Ischemia-reperfusion injury of the cochlea: effects of an iron chelator and nitric oxide synthase inhibitors.

Release of free iron from cellular stores and activation of nitric oxide synthase (NOS) has been implicated in a wide variety of cochlear injuries. In order to evaluate the effects of deferoxamine (a iron chelator), 3-bromo-7-nitroindazole (a relatively selective neuronal NOS (nNOS) inhibitor) or aminoguanidine (a relatively selective inducible NOS (iNOS) inhibitor) on the post-ischemic cochlear dysfunction, albino guinea pigs were subjected to 30 min ischemia, and the threshold shifts of the compound action potential (CAP) from pre-ischemic values were compared with those of control animals 4 h after the onset of reperfusion. A statistically significant reduction in the post-ischemic CAP threshold shift was observed in the animals treated with deferoxamine or 3-bromo-7-nitroindazole. However, aminoguanidine did not affect the post-ischemic CAP threshold shift. These results suggest that free iron and nNOS play deleterious roles in the cochlear injury induced by transient ischemia.

Effect of three flavonoids, 5,7,3',4'-tetrahydroxy-3-methoxy flavone, luteolin, and quercetin, on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophil.

The effect of three flavonoids, 5,7,3',4'-tetrahydoxy-3-methoxy flavone (THMF), luteolin, and quercetin, on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils were investigated. When the cells were preincubated with these flavonoids, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly suppressed, showing a dependence on amounts of the flavonoid. The suppressing effect of the flavonoid was THMF > luteolin > quercetin. These flavonoids also suppressed the superoxide generation induced by phorbol 12-myristate 13-acetate. In this case also, THMF was more effective than luteolin and quercetin. On the other hand, the superoxide generation induced by arachidonic acid was markedly suppressed by quercetin. The suppressing effect was quercetin >> THMF > luteolin. THMF, luteolin, and quercetin significantly suppressed tyrosyl phosphorylation of 80.1-, 58.0-, and 45.0-kDa proteins in fMLP-treated human neutrophils. The suppression depended on the concentration of the flavonoids, and the inhibition of tyrosyl phosphorylation was in parallel to that of the fMLP-induced superoxide generation, respectively. While luteolin and quercetin showed a weak hemolytic activity at 2.5 mM, THMF showed almost no hemolytic activity even at 5 mM, suggesting an advantage of THMF for its clinical use.

Medial septal microinfusion of scopolamine disrupts hippocampal activity and trace jaw movement conditioning.

This study investigated the effects of microinfusion of scopolamine into the medial septum (MS Scp) on hippocampal neurophysiology and learning of the rabbit's classically conditioned jaw movement response. The percentage of hippocampal theta slow waves (2-8 Hz) decreased after drug infusion in the MS Scp group but did not change in control groups that received infusion of saline into the MS or scopolamine into the cortex. Unit recordings from the MS Scp group showed significantly smaller conditioning-related hippocampal neural responses than seen in controls, and during conditioning, rabbits in the MS Scp group took significantly longer to reach learning criterion than either control group. Thus, the neural and behavioral impairments previously reported for systemic muscarinic blockade were reproduced by microinfusions restricted to the medial septal nucleus.

Scopolamine disruption of behavioral and hippocampal responses in appetitive trace classical conditioning.

Twelve young rabbits (3-6 months; Oryctolagus cuniculus) were classically conditioned in a trace jaw movement paradigm (300 ms tone, 450 ms trace, 200 ms intraoral water) after implantation of electrodes into area CA1 of dorsal hippocampus. Rabbits were divided into two groups and administered either 0.5 mg/kg scopolamine hydrobromide (HBr) or 0.5 mg/kg scopolamine methylbromide (MBr) subcutaneously before daily training sessions. Rabbits given HBr took significantly more trials to reach a behavioral criterion of eight conditioned responses in any nine consecutive trials than rabbits given MBr (P = 0.03). Conditioned, but not unconditioned, rhythmic jaw movement responses of the HBr group were of a lower frequency (Hz) than those of MBr rabbits (P = 0.02). The magnitude of hippocampal conditioning-related responses across the first 3 days of training was significantly smaller for HBr rabbits than for MBr rabbits (P = 0.02). These effects of central cholinergic blockade are similar to those reported for undrugged aging rabbits trained in the same paradigm (Seager MA, Borgnis RL, Berry SD. Neurobiol. Aging 1997;18(6):631 639).

Localization of xenobiotic-responsive element binding protein in rat hepatocyte nuclei after methylcholanthrene administration as revealed by in situ Southwestern hybridization.

Xenobiotic-responsive element binding protein (XRE-BP), a heterodimer of aryl hydrocarbon receptor (AhR) and its nuclear translocator (Arnt), regulates the transcription of cytochrome P-450 1A1 gene (CYP1A1) through XRE in response to xenobiotic inducers. For a better understanding of the regulatory mechanism of CYP1A1 through XRE, localization of XRE-BP was examined in liver sections or isolated hepatocyte nuclei from control and 3-methylcholanthrene (MC)-treated rats by in situ Southwestern hybridization, using synthetic XRE as a probe, and was observed by confocal laser scanning microscopy and electron microscopy. Gel mobility shift assay and competitive binding assay showed specificity of the synthetic XRE probe. XRE-BP was exclusively localized in hepatocyte nuclei in liver sections from animals 3 hr after MC injection, whereas the protein was absent in hepatocyte cytoplasm in MC-treated animals and in hepatocyte nuclei and cytoplasm in control animals. In isolated hepatocyte nuclei, XRE-BP began to accumulate in the central region between 0.5 and 3 hr, showed a peak between 3 and 6 hr, decreased gradually between 6 and 72 hr, and disappeared at 72 hr after MC injection. The protein was scarce in peripheral and nucleolar regions of the nucleus. Therefore, XRE-BP is formed in the nuclei of hepatocytes after MC stimulation. In addition, XRE-BP was found in isolated hepatocyte nuclei from control animals after preincubation with cytoplasmic lysate from MC-treated animals, although the protein was absent in the nuclei before the preincubation. These findings strongly suggest that AhR translocates from hepatocyte cytoplasm to the nucleus and forms XRE-BP in the nucleus after MC stimulation.

Quantitative analysis of endoplasmic reticulum and cytochrome P-450 in hepatocytes from rats injected with methylcholanthrene.

To examine whether the smooth endoplasmic reticulum (SER) proliferates in hepatocytes from animals treated with methylcholanthrene (MC) frequently used as an inducer for the enzymes of the microsomal mono-oxygenase system, we estimated the area of (smooth and rough) ER per unit cytoplasmic volume by morphometry in periportal, midzonal and perivenular hepatocytes from rats injected with 25 mg/kg MC once a day for 3 days. In addition, immunostaining intensity of major MC-inducible cytochrome P-450 (P-450) forms (1A1/1A2) and total P-450 content in the cytoplasm of hepatocytes in the three zones were measured by microphotometry to ascertain whether P-450 is sufficiently induced in each sublobular zone by the administration. In spite of significant increase in the staining intensity of P-450 1A1/1A2 and amount of total P-450, the proliferation of SER (and RER) did not occur in the three-zone hepatocytes from rats injected with MC. In perivenular hepatocytes, constitutive forms of P-450 other than 1A1/1A2 decreased (to 10%) instead of marked increase in P-450 1A1/1A2 (about 20 times), while the constitutive forms decreased to 50% in midzonal hepatocytes and remained unchanged in periportal hepatocytes after MC administration. In addition, the present results show divergence between biochemical and immumohistochemical results previously reported on MC-inducible P-450 after MC administration to be due primarily to a curvilinear relationship between content and intensity.

Emerin, deficiency of which causes Emery-Dreifuss muscular dystrophy, is localized at the inner nuclear membrane.

X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD) is an inherited muscle disorder characterized by the clinical triad of progressive wasting of humero-peroneal muscles, early contractures of the elbows, Achilles tendons and postcervical muscles, and cardiac conduction block with a high risk of sudden death. The gene for EDMD on Xq28 encodes a novel protein named emerin that localizes at the nuclear membrane of skeletal, cardiac and smooth muscles and some other non-muscle tissues. To investigate a possible physiological role for emerin, we examined the ultrastructural localization of the protein in human skeletal muscle and HeLa cells, using ultrathin cryosections. We found that the immune-labeled colloidal gold particles were localized on the nucleoplasmic surface of the inner nuclear membrane, but not on the nuclear pore. Emerin stayed on the cytoplasmic surface of the nuclear lamina, even after detergent treatment that solubilizes membrane lipids and washes out membrane proteins. These results suggest that emerin anchors at the inner nuclear membrane through the hydrophobic stretch, and protrudes from the hydrophilic region to the nucleoplasm where it interacts with the nuclear lamina. We speculate that emerin contributes to maintain the nuclear structure and stability, as well as nuclear functions, particularly in muscle tissues that have severe stress with rigorous contraction-relaxation movements and calcium flux.

Relationship between immunostaining intensity and antigen content in sections.

We studied the relationship between staining intensity of immunohistochemical reaction and antigen content in sections. Alpha-fetoprotein (AFP) and albumin in sections cut from livers of newborn, 5-, 10-, 20-, and 60-day-old rats were examined as examples. First, we compared average immunostaining intensity (sum of specific absorbance in pixel/number of pixels) measured by image processing (IP), with antigen content measured by immunochemical assay to determine whether the intensity is proportional to antigen content. The intensity of AFP was proportional to the antigen content, whereas that of albumin was not. Subsequently, the antigen preservation test was carried out to determine whether the intensity was decreased by fixation and, if so, which type of decrease (proportional or disproportionate) occurred. Thereafter, antigen content in the same portion in the same immunostained section was measured by the microphotometric (MP) method followed by the IP method, because the MP method gives a low average antigen content when a decrease in antibody binding occurs in sections, whereas the average antigen content measured by the IP method is unchanged. The intensity of AFP decreased primarily by a proportional decrease in antigenicity during fixation. However, the intensity of albumin decreased not only by a proportional decrease during fixation but also by a disproportionate reduction in antibody binding during immunostaining or before fixation. The results indicate that AFP content in sections is measurable by quantitative immunohistochemical methods, whereas albumin content is not.

Peri- and postnatal changes in reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase content in hepatocytes of rats.

To study the process of the expression of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase (EC 1.6.2.4) in the liver during development, the amount of enzyme in the cytoplasm of periportal and perivenular hepatocytes in sections cut from livers of male rats was measured during peri- and postnatal growth by quantitative immunohistochemistry with a video image processor. In livers of 19-day-old foetuses, the reductase content in the cytoplasm of periportal and perivenular hepatocytes was 0.16 microM and 0.20 microM, respectively. From the 19th day of gestation to 5 days after birth, the enzyme content increased markedly in the cytoplasm of periportal (288%) and perivenular hepatocytes (301%). Subsequently, the content in the cytoplasm of periportal hepatocytes increased slightly (46%) from 5 to 20 days of age, remained unchanged from 20 to 45 days of age, and increased slightly (15%) from 45 to 90 days of age. However, the content in the cytoplasm of perivenular hepatocytes increased progressively (125%) between 5 and 90 days of age. Thus, the amount of cytochrome P-450 reductase increases markedly in periportal and perivenular hepatocytes during the perinatal period, and subsequently the enzyme content increases gradually in periportal hepatocytes and progressively in perivenular hepatocytes. The present results also suggest that the divergence between cytochrome P-450 expression and the cytochrome P-450-dependent drug metabolic activity in hepatocytes during the perinatal period, found in previous studies, can be attributed to a low cytochrome P-450 reductase density in the membrane of endoplasmic reticulum of periportal and perivenular hepatocytes.

Preparation of liposomes that mimic the membrane of endoplasmic reticulum of rat hepatocytes.

To examine the interaction between biomembranes and membrane-bound proteins, large unilamellar liposomes have been required. In the present study, we prepared liposomes from a mixture of phospholipids having a phospholipid composition similar to that in the endoplasmic reticula (microsomes) of rat hepatocytes by eight different methods. The resulting liposomes were examined by a combination of the freeze-fracture-replica procedure with biochemical methods. The freeze-thawing method of Pick (1981) gave the best results; large unilamellar liposomes that mimic the membrane of endoplasmic reticulum were obtained. Liposomes made by this method are thus suitable for analysis of the interaction between the endoplasmic reticulum membrane and membrane-bound proteins.

Endoplasmic reticulum proliferates without an increase in cytochrome P-450 in hepatocytes of mice treated with phenobarbital and cobalt chloride.

To determine whether endoplasmic reticulum (ER) proliferation in hepatocytes after phenobarbital (PB) administration relates closely to cytochrome P-450 (P-450) increase, we have measured the amount of total P-450 per unit cytoplasmic volume (P-450 content) by microphotometry and estimated the area of ER per unit cytoplasmic volume (ER area) by morphometry in periportal, midzonal, and perivenular hepatocytes of mice injected daily with PB (100 mg/kg), or with PB (100 mg/kg) plus cobalt chloride (50 mg/kg) for three days. After injection of PB, the P-450 content and ER area increased in hepatocytes of the three sublobular zones. In mice treated with PB plus cobalt chloride, however, the ER area increased, but the P-450 content decreased or remained unchanged in hepatocytes of the three zones. We conclude that cobalt chloride inhibits the increase in total P-450 but has no effect on the proliferation of ER of hepatocytes in mice treated with PB, indicating a dissociation of ER proliferation and P-450 increase after administration of PB.

Location of light-repressible, small GTP-binding protein of the YPT/rab family in the growing zone of etiolated pea stems.

YPT/rab proteins are ras-like small GTP-binding proteins that serve as key regulators of vesicular transport. The mRNA levels of two YPT/rab genes in pea plants are repressed by light, with the process mediated by phytochrome. Here, we examined the mRNA expression and the location of the two proteins, pra2- and pra3-encoded proteins, using monoclonal antibodies. The pra2 and pra3 mRNA levels were highest in the stems of dark-grown seedlings. The corresponding proteins were found in the cytosol and the membranes of the stems. Most of the pra2 protein was in the growing internodes, especially in the growing region, but the pra3 protein was widespread. These results suggest that the pra2 protein is important for vesicular transport in stems, possibly contributing to stem growth in the dark, and that the pra3 protein is important for general vesicular transport. The amounts of pra2 and pra3 proteins decreased with illumination. The decrease in these proteins may be related to the phytochrome-dependent inhibition of stem growth that occurs in etiolated pea seedlings.

Isolation, characterization and expression of cationic peroxidase isozymes released into the medium of cultured tobacco cells.

Three glycoproteins of 34, 38 and 40 kDa were isolated from the spent medium of suspension-cultured tobacco cells. The 38-kDa and 40-kDa proteins were highly cationic peroxidases with indistinguishable enzymic properties but their structural difference was confirmed by sequence analysis of the amino-terminal regions and the recognition specificity of monoclonal antibodies. The 34-kDa protein was a moderately cationic peroxidase with enzymic properties quite different from those of the 38-kDa and 40-kDa enzymes. They were undetectable in the spent medium during the cell-proliferation phase but became abundant in the medium during the cell-expansion phase. This was confirmed quantitatively with the 40-kDa protein using the 40-kDa-specific monoclonal antibody. The mRNA expression for 40-kDa protein was at a constant basal level in the cell-proliferation phase but increased in the cell-expansion phase.

Measurement of NADPH-cytochrome P-450 reductase content in rat liver sections by quantitative immunohistochemistry with a video image processor.

We have a quantitative light microscopic immunohistochemical method using video image processing. First, an antigen (NADPH-cytochrome P-450 reductase) content in homogenates of livers of rats was measured by enzyme immunoassay. Then frozen sections from rat livers were incubated with the anti-NADPH-cytochrome P-450 reductase antibody under saturation conditions by the indirect immunoperoxidase method. Subsequently, relative staining intensities in small portions and those in wide areas in the sections were measured with a video image processor. Finally, the resulting relative values obtained from the small portions were converted into absolute NADPH-cytochrome P-450 reductase contents using the results of enzyme immunoassay and the average relative staining intensity obtained from the wide areas in the sections. The reductase content in sections from rat livers measured by the image processing method coincided with the content measured by the microphotometric method using a nitrocellulose model system. The present image processing method is applicable to measurement of contents of antigens that can not be immobilized in model systems.

Significance of high glucose-6-phosphatase activity in rat oviduct epithelium.

To study the origin of glucose in the oviduct fluid, we cytochemically examined glucose-6-phosphatase (G6Pase) activity in rat oviduct. The activity in the whole oviduct was also assayed biochemically. During proestrous, estrous, and metestrous phases, staining reaction for the activity was moderate in the epithelium of the caudal isthmus (CaI) and uterotubal junction (UJ), whereas it was weak in that of the ampulla (A) and cephalic isthmus (CeI). In the diestrous phase, staining reaction in the epithelium of CaI and UJ became strong although it remained weak in that of A and CeI. Reaction product for the activity was localized in the endoplasmic reticulum and nuclear envelope of all cell types in the epithelium. The amount of reaction product in secretory cells was small to moderate in CaI and UJ, and small in A and CeI during proestrus, estrus, and metestrus. In diestrous the amount became abundant in CaI and UJ and moderate in A and CeI. However, the amount in ciliated cells remained small in the four segments during the four phases. The biochemical activity in diestrous was greater than that in proestrus, estrus, or metestrus. This shows that the activity is high in secretory cells in the epithelium of CaI and UJ in the diestrous phase and suggests that the role of the high activity is to release glucose into the oviduct fluid for use by the embryo passing down the CaI and UJ to the uterus.

Waldenström's macroglobulinemia associated with amyloidosis and membranous nephropathy.

A 61-year-old man with massive proteinuria and hyper gamma-globulinemia was admitted to hospital because of massive edema and pulmonary infection. He showed significantly high level of serum IgM (3244 mg/dl) with lambda-type M-protein and Bence Jones protein detected by immunoelectrophoresis. Renal biopsy specimen showed not only the diffuse amorphous amyloid deposition in mesangial area but global thickening of capillary wall with spike formation by silver staining which was similar to the spicular formation. Immunofluorescence disclosed find granular deposition of IgG and C3 along the capillary wall and the electromicroscopic findings clearly showed both massive amyloid fibril at mesangial area and diffuse epimembranous electron dense deposits. lambda-type Bence Jones protein in macroglobulinemia was suggested not only the cause of renal amyloidosis but also the antigenic origin of membranous nephropathy in this case.

Postnatal development and sublobular distribution of cytochrome P-450 in rat liver: a microphotometric study.

To study the process of expression of cytochrome P-450 (P-450) in hepatocytes during development, we measured microphotometrically the P-450 content in periportal and perivenular hepatocytes of male rats during peri- and postnatal growth. From Day 19 of gestation to Day 5 after birth, P-450 content in both periportal and perivenular hepatocytes increased markedly (periportal 1046%; perivenular 819%). The content in periportal hepatocytes remained unchanged from 5 to 20 days of age, and increased slightly (24%) from 20 to 45 days of age. However, the content in perivenular hepatocytes increased progressively (105%) between 5 and 45 days of age. The difference in P-450 content became apparent between periportal and perivenular hepatocytes after 7 days of age. The content in periportal or perivenular hepatocytes reached the adult level at 45 days of age. Therefore, the perinatal period is the time at which a marked increase in P-450 occurs in hepatocytes throughout the liver lobule. The subsequent period before weaning is the time at which the sublobular heterogeneous distribution of P-450 appears. The period after weaning is the time at which a slight increase in P-450 content in periportal hepatocytes and a marked increase in the enzyme in perivenular hepatocytes takes place.

Densities of NADPH-ferrihemoprotein reductase and cytochrome P-450 molecules in the endoplasmic reticulum membrane of rat hepatocytes.

In hepatocytes, NADPH-ferrihemoprotein reductase (reductase) has been hypothesized to exist as aggregates or micelles in endoplasmic reticulum (ER) membrane. However, if the number of reductase molecules per unit area of ER is low, this hypothesis cannot explain how a few reductase molecules efficiently reduce many P-450 molecules. To test this hypothesis, we estimated the numbers of reductase and P-450 molecules per unit ER area (reductase and P-450 densities) by microphotometry of the two enzymes in conjunction with morphometry of ER in periportal, midzonal, and perivenular rat hepatocytes. The reductase density in periportal, midzonal, and perivenular hepatocytes (107-179 molecules/microns 2 of ER) was high enough to efficiently reduce all P-450 molecules in the ER, although the value in perivenular hepatocytes was lowest owing to the relatively greater amount of ER in this region. The pattern of sublobular gradient in the reductase density was similar to that in the P-450 density. Consequently, the molar ratio of P-450 to reductase in ER was similar (about 40:1) in hepatocytes regardless of their positions within the liver lobule.

Postnatal changes in sublobular distribution of NADPH-cytochrome P-450 reductase in rat liver.

Immunohistochemical distribution of NADPH-cytochrome P-450 reductase (NADPH-ferrihaemoprotein reductase; EC 1.6.2.4.) in the liver lobule was examined during development of the rat. From the 19th day of gestation to 4 days after birth, the enzyme was distributed uniformly throughout the lobule. The immunostaining for the enzyme was weak before birth, and became slightly stronger after birth. A slightly uneven distribution of immunoreactivity, stronger in perivenular zones, appeared at 5 days after birth. Then, the staining intensity in perivenular zones became progressively stronger with age, except for a slight increase between 10 and 20 days of age. The intensity in periportal zones also increased gradually, although it remained weaker than that in perivenular zones. Around 30 days of age, the distribution of the immunostaining, stronger in perivenular than in periportal zones, was similar to that seen in the lobules of adult animals. Thus, heterogeneity among hepatocytes with respect to the enzyme content is not present in fetal and newborn rats but develops gradually during postnatal development; the postnatal growth of the liver is accompanied by a change in the pattern of the distribution of this enzyme within the lobule.

Relation between cytochrome P-450 increase and endoplasmic reticulum proliferation in hepatocytes of mice treated with phenobarbital: a microphotometric and morphometric study.

To obtain detailed information on phenobarbital (PB)-induced cytochrome P-450 (P-450) increase and endoplasmic reticulum (ER) proliferation in hepatocytes, we estimated microphotometrically the amount of P-450 per unit cytoplasmic volume and morphometrically the area of ER per unit cytoplasmic volume in hepatocytes adjacent to the portal area or central venule (1 periportal or 1 perivenular cells) and in the second and third layers from the portal area or central venule (2, 3 periportal or 2, 3 perivenular cells) from mice injected with 35, 50, 100, or 150 mg/kg PB once a day for 3 days. By dividing the P-450 amount by the ER area, the number of P-450 molecules per unit ER area was also calculated. In 1 and 2, 3 perivenular cells, except for 2, 3 perivenular cells after injection of 150 mg/kg PB, the amount of P-450 increased with ER proliferation and the number of P-450 molecules in ER remained unchanged after injection of 50, 100, or 150 mg/kg PB. In 2, 3 periportal cells, however, the P-450 amount and the number of P-450 molecules in ER increased markedly without or with some ER proliferation after injection of 50, 100, or 150 mg/kg PB; the P-450 increase appears to be generally independent of ER proliferation. The 1 periportal cells are probably exceptional hepatocytes that usually did not respond to PB stimulation.