ and not invasive, and also that there is a risk of under‐diagnosis.     

AtNUFIP, an essential protein for plant development, reveals the impact of snoRNA gene organisation on the assembly of snoRNPs and rRNA methylation in Arabidopsis thaliana

In all eukaryotes, C/D small nucleolar ribonucleoproteins (C/D snoRNPs) are essential for methylation and processing of ribosomal RNAs. They consist of a box C/D small nucleolar RNA (C/D snoRNA) associated with four highly conserved nucleolar proteins. Recent data in HeLa cells and yeast have revealed that assembly of these snoRNPs is directed by NUFIP protein and other auxiliary factors. Nevertheless, the precise function and biological importance of NUFIP and the other assembly factors remains unknown. In plants, few studies have focused on RNA methylation and snoRNP biogenesis. Here, we identify and characterise the AtNUFIP gene that directs assembly of C/D snoRNP. To elucidate the function of AtNUFIP in planta, we characterized atnufip mutants. These mutants are viable but have severe developmental phenotypes. Northern blot analysis of snoRNA accumulation in atnufip mutants revealed a specific degradation of C/D snoRNAs and this situation is correlated with a reduction in rRNA methylation. Remarkably, the impact of AtNUFIP depends on the structure of snoRNA genes: it is essential for the accumulation of those C/D snoRNAs encoded by polycistronic genes, but not by monocistronic or tsnoRNA genes. We propose that AtNUFIP controls the kinetics of C/D snoRNP assembly on nascent precursors to overcome snoRNA degradation of aberrant RNPs. Finally, we show that AtNUFIP has broader RNP targets, controlling the accumulation of scaRNAs that direct methylation of spliceosomal snRNA in Cajal bodies.     

Development of a series of gateway binary vectors possessing a tunicamycin resistance gene as a marker for the transformation of Arabidopsis thaliana

We made two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing a UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene driven by the nopaline synthase promoter (Pnos) as a tunicamycin resistance marker for the transformation of Arabidopsis thaliana. The reporters and tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7, and TAP. Selection of transformants was successful on plates containing 0.15 mg/L of tunicamycin. These vectors were compatible with existing pGWB and R4pGWB vectors for kanamycin, hygromycin B, and BASTA? selection, and are useful new tools for making transgenic Arabidopsis.     

Possible evidence of contamination by catechins in deconjugation enzymes from Helix pomatia and Abalone entrails

β-Glucuronidase and sulfatase are the major deconjugating enzymes used in the cleavage of the glucuronate and sulfate moieties, respectively, from certain conjugated food factors including polyphenols. In the present study, we found that compounds having the same molecular weights as catechins were present in Helix pomatia- and/or Abalone entrails-derived β-glucuronidase and sulfatase by liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring methods. On the other hand, the same molecular weights as catechins were undetectable in Escherichia coli-derived β-glucuronidase and Aerobacter aerogenes-derived sulfatase. By high performance liquid chromatography, enzyme-derived catechins were not detected because of approximately 1,000-fold lower sensitivity as compared to LC-MS/MS. These results suggest that the catechins in these enzymes might be attributed to the diets of the organisms as the enzyme sources.