Platelet-activating factor mediates acid-induced lung injury in genetically engineered mice.

Adult respiratory distress syndrome (ARDS) is an acute lung injury of high mortality rate, and the molecular mechanisms underlying it are poorly understood. Acid aspiration-induced lung injury is one of the most common causes of ARDS, characterized by an increase in lung permeability, enhanced polymorphonuclear neutrophil (PMN) sequestration, and respiratory failure. Here, we investigated the role of platelet-activating factor (PAF) and the PAF receptor (PAFR) gene in a murine model of acid aspiration-induced lung injury. Overexpression of the PAFR gene in transgenic mice enhanced lung injury, pulmonary edema, and deterioration of gas exchange caused by HCl aspiration. Conversely, mice carrying a targeted disruption of the PAFR gene experienced significantly less acid-induced injury, edema, and respiratory failure. Nevertheless, the efficiency of PMN sequestration in response to acid aspiration was unaffected by differences in PAFR expression level. The current observations suggest that PAF is involved in the pathogenesis of acute lung injury caused by acid aspiration. Thus, inhibition of this pathway might provide a novel therapeutic approach to acute lung injury, for which no specific pharmaceutical agents are currently available.

Glycosylation at the Fab portion of myeloma immunoglobulin G and increased fucosylated biantennary sugar chains: structural analysis by high-performance liquid chromatography and antibody-lectin enzyme immunoassay using Lens culinaris agglutinin.

An antibody-lectin enzyme immunoassay technique which had been developed for the analysis of sugar chains of alpha-fetoprotein (N. Kinoshita et al., Clin. Chim. Acta, 179: 143-152, 1989) was used for analysis of sugar chains of myeloma immunoglobulin G (IgG). The IgG sugar chains of four of nine patients with myeloma were found to be highly reactive to Lens culinaris agglutinin as compared with those of six normal controls and 177 patients without myeloma. This reflected a high L. culinaris agglutinin/concanavalin A ratio. The IgGs of these patients were found to have highly sialylated, fucosylated, and bisected biantennary sugar chains at Fab portions as judged by the lectin-blotting technique as well as by high-performance liquid chromatography analysis. These results indicate that some of the myeloma IgG proteins undergo unusual glycosylation processes.

Overexpression of alpha1-6 fucosyltransferase in hepatoma cells suppresses intrahepatic metastasis after splenic injection in athymic mice.

Changes in oligosaccharide structures alter the biological functions of cancer cells. Alpha1-6 fucosyltransferase (alpha1-6FucT) catalyzes the transfer of fucose to the innermost GlcNAc in N-glycans. Although alpha1-6FucT is barely detected in normal liver, it is enhanced during rat hepatocarcinogenesis and in human hepatoma. To understand the biological meaning of the alpha1-6FucT in hepatoma, especially in terms of metastasis, we established human hepatoma cell lines, which express high levels of alpha1-6FucT by transfection of the alpha1-6FucT gene and investigated intrahepatic metastasis after splenic injection to athymic mice. Tumor formation in the liver was dramatically suppressed in the alpha1-6FucT transfectants (1 of 9 and 1 of 10 in alpha1-6FucT transfectants versus 6 of 9 and 6 of 9 in controls). Although there were no differences in terms of cell invasiveness to a Matrigel or in terms of cytotoxicity to interleukin 2-treated lymphocytes between alpha1-6FucT transfectants and control cells, cell adhesion to mice hepatocytes and nonparenchymal liver cells in culture was significantly inhibited in alpha1-6FucT transfectants, compared to the controls. Attachment of alpha1-6FucT transfectants to a fibronectin-coated dish was decreased compared to controls because alpha5beta1 integrin was more strongly alpha1-6 fucosylated in the alpha1-6FucT transfectants. Two-dimensional electrophoresis followed by lectin blot showed that certain glycoproteins (Mr 50,000-150,000, pI 4.8-5.5) were alpha1-6 fucosylated and might be linked to suppression of intrahepatic metastasis. This is the first demonstration of the biological significance of alpha1-6 fucosylation on N-glycans in hepatoma cells under in vivo conditions.

The alpha1-6-fucosyltransferase gene and its biological significance.

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1-6-fucosyltransferase (alpha1-6FucT) catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycoproteins. This enzyme was purified from a human fibroblast cell line, porcine brain, a human gastric cancer cell line and human blood platelets. cDNA cloning of porcine and human alpha1-6FucT was performed from a porcine brain and gastric cancer cell cDNA libraries, respectively. Their homology is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence. No homology to other fucosyltransferases such as alpha1-2FucT, alpha1-3FucT and alpha1-4FucT was found except for a region consisting of nine amino acids. The alpha1-6FucT gene is located at chromosome 14q24.3, which is also a different location from other fucosyltransferases reported to date. The alpha1-6FucT gene is the oldest gene family in the phylogenic trees among the nine cloned fucosyltransferase genes. alpha1-6FucT is widely expressed in various rat tissues and the expression of alpha1-6FucT in the liver is enhanced during hepatocarcinogenesis of LEC rats which develop hereditary hepatitis and hepatomas. In cases of human liver diseases, alpha1-6FucT is expressed in both hepatoma tissues and their surrounding tissues with chronic liver disease, but not in the case of normal liver. Serum alpha1-6-fucosylated alpha-fetoprotein (AFP) has been employed for an early diagnosis of patients with hepatoma. The mechanisms by which alpha1-6 fucosylation of AFP occurs in the hepatoma is not due to the up-regulation of alpha1-6FucT alone. Interestingly, when the alpha1-6FucT gene is transfected into Hep3B, a human hepatoma cell line, tumor formation in the liver of nude mice after splenic injection is dramatically suppressed. In this review, we focus on alpha1-6FucT and summarize its properties, gene expression and biological significance.

Protective role for cytosolic phospholipase A2alpha in autoimmune diabetes of mice.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) plays an important role in arachidonate pathway. To investigate the contribution of cPLA(2)alpha to autoimmune diabetes, we established non-obese diabetic (NOD) mouse, an excellent model for human type 1 diabetes, deficient in cPLA(2)alpha. These mice showed severe insulitis and a higher incidence of diabetes. In their macrophages, decreased prostaglandin E(2) (PGE(2)) induced by cPLA(2)alpha deficiency, and the increase in production of tumor necrosis factor (TNF)-alpha were observed. These results suggested that cPLA(2)alpha plays a protective role in progression of insulitis and development of autoimmune diabetes by suppression of TNF-alpha production from macrophages.

Fragmentation of ceruloplasmin following non-enzymatic glycation reaction.

Bovine ceruloplasmin underwent fragmentation following non-enzymatic glycosylation. Western blot and ELISA analyses indicated that a polyclonal rabbit antiserum to hexitolysine reacted with bovine ceruloplasmin after incubation with 0.1 M glucose. The same fragmentation was seen upon exposure of the protein to a hydrogen peroxide bolus. Both catalase and EDTA blocked peroxide-dependent fragmentation. Incubation with glucose resulted in a time-dependent release of Cu2+. The released Cu2+ appeared to participate in a Fenton-type reaction to produce hydroxyl radicals, which effected the fragmentation. Hydroxyl radical scavengers such as thiourea, mannitol, methionine, and formate inhibited this cleavage. ESR spectral studies also supported participation of hydroxyl radicals. Inhibition by EDTA of the fragmentation induced by an H2O2 bolus also supports a role for copper in a Fenton-type reaction. Taken together these results suggest that reactive oxygen species, such as superoxide anion and H2O2, were formed by the Maillard reaction which led to hydroxyl radicals being produced by a copper-dependent Fenton-type reaction. Both processes are likely to be involved in the fragmentation of ceruloplasmin.

Purification and cDNA cloning of GDP-L-Fuc:N-acetyl-beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha1-6 FucT) from human gastric cancer MKN45 cells.

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha1-6 FucT), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a culture supernatant of human gastric cancer cell line MKN45. The purification procedures included chromatographies on Q-Sepharose Fast Flow, synthetic GDP-hexanolamine-Sepharose, and GnGn-bi-Asn-Sepharose columns. SDS-PAGE of the purified enzyme gave a major band corresponding to an apparent molecular mass of 60 kDa. The enzyme was recovered in a 12% final yield with an approximately 4,600-fold increase in specific activity. The pH optimum was 7.5, and the enzyme was fully active in the presence of 5 mM EDTA and did not require divalent cations, Mg2+ and Ca2+. Oligonucleotide primers designed from partial amino acid sequences were used to amplify and clone alpha1-6 FucT cDNA from a cDNA library of MKN45 cells. The cDNA encodes 575 amino acids in length, and contains the predicted N-terminal and internal amino acid sequences derived on lysyl endopeptidase digestion. The homology to porcine brain alpha1-6 FucT is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence of the human MKN45 cell enzyme or that of porcine brain. Thus, the enzyme is distinct from other fucosyltransferases which catalyze alpha1-2, alpha1-3, and alpha1-4 fucose addition.

A fluorescent assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1-6fucosyltransferase activity, involving high performance liquid chromatography.

An assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1-6fucosyltransferase (alpha 1-6FucT; EC 2.4.1.68) activity has been developed, involving a fluorescent pyridylaminated substrate. A glycopeptide derived from bovine gamma-globulin was coupled with 4-(2-pyridylamino)butylamine (PABA) through the peptide bond, and the following substrate was obtained. [equation: see text] The substrate and guanosine diphospho-fucopyranoside (GDP-Fuc) were incubated with a crude enzyme extract for 2 h, and then the enzymatic product was separated by reversed phase HPLC. Quantitation of the product involved measurement of the fluorescence intensity of the fucosylated pyridylaminated sugar. The structures of both synthesized GnGn-bi-Asn-PABA (substrate), and synthesized GnGnF-bi-Asn-PABA (product) were analyzed by 1H NMR. The enzymatic product was also analyzed by 1H NMR and was found to have alpha 1-6fucose at the reducing end GlcNAc. This method is highly specific for alpha 1-6FucT and is applicable for various experiments, including purification and cell culture ones.

Light activation of expression associated with the tomato rbcS promoter in transformed tobacco cell line BY-2.

The promoter (2.3 kb) of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS3B) from tomato was inserted into the upstream multi-cloning site of beta-glucuronidase (GUS) gene. The construction was introduced into the tobacco suspension cultured cell, BY-2. The transformed tobacco cell exhibited greater GUS activity (10-fold) under the light condition than the dark condition. The specific GUS activity increased with increasing culture time under the light condition. In fed-batch culture, high density culture was achieved, 47.6 g l-1 dry weight, which was 6.8-fold greater than the batch culture, whereas the GUS was produced 1.7-fold in specific activity compared with the batch culture. Growth of BY-2 and GUS gene expression during the culture were simulated by a kinetic model.

Mice deficient in cytosolic phospholipase A2 are less susceptible to cerebral ischemia/reperfusion injury.

To determine the role of cytosolic phospholipase A2 (cPLA2) in infarct development, wild-type and cPLA2 knock-out mice were subjected to focal cerebral ischemia for 75 min by occluding the middle cerebral artery using nylon filament and subsequent reperfusion by withdrawing the filament. The neurological deficit severity was evaluated by a modified 4-point scale. After the reperfusion period (72 h), mice were killed, and the brains were cut into four 2 mm coronal sections using a rodent brain matrix. Sections were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC). The infarct volume was 87.19 +/- 27.54 mm3 (mean +/- SD, n = 11) in the wild-type mice and 48.20 +/- 31.32 mm3 (n = 10; P < 0.01 vs. wild-type) in the knock-out mice. Less severe functional neurological deficits were observed in knock-out mice at 72 h after ischemia when compared with wild-type. Thus, disruption of cPLA2 resulted in significant reduction of infarct area and neurological deficit severity in the MCA occlusion model. These data indicate a critical role for cPLA2 in the pathogenesis of cerebral ischemia/ reperfusion injury.

Effect of aldosterone antagonist on the DC potential in the endolymphatic sac.

The effect of aldosterone and canrenoate, known as an aldosterone antagonist, on the DC potential in the endolymphatic sac (ESP) was examined in the guinea pig. Intravenous administration of aldosterone (1 mg/kg) induced no change in the ESP for 60 minutes after the injection. However, canrenoate, an aldosterone antagonist, produced a dose-dependent decrease in the ESP. Pretreatment with aldosterone attenuated the decrease in the ESP by canrenoate. The results suggest the possibility that aldosterone may play a role in the endolymph absorption of the endolymphatic sac.

Interaction of catecholamine and acetazolamide in the action on the endolymphatic sac direct current potential.

Our recent studies have revealed that catecholamines depress the endolymphatic sac DC potential (ESP) by beta-adrenergic action, and that acetazolamide (ACTZ), a potent carbonic anhydrase inhibitor, decreases the ESP. The interaction of ACTZ and isoproterenol (Iso), a nonselective beta agonist, in their action on the ESP was examined in the guinea pig. Intravenous administration of Iso (6.25 micrograms/kg/min) and ACTZ (10 mg/kg) reduced the ESP amplitude by 38.5 +/- 5.9% (n = 8) and 39.8 +/- 3.7% (n = 8), respectively. Co-administration of both agents reduced the ESP amplitude by 62.3 +/- 3.3% (n = 8). The ESP change induced by co-administration was significantly larger than that by the administration of each agent alone. Co-treatment with Iso and ACTZ at doses producing near-maximum reduction of the ESP depressed almost all parts of oxygen-dependent components of the ESP. The results suggest that Iso and ACTZ decrease the ESP via mechanisms different from each other, and that oxygen-dependent components of the ESP are composed of Iso- and ACTZ-sensitive parts.

Response of the endolymphatic sac d.c. potential to asphyxia.

The effect of asphyxia on the endolymphatic sac d.c. potential (ESP) was examined in the guinea pig. Asphyxia was caused for 1.5 min by stopping the respirator. The ESP decreased in amplitude during asphyxia. After the termination of asphyxia the ESP showed a diphasic recovery pattern. When respiration was resumed, the ESP decreased again following a transient recovery. Thereafter, the ESP showed a gradual recovery. beta-blocker (propranolol) inhibited a temporary decrease in the ESP after the resumption of respiration, but not alpha-blocker (phentolamine). The result indicates that the ESP decrease after the resumption of respiration is induced by beta-adrenergic action.

The effect of acetazolamide on the endolymphatic sac DC potential.

The effect of acetazolamide known as a potent carbonic anhydrase inhibitor of the endolymphatic sac DC potential (ESP) was examined in the guinea pig. Intravenous administration of acetazolamide produced a dose-dependent decrease in ESP amplitude at doses of less than 10 mg/kg. The effect of acetazolamide on ESP was saturated at doses exceeding 10 mg/kg. The maximum reduction of ESP induced by acetazolamide was approximately 50% of the original ESP amplitude. The results suggest that carbonic anhydrase is involved in the generation of ESP.

Effect of angiotensin II on the endolymphatic sac direct current potential.

The effect of angiotensin II (ANG II), known as a potent pressor hormone, on the endolymphatic sac direct current potential (ESP) was examined. Intravenous administration of ANG II produced a reversible decrease in the ESP at doses exceeding 0.1 microgram/kg. The effect of ANG II on the ESP was dose-dependent at doses of less than 3 micrograms/kg with a saturated effect at doses of more than 3 micrograms/kg. The maximum reduction of the ESP produced by ANG II was 33.8 +/- 2.9% (m +/- SE, n = 6) of the original amplitude. The action of ANG II on the ESP was blocked by propranolol (beta-adrenergic antagonist). The mechanism underlying the action of ANG II on the ESP is discussed.

Absorption of horseradish peroxidase in the endolymphatic sac: ultrastructural cytochemistry using a new electrophoretic technique.

Using a newly developed injection technique, the absorption of horseradish peroxidase (HRP) in the endolymphatic sac (ES) of the guinea pig was examined by light and electron microscopy. HRP (molecular weight: 40,000; molecular diameter: about 5-nm) was directly injected into the lumen of the ES by electrophoresis after the recording of a direct current potential in the ES lumen. Both the macrophages floating in the ES lumen and the epithelial cells in the intermediate portion of the ES absorbed intraluminal HRP. The macrophages internalized the intraluminal HRP at a higher rate than the epithelial cells, suggesting that macrophages play a major role in macromolecular absorption in the ES. It was considered that the macrophages took up intraluminal HRP by phagocytosis, while the epithelial cells of the intermediate portion took it up by pinocytosis. In contrast, the epithelial cells in the proximal portion of the ES absorbed little HRP. No penetration through the junctional complexes between epithelial cells was observed in either the intermediate or the proximal portion at any interval after the injection of HRP. This finding indicates that these junctional complexes are impermeable to intraluminal HRP.

[Appropriate administration of granulocyte colony stimulating factor for malignant lymphoma of the head and neck].

The effects of granulocyte colony stimulating factor (G-CSF) were evaluated in 9 patients with malignant lymphoma of the head and neck. The effects of 31 cycles of cytotoxic chemotherapy were treated with G-CSF. G-CSF was given by one of the following three routes: 1) administration before or with cytotoxic chemotherapy, 2) administration after cytotoxic chemotherapy with leukocyte counts of more than 2000/mm3, and 3) administration after leukocyte counts had dropped to less than 2000/mm3. The first group consisted of one cycle of CHOP therapy and 2 cycles of VAMA therapy. The second group consisted of 10 cycles of CHOP therapy. The third group consisted of 13 cycles of CHOP therapy and 5 cycles of VAMA therapy. Leukocyte nadirs occurred on around day 14 for CHOP therapy and around day 21 for VAMA therapy without G-CSF treatment. In the first group, the leukocyte nadirs occurred earlier with G-CSF treatment. Additional G-CSF treatments were given in two of the three cycles. In the second group, the leukocyte counts did not drop below 2000/mm3 in three of the ten cycles. Additional G-CSF treatments were given in five of the remaining seven cycles. The two other cycles went without additional treatment. The mean volume of G-CSF was 305 +/- 86 micrograms. In the third group, the leukocyte counts increased to more than 2000/mm3 immediately after G-CSF administration in CHOP therapy. The mean volume was 227 +/- 78 micrograms, significantly less than that of the second group. The leukocyte counts also exceeded 2000/mm3 3-5 days after G-CSF administration in 5 cycles of VAMA therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Roles of tandem-pore K+ channels in plants - a puzzle still to be solved.

The group of voltage-independent K(+) channels in Arabidopsis thaliana consists of six members, five tandem-pore channels (TPK1-TPK5) and a single K(ir)-like channel (KCO3). All TPK/KCO channels are located at the vacuolar membrane except for TPK4, which was shown to be a plasma membrane channel in pollen. The vacuolar channels interact with 14-3-3 proteins (also called General Regulating Factors, GRFs), indicating regulation at the level of protein-protein interactions. Here we review current knowledge about these ion channels and their genes, and highlight open questions that need to be urgently addressed in future studies to fully appreciate the physiological functions of these ion channels.