Recovery from diabetes in neonatal mice after a low-dose streptozotocin treatment

Administration of streptozotocin (STZ) induces destruction of β-cells and is widely used as an experimental animal model of type I diabetes. In neonatal rat, after low-doses of STZ-mediated destruction of β-cells, β-cells regeneration occurs and reversal of hyperglycemia was observed. However, in neonatal mice, β-cell regeneration seems to occur much slowly compared to that observed in the rat. Here, we described the time dependent quantitative changes in β-cell mass during a spontaneous slow recovery of diabetes induced in a low-dose STZ mice model. We then investigated the underlying mechanisms and analyzed the cell source for the recovery of β-cells. We showed here that postnatal day 7 (P7) female mice treated with 50 mg/kg STZ underwent the destruction of a large proportion of β-cells and developed hyperglycemia. The blood glucose increased gradually and reached a peak level at 500 mg/dl on day 35–50. This was followed by a spontaneous regeneration of β-cells. A reversal of non-fasting blood glucose to the control value was observed within 150 days. However, the mice still showed impaired glucose tolerance on day 150 and day 220, although a significant improvement was observed on day 150. Quantification of the β-cell mass revealed that the β-cell mass increased significantly between day 100 and day 150. On day 150 and day 220, the β-cell mass was approximately 23% and 48.5% of the control, respectively. Of the insulin-positive cells, 10% turned out to be PCNA-positive proliferating cells. Our results demonstrated that, β-cell duplication is one of the cell sources for β-cell regeneration.     

Synchronous culture of Magnetospirillum sp. AMB-1 by repeated cold treatment

Abstract We established a synchronous culture of Magnetospirillum sp. AMB-1 by repeated cold treatment at 5 °C. This is the first reported synchronous culture of a magnetic bacterium. Cold treatment did not affect magnetic particle synthesis or cell morphology. Iron uptake was observed both before and during cell division. The amount of iron uptake was almost equivalent to that of magnetite formation. The proportion of magnetosensitive cells did not change during cell division.     

Fate maps of ventral and dorsal pancreatic progenitor cells in early somite stage mouse embryos

The origins of liver progenitor cells have been extensively studied, but evidence on the origin of pancreatic precursor cells is currently limited. Pancreatic and duodenal homeobox gene 1 (Pdx1) is one of the earliest known markers for the pancreas. A transgenic mouse line expressing green fluorescent protein (GFP) under the control of the Pdx1 promoter showed that Pdx1/GFP expression was first observed in the mid-region of the anterior intestinal portal (AIP) lip at embryonic day (E) 8.5 at the 5-6 somite stage (ss). The liver progenitors were confirmed to originate from separate domains at the lateral endoderm and the inner part of the medial AIP as previously reported (Tremblay and Zaret, 2005), which turned out to lie caudally to the Pdx1/GFP-expressing domain. To confirm if the early Pdx1/GFP-positive cells give rise to the pancreatic bud, we labeled the cells on the lip of the AIP using the carbocyanine dye CM-DiI and traced their fates in 1-4 ss, 5-6 ss and 7-9 ss E8.5 embryos using an ex utero whole embryo culture method. At 1 ss, the ventral pancreas progenitors were observed in the lateral endoderm, not yet being segregated from the liver or gut progenitors. Cells that contributed solely to the ventral pancreas first appeared at the AIP lip from 5 ss. At 5-6 ss, cells from the medial of the AIP lip contributed to the ventral pancreas. The pancreas fate region become narrower as development progresses. At 7-9 ss, the cells contributing to the ventral pancreas resided in a narrow region of the AIP lip. From 5 ss, the right flanking region contributes to the posterior gut, and the left flanking region contributes to the anterior gut. Dorsal pancreatic progenitors originate from the dorsal endoderm at the 3-6 somite level at 7-9 ss, though they have not yet diverged from the dorsal gut progenitors at this stage.     

Identification of a PDZ protein, PIST, as a binding partner for Rho effector Rhotekin: biochemical and cell-biological characterization of Rhotekin-PIST interaction

Among various effector proteins for the small GTPase Rho, the function(s) of Rhotekin is (are) almost unknown. We have identified PIST [PDZ (PSD-95, Discs-large and ZO-1) domain protein interacting specifically with TC10 (a Rho-family small GTPase)] as a binding partner for Rhotekin, using yeast two-hybrid screening. Rhotekin was found to associate with PIST in vitro and in both polarized and non-polarized MDCK (Madin-Darby canine kidney) cells. The C-terminal SPV (Ser-Pro-Val) motif of Rhotekin exhibited binding to the PDZ domain of PIST. The binding was markedly inhibited by an activated version of Rho and partially by that of Rac or Cdc42 in COS7 cells. In contrast, TC10 had no effects on the binding. Immunofluorescence analyses revealed the co-localization of PIST and Rhotekin at the Golgi apparatus in non-polarized fibroblast-like MDCK cells and AJs (adherens junctions) in the fully polarized cells. PIST and Rhotekin are recruited from the cytosol to AJs as the cell becomes polarized. Expression of constitutively active Rho or prevention of Rhotekin-PIST interaction induced diffuse cytoplasmic distribution of Rhotekin in polarized MDCK cells. These results suggest that there is (1) Rho-dependent regulation of Rhotekin-PIST interaction, (2) involvement of PIST in the recruitment of Rhotekin to AJs and (3) a possible role(s) for these two proteins in cell-polarity development and/or maintenance.     

The plasma membrane shuttling of CAPRI is related to regulation of mast cell activation

The Ca(2+)-promoted Ras inactivator (CAPRI), a Ras GTPase-activating protein, is involved in the inactivation of mitogen-activated protein kinase pathway. However, a precise role of CAPRI in immune responses is still unknown. Here we showed that overexpression of CAPRI suppresses antigen-induced degranulation and cytokine production in mast cells (RBL cells). Antigen elicited the translocation of CAPRI to the plasma membrane from the cytoplasm, which was concomitant with the increase in the intracellular Ca(2+) concentration. The nuclear import of extracellular signal-regulated kinase 2 (ERK2) occurred after the re-localization of CAPRI to the cytoplasm in the mast cells, suggesting that the early phase of ERK2 activation is eliminated. A mutant of GAP-related domain, CAPRI(R472S), showed a feeble translocation to the plasma membrane but did not affect the degranulation, ERK2 activation, and cytokine production. The results suggested that the translocation of CAPRI to the plasma membranes regulates crucially cellular responses in mast cells.     

Identification and characterisation of a leucine aminopeptidase from the hard tick Haemaphysalis longicornis

Aminopeptidases responsible for blood digestion have yet to be identified in haematophagous ticks. We report here the cloning and molecular characterisation of a cDNA encoding leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from the hard tick Haemaphysalis longicornis (HlLAP). Endogenous HlLAP was detected in the soluble fraction of adult tick extracts by immunoblotting. Immunohistochemical studies demonstrated that endogenous HlLAP expression mainly took place in the cytosol of midgut epithelial cells. Furthermore, expression of HlLAP was induced by a blood-feeding process. A functional recombinant HlLAP expressed in Escherichia coli efficiently hydrolyses synthetic substrates for aminopeptidase, a leucyl (with the Km value 0.19 +/- 0.011 mM and Vmax value 157.2 +/- 3.17 nmol/min/mgprotein) and a methionyl substrate (with the Km value 0.12+/-0.0052 mM and Vmax value 171.9 +/- 2.31 nmol/min/mgprotein). Enzyme activity was found to be optimum at pH 8 and 35 degrees C. The recombinant HlLAP enzyme activity was strongly dependent on metal divalent cations, Mn2+, and was inhibited by bestatin. These results indicate that HlLAP play an important role for host's blood digestion process.     

Role of the carboxy-terminal region of the outer membrane protein AatA in the export of dispersin from enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen in both developing and industrialized countries. AatA, an outer-membrane protein that is a homolog of E. coli TolC, facilitates the export of the dispersin protein Aap across the outer membrane in EAEC. To identify which amino acids are important for this export activity, site-directed mutagenesis of the carboxy terminus was performed. An insertional mutant of aatA was complemented with each of several deletion mutants, and was examined for Aap secretion. The results showed that three nonpolar amino acids at positions 381-383 (Phe-Leu-Leu) were required for the activity, and these residues were located at the base of carboxy-terminal elongation in the equatorial domain of AatA.