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The glutathione transferases (GSTs) are a family of ubiquitous enzymes that catalyze the conjugation of reduced glutathione (GSH) with reactive electrophiles. Rat vascular tissue contains GST isoforms that represent a major cellular defense mechanism against atherogenic alpha,beta-unsaturated aldehydes (Misra et al., Toxicol. Appl. Pharmacol. 133, 27-33, 1995). In this study we examined the role of GSTs in providing protection to cultured neonatal vascular smooth muscle cells (VSMCs) from the alpha,beta-unsaturated carbonyl cardiovascular toxins, allylamine and its metabolite, acrolein. Confluent cultured cells were exposed to 2 to 10 microM allylamine (a cardiovascular toxin that is metabolized in vivo and in vitro by VSMCs to the reactive aldehyde, acrolein) or to acrolein (2-10 microM) for 48 h; dose-cytotoxicity curves were generated utilizing a tetrazolium-dependent cytotoxicity assay. Concommittant treatment with sulfasalazine, an established inhibitor of GST, was found to markedly increase allylamine- or acrolein-induced cytotoxicity, decreasing the LC50 by two- to threefold at 50 to 100 microM sulfasalazine. A clonogenic survival assay in VSMCs exposed to these compounds for 4 h confirmed lethal toxicity and enhanced toxicity following cotreatment with sulfasalazine. Isobologram analysis (which statistically defines the limits of additivity of two independent treatments) showed that the sulfasalazine effect on both allylamine and acrolein cytotoxicity was supraadditive, or synergistic. Sulfasalazine was not cytotoxic to VSMCs in the range of concentrations that augmented acrolein or allylamine cytoxicity; total GST activity was inhibited, however, in a dose-dependent manner in that range. GST purified by GSH-affinity chromatography from pelleted untreated cells gave specific activities and kinetic constants consistent with those previously reported for rat aorta total GSTs. The catalytic efficiency (Kcat/Vm) was found to be much greater for 4-hydroxy-2-nonenal than for 1-chloro-2,4-dinitrobenzene (0.058 vs 0.4 s-1 mM-1). Western blot of purified total GSTs using antibodies against rec-mGSTA4-4 revealed a single band at 25 kDa, confirming the presence of a GST isozyme immunologically similar to rat GST8-8, which is known to utilize alpha,beta-unsaturated carbonyls as preferred substrates. Our data indicate that GSTs are an important defense in the vascular media, protecting blood vessels against alpha,beta-unsaturated carbonyl cardiovascular toxins that are involved in initiating atherosclerotic lesions.
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PMID:The role of glutathione S-transferases as a defense against reactive electrophiles in the blood vessel wall. 977 3

Apocrine sweat glands in the circumanal glands of the dog are not connected morphologically with the lobules of the circumanal glands. However, an apparent functional association has been demonstrated and it is possible that the apocrine sweat glands might serve as excretory ducts for degenerated polyhedral cells of the circumanal glands. In this study, we examined the ultrastructure of the apocrine sweat glands in the circumanal glands of the dog in an effort to define more precisely the relationship between the apocrine sweat glands and the circumanal glands. Paraffin sections stained with azan and sections after immunohistochemical staining with antibodies against actin were examined by light microscopy. Samples fixed by aldehyde perfusion were examined with the electron microscope. Diameters of apocrine sweat glands and height of cells in the secretory epithelium varied considerably. Immunohistochemical staining for actin was weakly positive in the supranuclear regions of secretory cells and very intense in myoepithelial cells. In secretory cells, the endoplasmic reticulum was well-developed. Multivesicular bodies were abundant and were discharged into lumens. Apocrine secretion and exocytosis were observed at luminal surfaces of secretory cells. There were three types of large granule in the cytoplasm: giant mitochondria without cristae; membrane-enclosed globules with or without myelin-like contents; and electron-dense, homogeneous, globular structures. Luminal surfaces were always covered with microvilli, and extensive folding of the cell membrane was found in basal regions. Bundles of actin filaments were dispersed throughout the cytoplasm. In the lumens of apocrine tubules, we observed shed secretory cells with well-preserved normal fine structures. We also noted the differentiation of secretory cells that was due to cell renewal. Apocrine sweat glands in the circumanal glands of the dog appear to be more active than those on the general body surface in terms of apocrine secretion, exocytosis, and the release of multivesicular bodies. Shed secretory cells containing large granules, as well as degenerated polyhedral cells from the circumanal glands, might contribute, to some extent, to the subtle composition of sweat from these apocrine sweat glands.
Anat Rec 1998 11
PMID:Apocrine sweat glands in the circumanal glands of the dog. 981 Dec 18

The excurrent duct system of the rat submandibular gland consists of a number of distinct segments. Using the direction of salivary flow as a reference point, these segments are, in order, intercalated duct, granular convoluted tubule, striated duct, excretory duct, main excretory duct (MED), and salivary bladder (which is an expanded portion of the MED). Because these ducts (with the exception of the MED and the salivary bladder) are encased in secretory endpieces, they are difficult to locate and to observe by scanning electron microscopy. A simple method has been devised to rid the gland of these obscuring endpieces so that the detailed architecture of the duct system can be examined. Rat submandibular glands were fixed initially by vascular perfusion with half-strength Karnovsky's fixative. The connective tissue capsule was removed from extirpated glands and the glands remained in fixative for varying lengths of time. For our purposes, a 30-minute immersion in the aldehyde mixture was optimum. After the sublingual gland was removed, the submandibular gland was softly struck with forceps having rounded tips, then shaken in fixative or buffer. The tissue that remained was postfixed in osmium tetroxide. This method results in the complete divestment of nonductular parenchyma from the rat submandibular gland, leaving the duct system clean and ready for microscopic examination.
Anat Rec 1999 01
PMID:Simple method to isolate the intact duct system of the rat submandibular gland. 989 19

Naked-backed bats of the genus Pteronotus (family Mormoopidae) occur in the Neotropics from Mexico through northern South America. These are relatively small-sized insectivorous species that frequently roost in caves. Eight specimens of naked-backed bats (Pteronotus parnellii) were live-trapped in Suriname and one in Cuba (P. quadridens). Their parotid glands were fixed in an aldehyde mixture designed for field work and postfixed in the laboratory with osmium tetroxide. Tissues were further prepared for electron microscopy by conventional means. The parotid glands of the two species of Pteronotus closely resemble each other except for the substructure of their serous secretory granules. Serous granules in P. parnellii are bizonal, with a moderately dense inner matrix and an outer, denser corona or crescent. The matrix is occupied by laminae, flakes, and filaments in random array. In contrast, serous granules in P. quadridens consist of a uniform matrix that contains dense, usually stacked toroids or tubules either in random array or packed in bundles. A parotid gland from one specimen of P. parnellii contained an endpiece that consisted of cells that contained giant (up to 9 pm in diameter) serous granules. Serous cells in both species contain aggregates of small, uniformly dense, rod-like, membrane-delimited organelles as well as occasional bundles of cytofilaments. The endpieces are separated from intercalated ducts by a ring of granulated cells that contain secretory granules that often have a bull's-eye configuration. Intercalated and striated ducts are typical in appearance, except that many of the cells in the latter contain small, dense secretory granules in their apical cytoplasm. The parotid glands in the two species of naked-baked bats differ slightly in terms of acinar secretory granule ultrastructure, but otherwise are fairly conservative. It is thought that the glands in these particular bats might represent the "basal" condition of the salivary glands of insectivorous bats and thus can serve as a reference point for making comparisons to the highly diversified (in terms of diet) phyllostomid bats.
Anat Rec 1999 06 01
PMID:Ultrastructure of the parotid gland in two species of naked-backed bats. 1035 12

Using a recently developed fixation technique for parietal cells (Sugai et al., Acta Anat Nippon 1995:70:S79, 1999:74:S101), we have reinvestigated the organization of the cytoplasmic membrane system in the resting stomach by ultra-high-resolution scanning electron microscopy (SEM). Rat gastric mucosae were microwave-fixed in cacodylate buffer [334 milliosmoles/kg H(2)O (mOsm)], to which 1.0% glutaraldehyde and 0.5% formaldehyde were added. Specimens examined by transmission electron microscopy (TEM) of thin sections revealed cytoplasm packed with tubular membranes similar to images detected by rapid-freeze/freeze-substitution fixation which is generally considered to cause minimal structural alterations. To render the cytoplasmic membranes visible by SEM, fixed mucosae were frozen, fractured, and the exposed cytoplasm of parietal cells was macerated by the aldehyde-osmium-DMSO-osmium procedure. With much of the cell matrix and filaments removed, SEM revealed numerous 30-60 nm tubules which formed a meshwork and also small cisternae. The cytoplasmic surface of the tubules was smooth while some cisternal areas had attached polyribosomes. Vesicles or isolated tubules were not found in appropriately macerated parietal cells. The cytoplasmic surface of the intracellular canaliculus was smooth except for round openings representing the bases of macerated microvilli. In favorable sites connections of the tubular membranes to the canaliculi were clearly visible. Stereo pair views were particularly useful to demonstrate these continuities. Connections between these two membrane compartments suggest the probability of rapid membrane transposition.
Anat Rec 2000 01 01
PMID:Scanning EM of resting gastric parietal cells reveals a network of cytoplasmic tubules and cisternae connected to the intracellular canaliculus. 1060 44

The mammalian nasal cavity is lined by an olfactory mucosa (OM) and a respiratory mucosa (RM). The principal OM cell type is the olfactory receptor neuron (ORN). However, little is known about ORNs in the life histories of primates. The RM, similar to the RM in the tracheobronchial tract (TBT), is dominated by ciliated columnar cells. Neuroendocrine cells (NECs) are essential in the TBT; little is known about nasal NECs. This study examined the immunolabeling characteristics of primate OM and RM for three important proteins-calretinin (CR), olfactory marker protein (OMP), and protein gene product 9.5 (PGP). Tissues from newborn to 15-year-old macaques were analyzed to determine the expression of these proteins during various stages of development. Standard immunocytochemistry on aldehyde-fixed tissues was applied, utilizing the avidin-biotin peroxidase (ABC) method. Immuno-electron microscopy confirmed the immunoreactive cell types. ORNs were immunoreactive for CR, OMP, and PGP at all ages studied. Immunoreactivity for PGP also was displayed in a subset of ciliated, columnar epithelial cells in the RM and in an extensive network of subepithelial fibers spread throughout both mucosae. The results suggest that macaque ORNs express three important proteins over a wide life history, and that the macaque may be a reliable model for studying primate/human olfaction during aging. The PGP-labeling results also suggest that the macaque nasal peptidergic fibers express PGP and that the respiratory epithelium contains NECs with labeling characteristics similar to those in the TBT.
Anat Rec 2000 06 01
PMID:Immunocytochemical characteristics of cells and fibers in the nasal mucosa of young and adult macaques. 1082 Mar 23

Motility patterns and their structural basis were studied by video analysis, light and electron microscopy on the physiologically distended gut from normal and W/W(v) suckling mice and normal adult mice. Empty or diltiazem-relaxed intestine were used as references. In contrast to conventional primary aldehyde fixation, a brief primary fixation with osmic acid before aldehydes preserved the visible contraction patterns and revealed dynamic increases in the number of peg-and-socket junctions coupling muscle cells mutually and with interstitial cells of Cajal (ICC). In tissue engaged in segmentation, the major increase was in the circular muscle and involved the ICC-DMP (integrated in the circular muscle layer at the site of the deep muscular plexus), whereas the increase during sleeve contractions was in the longitudinal muscle and involved the ICC-AP (located at the site of Auerbach's plexus). The number and distribution of gap junctions were unaffected. Area analysis of cell profiles supported the involvement of circular muscle in segmentation, but longitudinal muscle alone in sleeve contractions. The gut of both normal and W/W(v) sucklings (and adults) contracted during segmentation at frequencies close to reported slow-wave frequencies. In W/W(v) sucklings, ICC-AP were absent whereas ICC-DMP were present in adult configuration. Before Day 8 pp gap junctions were seen only between ICC-DMP. In the sucklings ICC-DMP may be responsible for rapid circumferential coordination and pacemaking of ring contractions. The geometry, organization, and dynamic regulation of peg-and-socket junctions strongly suggest a crucial role in coordination of smooth muscle and pacemakers, probably as stretch sensors, mediating a 'stretch-coupling' in the system.
Anat Rec 2001 01 01
PMID:Toward a concept of stretch-coupling in smooth muscle. I. Anatomy of intestinal segmentation and sleeve contractions. 1114 34

Serum macromolecules are transported through the vascular endothelial layer to the interstitium via the caveolae and interendothelial clefts, but the nature of the permeability of these structures is unknown, and the manner of caveola-vesicle transport is controversial. We have developed a method of detecting macromolecular channels using an in situ HRP perfusion into arteries previously perfused with aldehyde and random conventional sectioning for electron microscopy. Using unbiased morphometry, 4.75% of the abluminal caveolae and 15.13% of the intercellular clefts were the tracer-positive in rat aortic endothelium. In rat aortas treated with N-ethylmaleimide, all caveolae and most free vesicles in the cytoplasm except those around the Golgi area were HRP-positive in the endothelial cells; 1.48% of abluminal caveolae were structurally recognized as caveolar channels through the endothelial layer in a plane of single section. The length density of the abluminal caveolae was decreased to about 80% to the physiological control level whereas the larger invaginations were more frequently observed. Moreover 96.17% of the intercellular clefts were HRP-positive. We suggest that a flexible channel-system functions extensively as a macromolecular transport pathway in the arterial endothelium in vivo because the tracer-labeled abluminal caveolae and intercellular clefts should be opened to the luminal surfaces methodologically. We therefore propose that caveolar channels, rather than transcytosis, provide a mechanism of caveola-vesicle transport in the endothelial cells, because free vesicles involved in transcytosis were few in number.
Anat Rec 2001 09 01
PMID:Caveolar and intercellular channels provide major transport pathways of macromolecules across vascular endothelial cells. 1150 69

Pulmonary intravascular macrophages (PIMs) contain a unique electron-dense globular surface-coat which is sensitive to heparin treatment, halothane anesthesia, and the digestive effect of lipolytic lipase (LPL), suggesting that the coat is predominantly composed of lipoproteins. In the present study, evidence is presented that heparin, when administered intravenously in goats, potentiated both the translocation of the surface-coat into the vacuolar system and the expansion of the Golgi apparatus. Sequentially, these changes were followed by proliferation of peroxisomes in combination with peroxisomal reticulum (PR), a transient precursor of this organelle. The peroxisomes, as well as PR, reacted positively for catalase after aldehyde fixation and 3,3'-diaminobenzidine (DAB) staining. In addition to their role as phagocytes, the ultrastructural and cytochemical detection of peroxisomes suggests a functional capacity of the PIMs, which may be adaptable to the circulating level of free fatty acids (FAAs).
Anat Rec 2002 01 01
PMID:In situ heparin-induced peroxisomal reticulum and biogenesis of peroxisomes in pulmonary intravascular macrophages (PIMs) of caprine lung: an ultrastructural and cytochemical study. 1174 73

Radical reactions mediated by Schwartz reagent and zirconocene(alkene) complex are firstly described. Schwartz reagent is a promising alternative to tributyltin hydride and the first transition metal hydrido complex used as a radical mediator in organic synthesis. A zirconocene(alkene) complex effects single electron transfer to alkyl halide to generate the corresponding alkyl radical. Secondly, serendipitous allylic C-H bond activation of the coordinating alkene of zirconocene(alkene) complex and its application to organic synthesis are summarized. By utilizing equilibrium between zirconocene(alkene) and zirconocene 2-alkenyl hydride, reaction of acid chloride with zirconocene(alkene) provides the corresponding homoallylic alcohol by sequential attacks of the hydride and 2-alkenyl moieties. A set of hydride and 2-alkenyl attacks on 1,4-diketone yields 6-heptene-1,4-diol derivative in high yield with high stereoselectivity. Selective capture of the hydride with diisopropyl ketone gives zirconocene 2-alkenyl alkoxide, which is a useful reagent for stereoselective allylation of aldehyde and imine. alpha-Halo carbonyl compounds undergo radical allylation with the zirconocene 2-alkenyl alkoxide which serves as a substitute for allyltin.
Chem Rec 2004
PMID:Innovative reactions mediated by zirconocene. 1507 78


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