Immunologic protection afforded by sun screens.
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Humans↗
Biomedical subjects
J F Morris
Publications and source records attributed to J F Morris.
Characterization of monoclonal antibodies directed to the amino-terminus of the WT1, Wilms' tumor suppressor protein.
We have produced and characterized three monoclonal antibodies (MAbs) directed to the amino terminus of the WT1 Wilms' tumor suppressor transcription factor and compared their properties to rabbit polyclonal sera raised to the same immunogen. A recombinant protein consisting of amino acids 1-181 of human WT1 was overexpressed in E. coli, purified, and used as the immunogen. Three MAbs designated 6F-H2, 6F-H7, and 6F-HC-17--all of the IgG1 subclass--were selected and further characterized. Each recognized all isoforms of the full-length WT1 protein in Western blot assays and immunoprecipitated WT1 in both physiologic buffers and under high detergent/high salt (RIPA) conditions. Preliminary epitope mapping suggests that all three MAbs recognize a region in the amino terminal 84 amino acids of WT1 and that the MAbs do not recognize the polyglycine or polyproline regions of the protein. The WT1 antibodies do not recognize the structurally and functionally related early growth response (EGR)1, EGR2, EGR3, or EGR4 proteins. All WT1 MAbs recognize the murine WT1 protein and immunohistochemical staining of murine embryonic and newborn kidney sections show strong staining of condensing metanephric mesenchyme and primitive podocytes in developing glomeruli. These WT1-specific MAbs should be useful in characterizing the biochemical and developmental roles of WT1 and in defining the emerging role of WT1 as a diagnostic and/or prognostic marker in mesothelioma, leukemias, and breast cancer.
Amino Acid Sequence↗
Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm.
Lipocortin 1 (annexin I) is a calcium- and phospholipid-binding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.
Adenocarcinoma↗
Macrophages and pancreatic islet amyloidosis.
Islet amyloid formed from islet amyloid polypeptide (IAPP, amylin) is found in spontaneously diabetic monkeys and cats. Islet amyloidosis is progressive, apparently irreversible and is associated with destruction of insulin-secreting cells. The role of macrophages in the destruction and removal of islet amyloid is unknown. Therefore, the presence and morphology of macrophages were determined by electron and quantitative light microscopy in islets of diabetic and nondiabetic man and monkeys and in transgenic mice expressing the gene for human IAPP. Tissue macrophages were present in all pancreatic sections and tissue distribution was similar in exocrine and endocrine areas. There was no difference in macrophage density in amyloidotic and amyloid-free islets in monkeys and man. Macrophage density was similar in islets of transgenic mice expressing human IAPP which do not contain amyloid in vivo but in which fibrils are formed in vitro following islet isolation compared to islets from mice expressing rat IAPP which is not amyloidogenic. IAPP amyloid fibrils were visible by electron microscopy in lysosomes of pancreatic macrophages in man, monkeys and human IAPP transgenic mice. Thus, human IAPP is internalised but inefficiently degraded by tissue macrophages. Diabetes-associated amyloidosis is not associated with visible recruitment of macrophages for removal of amyloid or islet debris.
Aged↗
Local protein synthesis in magnocellular dendrites. Basic elements and their response to hyperosmotic stimuli.
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Animals↗
Ouabain-sensitive Na+,K(+)-ATPase activity in toad brain.
Toads of the genus Bufo are highly resistant to the toxic effects of digitalis glycosides, and the Na+,K(+)-ATPase of all toad tissues studied to date has been relatively insensitive to inhibition by digitalis and related compounds. In studies of brain microsomal preparations from two toad species, Bufo marinus and Bufo viridis, inhibition of ATPase activity and displacement of [3H]ouabain from Na+,K(+)-ATPase occurred over broad ranges of ouabain or bufalin concentrations, consistent with the possibility that more than one Na+,K(+)-ATPase isoform may be present in toad brain. The data could be fitted to one- or two-site models, both of which were consistent with the presence of Na+,K(+)-ATPase activity with high sensitivity to ouabain and bufalin. Ki (concentration capable of producing 50% inhibition of activity) values for ouabain in the one-site model were in the 0.2 to 3.7 microM range, whereas Ki1 values in the two-site model ranged from 0.085 to 0.85 microM, indicating that brain ATPase was at least three orders of magnitude more sensitive to ouabain than B. marinus bladder ATPase (Ki = 5940 microM). Ouabain was also an effective inhibitor of 86Rb+ uptake in B. marinus brain tissue slices (Ki = 3.1 microM in the one-site model; Ki1 = 0.03 microM in the two-site model). However, the relative contribution of the high ouabain-sensitivity site to the total activity was 17% in the transport assay as compared with 63% in the Na+,K(+)-ATPase enzymatic assay. We conclude that a highly ouabain-sensitive Na+,K(+)-ATPase activity is present and functional in toad brain but that its function may be partially inhibited in vivo.
Animals↗
Age-dependent accumulation of hybrid vasopressin-oxytocin gene products but not hybrid oxytocin-vasopressin products in the endoplasmic reticulum of Brattleboro rats.
The age-dependence of the incidence of magnocellular neurosecretory neurons containing abnormal accumulations of peptide in the rough endoplasmic reticulum was examined in homozygous Brattleboro rats and in their wild-type Long Evans counterparts. Neurons in which the immunophenotype of the peptide aggregates indicate that somatic cross-over mutations involving the 5' end of the vasopressin gene and the 3' end of the oxytocin gene have occurred, increased with age in homozygous Brattleboro rats, reaching a maximum of 24 cells per hypothalamus (approximately 0.6% of the vasopressin neurons). The increase occurred in both male and female animals but was significantly greater in females. The average incidence of such cells was 6 times greater in the supraoptic than in the paraventricular nucleus. No such cells could be detected in either nucleus of Long Evans rats despite the evidence for hybrid mRNA in these animals. Moreover, no accumulation of peptide translated from the hybrid mRNAs derived from the 5' end of the oxytocin gene and the 3' end of the vasopressin gene could be detected in either Brattleboro or Long Evans animals. These results strongly suggest that the accumulation of peptide in the rough endoplasmic reticulum of vasopressin neurons in homozygous Brattleboro rats is due to an abnormality other than the somatic crossing-over mutation. A second type of abnormal magnocellular neuron with accumulations of peptide in the rough endoplasmic reticulum, in which the immunophenotype of the peptide reveals products derived only from the oxytocin precursor, was present in both Long Evans and Brattleboro rats, but did not increase with age in Brattleboro rats. The incidence of these cells was similar in the supraoptic and paraventricular nuclei.
Aging↗
Immunologic protection afforded by sunscreens in vitro.
Several studies have suggested a lack of correlation between sunscreen sun protection factor and protection of the skin immune system, potentially allowing greater damage to the skin by removing the natural protective erythemal response to sun exposure. Despite this, routine testing of immune protection afforded by sunscreens is not performed by industry. Current laboratory methods for investigating the efficacy of sunscreen protection of epidermal immune function use the induction of contact hypersensitivity or epidermal cell alloantigen presentation. Animal models, cell culture systems, and in vivo human studies are commonly employed, but all these systems have significant drawbacks for use in routine testing. The purpose of this study was to develop an in vitro system for testing the immunologic protection afforded by sunscreens in human skin. Five test sunscreens plus a vehicle control were tested in a "blind" fashion for their in vitro level of immune protection. Creams were applied in a standard manner to human whole skin explants and were irradiated over a range of physiologic doses using an Oriel solar simulator. A mixed epidermal lymphocyte reaction was used to quantify epidermal alloantigen-presenting capacity, in the presence or absence of test cream, for five explants. Results consistently demonstrated that all the test sunscreens protected beyond their designated sun protection factors, whereas the vehicle conferred no protection. The explant-mixed epidermal lymphocyte reaction system gave consistent, reproducible results and may prove useful for the allocation of an immune protection factor to all sunscreens.
Administration, Topical↗
Characterization and localization of lipocortin 1-binding sites on rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy.
Lipocortin 1 (LC1) is an important mediator of glucocorticoid action in the anterior pituitary gland, where it appears to act via cell surface binding sites to suppress peptide release. We have exploited a combination of fluorescence-activated cell (FAC) analysis/sorting and electron microscopy to detect, characterize, and localize LC1-binding sites on the surface of dispersed rat anterior pituitary cells, using human recombinant LC1 (hu-r-LC1) as a probe. High affinity (Kd = 14 +/- 3 nM) hu-r-LC1-binding sites were detected on approximately 80% of anterior pituitary cells dispersed with collagenase. The binding characteristics of the ligand resembled those observed in leukocytes, in that it was saturable; concentration, Ca2+, and temperature dependent; and abolished by trypsin. Functional studies demonstrated an excellent correlation between the presence of the cell surface binding protein and the capacity of an anti-LC1 monoclonal antibody to abrogate the inhibitory actions of dexamethasone (10 nM) on the release of ACTH initiated in vitro by CRH-41 (1 nM). Morphological analysis of cells harvested by FAC sorting showed that 1) somatotrophs, corticotrophs, lactotrophs, thyrotrophs, and gonadotrophs were all included in the population expressing LC1 binding sites; and 2) the LC1-binding sites assume a punctate distribution across the cell surface. These data show that anterior pituitary cells express high affinity surface LC1-binding protein(s); they thus provide further evidence for a specific membrane mechanism of action of LC1 in regulating the endocrine function of the anterior pituitary.
Adrenocorticotropic Hormone↗
An antisense oligodeoxynucleotide to lipocortin 1 reverses the inhibitory actions of dexamethasone on the release of adrenocorticotropin from rat pituitary tissue in vitro.
Our previous studies have demonstrated that lipocortin 1 (LC1, also called annexin 1) is an important mediator of glucocorticoid action in the neuroendocrine system, particularly with regard to the powerful inhibitory actions of the steroids on the secretion of ACTH and its hypothalamic releasing hormones. In the present study, we have used an antisense oligodeoxynucleotide (ODN) unique to LC1 to investigate further the role of this protein in the regulatory effects of dexamethasone on ACTH release in vitro from rat anterior pituitary cells. Pituitary cells dispersed with collagenase retained their functional and morphological integrity in vitro and sequestered ODNs in a time-dependent manner from the incubation medium. LC1 was readily detected in the cells by Western blot analysis or by immunoprecipitation/autoradiography after preloading with 35S-methionine/cysteine; the bulk of the protein was contained within an intracellular pool but a small amount was attached to the outer cell surface (pericellular). Dexamethasone (100 nm, 2.5 h) initiated de novo synthesis of LC1; it also increased the amount of LC1 in the pericellular pool detected by either method and caused a concomitant decrease in intracellular LC1. The responses to the steroid were prevented by the inclusion in the medium of an LC1 antisense ODN (50 nM, 3.5 h) but the corresponding sense and scrambled ODN sequences were inert. None of the ODN sequences tested influence the expression of annexin 5 in the pituitary tissue. CRH-41 (100 pM-1 mM), forskolin (1 nM-1 mM) and an L-Ca2+-channel opener BAY K8644 (100 pM-1 microM) initiated concentration dependent increases in immunoreactive- (ir-) ACTH release from the pituitary cells that were reduced (P < 0.01) by preincubation with dexamethasone (100 nM, 2.5 h). The inhibitory effects of the steroid were reversed by the LC1 antisense ODN (50 nM, P < 0.01), whereas the LC1 sense and scrambled control sequences (50 nM) were both ineffective in this respect (P > 0.05). The results add further support to the view that the acute inhibitory effects of glucocorticoids on the secretion of ACTH by the pituitary gland are dependent on the generation of lipocortin 1.
Adrenocorticotropic Hormone↗
A novel bufadienolide, marinosin, in the skin of the giant toad, Bufo marinus.
We have identified a novel cardiac steroid, 11,19-epoxy-19-methoxytelocinobufagin, named marinosin (1), in the skin of the toad, Bufo marinus (L.) Schneider. The treatment of compound 1 with 50% CH3CN containing 0.1% trifluoroacetic acid yielded a 11 alpha-hydroxyhellebrigenin (2), which has not previously been isolated from animals or plants. The structures of both compounds were established from spectral data obtained by NMR and MS, which were compared with those of a reference bufadienolide, 11 alpha-hydroxytelocinobufagin. Compounds 1 and 2 have A/B cis and C/D cis configuration, which is characteristic of bufadienolides such as bufalin and marinobufagin. However, the stereo-structure of compound 1 was characterized by a boat form of the B ring, which is different from the chair form in typical bufadienolides such as compound 2. Compounds 1 and 2 both exhibited activity, as demonstrated by inhibition of Na+, K(+)-ATPase enzymatic activity and by inhibition of the binding of [3H]ouabain to Na+, K(+)-ATPase; however, marinosin (1) was as less effective inhibitor than 2, 11 alpha-hydroxyhellebrigenin. We have identified compound 2 in toad venom, but not in the skin.
Animals↗
Lesbian coming out as a multidimensional process.
Coming out for lesbians is discussed as a multidimensional process. Four dimensions are presented and explored. First is sexual identity formation which encompasses development of lesbian sexuality and awareness of being a lesbian. Second is disclosure of sexual orientation to others. Third is sexual expression and behavior. Fourth is lesbian consciousness which refers to how lesbians see themselves in relation to the social environment, including lesbian and gay communities. These dimensions are examined in relation to existing theories and research about lesbian coming out processes. Further, there is a discussion of coming out and demographic differences among lesbians. The possible impact of age, race, ethnicity, religion, geographic location, income, employment, and education on lesbian coming out are considered.
Female↗
Dissociation of nitric oxide synthase immunoreactivity and NADPH-diaphorase enzyme activity in rat pituitary.
The relationship between nitric oxide synthase (NOS) immunocytochemistry and NADPH-diaphorase (NADPH-d) histochemistry was investigated in the anterior and posterior pituitary of ovariectomized rats. NADPH-d activity was present throughout the posterior pituitary but could not be detected in the anterior pituitary. By contrast, an antibody raised against whole recombinant rat neuronal NOS revealed strongly NOS-immunoreactive gonadotrophs and folliculo-stellate cells scattered throughout the anterior pituitary in addition to immunoreactive fibres in the posterior pituitary. The study demonstrates that NADPH-d histochemistry is not always coincident with NOS and provides evidence for an as yet uncharacterized subtype of NOS in the rat anterior pituitary.
Animals↗
Hematopoietic transcriptional regulation by the myeloid zinc finger gene, MZF-1.
Transcriptional regulators control much of hematopoiesis. One such transcriptional regulator is the myeloid zinc finger gene MZF-1. MZF-1 has been localized to the telomere of chromosome 19q, where a large number of related zinc finger genes reside. It has been found to be essential in granulopoiesis. It is a bi-functional transcriptional regulator, repressing transcription in non-hematopoietic cells, and activating transcription in cells of hematopoietic origins. Its consensus DNA binding site has been isolated, and sites in several promoters of myeloid-specific genes, such as CD34, lactoferrin, and myeloperoxidase, have been defined. In co-transfection experiments MZF-1 has been found to regulate transcription from the CD34 promoter.
Base Sequence↗
Synaptic relationship of substance P-like-immunoreactive nerve fibers with gland cells of the anterior pituitary in the rat.
Our previous studies have proved the presence of substantial amounts of peptidergic nerve fibers in the anterior pituitary of human, monkey, dog, and rat, and synaptic contacts have been demonstrated on corticotrophs and somatotrophs in the dog. The present study is aimed at investigating the synaptic relationship between axons and gland cells in the rat. The substance P-like-immunoreactive nerve fibers were studied immuno-electron-microscopically. They were nonmyelinated and varicose. Typical synaptic contacts between the fibers and every type of the hormone-secreting gland cell could be identified, most frequently on lactotrophs. No synaptic contacts were found on the folliculo-stellate cell. The synaptic contacts were of asymmetrical type with clusters of small round clear vesicles at the presynaptic site and scattered large dense-cored vesicles. This pattern of synaptic relationships was markedly different from that in the dog. The presence of synapses in the anterior pituitary of the rat implies a direct neural regulation of certain cells and strongly supports our hypothesis of neural-humoral dual regulation of this gland.
Animals↗
Development of microglia and macrophages in the postnatal rat pituitary.
Microglia and macrophages, immunolabelled with F4/80 (which binds a 160-kDa plasmalemmal glycoprotein) and OX-42 (which labels the complement type 3 receptor, CR3), were identified in the neuro- and adenohypophyses, respectively, of postnatal rats from day 1 to adulthood. In the neurohypophysis, the numerical density (cells/mm2) of microglia increased from postnatal day 1 to day 7 but was then unchanged from the adult density. In the adenohypophysis, the numerical density of macrophages increased from postnatal day 1 to day 21. The increasing size of the pituitary meant that the total number of such cells increased rapidly in the neurohypophysis up to day 14, but was then essentially unchanged; in the adenohypophysis macrophages increased in proportion to the increasing size of the gland up to day 21. Proliferation of the mononuclear cells was analysed by the immunodetection of bromodeoxyuridine incorporation into the nuclei of microglia and macrophages. F4/80-immunoreactive cells incorporating bromodeoxyuridine were found on all the postnatal days studied. The proportion of such cells in the neurohypophysis was high from postnatal day 1 to day 14 and in the adenohypophysis was maximal on day 14, decreasing in both parts of the pituitary by day 21. The estimated total number of proliferating cells was maximal in both parts of the pituitary on day 14. In both parts, OX-42-immunoreactive cells were less numerous than F4/80-immunoreactive cells up to postnatal day 14; CR3 expression may therefore be associated with maturation of these cells. Neurohypophysial microglia increased in size to postnatal day 7, consistent with the assumption of a 'compact' microglial morphology; adenohypophysial macrophages did not change in size over the postnatal period. Throughout the period studied, neurohypophysial microglia were significantly more densely distributed and larger in size than adenohypophysial macrophages. Neurohypophysial microglia phagocytose terminals of neurosecretory neurons from day 7, concurrent with the development of a distinct perivascular space. In the adenohypophysis, the perivascular space was present from birth and macrophages were not phagocytic. There are, therefore, considerable differences in the density, morphology and activity between the populations of myelomonocytic cells in the postnatal rat neuro- and adenohypophyses.
Animals↗
Presence of neuronal nitric oxide synthase in the suprachiasmatic nuclei of mouse and rat.
Nitric oxide has recently been identified as a major neural regulator. It is synthesized by the enzyme nitric oxide synthase. Whilst considerable functional evidence has pointed to an involvement of nitric oxide in circadian regulation, all previous morphological studies have failed to demonstrate the presence of nitric oxide synthase in the mammalian suprachiasmatic nucleus. By use of an antibody directed against whole recombinant rat neuronal nitric oxide synthase we have identified the presence of immunoreactivity for this enzyme in the suprachiasmatic nucleus of mouse and rat, to provide the first detailed report of this enzyme in the suprachiasmatic nucleus for any species of mammal. Immunoreactivity for neuronal nitric oxide synthase was found in neurons throughout the suprachiasmatic nucleus of mice and no difference could be detected between the ventrolateral and dorsomedial parts of the nucleus in terms of the optical density of the immunostaining. Very small, rounded neuronal cell bodies were immunopositive. Electron microscopy revealed that these neurons had relatively large nuclei and scant cytoplasm containing relatively few organelles, which sometimes included some 100 nm dense-cored peptidergic vesicles. Only about 5% of such neurons were not detectably immunoreactive. By contrast, in the rat suprachiasmatic nucleus, a much smaller number of neurons were immunopositive and these cells were aggregated in the ventrolateral part of the nucleus. The immunoreactive neurons were bipolar cells with scanty cytoplasm. Electron microscopy revealed diffuse immunoreactivity in the cytosol, but not within any organelles. In the surrounding neuropil immunoreactive dendrites and axons mingled with much larger numbers of immunonegative processes, but immunoreactive boutons were only identified just outside the dorsal margin of the nucleus. Astrocytes, oligodendrocytes and endothelial cells in the suprachiasmatic nucleus were immunonegative. NADPH-diaphorase activity was not detectable in the suprachiasmatic nucleus of either mouse or rat. This morphological evidence for nitric oxide synthase-immunoreactive cells in the suprachiasmatic nucleus supports the existing functional evidence for an involvement of nitric oxide in the transmission of light-induced signals to the suprachiasmatic nucleus in mammals.
Animals↗
Heterogeneity and lability of endogenous digitalis-like substances in the plasma of the toad, Bufo marinus.
Three major groups of endogenous digitalis-like substances (EDLS) have been identified in the plasma of the toad, Bufo marinus. One group of compounds, present in fresh plasma, is composed of chromatographically homogeneous polar conjugates, principally bufadienolide 3-sulfates, which exhibit relatively weak Na(+)-K(+)-adenosinetriphosphatase (ATPase) inhibitory activity. A second and larger group of compounds, also found in fresh plasma, includes chromatographically heterogeneous conjugates, which are effective inhibitors of Na(+)-K(+)-ATPase; these compounds possess properties similar to those of bufotoxins. The third group of EDLS consists of free unconjugated bufadienolides, which are also effective Na(+)-K(+)-ATPase inhibitors. These unconjugated bufadienolides are present in relatively low concentrations in fresh toad plasma, but appreciable quantities are enzymatically generated from conjugates (believed to consist principally of bufotoxins) during the in vitro incubation of plasma. We suggest that the extent to which circulating polar EDLS are enzymatically deconjugated in vivo may be important in the regulation of the digitalis-sensitive Na(+)-K(+)-ATPase of toad brain, the only known digitalis-sensitive Na(+)-K(+)-ATPase in the toad.
Animals↗