Generation of phenoxy radicals by methemoglobin-hydrogen peroxide studied by electron paramagnetic resonance

The free radical intermediates of phenol derivatives, produced by the methemoglobin-hydrogen peroxide system at pH 5 and 7, are detected by electron paramagnetic resonance equipped with a continuous-flow apparatus. All the radicals from phenols are the phenoxy radicals, as identified by analyzing the observed hyperfine structures of the spectra with the aid of SCF-LCAO MO calculations. Comparing with the reaction of Fenton's reagent, it is concluded that free OH radical, even if it exists, is not liberated into the solution in the methemoglobin-hydrogen peroxide system.     

A point mutation of c-Ki-ras gene was found in human esophageal carcinoma cell lines but not in primary esophageal carcinomas.

Activation of the ras oncogene in primary human esophageal carcinomas and cell lines established from such carcinomas was analyzed using the polymerase chain reaction (PCR)-direct sequencing method. This analysis revealed a GC----AT transition at the second base in the 12th codon of the c-Ki-ras gene in TE1 and TE2 esophageal carcinoma cell lines. In contrast, no point mutation was detected in the 12th, 13th, and 61st codon of the c-Ki-ras and c-Ha-ras gene in 31 primary esophageal carcinomas including those from which TE1 and TE2 cell lines were established. These results demonstrate that while activation of the c-Ki-ras gene by point mutation occurred in a subset of esophageal carcinoma cell lines during establishment of the cell lines, the activation events are not important in the transformation of human esophageal epithelial cells.     

Detection of nitric oxide production in lipopolysaccharide-treated rats by ESR using carbon monoxide hemoglobin.

Release of nitric oxide (NO), from macrophages activated with E. coli lipopolysaccharide (LPS) and endothelial cells, has been proposed using chemiluminescence and spectrophotometry. However these methods can not distinguish NO from NO2-. The present study was aimed to prove in vivo production of NO, by ESR using CO-hemoglobin (HbCO) as a trapping agent of NO in the peritoneal cavity of rats treated with LPS. We detected a broad signal in the recovered HbCO solution. Inositol hexaphosphate induced a three-line hyperfine structure, characteristic of NO-hemoglobin (HbNO). In the arterial blood, ESR signal of HbNO with faint hyperfine structure was detected. NG-Monomethyl-L-arginine inhibited the formation of HbNO. HbNO was not detected in the peritoneal cavity of the LPS-untreated rat given i.p. both NO2- and HbCO. HbNO was, therefore, derived from NO, not from NO2-. These results show that free NO is produced in vivo by the stimulation of LPS.     

The existence of two different forms of apo-electron-transferring flavoprotein

The association process of FAD and apo-electron-transferring flavoprotein (apoETF) from hog kidney was investigated. The reaction schemes which involve the association-dissociation of the protein species could be excluded by the light scattering data, which indicated that the molecular weights of apoETF and holoETF are identical. The binding reaction between FAD and a large excess of apoETF was monophasic and obeyed pseudo-first order kinetics. On the other hand, the reaction between apoETF and a large excess of FAD was biphasic: the fast phase obeyed a pseudo-first order reaction, and the rate of the slow phase was almost independent of FAD concentration. These results suggest the existence of two different forms of apoETF, as represented in the following reaction scheme: [formula: see text] where "F" is FAD, "H" is holoETF, and "A" and "A" are the different forms of apoETF. The kinetic parameters were determined as k-1 = 3.9 x 10(4) M-1.s-1, k-1 approximately 10(-5) s-1, k+2 = 1.0 x 10(-3) s-1, and k-2 = 3.1 x 10(-3) s-1, in 50 mM potassium phosphate buffer, pH 7.6, containing 0.3 mM EDTA, and 5% v/v glycerol, at 7 degrees C. The elution patterns of apoETF on molecular sieve chromatography were very different from that of holoETF although the true molecular weights were identical. This result suggests that the structure of apoETF differs greatly from that of holoETF.     

Isomerization of delta 1-piperideine-2-carboxylate to delta 2-piperideine-2-carboxylate on complexation with flavoprotein D-amino acid oxidase.

The 1,646 cm-1 band in a resonance Raman spectrum obtained with excitation in the charge-transfer band of the complex of oxidized D-amino acid oxidase (DAO) with the oxidation product of D-lysine catalyzed by DAO shifted to 1,617 cm-1 upon 2-13C substitution of lysine. Thus, the band is assigned to a C(2) = C(3) stretching mode of the enamine, delta 2-piperideine-2-carboxylate (En). In the enzyme-free solution, the product is preferentially in the cyclic imine form, delta 1-piperideine-2-carboxylate (Im). Thus, DAO has a higher affinity for the enamine form than for the imine form. The pH effects on the affinity of DAO for the product and on the molar absorption coefficient at 630 nm in the charge-transfer band, suggest that the enzyme-bound product is En in the neutral form at the N atom. As the value of observed rate constant between DAO and the product was constant at high product concentrations, the binding mechanism can be explained as follows; E + Im in equilibrium with EIm in equilibrium with EEN: rapid bimolecular and slow unimolecular processes. The isomerization of the imine form to the enamine form proceeds in the slow process. The low affinity of Im for DAO may be due to a steric repulsion of the hydrogen atoms of Im at C(3) in the active site. The hydrogen atoms of a substrate D-amino acid at C(3), which correspond to the C(3) hydrogens of Im, may act repulsively in the active site and the repulsive energy may induce strain or distortion of the substrate and the enzyme, accelerating the catalytic reaction.     

A novel gene activated in regenerating islets

Administration of poly(ADP-ribose) synthetase inhibitors such as nicotinamide to 90% depancreatized rats induces regeneration of pancreatic islets, thereby ameliorating the surgical diabetes (Yonemura, Y., Takashima, T., Miwa, K., Miyazaki, I., Yamamoto, H., and Okamoto, H. (1984) Diabetes 33, 401-404). In screening the regenerating islet-derived cDNA library, we came across a novel gene encoding a 165-amino acid protein. The gene was expressed in regenerating islets but not in normal pancreatic islets, insulinomas, or regenerating liver. In 90% depancreatized and nicotinamide-injected rats, the expression of the gene was increased 1 month after the partial pancreatectomy and reached a peak 3 months after the operation. The increase in expression of the gene was temporally correlated with the increase in size of regenerating islets and the decrease in urinary glucose level. The gene was also found to be activated in hyperplastic islets of aurothioglucose-treated mice. Thus, the expression of the gene in both regenerating and hyperplastic islets suggests possible roles for this gene in replication, growth, and maturation of islet beta-cells. We also found that a human pancreas-derived cDNA library contained a homologue to the gene.     

Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified proteins

Long term incubation of proteins with glucose, named the Maillard reaction (Maillard, L. C. (1912) C. R. Acad. Sci. (Paris) 154, 66-68), gives rise to advanced glycosylation end product (AGE) with fluorescence, color, as well as cross-linked properties. The receptor-mediated endocytosis of AGE-proteins by macrophages was reported (Vlassara, H., Brownlee, M., and Cerami, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5588-5592). The present study on the binding of AGE-bovine serum albumin (BSA) to rat peritoneal macrophages and sinusoidal liver cells demonstrated the presence of a saturable, high affinity receptor for AGE-BSA with Kd = 2.4 x 10(-7) M (macrophages) and 2.1 x 10(-7) M (sinusoidal cells). The cellular binding of AGE-BSA and its endocytic uptake by these cells were competitively inhibited by BSA preparations modified with aliphatic aldehydes such as formaldehyde or glycolaldehyde, ligands known to be specific for a scavenger receptor for aldehyde-modified proteins (Horiuchi, S., Murakami, M., Takata, K., and Morino, Y. (1986). J. Biol. Chem. 261, 4962-4966). These ligands also had a profound in vivo effect on the plasma clearance of 125I-AGE-BSA as well as its hepatic uptake. Thus, endocytic uptake of AGE-proteins by macrophages appeared to be mediated by a scavenger receptor for aldehyde-modified proteins. This provides evidence for the biological importance of the scavenger receptor in eliminating senescent macromolecules from the circulation.     

Identification of early developing axon projections from spinal interneurons in the chick embryo with a neuron specific beta-tubulin antibody: evidence for a new 'pioneer' pathway in the spinal cord

The early development of interneurons in the chick embryo spinal cord was studied using a monoclonal antibody against a neuron-specific beta-tubulin isoform. Early developing interneurons were divided into two cell groups on the basis of their location and the pattern of growth of their axons. One group is composed of cells that establish a primitive longitudinal pathway (PL-cells), whereas the other group contains cells constituting a circumferential pathway (C-cells). The onset of axonal development in both cell groups occurs at stage (st.) 15 (embryonic day, (E), 2) in the branchial segments, which is prior to axonogenesis of motoneurons. PL-cells develop in the region between the floor plate and the motoneuron nucleus. Their axons are the first neuronal processes ('pioneer axons') to arrive in the ventrolateral marginal zone and they project both rostrally and caudally to establish a primitive longitudinal association pathway at the ventrolateral surface of the neural tube. This pathway is formed before axons of C-cells arrive in the ventrolateral region. The first C-cells are initially located in the most dorsal portion of the neural tube, whereas later appearing C-cells are also located in both intermediate and ventral regions of the neural tube. The axons of C-cells project ventrally, without fasciculating, along the lateral border of the neural tube. Some of their axons enter the ipsilateral ventrolateral longitudinal pathway at st. 17. We often observed apparent contacts and interactions between preexisting axons of PL-cells and newly arriving axons of C-cells. The axons of commissural C-cells first enter the floor plate at st. 17 and cross the midline at st. 18. Axons of C cells begin to join the contralateral ventrolateral longitudinal pathway at st. 18+ to st. 19. In the floor plate region, contacts between growth cones and axons were often observed. However, axons in the floor plate at these stages were not fasciculated. These observations establish the timing and pattern of growth of axons from two specific populations of early developing interneurons in the chick spinal cord. Additionally, we have identified an early and apparently previously undescribed 'pioneer' pathway that constitutes the first longitudinal pathway in the chick spinal cord.     

Mast cells are important in the development of hypersensitivity pneumonitis. A study with mast-cell-deficient mice

We have previously reported that C57B1/6 mice develop lung lesions similar to human hypersensitivity pneumonitis (HP) by repeated transnasal administration of Thermoactinomyces vulgaris antigen. Since the HP-like lesions were induced via respiratory route and by the causative antigen in human HP (farmer's lung), it seems that this murine model is useful for investigating the cell-to-cell interactions in human HP. To clarify the involvement of mast cells (MC) in the development of HP, T. vulgaris (90 micrograms/day) was transnasally administered to MC-deficient WBB6F1-W/Wv mice (W/Wv) and their littermates (+/+) five times a wk for 3 wk. When the lungs were examined by scoring pathological findings and lung indexes, HP-like lesions were significantly less severe in W/Wv than in +/+, whose lesions were equivalent to those of C57B1/6. Bone-marrow-derived cultured MC from +/+ mice (98% purity) were obtained by in vitro culture mixed with WEHI-3B-derived conditioned medium which contained IL-3. When these MC were adoptively transferred to W/Wv mice (10(7) cells/mouse), the HP-like lesions in W/Wv mice were enhanced to be as severe as those in +/+. Importantly, significant numbers of MC were found in the lungs of MC-transferred W/Wv mice. These results suggest that MC play an important role in the development of the murine experimental HP.