Hirokazu Komatsu

Quantum dot FRET biosensors that respond to pH, to proteolytic or nucleolytic cleavage, to DNA synthesis, or to a multiplexing combination

Fluorescent acceptors have been immobilized on nanoparticulate quantum dots (QDs), which serve in turn as their FRET donors. The broad excitation and narrow emission bands of QDs mark them as having excellent potential as donors for FRET and, in principle, differently colored QDs could be excited simultaneously. The present work describes the preparation and operation of FRET-based QD bioprobes individually able to detect the actions of protease, deoxyribonuclease, DNA polymerase, or changes in pH. In addition, two such QD-mounted biosensors were excited at a single wavelength, and shown to operate simultaneously and independently of each other in the same sample solution, allowing multiplex detection of the action of a protease, trypsin, in the presence of deoxyribonuclease.

Newly developed Mg2+-selective fluorescent probe enables visualization of Mg2+ dynamics in mitochondria.

Mg2+ plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg2+ regulation and the Mg2+ concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg2+ in mitochondria in intact cells. Here, we have developed a novel Mg2+–selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg2+ concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg2+, KMG-104, enabled us to compare the dynamics of Mg2+ in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)–induced Mg2+ mobilization from mitochondria to the cytosol was visualized. Although a FCCP–induced decrease in the Mg2+ concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg2+ and Parkinsons disease was analyzed in a cellular model of Parkinsons disease by using the 1-methyl-4-phenylpyridinium ion (MPP+). A gradual decrease in the Mg2+ concentration in mitochondria was observed in response to MPP+ in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg2+ dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)).

Muscarinic acetylcholine receptors in rat gastric mucosa

SummaryThe muscarinic cholinergic innervation of the rat gastric mucosa was investigated by localizing the muscarinic receptors using a tritiated muscarinic antagonist, pirenzepine. Radioautography was performed by freeze drying stomach tissue, which was then embedded in Epon and wet sectioned with ethylene glycol, and dry mounting on emulsion film by the wire-loop method to prevent loss of the labelled substance during fixation and the radioautographic procedure. Light and electron microscopy showed that the specific pirenzepine-binding sites were localized predominantly on parietal cells, chief cells and perivascular plexuses. Analysis of the grain distribution on parietal cells revealed that the silver grains corresponding to the pirenzepine-binding sites were mainly on the basolateral plasma membrane. On the other hand, the surface mucous or mucous neck cells had few pirenzepine-binding sites.

SPR sensor signal amplification based on dye-doped polymer particles

Abstract A novel amplification method was prepared based on dye-doped polymer particles for SPR signals originating from antigen–antibody (BSA–anti-BSA) interactions. Coloring the particles enhances the change of SPR signals based on the change of the imaginary part of the refractive index. The signal amplification effect in the reflectance mode was 31-fold stronger compared to the effect obtained with white latex particles and the combined amplification effect due to the presence of polymer particles and the colorant was over 100-fold compared to non-amplified SPR signals originating from BSA–anti-BSA interactions. The amplification method based on the dye-doped particles is widely applicable for the analysis of antibody–antigen interactions and DNA interactions at low concentrations.

Autoradiographic demonstration of gastrin-releasing peptide-binding sites in the rat gastric mucosa

The location of [125I]iodotyrosyl gastrin-releasing peptide-binding sites in the rat fundic mucosa was studied. Peptide specificity was demonstrated by competitive binding studies using the addition of a large amount of cold gastrin-releasing peptide or substance P. Autoradiography of the stomach tissue was carried out by freeze-drying, embedding in Epon, wet-sectioning with ethylene glycol, and dry-mounting the emulsion film by the wire-loop method to prevent loss of the labeled substance. Specific binding sites of gastrin-releasing peptide were found on D cells, surface mucus cells, and parietal cells, whereas few binding sites were seen on the chief or mucus neck cells.

Detection of ethanol in alcoholic beverages or vapor phase using fluorescent molecules embedded in a nanofibrous polymer.

An alcohol sensor was developed using the solid-state fluorescence emission of terphenyl-ol (TPhOH) derivatives. Admixtures of TPhOH and sodium carbonate exhibited bright sky-blue fluorescence in the solid state upon addition of small quantities of ethanol. A series of terphenol derivatives was synthesized, and the effects of solvent polarities and the structures of these π-conjugated systems on their fluorescence were systematically investigated by using fluorescence spectroscopy. In particular, π-extended TPhOHs and TPhOHs containing electron-withdrawing groups exhibited significant solvatochromism, and fluorescence colors varied from blue to red. Detection of ethanol contents in alcohol beverages (detection limit ∼ 5 v/v %) was demonstrated using different TPhOHs revealing the effect of molecular structure on sensing properties. Ethanol contents in alcoholic beverages could be estimated from the intensity of the fluorescence elicited from the TPhOHs. Moreover, when terphenol and Na2CO3 were combined with a water-absorbent polymer, ethanol could be detected at lower concentrations. Detection of ethanol vapor (8 v/v % in air) was also accomplished using a nanofibrous polymer scaffold as the immobilized sensing film.

Autoradiographic demonstration of gastrin binding sites in rat gastric mucosa.

The location of 125I-iodotyrosyl gastrin I binding sites in rat gastric mucosa was studied. Peptide specificity was demonstrated by competitive binding studies through the addition of a large dose of cold human gastrin I or cholecystokinin-octapeptide. Autoradiography of the stomach tissue was carried out by freeze-drying, embedding in Epon, wet-sectioning with ethylene glycol, and dry-mounting the emulsion film by means of the wire-loop method to prevent loss of the labeled substance. Specific binding sites for gastrin were found on parietal and chief cells, whereas few binding sites were seen on the surface mucous or mucous neck cells. Binding sites on the parietal cells were dispersed in the cytoplasm, while those on the chief cells were found near the basal plasma membrane.

Involvement of autonomic nervous system in gastric mucosal defense mechanism.

This article describes the histochemical, immunohisto-chemical, radioautographic, and ultrastructural localizations of aminergic and peptidergic nerves, neurotransmitter receptors, and their binding sites in the stomach wall. Cholinergic and vasoactive intestinal polypeptide (VIP)-ergic nerve fibers are distributed along the gastric microvasculature, within the myenteric and submucosal plexuses, and in the muscularis mucosae and circular muscle layer. In the mucosa, both nerve fibers evenly extend along the capillaries in association with the epithelial cells up to the mucosal surface. In particular, cholinergic nerves are proved to doubly innervate the mucosal capillaries and nonmuscular venules as well as the parietal cells. Adrenergic and neuropeptide Y (NPY)-containing nerves are distributed primarily along the arterioles of the gastric micro vasculature, within the myenteric plexuses, and in the circular muscle layer. These nerve fibers extend up to the basal portion of the mucosa in close association with small arterioles, capillaries, and epithelial cells. Some of the adrenergic nerve axons are coexistent with the cholinergic nerve axons within the Schwann cell. Histamine H1 receptors are widely located on the walls of arterioles, capillaries and venules, while H2 receptors are evident not only on the parietal cells but also on the walls of the collecting venules and surrounding capillaries in the mucosa. Dopamine D1 receptors are predominantly located on the smooth muscle cells of the arterioles near the muscularis mucosae, while D2 receptors are present on the walls of postcapillary venules and collecting venules. Functional coordination of both intramural peptidergic nerves as intrinsic origin and aminergic nerves as extrinsic origin is considered to be essential for maintaining the gastric mucosal defense mechanism against a variety of aggressive factors.

Wound healing of acetic acid-induced gastric ulcer in rats and the effects of cimetidine and calcitonin, with special reference to prolylhydroxylase and collagenase enzyme activity

The healing of acetic acid-induced gastric ulcer in rats and the effects of cimetidine and calcitonin were investigated with reference to the enzyme activity of both prolylhydroxylase and collagenase as related to histological findings. The rats were observed by endoscopy on the 3rd day after the subserosal injection of acetic acid; rats with ulcers were divided into three groups: non-treated, and cimetidine- and calcitonin-treated. The latter two groups were treated for 7 days. Prolylhydroxylase activity in active ulcers in the non-treated group was slightly higher on the 3rd day and significantly higher on the 10th day than the activity in control rats that had received subserosal injections of physiological saline solution on the respective days. In non-treated rats, the healed ulcer on the 10th day showed lower prolylhydroxylase activity than that in the active ulcer on the same day. Cimetidine did not affect prolylhydroxylase activity, but, with calcitonin, there was higher prolylhydroxylase activity in the healed than in the active ulcer, although the difference was not significant. Interstitial collagenase showed the highest activity on the 3rd day and decreased on the 10th day in non-treated rats. Collagenase activity was higher in the cimetidine-treated group, than that in the non-treated group, and numerous peroxidase-positive granulocytes were seen in the mucosa and submucosa. Calcitonin did not affect collagenase activity. The participation of both enzymes is indispensable in the healing process and the effects of anti-ulcer agents on these enzymes must be considered.

Determination of blood potassium using a fouling-resistant PVDF–HFP-based optode

Monitoring potassium levels in blood is a significant aspect of clinical analysis. For this reason, polymeric bulk optodes have received much attention for their use in portable and easy-to-use analysis systems in situ determination without additional calibration. However, blood contamination on the detection area of the sensor can hinder accurate sensing and also increases risk of infection from the wounds. In this paper, we report a system for determination of potassium in blood which has the additional advantage of being blood-fouling resistant. We have replaced the generally used poly(vinyl chloride) (PVC) with hydrophobic fluorinated poly(vinylidene fluoride–hexafluoropropylene) (PVDF–HFP) for preparation of a polymeric bulk optode. Sensing ability in the visual range of the polymeric bulk optode was retained despite the variation of the polymer matrix. These polymeric bulk optodes are suitable for potassium determination in blood with the PVDF–HFP-based optode possessing better blood antifouling properties than the PVC-based optode. The blood monitoring system described here represents the basis for functionalization of the optode toward safe and easily implementation in blood and in situ sensing applications.