Aldehyde dehydrogenase 2 partly mediates hypotensive effect of nitrite on L-NAME-induced hypertension in normoxic rat.

Nitrite has become a topic of interest in the field of medical research because of its potential therapeutic role as an alternative source of nitric oxide (NO). While the bioconversion of nitrite to NO occurs via either nonenzymatic or enzymatic reduction under acidic or hypoxic conditions, little is known about its conversion to NO under normoxic conditions. Because of a recent report of aldehyde dehydrogenase 2 (ALDH2)-catalyzed glyceryl trinitrate (GTN) vasorelaxation by denitration of GTN to 1,2-glyceryl dinitrate (1,2-GDN) and nitrite, we therefore investigated a catalytic activity of ALDH2 for nitrite reduction and subsequent effect on N(ω)-nitro-l-arginine methyl ester (l-NAME)-induced hypertension in normoxic rat. Male Sprague-Dawley rats treated with l-NAME in drinking water for 3 weeks developed hypertension with significantly reduced plasma levels of nitrite and nitrate. The intravenous injection of sodium nitrite lowered the arterial pressure in a dose-dependent manner (17, 50 and 150 µmol/kg). Pretreatment with ALDH2 inhibitors (cyanamide and chloral hydrate) partially inhibited the hypotensive responses to sodium nitrite. In addition, cyanamide significantly delayed the nitrite clearance from plasma and most of the organs examined during the experimental period. These results suggest that ALDH2 may be at least in part involved in nitrite-mediated hypotensive effects and nitrite catalysis in many organs of normoxic rats.

Development of novel rat model for high-fat and high-cholesterol diet-induced steatohepatitis and severe fibrosis progression in SHRSP5/Dmcr.

OBJECTIVES: Patients with nonalcoholic fatty liver disease are increasing worldwide, and preventive measures are an urgent need and primary concern today. AIM: This study aimed to develop and clarify the usefulness of the SHRSP5/Dmcr rat, derived from a stroke-prone spontaneously hypertensive rat, as a novel animal model for time-course analysis of steatohepatitis and the severe fibrosis progression often observed in the disease. METHODS: Ten-week-old male SHRSP5/Dmcr rats were divided into six groups: half were fed a high-fat and high-cholesterol-containing diet (HFC diet), and the others the control, stroke-prone (SP) diet for 2, 8, and 14 weeks. RESULTS: The HFC diet significantly increased serum transaminase and gamma glutamyl transpeptidase activities, tumor necrosis factor alpha levels, and serum and hepatic total cholesterol levels over time. In contrast, this diet decreased serum albumin, glucose, and adiponectin levels throughout or the later stage of the feeding period, but did not influence serum insulin levels. Histopathologically, the HFC diet increased microvesicular steatosis, and focal or spotty necrosis with lymphocyte infiltrations were observed in the liver at 2 weeks, macrovesicular steatosis, ballooned hepatocytes with Mallory-Denk body formation in some, and multilobular necrosis and fibrosis at 8 weeks. Interestingly, this fibrosis formed a honeycomb network at 14 weeks. These changes are very similar to those observed in patients with non-alcoholic steatohepatitis. CONCLUSIONS: SHRSP5/Dmcr rats appear to be a useful model for analyzing the time-dependent changes of HFC diet-induced steatohepatitis and fibrosis progression.

Permethrin may disrupt testosterone biosynthesis via mitochondrial membrane damage of Leydig cells in adult male mouse.

Permethrin, a popular synthetic pyrethroid insecticide used to control noxious insects in agriculture, forestry, households, horticulture, and public health throughout the world, poses risks of environmental exposure. Here we evaluate the reproductive toxicity of cis-permethrin in adult male ICR mice that were orally administered cis-permethrin (0, 35, or 70 mg/kg d) for 6 wk. Caudal epididymal sperm count and sperm motility in the treated groups were statistically reduced in a dose-dependent manner. Testicular testosterone production and plasma testosterone concentration were significantly and dose-dependently decreased with an increase in LH, and a significant regression was observed between testosterone levels and cis-permethrin residues in individual mice testes after exposure. However, no significant changes were observed in body weight, reproductive organ absolute and relative weights, sperm morphology, and plasma FSH concentration after cis-permethrin treatment. Moreover, cis-permethrin exposure significantly diminished the testicular mitochondrial mRNA expression levels of peripheral benzodiazepine receptor (PBR), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side-chain cleavage (P450scc) and enzyme and protein expression levels of StAR and P450scc. At the electron microscopic level, mitochondrial membrane damage was found in Leydig cells of the exposed mouse testis. Our results suggest that the insecticide permethrin may cause mitochondrial membrane impairment in Leydig cells and disrupt testosterone biosynthesis by diminishing the delivery of cholesterol into the mitochondria and decreasing the conversion of cholesterol to pregnenolone in the cells, thus reducing subsequent testosterone production.

Weakness in intercellular association of keratinocytes in severely brittle nails.

The brittle fingernail is a common complaint, but the features of the cellular structure of the nail plate remain unclear. In this study, clipped nailplates from two persons with severely brittle nails, one female aged 26 years and one male aged 82 years, were observed by light and electron microscopy and compared with normal nail plates. Numerous cracks were observed in clipped brittle nails, but not in normal nails, on light microscopy. When the deep areas of nail plates of the clipped normal nails were observed by electron microscopy, intercellular boundaries appeared intermingled, and two thin, electron-dense layers were observed in a narrow intercellular gap. In contrast, in brittle nails, marked dilatation of intercellular spaces was frequently observed and electron-dense layers were either not seen or were disrupted. When clipped normal nails were dehydrated in a desiccation chamber, similar dilatations - though not so severe -were observed, without evident cracks. These results suggest that dilatation of the intercellular space between nail keratinocytes is correlated with brittle nails and that dehydration may result in such intercellular dilatation.

Effects of estradiol administration on feto-placental growth in rat.

BACKGROUND: To clarify the effect of estradiol benzoate on placental structure and its consequences for fetal survival and fetoplacental growth. STUDY DESIGN: Estradiol benzoate (0, 0.1, 1, 10, 100 microg/day) was infused intraperitoneally into pregnant Wistar rats from 12 to 19 days' gestation. Survival rate, weight of pups and placentas at 20 days' gestation, and plasma levels of estrogen and progesterone were measured. Pathological changes in the placenta were also examined. RESULTS: Estradiol benzoate reduced fetal survival (1 microg/day: 100%, 10 microg/day: 70%, 100 microg/day: 14.6%) and the weights of the pups and placentas in a dose-dependent manner. Maternal estradiol concentration was raised 23-fold with 100microg/day of estradiol benzoate. Trophoblast degeneration, including apoptosis and destruction of placental labyrinth was induced but the structures of the maternal kidney and liver were not affected. CONCLUSIONS: In pregnant rats, estradiol benzoate causes fetal mortality at a pharmacological dose (more than 10 microg/day) and fetoplacental growth retardation via trophoblastic degeneration and destruction of the placental labyrinth even at a physiological dose (1 microg/day).

Collagen of chronically inflamed skin is over-modified and upregulates secretion of matrix metalloproteinase 2 and matrix-degrading enzymes by endothelial cells and fibroblasts.

In order to investigate the properties of collagen in chronically inflamed tissue, we isolated collagen from the ear skin of mice with chronic contact dermatitis and examined its biochemical characteristics and the functions that regulate the secretion of matrix metalloproteinase 2 and collagen-degrading enzymes from endothelial cells and fibroblasts. Collagen in skin with chronic contact dermatitis comprised 60% type I collagen and 40% type III collagen, which latter is higher than the content of type III collagen in control skin (35%). The denaturation temperature was higher (42 degrees C) than that of control skin (39 degrees C). The alpha2 chain of type I collagen was over-hydroxylated at both proline and lysine residues. Segment-long-spacing crystallites of type I collagen were unusually connected in tandem. Collagen of chronically inflamed skin was less susceptible to matrix metalloproteinase 2 after heat denaturation. Endothelial cells and fibroblasts secreted an increased amount of matrix metalloproteinase 2 when cultured on a gel formed from the collagen of chronically inflamed skin. Collagen-degrading activity secreted from fibroblasts was also upregulated when cells were in contact with collagen of chronically inflamed skin. These results suggest that the collagen in chronically inflamed tissue has altered biochemical characteristics and functions, which may affect the pathogenesis of the chronic skin disease.

Structural changes in the glycosaminoglycan chain of rat skin decorin with growth.

Decorin controls collagen fibrilogenesis in skin, and its molecular weight changes in wound healing and with age. In this report, the quantitative and structural changes of decorin were investigated with growth in rat skin from the fetus to the young adult. A northern blot analysis showed that the highest level of skin decorin mRNA was at post partus 0.5 days; this level was about 3.7 times the level at embryo 16.5 days. The mRNA level in the rat skin decreased by 1/5 from post partus 0.5 days until 90 days of age. Western blotting showed that the amount of decorin increased with age in protein level. The molecule size of decorin at embryo 18.5 days was about 110 kDa, and that at post partus 90 days was about 70 kDa. There were no changes in molecular size of its core protein, so the reduction in the size of decorin was dependent on the size of the glycosaminoglycan (GAG) as shown by western blot analyses. Electron micrography of the rat skin with cupromeronic blue staining showed that the length of GAG at embryo 18.5 days was about 78.58 +/- 13.94 nm, and that at post partus 90 days was about 54.05 +/- 4.79 nm. The reduction in length of decorin GAG with age shrunk the distance between the collagen fibrils. We suggested that decorin changes the GAG length in order to control skin reconstruction in response to inflammation and injury.

Morphological study of disordered myelination and the degeneration of nerve fibers in the spinal cord of mice lacking complex gangliosides.

Gangliosides, a family of glycosphingolipids that contain sialic acid, are abundant on the neuronal cell membranes, but their precise functions in the central nervous system remain largely undefined. In a previous study of GalNAc-T(-/-) mice engineered to lack beta1,4-N-acetylgalactos-aminyltransferase (GM2/GD2 synthase) to abolish any, complex gangliosides, we observed the reduction of nerve conduction velocity but did not find any obvious morphological change in the brain. In the present study, we observed morphological changes in the nerve fiber tracts of the spinal cord in these mice. In GalNAc-T(-/-) mice, the number of degenerated axons was markedly increased in the dorsal funiculus, tract of Lissauer, and dorsolateral funiculus of the cervical segment of the spinal cord as well as the dorsal funiculus and tract of Lissauer of the lumbar segment of the spinal cord. There were also increased numbers of unmyelinated fibers in GalNAc-T(-/-) mice. Loosened myelin sheaths and myelin sheaths separated from axons by wide spaces were also observed in GalNAc-T(-/-) mice. These results provide a morphological basis for the previously observed reduction in the nerve conduction velocity and suggest that complex gangliosides are essential for the maintenance of myelin and the integrity of nerve fibers of the spinal cord.

Innervation and functional characteristics of connective tissues, especially elastic fibers, in human fetal thoracic intervertebral articular capsule and its surroundings.

The articular capsules between the thoracic vertebrae, which have physiologically different functions from those of other levels of the vertebrae, have yet to be subjected to neuro-anatomical and fine structural analysis. In the present study, we analyzed serial frozen sections of decalcified thoracic vertebrae in human fetuses, and identified the articular capsule tissue with its unique distribution of elastic fibers. The fine structure of the elastic fibers was studied by transmission electron microscopy. In the early-stage fetus, the fibrous membrane forming the lateral intervertebral articular capsule contained abundant thin elastic fibers consisting of microfibrils. In the late-stage fetus, the lateral capsule of fibrous membrane was occupied by thick elastic fibers. A medial articular capsule, namely the ligamenta flava, contained numerous thick elastic fibers in both early and late-stage fetuses. The distributional differences in nerve fibers between early and late-stage fetuses were determined by immunostaining, using antibodies raised against protein gene product 9.5 (PGP 9.5; ubiquitin carboxyl-terminal hydrolase). Innervation by PGP 9.5 immunoreactive fibers was limited to the areas of the articular capsules near the blood vessels, which may indicate their functional relation with blood flow. No PGP 9.5 immunoreactive fibers were found in the ligamenta flava of the late-stage fetus. Innervation might be directly involved in the development of the intervertebral articular capsules in normal human fetuses.

Ultrastructural localization of aminopeptidase A/angiotensinase and placental leucine aminopeptidase/oxytocinase in chorionic villi of human placenta.

AIMS: Membrane-bound aminopeptidases in human placenta are thought to be involved in maintaining homeostasis during pregnancy by metabolizing bioactive peptides such as oxytocin and angiotensin at the interface between the fetus and mother. Because determining the precise localization of these enzymes is required to support this notion, we investigated the ultrastructural localization of two principal enzymes, aminopeptidase A (APA; EC 3.4.11.7)/angiotensinase and placental leucine aminopeptidase (P-LAP; EC 3.4.11.3)/oxytocinase in human first trimester and full-term placenta. METHODS: Immunohistochemical analysis using anti-P-LAP and anti-APA antibodies was performed on ultrathin frozen sections of fixed human placental villi. RESULTS: Transmission immunoelectron microscopy revealed that both enzymes were expressed on the surface of apical microvilli of syncytiotrophoblast cells and, to a lesser extent, on the basal infoldings. The location of the two enzymes did not vary between the first trimester and full-term placenta sections, while the staining intensities were slightly enhanced in full-term villi. CONCLUSIONS: Our observation that P-LAP and APA are present on the microvilli, which is a site of interaction between the mother and fetus, suggests possible involvement of these enzymes in cleaving peptide hormones from the fetus and mother in order to regulate bioactivity.

Characterization of a holin (HolNU3-1) in methicillin-resistant Staphylococcus aureus host.

The gene encoding holin protein HolNU3-1 from a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) NU3-1 was cloned and expressed in S. aureus RN4220. HolNU3-1 encoded by the holNU3-1 gene, which is located upstream of the deleted endolysin gene, was functional. Expression of the holNU3-1 gene induced a decrease in culture turbidity and formation of translucent (empty ghost) cells in S. aureus. We found heterogeneity of the holin genes and diversity of the two-component lysis system, which consists of holin and endolysin, in MRSA hosts. We suggest that this diversity is important in the identification of the evolution of clinical isolates of S. aureus.

Size control of decorin dermatan sulfate during remodeling of collagen fibrils in healing skin.

Recently it has been reported that the molecular size of decorin dermatan sulfate (DS) was increased in healing skin after hapten application and that the elongated DS was distributed in enlarged interfibrillar space among thin collagen fibrils in situ. Here we show that such modulation of the length of decorin DS is temporary. Although the size of decorin DS was evidently increased on day 15, it decreased to almost normal size on day 35 when the altered disaccharide composition of DS was also recovered. Electron microscopic observation revealed that elongated decorin DS was localized among thin collagen fibrils packed loosely in hapten-treated skin on day 15. In contrast, decorin DS of normal size was distributed among thick collagen fibrils packed tightly on day 35. These results suggest that size control of decorin DS plays important roles in organization of collagen fibrils into bundles by regulating interfibrillar space in healing skin, particularly in maturation of collagen fibrils through shortening of decorin DS in later stages of healing.

[Ultrastructural changes of collagen fibrils in mouse dermal connective tissue after moxibustion treatment].

Morphological changes in mouse back skin, especially dermal connective tissue, were observed after moxibustion treatment. Various amounts of direct moxibustion and indirect moxibustion (column) were performed and the exact skin area was processed for light and electron microscopy. Just after treatment, epidermal cell layer was degenerated and increased amounts of mast cells were observed. Many unwinding collagen fibrils were prominent. Twenty-four hours after treatment, the number of unwinding fibrils decreased. Instead, tightly winding fibrils, sometimes thicker, were observed in a similar area. D-period of collagen fibrils did not change in any situation. Physical reaction to moxibustion varies depending on stimulation rate and/or approach, which includes the change of interstitial connective tissues as well as cellular profiles. It is also important to survey the time-dependent changes in moxibustion treatment.

Antibacterial activities of beta-lactamase inhibitors associated with morphological changes of cell wall in Helicobacter pylori.

BACKGROUND: Recent study has demonstrated that beta-lactamase inhibitors including clavulanate, sulbactam and tazobactam have an vitro antibacterial effect on Helicobacter pylori. Here we describe the relationship between viability and cell profiles of H. pylori exposed to beta-lactamase inhibitors and some antibiotics in a short-time course. MATERIALS AND METHODS: The antibacterial effects of beta-lactamase inhibitors including clavulanate, sulbactam and tazobactam on the bacterial viability of and morphological changes in H. pylori ATCC43504 were examined. RESULTS: The beta-lactamase inhibitors such as clavulanate and sulbactam alone decreased the viable counts of H. pylori, depending on the antibiotic concentrations. Exposure to these beta-lactamase inhibitors resulted in morphological changes of cell shape, cell-wall disintegration and cell lysis. Among these beta-lactamase inhibitors, clavulanate was the most active, causing a decrease in viable counts and morphological changes such as short filamentous to sphaeroplast formation and lysis. One x minimum inhibitory concentration (MIC) of amoxicillin plus 1 x MIC of clavulanate decreased viable counts effectively compared with 1 x MIC of amoxicillin or 1 x MIC of clavulanate alone, and induced morphological changes of cell shape and cell wall. CONCLUSION: Our results suggest that the beta-lactamase inhibitors alone have concentration-dependent antibacterial activities against H. pylori and affect the morphology of the cell shape and the cell wall in vitro.

Separation of corneal stroma and Descemet's membrane during deep lamellar keratoplasty.

PURPOSE: To study the interface of the corneal stroma and Descemet's membrane and to report the histologic findings of the deep corneal stromal tissue removed during deep lamellar keratoplasty (DLKP). METHODS: The deep stromal tissues of four corneas were removed during DLKP and were examined histologically by light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: Descemet's membrane appeared as a smooth surface under the surgical microscope during the removal of deep corneal stroma. Two of the four excised tissues showed a thin layer attachment to the deep stromal tissue by LM and TEM. The layer measured approximately 3.0 to 4.0 microm and was observed as a striated structure attached to the stromal tissue of both specimens. The other two specimens contained only stromal tissue. CONCLUSIONS: Separation of the deep stromal tissue from Descemet's membrane may occur within the Descemet's membrane, and the separation is probably between the anterior banded and the posterior nonbanded layer of Descemet's membrane in some cases during DLKP. The lamellar structure of the delaminated region suggests a mechanically weak segment of Descemet's membrane. The smooth surface of Descemet's membrane observed under a surgical microscope is not the actual interface of corneal stroma and Descemet's membrane.

Relationship between morphological changes and endotoxin release induced by carbapenems in Pseudomonas aeruginosa.

The relationship between morphological changes and endotoxin release induced in vitro by carbapenems in a clinical isolate of Pseudomonas aeruginosa was examined. The time-course and magnitude of endotoxin release induced varied among imipenem, panipenem, meropenem and biapenem and related to the morphological changes caused by these agents which variously affected cell shape, cell-wall disintegration and cell lysis. The amount of endotoxin released by carbapenem-treated cells correlated with both the cell-wall morphology and bacterial shape immediately before lysis. Meropenem and biapenem caused markedly increased endotoxin release during cell lysis and cell-wall disintegration, whereas imipenem and panipenem caused much less release of endotoxin.