Natural hybridization of Japanese <Emphasis Type="Italic">Rhododendron</Emphasis> section <Emphasis Type="Italic">Brachycaryx</Emphasis> in Mount Kintoki in eastern Japan and concerns for genetic diversity in restoring their habitat

Native Rhododendrons section Brachycaryx in eastern Japan are decreasing in their natural habitats and the need to restore these habitats is increasing. Conservation of genetic diversity in restoring habitat requires clarification of the balance of interspecies genetic exchange which occurs in their natural habitats. In well-preserved natural habitats of Rhododendron dilatatum, R. kiyosumense, and R. wadanum and their natural hybrids R.×kuratanum and R.×hasegawai we investigated their geographical distribution, frequency, and flowering period. DNA analysis of the internal transcribed spacer (ITS) region was also conducted to confirm the species related to hybridization. Our findings in the field survey were: (1) Hybridizations occur in the overlap zones of related species. (2) R.×hasegawai occurs more frequently than R.×kuratanum, probably because the flowering seasons of R. kiyosumense and R. wadanum overlap longer than those of R. dilatatum and R. kiyosumense. (3) Natural hybrid occurrence is, nevertheless, under 9% of all related Rhododendrons section Brachycalyx. Analysis of the ITS region suggested that the two hybrids are generated from interspecific gene exchange, i.e., (4) R. dilatatum and R. kiyosumense relate to the formation of R.×kuratanum. (5) R.×hasegawai is a hybrid of R. wadanum and some species other than R. wadanum. On the basis of these findings we delineated several guidelines for restoring habitats of Rhododendrons of Section Brachycaryx with concerns for genetic diversity: (1) Before use, identify plant materials by morphological traits to determine whether they are original species or hybrids. (2) Investigate the distribution of remnant Rhododendrons section Brachycaryx before restoration. (3) Combine plant materials of original species in the natural distribution.     

A central kinase domain of type I phosphatidylinositol phosphate kinases is sufficient to prime exocytosis: isoform specificity and its underlying mechanism

Exocytosis, a critical process for neuronal communication and hormonal regulation, involves several distinct steps including MgATP-dependent priming (which involves the synthesis of phosphatidylinositol 4,5-bisphosphate). Type I phosphatidylinositol phosphate kinases (PIPKIs) were purified biochemically as a priming factor. PIPKI consists of three domains: the N-terminal region, the central kinase domain, and the C-terminal region. Three isoforms (alpha, beta, and gamma) of PIPKI have been identified, and each is alternatively spliced at the C-terminal region. In the present study, we conducted a structure/function analysis of PIPKIs in the priming of exocytosis, and we found that recombinant PIPKIalpha and PIPKIgamma had priming activity. However, an unexpected finding of these results was that PIPKIbeta did not prime exocytosis. The N- or C-terminal region of PIPKIalpha and PIPKIgamma was not required for priming, which indicates that the central kinase domain is sufficient for this process. Alternative splicing in each isoform did not affect the isoform specificity in priming. Priming activity by isoforms is strongly correlated with their phosphatidylinositol phosphate kinase activity because PIPKIalpha and PIPKIgamma had higher kinase activity than PIPKIbeta. These results suggest that PIPKIalpha and PIPKIgamma are the critical priming factors for exocytosis; it also suggests that the levels of phosphatidylinositol phosphate kinase activity in producing phosphatidylinositol 4,5-bisphosphate specify the function of PIPKI isoforms in priming.     

Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator

We have identified cDNA encoding a new member of the adrenomedullin (AM) family, AM2, for the first time in mammals (mouse, rat and human). The predicted precursor carried mature AM2 in the C-terminus, which had an intramolecular ring formed by an S-S bond and a possibly amidated C-terminus. Phylogenetic analyses clustered AM2 and AM into two distinct but closely related groups. Similarity of exon-intron structure and synteny of neighboring genes showed that mammalian AM2 is an ortholog of pufferfish AM2 and a paralog of mammalian AM. AM2 mRNA was expressed in submaxillary gland, kidney, stomach, ovary, lymphoid tissues and pancreas of mice, but not in adrenal and testis. Intravenous injection of synthetic mature AM2 decreased arterial pressure more potently than AM, and induced antidiuresis and antinatriuresis in mice. These results show that at least two peptides, AM and AM2, comprise an adrenomedullin family in mammals, and that AM2 may play pivotal roles in cardiovascular and body fluid regulation.     

RalA-exocyst interaction mediates GTP-dependent exocytosis

Many secretory cells utilize a GTP-dependent pathway, in addition to the well characterized Ca2+-dependent pathway, to trigger exocytotic secretion. However, little is currently known about the mechanism by which this may occur. Here we show the key signaling pathway that mediates GTP-dependent exocytosis. Incubation of permeabilized PC12 cells with soluble RalA GTPase, but not RhoA or Rab3A GTPases, strongly inhibited GTP-dependent exocytosis. A Ral-binding fragment from Sec5, a component of the exocyst complex, showed a similar inhibition. Point mutations in both RalA (RalA(E38R)) and the Sec5 (Sec5(T11A)) fragment, which abolish RalA-Sec5 interaction also abolished the inhibition of GTP-dependent exocytosis. Moreover, transfection with wild-type RalA, but not RalA(E38R), enhanced GTP-dependent exocytosis. In contrast the RalA and the Sec5 fragment showed no inhibition of Ca2+-dependent exocytosis, but cleavage of a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein by Botulinum neurotoxin blocked both GTP- and Ca2+-dependent exocytosis. Our results indicate that the interaction between RalA and the exocyst complex (containing Sec5) is essential for GTP-dependent exocytosis. Furthermore, GTP- and Ca2+-dependent exocytosis use different sensors and effectors for triggering exocytosis whereas their final fusion steps are both SNARE-dependent.     

CD1d-independent NKT cells in beta 2-microglobulin-deficient mice have hybrid phenotype and function of NK and T cells

Unlike CD1d-restricted NK1.1(+)TCRalphabeta(+) (NKT) cells, which have been extensively studied, little is known about CD1d-independent NKT cells. To characterize their functions, we analyzed NKT cells in beta(2)-microglobulin (beta(2)m)-deficient B6 mice. They are similar to NK cells and expressed NK cell receptors, including Ly49, CD94/NKG2, NKG2D, and 2B4. NKT cells were found in normal numbers in mice that are deficient in beta(2)m, MHC class II, or both. They were also found in the male HY Ag-specific TCR-transgenic mice independent of positive or negative selection in the thymus. For functional analysis of CD1d-independent NKT cells, we developed a culture system in which CD1d-independent NKT cells, but not NK, T, or most CD1d-restricted NKT cells, grew in the presence of an intermediate dose of IL-2. IL-2-activated CD1d-independent NKT cells were similar to IL-2-activated NK cells and efficiently killed the TAP-mutant murine T lymphoma line RMA-S, but not the parental RMA cells. They also killed beta(2)m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. IL-2-activated NKT cells expressing transgenic TCR specific for the HY peptide presented by D(b) killed RMA-S, but not RMA, cells. They also killed RMA (H-2(b)) cells that were preincubated with the HY peptide. NKT cells from beta(2)m-deficient mice, upon CD3 cross-linking, secreted IFN-gamma and IL-2, but very little IL-4. Thus, CD1d-independent NKT cells are significantly different from CD1d-restricted NKT cells. They have hybrid phenotypes and functions of NK cells and T cells.     

Glycyrrhizin inhibits the manifestations of anti-inflammatory responses that appear in association with systemic inflammatory response syndrome (SIRS)-like reactions

In association with the systemic inflammatory response syndrome (SIRS), anti-inflammatory response syndrome is commonly manifested in patients with trauma, burn injury, and after major surgery. These patients are increasingly susceptible to infection with various pathogens due to the excessive release of anti-inflammatory cytokines from anti-inflammatory effector cells. Recently, CC-chemokine ligand 2 (CCL2) found in the sera of mice with pancreatitis was identified as an active molecule for SIRS-associated anti-inflammatory response manifestation. Also, the inhibitory activity of glycyrrhizin (GL) on CCL2 production was reported. Therefore, the effect of GL on SIRS-associated anti-inflammatory response manifestation was investigated in a murine SIRS model. Without any stimulation, splenic T cells from mice 5 days after SIRS induction produced cytokines associated with anti-inflammatory response manifestation. However, these cytokines were not produced by splenic T cells from SIRS mice previously treated with GL. In dual-chamber transwells, IL-4-producing cells were generated from normal T cells cultured with peripheral blood polymorphonuclear neutrophils (PMN) from SIRS mice. However, IL-4-producing cells were not generated from normal T cells in transwell cultures performed with PMN from GL-treated SIRS mice. CCL2 was produced by PMN from SIRS mice, while this chemokine was not demonstrated in cultures of PMN from SIRS mice treated with GL. These results indicate that GL has the capacity to suppress SIRS-associated anti-inflammatory response manifestation through the inhibition of CCL2 production by PMN.     

Contribution of comparative fish studies to general endocrinology: structure and function of some osmoregulatory hormones

Fish endocrinologists are commonly motivated to pursue their research driven by their own interests in these aquatic animals. However, the data obtained in fish studies not only satisfy their own interests but often contribute more generally to the studies of other vertebrates, including mammals. The life of fishes is characterized by the aquatic habitat, which demands many physiological adjustments distinct from the terrestrial life. Among them, body fluid regulation is of particular importance as the body fluids are exposed to media of varying salinities only across the thin respiratory epithelia of the gills. Endocrine systems play pivotal roles in the homeostatic control of body fluid balance. Judging from the habitat-dependent control mechanisms, some osmoregulatory hormones of fish should have undergone functional and molecular evolution during the ecological transition to the terrestrial life. In fact, water-regulating hormones such as vasopressin are essential for survival on the land, whereas ion-regulating hormones such as natriuretic peptides, guanylins and adrenomedullins are diversified and exhibit more critical functions in aquatic species. In this short review, we introduce some examples illustrating how comparative fish studies contribute to general endocrinology by taking advantage of such differences between fishes and tetrapods. In a functional context, fish studies often afford a deeper understanding of the essential actions of a hormone across vertebrate taxa. Using the natriuretic peptide family as an example, we suggest that more functional studies on fishes will bring similar rewards of understanding. At the molecular level, recent establishment of genome databases in fishes and mammals brings clues to the evolutionary history of hormone molecules via a comparative genomic approach. Because of the functional and molecular diversification of ion-regulating hormones in fishes, this approach sometimes leads to the discovery of new hormones in tetrapods as exemplified by adrenomedullin 2.     

Mouse epidermal keratinocytes in three-dimensional organotypic coculture with dermal fibroblasts form a stratified sheet resembling skin

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal keratinocytes and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified keratinocytes and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the keratinocyte sheet transiently increased and then decreased to two cell layers at 2 weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1alpha and 1beta in the keratinocytes and of keratinocyte growth factor 1 and 2 in the fibroblasts correlated with keratinocyte stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.     

Cell type-specific activation of metabolism reveals that beta-cell secretion suppresses glucagon release from alpha-cells in rat pancreatic islets

Abnormal glucagon secretion is often associated with diabetes mellitus. However, the mechanisms by which nutrients modulate glucagon secretion remain poorly understood. Paracrine modulation by beta- or delta-cells is among the postulated mechanisms. Herein we present further evidence of the paracrine mechanism. First, to activate cellular metabolism and thus hormone secretion in response to specific secretagogues, we engineered insulinoma INS-1E cells using an adenovirus-mediated expression system. Expression of the Na+-dependent dicarboxylate transporter (NaDC)-1 resulted in 2.5- to 4.6-fold (P < 0.01) increases in insulin secretion in response to various tricarboxylic acid cycle intermediates. Similarly, expression of glycerol kinase (GlyK) increased insulin secretion 3.8- or 4.2-fold (P < 0.01) in response to glycerol or dihydroxyacetone, respectively. This cell engineering method was then modified, using the Cre-loxP switching system, to activate beta-cells and non-beta-cells separately in rat islets. NaDC-1 expression only in non-beta-cells, among which alpha-cells are predominant, caused an increase (by 1.8-fold, P < 0.05) in glucagon secretion in response to malate or succinate. However, the increase in glucagon release was prevented when NaDC-1 was expressed in whole islets, i.e., both beta-cells and non-beta-cells. Similarly, an increase in glucagon release with glycerol was observed when GlyK was expressed only in non-beta-cells but not when it was expressed in whole islets. Furthermore, dicarboxylates suppressed basal glucagon secretion by 30% (P < 0.05) when NaDC-1 was expressed only in beta-cells. These data demonstrate that glucagon secretion from rat alpha-cells depends on beta-cell activation and provide insights into the coordinated mechanisms underlying hormone secretion from pancreatic islets.