Kentaro Sakai

Pulmonary surfactant is a potential endogenous inhibitor of proteolytic activation of Sendai virus and influenza A virus

The pathogenicities of influenza viruses and paramyxoviruses have been proposed to be primarily determined by a host cell protease(s) that activates viral infectivity by proteolytic cleavage of the envelope glycoproteins. We recently isolated a trypsin‐type endoprotease, named tryptase Clara, from rat bronchial and bronchiolar epithelial Clara cells, which is secreted into the airway lumen and activates Sendai virus and influenza A virus proteolytically. We report here that surfactant in the bronchial fluid inhibited tryptase Clara specifically, having a K i value of 0.13 μM, and inhibited the proteolytic activations by tryptase Clara in vitro and in organ cultures of rat lung. Intranasal infection of rats with Sendai virus was shown to stimulate secretion of tryptase Clara without changing the amount of surfactant in the bronchial lumen, resulting in a preferable condition for proteolytic viral activation and multiplication.

Electron immunohistochemical localization in rat bronchiolar epithelial cells of tryptase Clara, which determines the pneumotropism and pathogenicity of Sendai virus and influenza virus.

The intracellular localization in rat bronchiolar epithelial cells of a novel trypsin-like protease named tryptase Clara, a possible activator of inactive viral fusion glycoprotein of influenza A and wild-type Sendai virus in the respiratory tract, was examined by electron microscopy. In thin sections embedded in LR White, gold particles indicating immunoreactivity of tryptase Clara were detected specifically in secretory granules of Clara cells. No immunoreactivity was detected in bronchiolar ciliated cells, alveolar cells including epithelial Type I and II cells, or alveolar macrophages. Some granules enveloped in a thin membrane and labeled intensely with immunogold particles were seen protruding from peripheral and submembrane regions and a few were observed free in the airway lumen. Scanning electron microscopy also revealed smooth droplets along the main body of Clara cells. These data suggest that tryptase Clara is secreted into the bronchiolar lumen. These findings are the first to show the subcellular localization of tryptase Clara in rat bronchioles and suggest the site of proteolytic activation of the progeny of enveloped pneumotropic viruses, such as Sendai virus and influenza virus.

Determination of Antihypertensive Peptides from an Izumi Shrimp Hydrolysate

The izumi shrimp (Plesionika izumiae Omori, 1971) is an unused resource which can be caught off the southern coast of Tokushima Prefecture. We have previously found that an izumi shrimp hydrolysate significantly inhibited the age-associated spontaneous increase in blood pressure in stroke-prone spontaneously hypertensive rats. In this present study, two angiotensin I-converting enzyme inhibitory peptides were isolated from an izumi shrimp hydrolysate by using high-performance liquid chromatography, and their amino acid sequences were determined to be Val-Trp-Tyr-His-Thr and Val-Trp. A single oral administration of synthetic Val-Trp-Tyr-His-Thr or Val-Trp significantly decreased the blood pressure in stroke-prone spontaneously hypertensive rats. The antigenicity and allergenicity of the izumi shrimp hydrolysate against BALB/c mice were very low. These results demonstrate that the angiotensin I-converting enzyme inhibitory peptides isolated from the izumi shrimp hydrolysate had an anti-hypertensive effect on rats.

Peptic digestibility of raw and heat-coagulated hen's egg white proteins at acidic pH range

Allergenicity in food proteins is generally dependent on their heat stability and resistance to digestive enzymes together with the presence of IgE-recognizing epitopes on the molecules. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, we assessed peptic digestibility of raw and heat-coagulated hens egg white proteins at acidic pH range (1.5–4.0). Ovalbumin in raw egg white was slightly digested by pepsin at pH 1.5 and pH 2.0, and was almost resistant to the enzyme at pH 2.5 and over, which was altered in heat-coagulated egg white at the pH range from 1.5 to 2.5 where the protein was well digestive against the enzyme. Peptic digestibility of ovomucoid in raw egg white was good at the pH range from 1.5 to 2.5, but almost non-existent at pH 3.0 and over where the improvement of the digestibility of the protein was not found even in heat-coagulated egg white. As the stomach in new born infants shows a low amount of secretary pepsin and an out of optimum pH of peptic activity, low digestibility of ovalbumin and ovomucoid in raw and heat-coagulated egg white at over pH 3.0 is supposed to be responsible for their allergenicity and delayed outgrowth from hens egg allergy in patients with delayed maturation of stomach functions.

Sendai virus infection changes the subcellular localization of tryptase Clara in rat bronchiolar epithelial cells

Tryptase Clara activates the infectivity of Sendai and influenza viruses proteolytically. In this study, we investigated changes in the subcellular localization of tryptase Clara in rat bronchioles with progression of Sendai virus infection. Tryptase Clara and Sendai virus F2 antigen were localized by light and electron immunohistochemical studies. In the uninfected rat lung, tryptase Clara was specifically localized in the secretory granules of respiratory bronchiolar epithelial nonciliated cells, but not in bronchiolar ciliated, or alveolar cells. In the initial stage of Sendai virus infection with slight pathological changes, however, anti-tryptase Clara was highly reactive in luminal peripheral membranes of both nonciliated and ciliated epithelial cells of the bronchioles together with some Sendai virus envelope glycoprotein, F2 antigen. In the progressed stage, tryptase Clara was hard to detect, with heavy accumulation of F2 antigen in the epithelial cells. These immunohistochemical results support our previous findings that in the bronchial lavage fluid tryptase Clara is significantly increased both in amount and activity after viral infection. These results suggest that Sendai virus stimulates the secretion of tryptase Clara from nonciliated bronchiolar epithelial cells to the airway lumen. Accumulation of tryptase Clara on the luminal surface of the bronchiolar epithelial cells and/or in the airway lumen may produce favourable conditions for proteolytic viral activation and multiplication.

Inhibition of glucose absorption by phlorizin affects intestinal functions in rats

BACKGROUND To investigate the mechanism of regulation of intestinal disaccharidase activity and glucose absorption, the effect of dietary intake of phlorizin, a potent and specific inhibitor of intestinal glucose transport, on intestinal disaccharidase activity and Na(+)-dependent glucose transporter was examined in rats. METHODS Jejunal disaccharidase activity and the number of Na(+)-dependent glucose transporters were determined in rats maintained on a low-starch diet, a high-starch diet, or low-starch diets containing various amounts of phlorizin (0.1%-0.9% wt/wt). RESULTS Jejunal disaccharidase activity increased in a dose- and time-dependent manner. Stimulation of jejunal disaccharidase activity only occurred when phlorizin was added to starch-containing diets, not when it was added to a carbohydrate-free diet. Addition of the same amount of phloretin and glucose (constituents of phlorizin), to the diet failed to increase disaccharidase activity. The maximum binding of phlorizin to brush border membrane vesicles was increased in the rats fed phlorizin, whereas the dissociation constant remained unchanged, suggesting an increase of glucose transporter expression. CONCLUSIONS Dietary phlorizin increased the jejunal disaccharidase activity and Na(+)-dependent glucose transporter expression. The trigger for these changes may have been due to an increased luminal glucose content.

Molecular Cloning and Allergenicity of Pen j 1, a Major Allergen of Kuruma Prawn, Penaeus japonicus

Tropomyosins have been identified as a common allergen in crustaceans, but their allergenicity is not well understood. In the present study, we isolated an allergen, Pen j 1, a tropomyosin from kuruma prawn Penaeus japonicus, and determined its N-terminal amino acid sequence. The cDNA encoding the allergen was cloned by 5′- and 3′-rapid amplification of cDNA ends (RACE), and was found to code for a protein which consists of 284 amino acid residues. Sequencing analyses indicated for the first time that mature tropomyosin is formed by the elimination of a leader peptide of nine amino acid residues. To elucidate the binding sites of IgE antibodies in the sera of shrimp-sensitive patients, various recombinant peptides were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST), and the examined with regard to reactivity with IgE antibodies. The IgE-binding epitopes were found to locate over the whole sequence of the allergen, and the IgE antibodies in the sera were found to recognize strongly its C-terminal region.

Interleukin-3 Does Not Affect the Differentiation of Mast Cells Derived from Human Bone Marrow Progenitors

Although IL-3 is commonly used for culture of human progenitor-derived mast cells together with Stem cell factor (SCF) and IL-6, the effect of IL-3 on human mast cell differentiation has not been well elucidated. Human bone marrow CD34+ progenitors were cultured for up to 12 weeks in the presence of rhSCF and rhIL-6 either with rhIL-3 (IL-3 (+)) or without rhIL-3 (IL-3 (−)) for the initial 1-week of culture. Total cell number increased at 2 weeks in IL-3 (+), as compared to IL-3 (−), but changes in the appearance of mast cells were delayed. When IL-3 was present for the initial 1-week culture, granules looked more mature with IL-3 than without IL-3. However, tryptase and chymase contents, and surface antigen expression (CD18, CD51, CD54, and CD117) were not altered by IL-3. Surface expression and mRNA level of FcεRIα and histamine release by crosslinking of FcεRIα did not differ from one preparation to the next. GeneChip analysis revealed that no significant differences were observed between IL-3 (+) and IL-3 (−) cells either when inactivated or activated by aggregation of FcεRIα. These findings indicate that initial incubation of human bone marrow CD34+ progenitors with IL-3 does not affect the differentiation of mast cells.

Molecular Basis of Proteolytic Activation of Sendai Virus Infection and the Defensive Compounds for Infection

It has been proposed that the pathogenicity of Sendai virus is primarily determined by a host cellular protease(s) that activates viral infectivity by proteolytic cleavage of envelope fusion glycoproteins. We isolated a trypsin-like serine protease, tryptase Clara, localized in and secreted from Clara cells of the bronchial epithelium of rats. The enzyme specifically cleaved the precursor of fusion glycoprotein F0 of Sendai virus at residue Arg116 in the consensus cleavage motif, Gln(Glu)-X-Arg, resulting in the presentation of the membrane fusion domain in the amino-terminus of the F1 subunit. Administration of an antibody against tryptase Clara in the airway significantly inhibited the activation of progeny virus and multiple cycles of viral replication, thus reducing the mortality rate. These findings indicate that tryptase Clara in the airway is a primary determinant of Sendai virus infection and that proteolytic activation occurs extracellularly. We identified two cellular inhibitory compounds against tryptase Clara in bronchial lavage. One was a mucus protease inhibitor, a major serine protease inhibitor of granulocyte elastase in the lining fluids of the human respiratory tract, and the other was a pulmonary surfactant which may adsorb the enzyme, resulting in its inactivation. These compounds inhibited virus activation by tryptase Clara in vitro and in vivo, but did not themselves affect the hemagglutination and the infectivity of the virus. The functional domain of the mucus protease inhibitor against the enzyme, which is organized in two homologous N- and C-terminal domains, is located in the C-terminal. Administration of these compounds in the airway may be useful for preventing infection with Sendai virus.

Piezoelectric Photothermal Spectra of Co Doped ZnO Semiconductor

Piezoelectric photothermal spectroscopy (PPTS) is a powerful tool for investigating optical properties of semiconductors. Temperature dependence of the PPT signal intensity of the Co-doped ZnO semiconductor was measured from 4.2 to 300 K. The Co concentration dependence of the PPT signal intensity was also studied to clarify the effect of doping with transition metals. In the case of doping at a low level, Co atoms form deep impurity levels in the ZnO matrices in the spectral region from 1.2 to 1.7 eV. However, for samples doped at higher levels, many crystal field split-off levels from localized Co2+ ions appeared at approximately 0.7–1.2 and 1.7–2.4 eV and these two bands became dominant in the spectra. The doping effect was investigated from the point of view of nonradiative transition for the first time.