Control of body size by SMA-5, a homolog of MAP kinase BMK1/ERK5, in C. elegans

We have analyzed the sma-5(n678) mutant in C. elegans to elucidate mechanisms controlling body size. The sma-5 mutant is very small, grows slowly and its intestinal granules look abnormal. We found a 15 kb deletion in the mutant that includes a 226 bp deletion of the 3' end of the W06B3.2-coding sequence. Based on this result, rescue experiments, RNAi experiments and a newly isolated deletion mutant of W06B3.2, we conclude that W06B3.2 is the sma-5 gene. The sma-5 mutant has much smaller intestine, body wall muscles and hypodermis than those of the wild type. However, the number of intestinal cells or body wall muscle cells is not changed, indicating that the sma-5 mutant has much smaller cells. In relation to the smaller cell size, the amount of total protein is drastically decreased; however, the DNA content of the intestinal nuclei is unchanged in the sma-5 mutant. The sma-5 gene is expressed in intestine, excretory cell and hypodermis, and encodes homologs of a mammalian MAP kinase BMK1/ERK5/MAPK7, which was reported to control cell cycle and cell proliferation. Expression of the sma-5 gene in hypodermis is important for body size control, and it can function both organ-autonomously and non-autonomously. We propose that the sma-5 gene functions in a MAP kinase pathway to regulate body size mainly through control of cell growth.     

Beyond vasodilation: the antioxidant effect of adrenomedullin in Dahl salt-sensitive rat aorta

We have investigated the antioxidant effect of adrenomedullin (AM) on endothelial function in the Dahl salt-sensitive (DS) rat hypertension model. Dahl salt-resistant (DR) and DS rats were fed an 8% NaCl diet. In addition, the DS rats were subcutaneously infused with either saline or recombinant human AM for 4 weeks. Although systolic blood pressures measured weekly in AM- and saline-infused rats did not significantly differ, aortic O2*- levels were significantly (P<0.01) higher in the latter. Likewise, both endothelial nitric oxide synthase (eNOS) mRNA and protein were significantly higher in saline-infused DS rats. Infusion of AM reduced both O2*- and eNOS expression to levels comparable to those seen in DR rats. AM infusion also upregulated the gene expression of guanosine-5'-triphosphate cyclohydrolase I and downregulated the expression of p22(phox), suggesting that AM increased the NOS coupling and bioavailability of NO. AM possesses significant antioxidant properties that improve endothelial function.     

Transgenic rats overexpressing the human MrgX3 gene show cataracts and an abnormal skin phenotype

The human MrgX3 gene, belonging to the mrgs/SNSRs (mas related genes/sensory neuron specific receptors) family, was overexpressed in transgenic rats using the actin promoter. Two animal lines showed cataracts with liquification/degeneration and swelling of the lens fiber cells. The transient epidermal desquamation was observed in line with higher gene expression. Histopathology of the transgenic rats showed acanthosis and focal parakeratosis. In the epidermis, there was an increase in cellular keratin 14, keratin 10, and loricrin, as well as PGP 9.5 in innervating nerve fibers. These phenotypes accompanied an increase in the number of proliferating cells. These results suggest that overexpression of the human MrgX3 gene causes a disturbance of the normal cell-differentiation process.     

Wound healing ability of Xenopus laevis embryos. II. Morphological analysis of wound marginal epidermis

We previously showed that bisectional wounds made in Xenopus laevis embryos at the primary eye vesicle stage were rapidly closed. In this study, microscopic analyses, including scanning electron microscopy, on the morphology of the epidermis were conducted during wound closure in the half embryos. Bright fluorescence of Texas red-phalloidin showing actin filaments started to be visualized at the cut edge 10 min after wounding. It increased with time, forming a distinguished, though discontinuous, bundle along the wound margin. The wound closure was completely inhibited by 20 microm cytochalasin B, and almost completely by 50 mm 2,3-butanedione 2-monoxime, an inhibitor to myosin ATPase activity. Scanning electron microscopy revealed that the outer epidermal cells became extensively elongated in the radial direction, and the contour of the closing wound edge did not become smoother but remained ragged. Thus, a representative embryonic type of wound closure may be driven in Xenopus embryos by a complex mechanism, involving not only the actin 'purse-string' but also an inward movement of individual cells. Anyhow, the wound closure is a movement of the epidermal sheet maintaining cell-cell contact, and not involving locomotion of single cells separated from the wound edge.     

Inactivation of tensin3 in mice results in growth retardation and postnatal lethality

Tensin family is a group of focal adhesion proteins that interact with integrins, actin, and phosphotyrosine-containing proteins. To explore the in vivo functions of a new member of the family, tensin3, we have generated mutant mice with a disrupted tensin3 gene. Inactivation of tensin3 resulted in growth retardation and postnatal lethality in one third of the homozygous mutants. Histological analysis of those mutants showed incomplete development of the small intestine, lung, and bone. Villus formation in the small intestine was affected and cells migrated slower in the runt mutants. Their lungs also displayed enlarged air space suggesting defects in alveogenesis. In addition, the resting zone was thicker and fewer proliferating cells were present in the growth plates of tensin3(-/-) tibiae. These observations indicate that tensin3 is essential for normal development and functions of the small intestine, lung, and bone. These phenotypes of the runt tensin3(-/-) mice are similar to some clinical features of Silver-Russell syndrome (SRS) which is a genetically inherited defect. About 10% of SRS cases have been linked to abnormality in chromosome 7p11.2-13, where human tensin3 gene is located, suggesting a potential link between tensin3 and SRS.     

Green fluorescent protein labeling of primordial germ cells using a nontransgenic method and its application for germ cell transplantation in salmonidae

Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.     

Neorickettsia risticii is vertically transmitted in the trematode Acanthatrium oregonense and horizontally transmitted to bats

Potomac horse fever is known to be transmitted through the ingestion of caddisflies parasitized with Neorickettsia (formerly Ehrlichia) risticii-infected metacercaria. However, the species of trematode involved and how N. risticii is maintained in nature are unknown. In this study, gravid trematodes were recovered from the intestines of 12 out of 15 Eptesicus fuscus big brown bats and eight out of nine Myotis lucifugus little brown bats from various sites in Pennsylvania, USA. Trematode specimens isolated from six E. fuscus bats contained N. risticii DNA. The trematode was identified as Acanthatrium oregonense. N. risticii was detected within individual trematode eggs by polymerase chain reaction as well as by immunofluorescence labelling with an anti-N. risticii antibody, indicating that N. risticii is vertically transmitted (from adult to egg) in A. oregonense. Furthermore, N. risticii DNA was detected in the blood, liver or spleen of 23 out of 53 E. fuscus and M. lucifugus bats, suggesting that N. risticii can also be transmitted horizontally from trematode to bat. These results indicate that A. oregonense is a natural reservoir and probably a vector of N. risticii.     

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.     

Electrochemical evaluation of cellular physiological status under stress in Escherichia coli with the rpoS-lacZ reporter gene

We developed an electrochemical detection method for evaluating cellular physiological status based on the stringent response as a means to monitor cell viability. A reporter plasmid was constructed by inserting the beta-galactosidase gene (lacZ) under the control of the rpoS promoter, and then used to transform E. coli cells. Electrochemical responses from the products catalyzed by beta-galactosidase expressed by these E. coli cells were detected using the chronoamperometric technique in a nondestructive manner. Comparisons of response currents between the relA-positive strain and relA-negative strain revealed that increases in these currents were caused by the stringent response due to the stressful alcoholic environment, and thus as a model of stressful cultivating conditions. The current was proportional to the beta-galactosidase activity assayed by a conventional method that required the destruction of cells. The cellular physiological status, which depends on the stringent response as a viability marker, therefore, could then be evaluated online with a current using the rpoS-lacZ reporter gene in the relA-positive strain without pretreatment.     

Application of ultra-high magnetic field for saccharide molecules: 1H NMR spectra of 6-O-alpha-D-glucopyranosyl-cyclomaltoheptaose and -cyclomaltohexaose

1H NMR spectra of G1-alpha-CD and G1-beta-CD were recorded using a spectrometer equipped with a 21.6 T magnet. An ultra-high magnetic field was effective for detecting 1H NMR signals with a small difference in chemical shifts. Introducing a glucosyl group onto CDs as a branch caused deformation of equilibrated 1H signals of cyclodextrin. Particularly, 1H signals in branched glucose were shifted greatly.