Identification of a peptide mimic of bioactive gibberellins with affinity to GA 2-oxidase

Previously we reported the first example of peptide mimics of a small hydrophobic molecule, a phytohormone gibberellin. The second peptide mimic of gibberellin has been identified from random peptide libraries by its affinity to a type of catalyzing enzyme of gibberellins, which specifically recognizes bioactive gibberellins. These results suggest that even hydrophobic compounds can be mimicked by peptides.     

Recombination of the GFP gene to the BFP gene using a man-made site-selective DNA cutter

By using the recently developed man-made DNA cutter [a combination of Ce(IV)/EDTA and two DNA additives], green fluorescent protein (GFP) was converted to closely related blue fluorescent protein (BFP). The phosphodiester linkages at T196-A200 in the sense strand of GFP were hydrolyzed by the cutter, and the A1-T196 fragment in the product was selectively connected with the downstream fragment (C197-A720) of BFP by T4 DNA ligase. This recombination changed three codons in the GFP gene (TGC at 196–198, TAT at 199–201, and ACC at 502–504) to TCT, CAT, and ATC in BFP, and accordingly three amino acids in GFP (Cys65, Tyr66, and Thr167) were altered to Ser65, His66, and Ile167. The recombinant gene was successfully expressed in Escherichia coli and emitted blue fluorescence, confirming the absence of undesired side reactions (mutation, deletion, insertion, depurination, etc.) in the DNA manipulation. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

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.     

Characteristics of rat round spermatids differentiated from spermatogonial cells during co-culture with Sertoli cells, assessed by flow cytometry, microinsemination and RT-PCR

The present study was undertaken to investigate whether rat spermatogonial stem cells can differentiate into developmentally competent round spermatids during co-culture with Sertoli cells. Type-A spermatogonia and Sertoli cells were prepared from 7-d-old Wistar-strain male rats, and seeded at 4 x 10(6) cells/ 4 mL/35-mm dish (Day 0). They were co-cultured at 37 degrees C for 3 d and at 34 degrees C for the subsequent 7d in 5% CO(2)/air. Round spermatid-like cells (approximately 15 microm in diameter) were first observed on Day 5. A flow cytometric analysis showed that a single peak of haploid cells was detected in the cell populations harvested on Day 10. The participation of the spermatid-like cells to full-term development was examined by microinjection into activated oocytes. The oviductal transfer of 143 microinseminated oocytes resulted in only 8 implantation sites (6%), but no viable offspring. The expression of the round spermatid-specific marker gene, PRM-2, was confirmed in the Day 10 cell population by RT-PCR; however, no mRNA of two other haploid makers, TP1 or TP2, was detected. These results suggested that rat type-A spermatogonial cells underwent meiosis during the primary co-culture with the Sertoli cells, based on morphology, flow cytometry and PRM-2 expression, but the normality of the spermatid-like cells was not supported by microinsemination and TP1/2 expression.     

Fabrication of endothelialized tube in collagen gel as starting point for self-developing capillary-like network to construct three-dimensional organs in vitro

A possible strategy for creating three-dimensional (3D) tissue-engineered organs in vitro with similar volumes to the primary organs is to develop a capillary network throughout the constructs to provide sufficient oxygenation and nutrition to the cells composing them. Here, we propose a novel approach for the creation of a capillary-like network in vitro, based on the spontaneous tube-forming activity of vascular endothelial cells (ECs) in collagen gel. We fabricated a linear tube of 500 microm in diameter, the inner surface of which was filled with bovine carotid artery vascular endothelial cells (BECs), in type I collagen gel as a starting point for the formation of a capillary-like network. The BECs exposed to a medium containing vascular endothelial growth factor (VEGF) migrated into the ambient gel around the tube. After 2 weeks of VEGF exposure, the distance of the migration into the ambient gel in the radial direction of the tube reached approximately 800 microm. Cross-sections of capillary-like structures composed of the migrating BECs, with a lumen-like interior space, were observed in slices of the gel around the tube stained with hematoxylin-eosin (H&E). These results demonstrate that this approach using a pre-established tube, which is composed of ECs, as a starting point for a self-developing capillary-like network is potentially useful for constructing 3D organs in vitro.     

Anisotropic nucleation growth of actin bundle: a model for determining the well-defined thickness of bundles

Biopolymers such as DNA, F-actins, and microtubules, which are highly charged, rodlike polyelectrolytes, are assembled into architectures with defined morphology and size by electrostatic interaction with multivalent cations (or polycations) in vivo and in vitro. The physical origin to determine their morphology and size is not clearly understood yet. Our results show that the actin bundle formation consists of two stages: the thickness of actin bundles is determined nearly at the initial stage, while the length of actin bundles is determined later on. It is also found that the thickness of actin bundles decreases with the increase of polycation-mediated attraction between F-actins. From these results, we propose the anisotropic nucleation-growth mechanism, in which the thickness of actin bundles is determined by critical nucleus size, whereas the length of actin bundles is determined by the concentration of free actins relative to nucleus concentration. Observing that polycations are concentrated in some sites of actin bundles, which are thought to be nucleation sites to initiate the formation of actin bundles, supports this model. This anisotropic nucleation-growth mechanism of actin bundles can be broadly applied to the self-assembly of rodlike polyelectrolytes.     

FKBP133: a novel mouse FK506-binding protein homolog alters growth cone morphology

FK506-binding proteins are the peptidyl prolyl cis-trans isomerases that are involved in various intracellular events. We characterized a novel mouse FK506-binding protein homolog, FKBP133/KIAA0674, in the developing nervous system. FKBP133 contains a domain similar to Wiskott-Aldrich syndrome protein homology region 1 (WH1) and a domain homologous to FK506-binding protein motif. FKBP133 was predominantly expressed in cerebral cortex, hippocampus, and peripheral ganglia at embryonic day 18.5. FKBP133 protein was distributed in the axonal shafts and was partially co-localized with F-actin in the growth cones of dorsal root ganglion neurons (DRG). The number of filopodia was increased in the DRG neurons overexpressing FKBP133. In contrast, the overexpression of a mutant deleted the WH1 domain reduced the growth cone size and the number of filopodia. Furthermore, the neurons overexpressing FKBP133 became significantly resistant to Semaphorin-3A induced collapse response. These results suggest that FKBP133 modulates growth cone behavior with the WH1 domain.     

Molecular cloning of natriuretic peptide receptor A from bullfrog (Rana catesbeiana) brain and its functional expression

A comparative study of natriuretic peptide receptor (NPR) was performed by cloning the NPR-A receptor subtype from the bullfrog (Rana catesbeiana) brain and analyzing its functional expression. Like other mammalian NPR-A receptors, the bullfrog NPR-A receptor consists of an extracellular ligand binding domain, a hydrophobic transmembrane domain, a kinase-like domain and a guanylate cyclase domain. Sequence comparison among the bullfrog and mammalian receptors revealed a relatively low ( approximately 45%) similarity in the extracellular domain compared to a very high similarity ( approximately 92%) in the cytoplasmic regulatory and catalytic domains. Expression of NPR-A mRNA was detected in various bullfrog tissues including the brain, heart, lung, kidney and liver; highest levels were observed in lung. Functional expression of the receptor in COS-7 cells revealed that frog atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) elicited cyclic guanosine 3'5'-monophosphate production by stimulating the receptor in a dose-dependent manner from 10(-10) M concentrations. Rat ANP was also effective in stimulating the frog receptor whereas rat BNP and porcine BNP were less responsive to the receptor. On the other hand, frog C-type natriuretic peptide (CNP) as well as porcine CNP stimulated the receptor only at high concentrations (10(-7) M). This clearly indicates that the bullfrog receptor is a counterpart of mammalian NPR-A, and is specific for ANP or BNP but not for CNP.