Adsorption of microorganisms onto an artificial siderophore-modified Au substrate

The hydroxamate-type artificial siderophore, tris[2-{3-(N-acetyl-N-hydroxamino)propylamido}propyl]aminomethane (TAPPA) and its Fe(III) complex, Fe(III)-TAPPA were prepared and characterized by several spectroscopic methods. Fe(III)-TAPPA exhibits biological activity for the hydroxamate-type siderophore auxotrophic microorganism, Microbacterium flavescens, suggesting that Fe(III)-TAPPA can permeate the cell membrane of the microorganism. The adsorption of the Fe(III)-siderophore complex onto a deposited Au substrate was achieved by a stepwise self-assembling method. The modification of Fe(III)-TAPPA on the surface was confirmed from the cyclic voltammogram of the resultant Au electrode, Fe(III)-TAPPA/Au. The adsorption experiments of M. flavescens with Fe(III)-TAPPA/Au were monitored by optical, scanning electron, and atomic force microscopy and quartz crystal microbalance (QCM) measurements. These results clearly indicate that Fe(III)-TAPPA/Au can immobilize M. flavescens. This adsorption characteristic is due to the interaction between Fe(III)-TAPPA on an Au electrode and a receptor/binding protein within the cell membrane.     

SNAP23 deficiency causes severe brain dysplasia through the loss of radial glial cell polarity

In the developing brain, the polarity of neural progenitor cells, termed radial glial cells (RGCs), is important for neurogenesis. Intercellular adhesions, termed apical junctional complexes (AJCs), at the apical surface between RGCs are necessary for cell polarization. However, the mechanism by which AJCs are established remains unclear. Here, we show that a SNARE complex composed of SNAP23, VAMP8, and Syntaxin1B has crucial roles in AJC formation and RGC polarization. Central nervous system (CNS)–specific ablation of SNAP23 (NcKO) results in mice with severe hypoplasia of the neocortex and no hippocampus or cerebellum. In the developing NcKO brain, RGCs lose their polarity following the disruption of AJCs and exhibit reduced proliferation, increased differentiation, and increased apoptosis. SNAP23 and its partner SNAREs, VAMP8 and Syntaxin1B, are important for the localization of an AJC protein, N-cadherin, to the apical plasma membrane of RGCs. Altogether, SNARE-mediated localization of N-cadherin is essential for AJC formation and RGC polarization during brain development.     

Binding of an RNA aptamer and a partial peptide of a prion protein: crucial importance of water entropy in molecular recognition

It is a central issue to elucidate the new type of molecular recognition accompanied by a global structural change of a molecule upon binding to its targets. Here we investigate the driving force for the binding of R12 (a ribonucleic acid aptamer) and P16 (a partial peptide of a prion protein) during which P16 exhibits the global structural change. We calculate changes in thermodynamic quantities upon the R12–P16 binding using a statistical-mechanical approach combined with molecular models for water which is currently best suited to studies on hydration of biomolecules. The binding is driven by a water-entropy gain originating primarily from an increase in the total volume available to the translational displacement of water molecules in the system. The energy decrease due to the gain of R12–P16 attractive (van der Waals and electrostatic) interactions is almost canceled out by the energy increase related to the loss of R12–water and P16–water attractive interactions. We can explain the general experimental result that stacking of flat moieties, hydrogen bonding and molecular-shape and electrostatic complementarities are frequently observed in the complexes. It is argued that the water-entropy gain is largely influenced by the geometric characteristics (overall shapes, sizes and detailed polyatomic structures) of the biomolecules.     

Low-Intensity Pulsed Ultrasound Induces Angiogenesis and Ameliorates Left Ventricular Dysfunction in a Porcine Model of Chronic Myocardial Ischemia

Background

Although a significant progress has been made in the management of ischemic heart disease (IHD), the number of severe IHD patients is increasing. Thus, it is crucial to develop new, non-invasive therapeutic strategies. In the present study, we aimed to develop low-intensity pulsed ultrasound (LIPUS) therapy for the treatment of IHD.

Methods and Results

We first confirmed that in cultured human endothelial cells, LIPUS significantly up-regulated mRNA expression of vascular endothelial growth factor (VEGF) with a peak at 32-cycle (P<0.05). Then, we examined the in vivo effects of LIPUS in a porcine model of chronic myocardial ischemia with reduced left ventricular ejection fraction (LVEF) (n = 28). The heart was treated with either sham (n = 14) or LIPUS (32-cycle with 193 mW/cm² for 20 min, n = 14) at 3 different short axis levels. Four weeks after the treatment, LVEF was significantly improved in the LIPUS group (46±4 to 57±5%, P<0.05) without any adverse effects, whereas it remained unchanged in the sham group (46±5 to 47±6%, P = 0.33). Capillary density in the ischemic region was significantly increased in the LIPUS group compared with the control group (1084±175 vs. 858±151/mm², P<0.05). Regional myocardial blood flow was also significantly improved in the LIPUS group (0.78±0.2 to 1.39±0.4 ml/min/g, P<0.05), but not in the control group (0.84±0.3 to 0.97±0.4 ml/min/g). Western blot analysis showed that VEGF, eNOS and bFGF were all significantly up-regulated only in the LIPUS group.

Conclusions

These results suggest that the LIPUS therapy is promising as a new, non-invasive therapy for IHD.     

An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation

Recently, the Golgi phosphoprotein 3 (GOLPH3) and its yeast homolog Vps74p have been characterized as essential for the Golgi localization of glycosyltransferase in yeast. GOLPH3 has been identified as a new oncogene that is commonly amplified in human cancers to modulate mammalian target of rapamycin signaling. However, the molecular mechanisms of the carcinogenic signaling pathway remain largely unclear. To investigate whether the expression of GOLPH3 was involved in the glycosylation processes in mammalian cells, and whether it affected cell behavior, we performed a loss-of-function study. Cell migration was suppressed in GOLPH3 knockdown (KD) cells, and the suppression was restored by a re-introduction of the GOLPH3 gene. HPLC and LC/MS analysis showed that the sialylation of N-glycans was specifically decreased in KD cells. The specific interaction between sialyltransferases and GOLPH3 was important for the sialylation. Furthermore, overexpression of α2,6-sialyltransferase-I rescued cell migration and cellular signaling, both of which were blocked in GOLPH3 knockdown cells. These results are the first direct demonstration of the role of GOLPH3 in N-glycosylation to regulate cell biological functions.     

A Novel Function of Thrombin-activatable Fibrinolysis Inhibitor during Rat Liver Regeneration and in Growth-promoted Hepatocytes in Primary Culture

Thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits anti-fibrinolytic activity by removing C-terminal lysine residues from fibrin or plasminogen receptor proteins on the cellular surface, and plays an important role in the regulation of fibrinolysis. In this study, we examined the regulation of TAFI in hepatocytes during liver regeneration, and revealed its pivotal role in hepatocyte proliferation. In rat models, partial hepatectomy or carbon tetrachloride (CCl₄)-induced acute liver injury suppressed the levels of plasma TAFI activity and hepatic TAFI mRNA, whereas this operation markedly increased both the hepatic plasmin activity and the level of proliferating cell nuclear antigen. In primary cultures of rat hepatocytes, the TAFI mRNA level was decreased under growth-promoting culture conditions. Treatment of the hepatocytes with TAFI siRNA increased the amount of plasmin on the hepatocytes and promoted hepatocyte proliferation. We concluded that TAFI regulates plasmin activity through its enzymatic activity whereby it reduces the plasminogen-binding capacity of the hepatocytes. The TAFI gene expression is down-regulated in hepatocyte proliferation for producing a fibrinolytic microenvironment suitable for cell growth. This is the first report on the role of TAFI in the pericellular fibrinolysis necessary for cellular proliferation.Thrombin-activatable fibrinolysis inhibitor (TAFI)³ is a 60-kDa plasma glycoprotein secreted by hepatocytes as an inactive form. Activation of TAFI is known to be mediated by thrombin (1), thrombin-thrombomodulin complex (2), or plasmin (3). Activated TAFI down-regulates fibrinolysis by removing the plasminogen-anchoring structure from fibrin. This fibrin structure contains C-terminal lysine residues, to which plasminogen binds via its lysine-binding site (4). Thus, TAFI is thought to exhibit negative regulatory activity in the binding of plasminogen to fibrin or cell surfaces.It is well known that plasminogen on fibrin or a cell surface exerts its maximum activity there, because this binding is not only a prerequisite for plasminogen activators to convert plasminogen to plasmin efficiently, but also a guarantee for the plasmin to be protected from inactivation by specific inhibitors, such as α2-plasmin inhibitor (5). As such, plasminogen can fulfill its roles for both thrombolysis and pericellular proteolysis, when it is located on the surface (6).It has been reported that activation of plasminogen is observed at the early stage of liver regeneration in rats and that plasmin contributes to the priming of hepatocytes for proliferation through the reorganization of extracellular matrix (ECM) components (7). The impairment of liver regeneration and abnormalities in liver repair have been observed in plasminogen-deficient mice, when they have undergone partial hepatectomy (8, 9) or been treated with CCl₄ (10). We demonstrated that the plasminogen activator/plasmin system acts to enhance the formation of hepatocyte spheroids (11) and to promote the proliferation of hepatocytes in vitro (12). These results strongly suggest that plasmin(ogen) is a positive regulator for liver regeneration.Although the function of TAFI in fibrinolysis has been well investigated (13), its function, including the regulation of TAFI gene expression in pericellular fibrinolytic events, remains to be investigated. In studies using TAFI-deficient mice, it has been found that such mice develop normally, reach adulthood, and are fertile. No gross physical abnormalities are observed up to 24 months of age (14). On the other hand, abnormalities in the cutaneous wound healing with delayed skin closure due to the altered epithelial migration and colonic anastomosis with bleeding complications are observed in these TAFI-deficient mice (15). Regarding TAFI gene expression, there is a report by Boffa et al. (16) showing that the combination of interleukin-1 and interleukin-6, but neither alone, reduces the abundance of TAFI mRNA in cultured human hepatoma (HepG2) cells by decreasing the stability of the mRNA.In the present study, we investigated the roles of TAFI in liver regeneration. The gene expression of TAFI was found to be strictly controlled by factors responsible for hepatocyte growth such as growth factors or physical conditions. In other words, suppression of TAFI action enhanced the growth of hepatocytes by providing higher plasmin activity on their surfaces. We employed the RNA interference technique to clarify the substantial role of TAFI. In TAFI-silenced hepatocytes, plasmin activity on hepatocyte surface increased, and hepatocyte proliferation was enhanced proportionally. Thus we demonstrated for the first time that TAFI plays an important role in the proliferation of hepatocytes in vivo through its regulatory effect on the cell surface-bound plasmin.     

Design, synthesis, and BK channel-opening activity of hexahydrodibenzazepinone derivatives

In order to explore new scaffolds for large-conductance Ca2+ -activated K+ channel (BK channel) openers, we carried out molecular design and synthesis on the basis of the following two concepts: (1) introduction of a heteroatom into the dehydroabietic acid (BK channel opener) skeleton would allow easier introduction of substituents. (2) Because of the fourfold symmetrical structure of BK channels, dimeric compounds in which two pharmacophores are linked through a tether are expected to have a greater binding probability to the channels, resulting in increased channel-opening activity. Herein, we explore the usefulness of the hexahydrodibenzazepinone structure as a new scaffold for BK channel openers. The synthesized monomer compounds of hexahydrodibenzazepinone derivatives, which can be derived from dehydroabietic acid, were subjected to electrophysiological patch-clamp studies, followed by Magnus contraction-relaxation assay using rabbit urinary bladder smooth muscle strips to assess overall activities. Dimeric compounds were designed by linking the monomeric hexahydrodibenzazepinone derivatives through a diacetylenebenzene tether, and their channel-opening activities were evaluated by electrophysiological methods. Finally, we concluded that the critical structure for BK channel-opening activity is the hexahydrodibenzazepinone monomer substituted with a phenyl-bearing alkynyl substituent on the lactam amide.     

Distinct and overlapping roles of two gibberellin 3-oxidases in Arabidopsis development

Gibberellin (GA) 3-oxidase, a class of 2-oxoglutarate-dependent dioxygenases, catalyzes the conversion of precursor GAs to their bioactive forms, thereby playing a direct role in determining the levels of bioactive GAs in plants. Gibberellin 3-oxidase in Arabidopsis is encoded by a multigene family consisting of at least four members, designated AtGA3ox1 to AtGA3ox4. It has yet to be investigated how each AtGA3ox gene contributes to optimizing bioactive GA levels during growth and development. Using quantitative real-time PCR analysis, we have shown that each AtGA3ox gene exhibits a unique organ-specific expression pattern, suggesting distinct developmental roles played by individual AtGA3ox members. To investigate the sites of synthesis of bioactive GA in plants, we generated transgenic Arabidopsis that carried AtGA3ox1-GUS and AtGA3ox2-GUS fusions. Comparisons of the GUS staining patterns of these plants with that of AtCPS-GUS from previous studies revealed the possible physical separation of the early and late stages of the GA pathway in roots. Phenotypic characterization and quantitative analysis of the endogenous GA content of ga3ox1 and ga3ox2 single and ga3ox1/ga3ox2 double mutants revealed distinct as well as overlapping roles of AtGA3ox1 and AtGA3ox2 in Arabidopsis development. Our results show that AtGA3ox1 and AtGA3ox2 are responsible for the synthesis of bioactive GAs during vegetative growth, but that they are dispensable for reproductive development. The stage-specific severe GA-deficient phenotypes of the ga3ox1/ga3ox2 mutant suggest that AtGA3ox3 and AtGA3ox4 are tightly regulated by developmental cues; AtGA3ox3 and AtGA3ox4 are not upregulated to compensate for GA deficiency during vegetative growth of the double mutant.