Nasopharyngeal-associated lymphoreticular tissue (NALT) immunity: fimbriae-specific Th1 and Th2 cell-regulated IgA responses for the inhibition of bacterial attachment to epithelial cells and subsequent inflammatory cytokine production

To investigate the antibacterial activity of mucosal Th1 and Th2 immune responses induced nasally and orally, mice were immunized with mucosal vaccine containing fimbrial protein of Porphyromonas gingivalis, a causative agent for a destructive chronic inflammation in the periodontium, and cholera toxin (CT) as mucosal adjuvant. Nasal vaccine containing low doses of fimbriae (10 micrograms) and CT (1 microgram) induced Ag-specific Th1/Th2-type response in CD4+ T cells in mucosal effector tissues, including nasal passage and submandibular glands, which accounted for the generation of Ag-specific IgA-producing cells. In contrast, oral immunization required higher amounts of fimbriae and CT for the induction of Ag-specific IgA responses. Fimbriae-specific IgA mAbs generated from submandibular glands of nasally immunized mice inhibited P. gingivalis attachment to and reduced subsequent inflammatory cytokine production from epithelial cells. These findings suggest that nasal vaccination is an effective immunization regimen for the induction of Ag-specific Th1 and Th2 cell-driven IgA immune responses that possess the ability to inhibit bacterial attachment to epithelial cells and subsequent inflammatory cytokine production.     

Primary structure of Saccharomyces cerevisiae NADPH-cytochrome P450 reductase deduced from nucleotide sequence of its cloned gene

We isolated cDNA (pgCYR, about 2.1 kb) and genomic DNA (pgGYR, about 4 kb) clones coding for NADPH-cytochrome P450 reductase by immunoscreening of yeast Saccharomyces cerevisiae cDNA and genomic DNA libraries in phage lambda gt11. The clones were sequenced and found to encode a protein of 691 amino acid residues with a calculated molecular weight of 76,737 daltons. The amino-terminal sequence (excluding the initial methionine residue) deduced therefrom was in agreement with the protein sequence of the yeast reductase. In addition, the deduced sequence included the partial amino acid sequence determined with the papain-solubilized reductase. The total amino acid sequence of the yeast reductase showed 33-34% similarity with those of the rat, rabbit, pig, and trout reductases. In spite of low similarity in the total amino acid sequences, the possible functional domains related to binding of FAD, FMN, and NADPH were well conserved among all five species compared.     

Membrane potential in liposomes measured by the transmembrane distribution of 86Rb+, tetraphenylphosphonium or triphenylmethylphosphonium: effect of cholesterol in the lipid bilayer

Valinomycin-induced potassium diffusion potential (delta psi, inside negative) in the liposomes made of phosphatidylcholine and various amounts of cholesterol was measured by uptake of 86Rb+, tetraphenylphosphonium (TPP+) or triphenylmethylphosphonium (TPMP+). In any liposome, the values of membrane potential obtained by 86Rb+ uptake (delta psi Rb) agreed well with those calculated from the imposed potassium concentration gradient using the Nernst equation, and were not affected by the presence of cholesterol. However, both delta psi TPP and delta psi TPMP showed smaller values than delta psi Rb when the cholesterol content in liposomes increased. delta psi TPMP at a stationary state was much smaller than delta psi TPP. The orientational order parameter of the lipids' bilayer with various cholesterol content was estimated from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The results indicated that the permeation of TPP+ or TPMP+ into liposomes containing a large amount of cholesterol is strongly restricted by the high ordering of phosphatidylcholine acyl chains.     

On the Molecular Weight of Polypeptide Chain of Sweet Potato β-Amylase

A number of N-acyl-L-proline derivatives were synthesized and their biological activities were investigated by using lettuce (Lactuca sativa L. cv. Sacramento) seedling test. A wide variety of these compounds promoted root growth at 25°C both under light and in darkness. Of the compounds tested, N-(2-ftuorobenzoyl)-L-proline methyl ester (4) showed the highest activity and caused a 270% increase in the root elongation compared to the control. N-(2-Naphthoyl)-L-proline methyl ester (14) promoted the root growth, while N-(1-naphthoyl)-L-proline methyl ester inhibited it. L-Proline, benzoic acid, and 2-naphthoic acid had no significant effect on lettuce seedlings. Compounds 4 and 14, and N-(2-chlorobenzoyl)-L-proline methyl ester (7) reduced the inhibitory effect of 1 ppm ABA on the root growth, while the D-isomer of 4 was less activite than compound 4. Compounds 4, 7, and 14 did not show any rescue-activity for the complete inhibition of germination that was caused by treating 10 ppm of ABA.     

Differential metabolism of 4-n- and 4-tert-octylphenols in perfused rat liver

Octylphenols, widely used in a variety of detergents and plastics, are known to exhibit estrogenicity in vivo. The details of their metabolism are needed to better understand the endocrine disruptions. We have previously shown that alkylphenols, having short alkyl chains, are glucuronidated and readily excreted into the bile from the liver, while 4-n-nonylphenol, having longer alkyl chains, remains as the alkylphenol's glucuronide in the tissue. This study elucidated the dependence of the metabolism on the shape of the alkyl chains by comparing 4-n-octylphenol and 4-tert-octylphenols in a perfused rat liver. Both octylphenols were highly glucuronidated by the liver microsomal fractions. The Vmax value of 4-tert-octylphenol glucuronidation was twice as high as that of 4-n-octylphenol in the liver microsomes. On the other hand, the Km values, being measures of enzymatic activity against these chemicals, were similar. 4-n-Octylphenol and 4-tert-octylphenol were both glucuronidated by a UDP-glucuronosyltransferase isoform, UGT2B1, expressed in the liver. In the liver perfusion, almost all of the 4-n-octylphenol perfused was metabolized directly to the glucuronide, whereas a portion of 4-tert-octylphenol was hydroxylated and then glucuronidated. The glucuronide of 4-n-octylphenol accumulated in the liver tissue in the same manner as 4-n-nonylphenol, but 4-tert-octylphenol and the hydroxylated metabolites were excreted readily into the bile. Only a small amount of 4-n-octylphenol-glucuronide and glucuronides of 4-tert-octylphenol and its hydroxylated metabolites could be excreted into the bile of Eisai hyperbilirubinemic rats (EHBR). These animals are deficient in xenobiotic conjugate transporter, multidrug resistance-associated protein (MRP-2), indicating that the glucuronides of both octylphenols are transported by MRP-2. These results indicate that the differences in metabolism of these octylphenols are due to the shape of their alkyl chains, suggesting that the estrogenic activities of not only the parent chemicals but also these metabolites must be taken into consideration.     

Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway

The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization-induced neurite outgrowth and the expression of growth-associated protein-43 (GAP-43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP-43 and synapsin I. These increases were suppressed by inhibition of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII), PLD, or mitogen-activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization-induced neurite outgrowth, and the increase in GAP-43 and synapsin I expression. Depolarization evoked a Ca²⁺ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIIδ by siRNA blocked KCl-induced phosphorylation of proline-rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal-regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1-butanol and transfection of PLD2 siRNA, whereas it was enhanced by over-expression of wild-type PLD2. Depolarization-induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization-induced PLD2 activation was downstream of CaMKIIδ and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP-43 and synapsin I expression during depolarization-induced neuronal differentiation in PC12 cells.     

Melanocortins Contribute to Sequential Differentiation and Enucleation of Human Erythroblasts via Melanocortin Receptors 1, 2 and 5

In this study, we showed that adrenocorticotropic hormone (ACTH) promoted erythroblast differentiation and increased the enucleation ratio of erythroblasts. Because ACTH was contained in hematopoietic medium as contamination, the ratio decreased by the addition of anti-ACTH antibody (Ab). Addition of neutralizing Abs (nAbs) for melanocortin receptors (MCRs) caused erythroblast accumulation at specific stages, i.e., the addition of anti-MC2R nAb led to erythroblast accumulation at the basophilic stage (baso-E), the addition of anti-MC1R nAb caused accumulation at the polychromatic stage (poly-E), and the addition of anti-MC5R nAb caused accumulation at the orthochromatic stage (ortho-E). During erythroblast differentiation, ERK, STAT5, and AKT were consecutively phosphorylated by erythropoietin (EPO). ERK, STAT5, and AKT phosphorylation was inhibited by blocking MC2R, MC1R, and MC5R, respectively. Finally, the phosphorylation of myosin light chain 2, which is essential for the formation of contractile actomyosin rings, was inhibited by anti-MC5R nAb. Taken together, our study suggests that MC2R and MC1R signals are consecutively required for the regulation of EPO signal transduction in erythroblast differentiation, and that MC5R signal transduction is required to induce enucleation. Thus, melanocortin induces proliferation and differentiation at baso-E, and polarization and formation of an actomyosin contractile ring at ortho-E are required for enucleation.     

Novel autotrophic nitrogen removal system using gel entrapment technology

A pilot plant involving a nitritation-anammox process was operated for treating digester supernatant. In the preceding nitritation process, ammonium-oxidizing bacteria were immobilized in gel carriers, and the growth of nitrite-oxidizing bacteria was suppressed by heat-shock treatment. For the following anammox process, in order to maintain the anammox biomass in the reactor, a novel process using anammox bacteria entrapped in gel carriers was also developed. The nitritation performance was stable, and the average nitrogen loading and nitritation rates were 3.0 and 1.7 kg N m⁻³ d⁻¹, respectively. In the nitritation process, nitrate production was completely suppressed. For the anammox process, the startup time was about two months. Stable nitrogen removal was achieved, and an average nitrogen conversion rate of 5.0 kg N m⁻³ d⁻¹ was obtained. Since the anammox bacteria were entrapped in gel carriers, stable nitrogen removal performance was attained even at an influent suspended solids concentration of 1500 mg L⁻¹.