Electrocatalytic synthesis of ammonia by surface proton hopping.

Highly efficient ammonia synthesis at a low temperature is desirable for future energy and material sources. We accomplished efficient electrocatalytic low-temperature ammonia synthesis with the highest yield ever reported. The maximum ammonia synthesis rate was 30 099 µmol gcat-1 h-1 over a 9.9 wt% Cs/5.0 wt% Ru/SrZrO3 catalyst, which is a very high rate. Proton hopping on the surface of the heterogeneous catalyst played an important role in the reaction, revealed by in situ IR measurements. Hopping protons activate N2 even at low temperatures, and they moderate the harsh reaction condition requirements. Application of an electric field to the catalyst resulted in a drastic decrease in the apparent activation energy from 121 kJ mol-1 to 37 kJ mol-1. N2 dissociative adsorption is markedly promoted by the application of the electric field, as evidenced by DFT calculations. The process described herein opens the door for small-scale, on-demand ammonia synthesis.

Surface Protonics Promotes Catalysis.

Catalytic steam reforming of methane for hydrogen production proceeds even at 473 K over 1 wt% Pd/CeO2 catalyst in an electric field, thanks to the surface protonics. Kinetic analyses demonstrated the synergetic effect between catalytic reaction and electric field, revealing strengthened water pressure dependence of the reaction rate when applying an electric field, with one-third the apparent activation energy at the lower reaction temperature range. Operando-IR measurements revealed that proton conduction via adsorbed water on the catalyst surface occurred during electric field application. Methane was activated by proton collision at the Pd-CeO2 interface, based on the inverse kinetic isotope effect. Proton conduction on the catalyst surface plays an important role in methane activation at low temperature. This report is the first describing promotion of the catalytic reaction by surface protonics.

Histogenesis of gastric foveolar metaplasia following duodenal ulcer: a definite reparative lineage of Brunner's gland.

AIMS: We aimed to clarify the histogenesis of gastric metaplasia in the duodenal mucosa, particularly in association with a reparative lineage of Brunner's glands. METHODS AND RESULTS: Using immunohistochemical methods with recently developed antimucin monoclonal antibodies (mAbs) that distinguish foveolar and deep mucins of the gastric type, as well as mAb MIB-1, the histogenesis of gastric metaplasia was investigated in the duodenal wall of 20 surgically resected specimens. In duodenal ulcers extending into Brunner's glands with destruction of the muscularis mucosae, proliferating cells positive for MIB-1 were scattered in Brunner's glands. Interestingly, a group of proliferating cells was often seen next to the ulcerated surface. These cells were also positive for M1 (gastric-foveolar type mucin) but negative for M2 (deep gastric and Brunner glands' mucin). In regenerating ducts through granulation tissue, the proliferating cell zone was elongated, above which foveolar-type cells positive for M1 but negative for M2 were detected, indicating that the G-zone is newly established in Brunner's glands at the floor of an ulcer to produce gastric-foveolar cells. Subsequently, an organoid growth of the normal stomach mucosa is completed in the duodenum. CONCLUSIONS: This study indicates a possible histogenetic pathway of gastric metaplasia in close association with a reparative lineage of Brunner's glands, suggesting that the occurrence of the gastric-foveolar type epi-thelium is not a simple expansion of Brunner's duct but a true metaplasia.

Alternatively spliced EDA segment regulates fibronectin-dependent cell cycle progression and mitogenic signal transduction.

Fibronectin (FN) is comprised of multiple isoforms arising from alternative splicing of a single gene transcript. One of the alternatively spliced segments, EDA, is expressed prominently in embryonic development, malignant transformation, and wound healing. We showed previously that EDA+ FN was more potent than EDA- FN in promoting cell spreading and cell migration because of its enhanced binding affinity to integrin alpha5beta1 (Manabe, R., Oh-e, N., Maeda, T., Fukuda, T., and Sekiguchi, K. (1997) J. Cell Biol. 139, 295-307). In this study, we compared the cell cycle progression and its associated signal transduction events induced by FN isoforms with or without the EDA segment to examine whether the EDA segment modulates the cell proliferative potential of FN. We found that EDA+ FN was more potent than EDA- FN in inducing G1-S phase transition. Inclusion of the EDA segment potentiated the ability of FN to induce expression of cyclin D1, hyperphosphorylation of pRb, and activation of mitogen-activated protein kinase extracellular signal regulated kinase 2 (ERK2). EDA+ FN was also more potent than EDA- FN in promoting FN-mediated tyrosine phosphorylation of p130(Cas), but not focal adhesion kinase, which occurred in parallel with the activation of ERK2, suggesting that p130(Cas) may be involved in activation of ERK2. These results indicated that alternative splicing at the EDA region is a novel mechanism that promotes FN-induced cell cycle progression through up-regulation of integrin-mediated mitogenic signal transduction.

Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment.

Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA- FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin alpha5 and beta1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin alpha5beta1. In support of this conclusion, purified integrin alpha5beta1 bound more avidly to EDA+ FN than to EDA- FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA- FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin alpha5beta1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

[Analysis of microsatellite regions and DNA ploidy pattern in signet ring cell carcinomas of the stomach].

We examined microsatellite instability (MSI) and loss of heterozygosity (LOH) in regions of several important genes in 25 signet-ring cell carcinomas of the stomach. The relationship between microsatellite analysis and DNA ploidy pattern was also investigated. MSI was observed in 15% (2/13) of early carcinomas and in 17% (2/12) of advanced carcinomas. Although LOH in the region of APC gene was found in 16% (4/25) and LOH of p53 was found in 12% (3/25), 15% (2/13) of early carcinomas and 33% (4/12) of advanced carcinomas showed LOH in regions of E-cadherin gene. Cyto-fluorometrical study revealed that 85% (11/13) of early carcinomas were diploid pattern, and aneuploid components were demonstrated in 50% (6/12) of advanced carcinomas. However, no MSI-positive cases contained aneuploid components, and in contrast, all p53-LOH cases contained aneuploid components. Our results suggest that gene abnormalities which have been frequently reported in differentiated adenocarcinomas are rare events in signet-ring cell carcinomas other than those associated with cell adhesion, and that MSI is not related to the occurrence of aneuploid cells.

[Tissue of origin of herpes simplex virus type 1 reactivated in tear film].

In the present study, we tried to detect the tissue of origin of herpes simplex virus type 1 (HSV-1) reactivated in tear film after artificial reactivation. The combined treatment consisted of iontophoresis on postinoculation day 35, followed by topical epinephrine on 2 days, after which the rabbits were killed. The ocular tissues and trigeminal ganglia were immediately dissected. Their cell-free supernatants were inoculated on CV-1 (African green monkey kidney cell) monolayers for infectious HSV-1 detection. The percentage of recovery from the cell-free supernatants was 50% (5 out of 10 samples) from the cornea, 0% from conjunctiva or lacrimal glands, and 20% (2 out of 10 samples) from trigeminal ganglia. The percentage of HSV-1 reactivation in the tear film was 50%. No infectious virus was detected from tissues or tear film in the control group. Four eyes showed HSV-1 reactivation simultaneously from the cornea and tear film, but only one eye from the trigeminal ganglion and tear film. These results demonstrate that the cornea might be the tissue of origin of HSV-1 reactivated in tear film.

Molecular cloning and the COOH-terminal processing of gp64, a putative cell-cell adhesion protein of the cellular slime mold Polysphondylium pallidum.

The cellular slime mold Polysphondylium pallidum expresses a cell surface glycoprotein (referred to as gp64), which seems to be implicated in cell-cell adhesion. We identified a near full-length gp64 cDNA (1,104 base) upon screening a P. pallidum lambda gt11 library with a monoclonal antibody. The open reading frame encodes a protein of 320 amino acids with a molecular mass of 32,752 Da; the protein includes hydrophobic segments at both a NH2- and a COOH-terminal ends. By an Edman degradation analysis of S-pyridylethylated gp64 and its COOH-terminal peptide, it was found that the NH2- and COOH-terminal segments are both removed from the precursor protein of gp64. The COOH-terminal segment was isolated from a lysyl endopeptidase digest of gp64 by an affinity method. The COOH-terminal segment was identified at positions 266-279 in the primary sequence deduced from the cDNA sequence. The mature gp64 consisted of 279 amino acid residues and extremely rich in Cys residues (36 Cys/279 amino acids = 12.9%). Although there was already maximal accumulation of gp64 mRNA in vegetative cells, the protein reached a maximal level during aggregation stage, decreased, and then leveled off through the developmental cycle.

Distribution of epidermal growth factor in rat ocular and periocular tissues.

Using a sensitive radioimmunoassay (RIA) specific for rat epidermal growth factor (rEGF), we investigated the presence of rEGF in a variety of rat ocular and periocular tissues. Immunoreactive rEGF (IR-rEGF) was present in tear fluids (25.5 +/- 5.8 ng/ml), exorbital lacrimal gland (6.73 ng/g wet weight), intraorbital lacrimal gland (2.80 ng/g wet weight), Harderian gland (1.90 ng/g wet weight), and conjunctiva (0.16 ng/g wet weight). EGF was not detectable in aqueous humor, cornea, iris and ciliary body, lens, or the posterior part of the globe (retina, choroid, and vitreous body). Gel exclusion chromatography and reverse-phase high-performance liquid chromatography revealed that IR-rEGF in the above ocular fluids and tissues was indistinguishable from standard rEGF. Using enzyme-linked immunohistochemistry, rEGF was demonstrated to be localized in the duct epithelial cells of lacrimal glands. These findings reveal that EGF is preferentially localized in the ocular surface and lacrimal apparatus.

Herpes simplex virus latency in human cornea.

The authors attempted to demonstrate the latency of herpes simplex virus type 1 (HSV-1) in corneas obtained during penetrating keratoplasty from patients with herpetic stromal keratitis in the nonactive (subsided) stage. The subjects comprised 20 patients (9 men, 11 women; average age 43.5 years). Infectious virus was not detected in the supernatants following corneal homogenization and centrifugation. Latent virus was detected in the cultured supernatants of the corneal sections from 8 patients. Although the ganglion trigger theory by Hill et al is conventionally supported as the mechanism of recurrence of herpetic keratitis, the present results suggest a ganglion and skin trigger theory, in which proliferation from latent HSV-1 in the cornea (peripheral tissue) might stimulate the ganglion.

Endothelial decompensation in a schizophrenic patient receiving long-term treatment with tranquilizers.

Bilateral corneal edema occurred in a schizophrenic patient receiving tranquilizers. This particular corneal edema was rapidly resolved after tranquilizer discontinuation. Specular microscopic examination disclosed marked enlargement of the corneal endothelium. This endothelial impairment may have been due to long-term administration of tranquilizers.

[Effect of trigeminal denervation on rabbit corneal epithelium].

To investigate the effects of trigeminal denervation on the corneal epithelium, left postganglionic trigeminal neurotomy via intracranial approach was performed in 22 rabbits. Among 16 rabbits survived successfully, 13 denervated eyes (81%) showed corneal epithelial abnormalities that included 4 epithelial defects and 9 epithelial opacities. The remaining 3 eyes were normal. Histological examination showed many atrophic epithelial cells and the thinning of the corneal epithelial layer in the denervated eyes with corneal opacities. There was no abnormality in corneal stroma or endothelium. The ratio of epithelial to total corneal thickness calculated by a computer-assisted image analyzer was 6.5 +/- 2.1 (%) in control eyes and 3.8 +/- 1.9 (%) in the denervated eyes, the two values being significantly statistically different (p less than 0.001).

[Vitreous oxygen tension of proliferative diabetic retinopathy].

The authors investigated the vitreous oxygen tension in 30 eyes of 29 cases of proliferative diabetic retinopathy patients in order to determine the distribution of oxygen tension and the possible role of neovascular tissue in tissue oxygenation. Vitreous oxygen tension was measured using a polarographic oxygen electrode and a PO2 monitoring system (PO-2080). Prior to pars plana vitrectomy, the oxygen electrode was inserted into the vitreous cavity under microscopic observation with dim illumination transmitted fiberoptically. The respective oxygen tension at the mid-vitreous cavity, above the optic disc, above the macula, above the neovascular tissue, in the peripheral vitreous, above the photocoagulated retina and above the non-photocoagulated retina were 15.8 +/- 4.7 mmHg, 31.2 +/- 10.0 mmHg, 17.1 +/- 4.0 mmHg, 32.0 +/- 9.9 mmHg, 15.6 +/- 5.1 mmHg, 16.5 +/- 5.5 mmHg and 18.6 +/- 4.9 mmHg. The oxygen tension values above the neovascular tissue and above the optic disc showed statistically significantly higher values than that of midvitreous cavity. We assume this to be due to differences between the oxygen demand and supply on the neovascular tissue, because in these tissues there are large amounts of vessels and blood flow compared to oxygen consumption. Therefore residual oxygen causes oxygen flow from the neovascularization to the mid-vitreous. This outcome is one of the facts which supports the hypothesis that neovascular tissues develop in order to compensate for retinal ischemia by releasing oxygen.

Recombinant human epidermal growth factor and corneal neovascularization.

With a view toward possible future clinical application, we investigated whether recombinant human epidermal growth factor (hEGF) could induce corneal neovascularization. Ethylene-vinyl-acetate copolymer slow-release pellets containing either 250 ng, 500 ng or 1 microgram of hEGF or 250 ng of bovine serum albumin (BSA) were implanted into rabbit corneal stroma, and the corneas were examined by slitlamp biomicroscope for 3 weeks. The results indicated that less than 1 microgram of hEGF per pellet did not induce neovascularization in the cornea. However, when pellets containing 250 ng of basic fibroblast growth factor (bFGF) were implanted, corneal neovascularization toward these pellets occurred within 10 days. The same phenomenon occurred toward the 250 ng hEGF pellets embedded in the ipsilateral or contralateral cornea of the same animal, but was significantly less severe. When a pellet containing 250 ng bFGF was intramuscularly implanted in the animal's back, similar neovascularization was observed toward the pellets containing hEGF implanted in the same animal's cornea, but not toward implanted pellets containing BSA. These results suggest that less than 1 microgram of hEGF cannot initiate corneal angiogenesis, but can promote new vessel formation if the limbal vasculature is activated by a sprouting signal from a certain substance as trace amounts of bFGF.

Protective effects of anti-glycoprotein D monoclonal antibodies in murine herpetic keratitis.

The protective effects of passive immunization with two kinds of anti-glycoprotein D (anti-gD) monoclonal antibodies, having different antiviral activities, were investigated in murine herpetic keratitis. One monoclonal antibody, designated M1, had high virus-neutralizing antibody titers, along with undetectable levels of complement-dependent cytolysis (CDC) and antibody-dependent cellular cytotoxicity (ADCC); the other, designated M12, exhibited extremely low titers of virus-neutralization with high level of CDC and ADCC. When systemically administered 24 hours prior to virus inoculation to the cornea, both M1 and M12 almost completely prevented the development of stromal keratitis. The protective efficacy of both was observed to be dose-dependent. Pepsin-treated M1 retained its efficacy in suppressing stromal keratitis, whereas pepsin-treated M12 did not. When the administration of M1 and M12 were delayed, both provided significant (but less complete) protection, up to 24 hours after virus inoculation. These results suggest that both virus neutralization and CDC/ADCC play an important role in preventing virus growth in the corneal stroma during the early stage of corneal infection.