14-3-3ζ-Mediated Stimulation of Oxidative Phosphorylation Exacerbates Oxidative Damage Under Hypothermic Oxygenated Conditions in Human Renal Tubular Cells (HK-2).

Cellular survival and death are at least partially regulated by the phosphorylation of proteins. A chaperon protein, 14-3-3ζ, regulates the activity of many proteins by covering the phosphorylation site within a 14-3-3 binding motif. Therefore, regulation of 14-3-3ζ activity may affect the fate of cells subjected to cold preservation and/or hypothermic oxygenated conditions. The present study assessed whether 14-3-3ζ protects cells from hypothermic oxygenation-induced injury and clarified its role in mitochondrial functions. Human renal tubular cell line HK-2 or 14-3-3ζ-overexpressed HK-2 (ζHK-2) cells were subjected to 72 hours of normoxic cold preservation in UW solution with or without antioxidants and hydroperoxides. Cellular death, adenosine triphosphate (ATP) content, and MTT catabolism were evaluated. Deferoxamine treatment reduced cellular death and augmented ATP content in both cell types. These indices were higher in ζHK-2, regardless of deferoxamine treatment. Exposure to hydroperoxides did not affect cellular death in either cell type, whereas hydroperoxide supplementation significantly reduced ATP content, except for low-dose hydrogen peroxide in HK-2 cells. MTT assay at normal state showed higher values in ζHK-2 cells, whereas it was impaired by hydroperoxides in both cell types. These results suggest that accumulation of hydroperoxides as a byproduct of the augmented oxidative phosphorylation by 14-3-3ζ overexpression causes mitochondrial dysfunction. In conclusion, despite possessing many potentially protective functions, 14-3-3ζ exacerbates cellular injury under hypothermic oxygenated conditions. 14-3-3ζ accelerates mitochondrial functions together with iron-dependent oxidative damage. Although further investigations are necessary, upregulation of 14-3-3ζ could be a method to maintain mitochondrial function under hypothermic oxygenated conditions, as shown in hypothermic machine preservation of renal grafts, when appropriate antioxidant treatment is administered.

Allergen-specific basophil reactivity exhibits daily variations in seasonal allergic rhinitis.

It remains poorly understood how symptoms in allergic rhinitis are most severe during overnight or early in the morning. The circadian clock consisting of a network of several 'clock genes' including Clock drives daily rhythms in physiology. This study showed that allergen-induced surface CD203c expression on basophils in seasonal allergic rhinitis caused by Japanese cedar pollen exhibited a time-of-day-dependent variation associated with temporal variations in canonical circadian clock gene expression. We also found that bone-marrow-derived basophils (BM basophils) generated from wild-type mice exhibited a time-of-day-dependent variation in IgE-mediated IL-4 and histamine production, which was not observed in BM basophils generated from Clock-mutated mice. Therefore, allergen-specific basophil reactivity shows daily variations depending on the circadian clock activity in basophils, which could partly explain temporal symptomatic variations in allergic rhinitis. Additionally, circadian variations in CD203c expression should be considered for interpretation of this biomarker in clinical research.

High-dose rabeprazole-amoxicillin versus rabeprazole-amoxicillin-metronidazole as second-line treatment after failure of the Japanese standard regimen for Helicobacter pylori infection.

BACKGROUND: There is currently no optimal second-line treatment after failure of Helicobacter pylori triple therapy. AIM: To determine effective salvage therapy after failure of lansoprazole-amoxicillin-clarithromycin. METHODS: After failure of lansoprazole-amoxicillin-clarithromycin 123 out-patients were randomized to receive either 2-week rabeprazole (20 mg b.d.) + amoxicillin (1000 mg b.d.) (RA group) or 1-week rabeprazole (10 mg b.d.) + amoxicillin (750 mg twice b.d.) + metronidazole (250 mg b.d.) (RAM group). Eradication was assessed by the 13C-urea breath test. We also evaluated cytochrome p450 (CYP) 2C19 genotype status, determined by polymerase chain reaction - restriction fragment length polymorphism, and susceptibility to clarithromycin and metronidazole. RESULTS: On an intention-to-treat basis, H. pylori infection cure was achieved in 37 of 63 (59%) patients in the RA group and in 49 of 60 (82%) patients in the RAM group. Per protocol-based eradication rates in the RA and RAM groups were 66% (37/56) and 88% (49/56), respectively. In both analytic sets there were significant differences between the treatment groups (P < 0.01 in each). Mild adverse events were observed in eight and five patients from the RA and RAM groups, respectively. Genetic predisposition of CYP2C19 and antibiotic resistance did not influence the treatment outcome either regimen. CONCLUSIONS: The rabeprazole + amoxicillin + metronidazole therapy yielded satisfactory results. In contrast, the cure rate in high-dose rabeprazole + amoxicillin was below an acceptable level.

Nomogram for individualizing supplementary iron doses during erythropoietin therapy in haemodialysis patients.

AIMS: An adequate iron supplement is crucial not only for prompt erythropoiesis but also for the restoration of tissue iron reserves in haemodialysis patients receiving recombinant human erythropoietin (r-HuEPO). An attempt was made to establish a comprehensive nomogram that allows individualization of intravenous (i.v.) iron doses according to patients' body weights, the initial status of tissue iron reserves and desired increases in haemoglobin levels. MATERIALS AND METHODS: Clinical and laboratory data retrieved from 95 haemodialysis patients who received r-HuEPO with or without iron supplements for at least 12 weeks were used to construct the nomogram. It was assumed that the administered iron was either incorporated into newly synthesized haemoglobin and tissue iron reserves or eliminated from the body at a constant rate. Tissue iron reserves of the patients were estimated by serum ferritin levels using van Wyck's equation (Kidney Int., 1989, 35, 712). The rate of iron loss in the patients was estimated by the data obtained from 15 of the above patients who exhibited stable haemoglobin levels but decreases in serum ferritin levels with no iron supplements. The validity of the equation was ascertained by comparing the measured serum ferritin levels at the end of r-HuEPO therapy and those predicted by the nomogram. The proposed nomogram was then validated prospectively in 24 haemodialysis patients to determine whether the nomogram-recommended iron doses would increase both haemoglobin and serum ferritin levels within 12 weeks. RESULTS: The mean (+/-SD) iron loss of haemodialysis patients was calculated to be 10.5 +/- 7.4 mg/week. There was a significant correlation (r=0.77, P < 0.001) between the measured serum ferritin levels, an index of tissue iron reserves, at the end of r-HuEPO therapy and those predicted by the equations used for formulating the nomogram. The prospective study indicated that the nomogram-recommended supplementary iron doses attained haemoglobin and serum ferritin levels of > 95 g/L and >100 microg/L in 79 and 50% of the patients, respectively, within 12 weeks. CONCLUSION: The present nomogram may be useful for individualizing supplementary i.v. iron doses for haemodialysis patients undergoing r-HuEPO therapy.

[Therapeutic effects of beclomethasone dipropionate enemas on colon damage of inflammatory bowel disease model rats].

The effects of beclomethasone dipropionate (BDP) enemas on ulcerative colitis were investigated by administrating BDP enemas to Fischer male rats with an inflammatory bowel disease induced by 2,4,6-trinitrobenzensulfonic acid (TNB). After administration of a TNB ethanol solution to rats, diarrhea and melena were found in all rats, and the wet tissue weights of the colons in the rats increased by erosion and thickness of epithelial mucous membranes with edema. BDP enemas were administered to the rats one time a day at a dose of 20 or 50 micrograms of BDP for 4 or 11 days from the day 3 after TNB treatment, respectively. After dosing of BDP, the rate of rats developing diarrhea and melena decreased more with time in comparison with that of BDP-free rats, and the symptoms of all rats developing the diseases were improved on the day 4 after administration of a dose of 50 micrograms of BDP. A dose dependent recovery in the wet tissue weights and scores of damages, and the myeloperoxidase (MPO) activity in the tissue were found at the early days (until the day 4). However, their measurements on the day 11 at each dose of BDP were not different from those of control rats significantly, as the damages in the colons of the control rats were recovered naturally with time. In conclusion, the clinical usefulness of BDP enemas was supported by elucidating the dose dependent effect of BDP at the early days in the model rats with an inflammatory bowel disease induced by TNB.

Inhibition of myo-inositol transport causes acute renal failure with selective medullary injury in the rat.

Myo-inositol is a major compatible osmolyte in the renal medulla that is accumulated under hypertonic conditions via the Na+/myo-inositol cotransporter (SMIT). We have recently reported that SMIT is predominantly present in the thick ascending limb of Henle (TAL) and is strongly induced by acute NaCl loading, suggesting an important role of myo-inositol in this nephron segment. In the present study, we sought to examine in vivo effects of inhibition of myo-inositol transport using a transport inhibitor, 2-O, C-methylene-myo-inositol (MMI). Intraperitoneal injection of MMI caused acute renal failure in the rats. Serum creatinine and urea nitrogen were significantly increased 12 hours after MMI injection. Morphologic study revealed that the tubular cells were extensively injured in the outer medulla. A considerable number of the tubular cells were injured in the cortex as well. Immunohistochemical study for Tamm-Horsfall protein (THP), which was used for identification of the TAL cells, showed that THP-positive cells were predominantly injured. The tubular injury apparently appeared to worsen when high concentration of NaCl was injected with MMI. Administration of myo-inositol prevented acute renal failure and improved the tubular injury after MMI injection. Furthermore, supplementation of betaine, another osmolyte in the TAL cells, partially prevented the toxic effects of MMI. These results suggest that myo-inositol play a crucial role in the TAL regarding osmoregulation of the cells.

Expression of Na+/myo-inositol cotransporter mRNA in normal and hypertonic stress rat eyes.

We studied the localization of Na+/myo-inositol cotransporter (SMIT) mRNA in normal and hypertonic stress rat eyes by in situ hybridization histochemistry using cRNA probes. SMIT mRNA signals were observed in the iris-ciliary body, the lens epithelial cells, and the ganglion cell layer and the inner nuclear layer of the retina. There was a rapid increase on SMIT mRNA in the retina of hypertonic stress rats compared with control rats. These findings suggest that Na+/myo-inositol cotransporter gene expression is osmotically regulated in vivo to protect retinal neuronal function against hypertonic stress.

A case of generalized pustular psoriasis followed by bullous disease: an atypical case of bullous pemphigoid or a novel bullous disease?

We describe a 31-year-old Japanese woman with generalized pustular psoriasis treated with PUVA who subsequently developed a bullous disease. Throughout the disease course, there was no phase of psoriasis vulgaris. Although several reports describe coexistence of psoriasis vulgaris and bullous disease such as bullous periphigoid, coexistence of generalized pustular psoriasis without any phase of psoriasis vulgaris and bullous disease is rare. As for the bullous disease, direct immunofluorescence study showed IgG and C3 deposition along the basement membrane zone. Indirect immunofluorescence disclosed IgG antibasement membrane zone antibodies. Indirect immunofluorescence on 1 mol/l sodium chloride-split skin demonstrated linear IgG staining almost exclusively on the dermal side of the split. Western immunoblot analysis revealed that the antibody was directed to neither epidermolysis bullosa acquisita antigen nor bullous pemphigoid antigens. Considering the unusual clinical course, we suspect the possibility of a novel autoimmune blistering disease.

Cloning and expression of a bovine glutamate transporter.

We have isolated a 3845 base-pair cDNA (BNGLUAS) encoding a bovine glutamate transporter (bovine GLAST) by screening a bovine retina cDNA library with an oligonucleotide probe corresponding to conserved regions of known glutamate transporters. The cDNA sequence predicted a protein of 542 amino acids and displayed 96% and 97% amino acid identity with the rat GLAST/GluT-1 and human GLAST, respectively. Expression of the bovine GLAST in Xenopus oocytes revealed Na(+)-dependent [14C]L-glutamate uptake and electrogenic glutamate uptake.

Vigintiphobia, unbound bilirubin, and auditory brainstem responses.

OBJECTIVE: The management of nonhemolytic hyperbilirubinemia in term newborns is controversial. To evaluate the usefulness of serum unbound bilirubin concentrations (UBCs) in the management of hyperbilirubinemia, we compared the concentrations with abnormal auditory brainstem responses (ABRs). METHODS: ABRs and serum UBCs in 37 hyperbilirubinemic term newborns (total bilirubin concentrations [TBCs] > or = 20 mg/dL and direct bilirubin concentrations < 2 mg/dL) were measured before treatment with either phototherapy or exchange transfusions. Eight of these newborns had blood incompatibilities. These hyperbilirubinemic newborns were divided into three groups according to the findings of ABR: group A, normal ABR (n = 18); group B, prolonged latency of wave I only (n = 8); and group C, prolonged interpeak latency of wave I-III/I-V and/or poor amplitude (n = 11). RESULTS: The peak TBC was significantly different between groups A and C (22.8 +/- 2.2 mg/dL and 25.4 +/- 2.5 mg/dL, respectively; P < .05), though there were no differences between groups A and B and between groups B and C. The peak UBCs in groups B (1.27 +/- 0.7 micrograms/dL) and C (1.34 +/- 0.37 micrograms/dL) were significantly higher than in group A (0.78 +/- 0.26 microgram/dL) (P < .05 and P < .01, respectively), though there was no significant difference in the peak UBC between groups B and C. Abnormal ABR findings were more clearly associated with the level of UBC at 1.0 microgram/dL than that of TBC at 23 mg/dL by multiple logistic regression analysis (odds ratio = 16.6, P = .0026, vs 4.2, P = .1272). CONCLUSIONS: These results suggest that measuring UBC may help in evaluating the possible risk of bilirubin encephalopathy in full-term newborns when there is vigintiphobia (fear of 20).

Enzyme immunoassay of gonadotropin releasing hormone in the canine hypothalamus and plasma using monoclonal antibodies.

A monoclonal antibody against gonadotropin releasing hormone (GnRH)-BSA was used in the development of a sensitive enzyme immunoassay of GnRH in the canine hypothalamus and in plasma. The method had a limit of detection of 4 pg per sample. The intra- and interassay coefficients of variation were < 7.3% and < 11.0%, respectively. Acid extracts of hypothalamus produced a dose response curve which was parallel to that obtained with the synthetic GnRH standard. Checking cross reactivity of various fragments of GnRH revealed that the antibody was formed predominantly against the C-terminal end of GnRH. Thyrotropin-releasing hormone and other hypothalamic hormones did not appear to influence the assay. In male dogs, hypothalamic GnRH levels increased with age up to 4 months, then fell to a plateau from 6 months to 2 years. The time required for a 50% reduction in plasma levels following intravenous administration of synthetic GnRH to five adult male dogs was 2.2 +/- 0.1 (SEM) min.

Time course and inhibition of saponin-induced hemolysis.

Hemolytic activities of 3 steroid saponins reached plateaus within 5 min, whereas those of 4 triterpenoid saponins did not within 60 min. Erythrocytes pretreated with a low concentration of tigogenin were resistant to hemolysis of some of these saponins, but those pretreated with hecogenin or tomatidine were as sensitive as non-treated erythrocytes. Therefore, the ketone group of hecogenin or the amino group of tomatidine would weaken the interactions between the erythrocytes and these sapogenins. Furthermore, incubations of these saponins with a small amount of cholesterol diminished the hemolytic activities.

Identification of a novel tumor-associated Mr 110,000 gene product in human gastric carcinoma cells that is immunologically related to carcinoembryonic antigen.

A novel gene product which is immunologically related to carcinoembryonic antigen (CEA) and constitutively expressed by six of eight human gastric carcinoma cell lines is described. The antigen was initially identified by the differential binding patterns of four monoclonal antibodies (MAbs) which recognize the putative Mr 180,000 CEA and/or the Mr 90,000 CEA-related gene product, NCA (normal cross-reacting antigen). Western blot analyses of partially purified membrane fractions prepared from Hs 746T gastric carcinoma cells identified an Mr 110,000 antigen. Northern blot analyses using CEA- and NCA-specific complementary DNA probes did not identify any specific CEA or NCA transcripts in polyadenylate-selected mRNA isolated from the Hs 746T cells. Likewise, a probe designed to hybridize with different CEA-related family members failed to identify a CEA-related message in the Hs 746T cells. Subsequent studies revealed that interferon-gamma (IFN-gamma) treatment substantially increased the level of expression of the Mr 110,000 antigen on the Hs 746T and five other gastric cell types that constitutively expressed the antigen. IFN-gamma treatment also de novo induced the expression of the Mr 110,000 antigen on the surface of GaCa gastric carcinoma cells. A high percentage of Hs 746T (i.e., greater than 85%) and GaCa (approximately 75%) gastric carcinoma cells expressed the Mr 110,000 antigen after IFN-gamma treatment; yet, neither cell type expressed CEA or NCA as measured by the binding of the anti-CEA MAb, COL-1, or B6.2, an anti-NCA MAb. In contrast to CEA and NCA, phosphatidylinositol phospholipase C treatment failed to release the Mr 110,000 antigen from the surface of the Hs 746T or IFN-gamma-treated GaCa cells, suggesting that membrane attachment of this novel antigen is not via a glycosyl-phosphatidylinositol anchor. Finally, primers that amplify the 420 base pairs of the immunoglobulin-like domain of CEA and NCA detected an appropriately sized product in untreated as well as IFN-gamma-treated GaCa cells using the polymerase chain reaction method. Thus, a potentially novel gene product coding for an Mr 110,000 antigen that is strongly upregulated by IFN-gamma has been identified in human gastric carcinoma cells. Immunologically, the antigen shares reactive epitopes with CEA and its related NCA gene product; however, Northern blot analyses, polymerase chain reaction, and phosphatidylinositol phospholipase C results suggest that the antigen may be, at best, a distant relative of the CEA gene family.

Metabolism of drugs in the eye. Menadione-dependent reduction of tertiary amine N-oxide by preparations from bovine ocular tissues.

As described previously, the microsomes and cytosol from bovine ciliary body exhibited a significant reductase activity toward tertiary amine N-oxide such as imipramine N-oxide when supplemented with menadione. In the present study, the menadione-dependent N-oxide reduction was further examined with preparations of bovine ocular tissues. The reduction of imipramine N-oxide occurred much more significantly when the microsomes and cytosols from bovine ciliary body were supplemented with both menadione and NAD(P)H, compared with menadione alone. The cytosolic menadione-dependent reduction, but not the microsomal one, was markedly inhibited by dicumarol, suggesting the involvement of DT-diaphorase in the reaction. Localization of the menadione-dependent N-oxide reductase activity in bovine ocular tissues indicated that the highest activity resided in the ciliary body, followed by retinal pigment epithelium-choroid, iris, retina and cornea. When the cytosol from bovine ciliary body was fractionated with ammonium sulfate, the distribution of the menadione-dependent N-oxide reductase activity in the resultant fractions was parallel, but roughly, to that of DT-diaphorase activity, supporting the assumption that the flavoenzyme was involved in the cytosolic menadione-dependent N-oxide reduction. We proposed a new mechanism for the metabolic reduction of tertiary amine N-oxide in the eye: Menadione is reduced to the corresponding diol by quinone-reducing enzymes and then tertiary amine N-oxide is reduced by the diol to the corresponding amine nonenzymatically.

Metabolism of drugs in the eye. Drug-reducing activity of preparations from bovine ciliary body.

Drug-metabolizing activities, especially the reductase activities towards N-oxide, hydroxamic acid, sulfoxide and nitro compounds were comparatively examined with bovine ciliary body. As described previously, the cytosol from the ocular tissue exhibits the nicotinamide N-oxide reductase activity when supplemented with 2-hydroxypyrimidine, an electron donor of aldehyde oxidase. When the cytosol was fractionated with ammonium sulfate, followed by assays of aldehyde oxidase and nicotinamide N-oxide reductase activities in each fraction, the distribution of aldehyde oxidase activity in the resultant ammonium sulfate fractions was nearly parallel to that of nicotinamide N-oxide reductase activity. Furthermore, reductase activities towards drugs such as sulfoxide, hydroxamic acid and nitro compounds were observed with the cytosol in the presence of 2-hydroxypyrimidine or N1-methylnicotinamide. In general, these reductase activities of the fraction were markedly inhibited by menadione, an inhibitor of aldehyde oxidase. These results suggest that aldehyde oxidase present in ciliary body plays an important role in the reduction of a variety of xenobiotics in mammalian eyes. However, in the case of imipramine N-oxide, its reduction in the ocular tissue appears to be more readily catalyzed by a menadione-linked enzyme different from aldehyde oxidase.

Coexistence of substance P and neurotensin-like peptides in single neurons of the rat hypothalamus.

The coexistence of substance P with neurotensin-like immunoreactivity in certain neurons of the hypothalamus were demonstrated by the double immunofluorescence method. Substance P and neurotensin-like immunoreactivity coexisted within single neurons of some hypothalamic areas such as the medial preoptic area, perifornical area, anterior hypothalamic area, lateral hypothalamic area, periventricular nucleus and posterior hypothalamic nucleus, although they did not coexist in the majority of immunoreactive cells.

Coexistence of substance P- and enkephalin-like peptides in single neurons of the rat hypothalamus.

The distribution of substance P- and enkephalin-like immunoreactivity in single cells were examined by the double immunofluorescence method. Substance P- and leucine-enkephalin-like compounds coexisted within individual neurons of some hypothalamic areas such as the medial preoptic area, anterior hypothalamic area, perifornical area, lateral hypothalamic area, premammillary nuclei and posterior hypothalamic nucleus, although they did not coexist in the majority of immunoreactive cells.

Nicotinamide N-oxide reductase activity in bovine and rabbit eyes.

The nicotinamide N-oxide reductase activity of a variety of ocular tissues was investigated. The 9,000g supernatant of ciliary body, retinal pigment epithelium-choroid, iris, retina and cornea, but not lens, exhibited reductase activity under anaerobic conditions when supplemented with 2-hydroxypyrimidine, an electron donor of aldehyde oxidase. Among these tissues, the highest activity was observed with ciliary body. When the 9,000g supernatant of ciliary body was fractionated, the 2-hydroxypyrimidine-linked reductase activity was mainly associated with the cytosolic fraction and was markedly inhibited by menadione, an inhibitor of aldehyde oxidase. Similarly, in the presence of 2-hydroxypyrimidine, the cytosolic fraction of rabbit ciliary body exhibited nicotinamide N-oxide reductase activity which was susceptible to inhibition by menadione. These facts strongly suggest that aldehyde oxidase present in mammalian eyes is involved in the reduction of nicotinamide N-oxide to nicotinamide.