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Biomedical subjects

D Sun

Publications and source records attributed to D Sun.

At least 55 records · Page 3Linked to original sources
PubMed2001

Apical heterotrimeric g-proteins activate CFTR in the native sweat duct.

Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to test whether G-proteins control CFTR Cl- conductance (CFTR G(Cl)) in the native sweat duct (SD). We permeabilized the basolateral membrane with alpha-toxin so as to manipulate cytosolic nucleotides. We activated G-proteins and monitored CFTR G(Cl) activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-gamma-S (100 microm) also activates CFTR G(Cl) in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-gamma-S increased CFTR G(Cl) by 44 +/- 20 mS/cm(2) (mean +/- se; n = 7). GDP (10 mm) inhibited G-protein activation of CFTR G(Cl) even in the presence of GTP-gamma-S. The heterotrimeric G-protein activator (AlF(4-) in the cytoplasmic bath activated CFTR G(Cl) (increased by 51.5 +/- 9.4 mS/cm(2) in the presence of 5 mm ATP without cAMP, n = 6), the magnitude of which was similar to that induced by GTP-gamma-S. Employing immunocytochemical-labeling techniques, we localized Galphas, Galphai, Galphaq, and Gbeta at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G(Cl) in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G(Cl) activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt absorption by controlling CFTR G(Cl) activity.

Adenosine Triphosphate↗
PubMed2001

Requirement for math5 in the development of retinal ganglion cells.

math5 is a murine orthologue of atonal, a bHLH proneural gene essential for the formation of photoreceptors and chordotonal organs in Drosophila. The expression of math5 coincides with the onset of retinal ganglion cell (RGC) differentiation. Targeted deletion of math5 blocks the initial differentiation of 80% of RGCs and results in an increase in differentiated amacrine cells. Furthermore, the absence of math5 abolishes the retinal expression of brn-3b and the formation of virtually all brn-3b-expressing RGCs. These results imply that math5 is a proneural gene essential for RGC differentiation and that math5 acts upstream to activate brn-3b-dependent differentiation processes in RGCs.

Animals↗
PubMed2001

Expression of the p53 homologue p63 in early cervical neoplasia.

BACKGROUND: p63, a homologue of the tumor suppressor gene p53, is expressed in embryonic, adult murine, and human basal squamous epithelium and encodes both transactivating and dominant negative transcript isoforms. Mouse embryos functionally deficient in p63 fail to replenish basal squamous epithelial cells, resulting in multiple defects that include absent genital squamous epithelium. This study investigated the expression of p63 in the human cervical transformation zone and early cervical neoplasia. METHODS: Tissue localization of p63 was determined by immunohistochemistry in a wide range of epithelia. A correlation was also made between p63 expression and squamous basal cell (keratin 14), endocervical columnar cell (mucicarmine), and cell-cycle specific (Ki-67) markers. RESULTS: p63 expression by immunostaining delineated basal and parabasal cells of maturing ectocervical squamous mucosa, squamous metaplasia in the cervix, and basal and subcolumnar cells of the cervical transformation zone. In atrophic epithelia immunostaining for p63 was present in all cell strata. In early cervical neoplasia, p63 expression was inversely correlated with both squamous cell maturation and nonsquamous differentiation in CIN. This biomarker also identified basal cells in a subset of preinvasive cervical neoplasms with endocervical cell differentiation that were bcl-2 and keratin 14 negative. CONCLUSIONS: In the lower female genital tract, p63 is preferentially expressed in immature cells of squamous lineage and is not linked to cell proliferation. The broader range of p63 expression relevant to keratin 14 and bcl-2 indicates that p63 may identify additional subsets of benign and neoplastic epithelial basal cells in the cervical transformation zone and may be useful in studying cell differentiation in the early stages of neoplastic change in this region.

Adenocarcinoma↗
PubMed2001

Identification of a basal/reserve cell immunophenotype in benign and neoplastic endometrium: a study with the p53 homologue p63.

BACKGROUND: Metaplastic differentiation, including squamous, mucinous, and tubal (ciliated), is common in both benign and neoplastic endometrium, and the cell of origin for this pathway is poorly understood. In this study, expression of a marker for basal and reserve cells in cervical squamous mucosa, designated p63, was investigated in a spectrum of endometrial alterations. METHODS: One hundred ninety different endometria from 132 patients were examined, including fetal (6), premenarchal (3), benign cyclic (29) and noncyclic (54), hyperplastic (14), and neoplastic (93) endometrial glandular epithelia. The latter included conventional endometrioid carcinomas with and without mucinous, ciliated, and squamous metaplasia, and uterine papillary serous carcinoma (UPSC). RESULTS: p63 expression was identified in basal/subcolumnar cells in the fetal endometrium in a distribution similar to that in basal/reserve cells of the cervix. Staining was confined to individual scattered basal and suprabasal cells in cycling endometrium. In polyps and postmenopausal endometria, focal clusters of p63-positive cells were identified in inactive glands or surface epithelium. Metaplastic (squamous or mucinous) epithelia, either alone or in conjunction with hyperplasias or carcinomas, exhibited the most intense staining, primarily in basal or subcolumnar cells. In some cases, immediately adjacent nonmetaplastic columnar epithelium also stained positive. UPSCs contained only rare scattered p63-positive cells. CONCLUSIONS: Cells with a basal or reserve cell phenotype exist in the endometrium during fetal life, are not conspicuous during the reproductive years, but may emerge during shifts in differentiation. Whether these cells signify specialized multipotential endometrial cells is not clear, but the similarity of these cells to basal/reserve cells of the cervix and their association with neoplasia merit further study.

Animals↗
PubMed2001

Endothelial K(ca) channels mediate flow-dependent dilation of arterioles of skeletal muscle and mesentery.

The role of Ca(2+)-activated potassium channels (K(Ca)) in flow-initiated intracellular events in microvessels is not known. We hypothesized that K(Ca) channels in the arteriolar endothelium are responsible for the mechanotransduction of flow/shear stress-induced arteriolar dilation in skeletal muscle and mesentery of rats. The active diameter of arterioles isolated from gracilis (80 mm Hg) and cremaster (60 mm Hg) muscles and mesentery (80 mm Hg) at a constant intraluminal pressure was 53 +/- 3, 77 +/- 5, and 72 +/- 6 microm, respectively. Their passive diameter (in Ca(2+)-free solution) was 113 +/- 3, 152 +/- 12, and 121 +/- 7 microm, respectively. At a constant intraluminal pressure stepwise increases in perfusate flow (25, 40, and 14 microL/min in 5, 10, and 2 microL/min steps) elicited a gradual increase in diameter of all three groups of arterioles up to 93 +/- 5, 137 +/- 11, and 102 +/- 7 microm, respectively. Flow-induced dilations of arterioles were eliminated by intraluminal administration of iberiotoxin (ibTX 10(-9) M), an inhibitor of high conductance K(Ca) channels (BK(Ca)). In contrast, arteriolar dilations to acetylcholine and sodium nitroprusside were not altered by this agent, indicating that BK(Ca) channels are not involved in the receptor-mediated endothelial synthesis of nitric oxide (NO) and that the inhibitor did not affect the action of NO on smooth muscle. Abluminal application of ibTX (10(-8) M) did not affect flow-dependent dilation. We conclude that in arterioles of several tissues activation of endothelial BK(Ca) channels is an obligatory step in the transduction of the signal initiated by changes in intraluminal flow/shear stress, leading to the release of endothelial factors evoking dilation.

Animals↗
PubMed2001

Low viscosity Ektacytometry and its validation tested by flow chamber.

The flow chamber was used to observe the orientation and small deformation of red blood cells (RBCs) in a shear flow of low viscosity. With the aid of computer software, the percentage of RBCs oriented to the C=0 orbit (OI)(F) and the degree of deformation (DI)(F) of such RBCs were calculated by processing the photographs. It was found that these parameters were highly correlated, respectively, to the orientation index (OI)(E) and the small deformation index (DI)(E) obtained by our low viscosity Ektacytometry (LVE). Thus, our flow chamber research has provided direct evidence to validate the use of this low viscosity Ektacytometry. Although there are relative merits for the flow chamber method using low viscosity medium, the LVE is more likely to be applied in clinic for its simplicity and convenience.

Animals↗
PubMed2001

Regulation of intracellular calcium in dispersed fat body trophocytes of the cockroach, Periplaneta americana, by hypertrehalosemic hormone.

Incubation of trophocytes from dissaggregated fat body of Periplaneta americana with either of the hypertrehalosemic hormones, HTH-I or HTH-II, leads to an increase in the cytosolic concentration of Ca(2+) from approximately 80 to approximately 310nM with a rise time of approximately 110s. The Ca(2+) concentration then declines to the resting level during the ensuing 5min. In the absence of extracellular Ca(2+) the increase in [Ca(2+)](i) due to HTH is limited to approximately 100nM. The calmodulin inhibitors calmidazolium and W-7 also limit to a similar degree the ability of HTH to increase [Ca(2+)](i). Phorbol 12-myristate 13-acetate, an activator of protein kinase C, was shown to block Ca(2+) entry through the plasma membrane. Additional evidence to support the view that HTH enhances Ca(2+) influx has been obtained by measuring the quenching of fura-2 fluorescence when Ca(2+) is replaced with Mn(2+).

Journal Article↗
PubMed2001

Computer simulation of temperature changes in a wheat storage bin.

A mathematical model describing the transient temperature distribution of grain in a cylindrical storage bin with mixed boundaries is presented and solved using the finite element method. Using the typical meteorological data of a region such as temperate China, temperature changes of wheat in a storage bin are simulated and analysed in detail. Simulated results indicate that high temperature accumulation areas of the grain are in the bin centre and near the bin bottom or at the top surface of the grain throughout the whole year and ventilation under appropriate weather conditions is necessary. This method can be used to develop and evaluate aeration control strategy and reduce the need for chemical treatments of grain during storage.

Journal Article↗
PubMed2001

In vitro reaction to orthopaedic biomaterials by macrophages and lymphocytes isolated from patients undergoing revision surgery.

Periprosthetic tissues observed at sites of loose total joint implants exhibit abundant macrophages, lymphocytes, fibroblasts and particulate debris. Macrophages phagocytose orthopaedic debris and release proinflammatory cytokines, chemokines, matrix metalloproteinases and other substances. In addition, other cell types present in tissues harvested from the bone-implant interface are thought to influence periprosthetic bone resorption. The present study examined the effects of polymethylmethacrylate (PMMA), cobalt chrome molybdenum alloy (CoCr), and titanium-alloy particle challenge on macrophages co-cultured with lymphocytes in vitro. Potential synergistic effects of lymphocytes on macrophage activation were determined by measuring interleukin-6 and tumor necrosis factor-alpha release following exposure to orthopaedic biomaterial particles. Exposure of macrophages or macrophages co-cultured with lymphocytes to all three types of particles resulted in increased release of interleukin-6 and tumor necrosis factor-alpha at 48 h, when compared to macrophages or macrophages co-cultured with lymphocytes, respectively, cultured in the absence of particles. Lymphocytes isolated from periprosthetic tissues secreted increased basal levels of cytokines relative to peripheral blood lymphocytes. Higher doses of PMMA and titanium-alloy particles stimulated increased levels of cytokine release in the macrophage and macrophage/lymphocyte groups. In contrast, a higher dose of CoCr particles (0.075% v/v) was not as effective as the 0.015% v/v dose, indicating probable CoCr toxicity. The macrophage/lymphocyte co-culture did not show synergism between the two types of cells with respect to cytokine release. T-cells at the bone-implant interface may alter the biological response to particulate debris.

Biocompatible Materials↗
PubMed2001

Interleukin-10 inhibits polymethylmethacrylate particle induced interleukin-6 and tumor necrosis factor-alpha release by human monocyte/macrophages in vitro.

Periprosthetic membranes commonly observed at sites of total joint implant loosening exhibit abundant macrophages and particulate debris. Macrophages phagocytose orthopedic debris and release the pro-inflammatory mediators interleukin-1, interleukin-6, tumor necrosis factor-alpha, and prostaglandin E2. Populations of activated lymphocytes are often seen in periprosthetic membranes. These lymphocytes may modulate the monocyte/macrophage response to particulate debris and influence aseptic loosening. In addition, other immunologic agents, such as interleukin-10, are present in tissues harvested from the bone-implant interface of failed total joint arthroplasties. The present study examined the effects of interleukin-10 on polymethylmethacrylate (PMMA) particle challenged human monocyte/macrophages in vitro. Human monocyte/macrophages isolated from buffy coats of five healthy individuals were exposed to 1-10 microm PMMA particles. Interleukin-10 was added to the monocyte/macrophages with and without the addition of PMMA particles. Interleukin-10-induced alterations in monocyte/macrophage metabolism were determined measuring interleukin-6 and tumor necrosis factor-alpha release by the cells following exposure to PMMA particles. Exposure of the monocyte/macrophages to PMMA particles resulted in a dose-dependent release of interleukin-6 and tumor necrosis factor-alpha at 48 h. Interleukin-10 reduced the levels of interleukin-6 and tumor necrosis factor-alpha release by macrophages in response to PMMA particles in a dose-dependent manner. At 48 h, particle-induced interleukin-6 release was inhibited by 60 and 90% with 1.0 and 10.0 ng/ml treatments of interleukin-10, respectively. At 48 h, particle-induced tumor necrosis factor-alpha release was inhibited by 58 and 88% with 1.0 and 10.0 ng/ml treatments of interleukin-10, respectively. Interleukin-10 challenge alone did not significantly alter basal interleukin-6 or tumor necrosis factor-alpha release relative to control cultures. The data presented in this study demonstrate that the anti-inflammatory cytokine, interleukin-10, inhibits monocyte/macrophage release of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in response to PMMA particle challenge in vitro.

Biocompatible Materials↗
PubMed2001

Inositol trisphosphate mediates the action of hypertrehalosemic hormone on fat body of the American cockroach, Periplaneta americana.

The rate of synthesis of inositol trisphosphate (InsP(3)) in trophocytes derived from disaggregated cockroach (Periplaneta americana) fat body increases following treatment of the cells with hypertrehalosemic hormone I or II (HTH-I, -II) in vitro. Trophocytes preloaded with [3H]inositol display a significant increase in InsP(3) synthesis as early as 15 s after addition of the hormone. When the trophocytes are pre-incubated with LiCl and subsequently incubated with HTH the [3H] content of the InsP(3) fraction is greater than that found with HTH alone. This is taken as evidence that inositol monophosphate phosphatase is part of the mechanism for clearing InsP(3) from the cytosol. In contrast to HTH, octopamine, which is also capable of exerting a hypertrehalosemic effect in the cockroach, does not increase the synthesis of InsP(3). 1-Octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3)), a potent and selective inhibitor of phosphatidylinositol phospholipase C, blocks the activation of phosphorylase by HTH-I as well as the hypertrehalosemic effect induced by the hormone.

Animals↗
PubMed2001

Crystal structure of rat biliverdin reductase.

Biliverdin reductase (BVR) is a soluble cytoplasmic enzyme that catalyzes the conversion of biliverdin to bilirubin using NADH or NADPH as electron donor. Bilirubin is a significant biological antioxidant, but it is also neurotoxic and the cause of kernicterus. In this study, we have determined the crystal structure of rat BVR at 1.4 A resolution. The structure contains two domains: an N-terminal domain characteristic of a dinucleotide binding fold (Rossmann fold) and a C-terminal domain that is predominantly an antiparallel six-stranded beta-sheet. Based on this structure, we propose modes of binding for NAD(P)H and biliverdin, and a possible mechanism for the enzyme.

Amino Acid Sequence↗
PubMed2001

Fluorescence, reflectance, and light-scattering spectroscopy for evaluating dysplasia in patients with Barrett's esophagus.

BACKGROUND & AIMS: The aim of this study was to assess the potential of 3 spectroscopic techniques (fluorescence, reflectance, and light-scattering spectroscopy) individually and in combination, for evaluating low- and high-grade dysplasia in patients with Barrett's esophagus (BE). METHODS: Fluorescence spectra at 11 excitation wavelengths and a reflectance spectrum were acquired in approximately 1 second from each site before biopsy using an optical fiber probe. The measured fluorescence spectra were combined with the reflectance spectra to extract the intrinsic tissue fluorescence. The reflectance spectra provided morphologic information about the bulk tissue, whereas light-scattering spectroscopy was used to determine cell nuclear crowding and enlargement in Barrett's epithelium. RESULTS: Significant differences were observed between dysplastic and nondysplastic BE in terms of intrinsic fluorescence, bulk scattering properties, and levels of epithelial cell nuclear crowding and enlargement. The combination of all 3 techniques resulted in superior sensitivity and specificity for separating high-grade from non-high-grade and dysplastic from nondysplastic epithelium. CONCLUSIONS: Intrinsic fluorescence, reflectance, and light-scattering spectroscopies provide complementary information about biochemical and morphologic changes that occur during the development of dysplasia. The combination of these techniques (Tri-Modal Spectroscopy) can serve as an excellent tool for the evaluation of dysplasia in BE.

Barrett Esophagus↗
PubMed2001

Histologic and immunophenotypic classification of cervical carcinomas by expression of the p53 homologue p63: a study of 250 cases.

Recent studies of the p53 homologue p63 indicate that this gene is preferentially expressed in basal and immature cervical squamous epithelium. This study correlated p63 expression with morphologic phenotype and human papillomavirus (HPV) type in a wide range of cervical neoplasms. Two hundred fifty cases of cervical carcinoma, including squamous cell carcinoma (SCCA; n = 178), adenocarcinoma (ADCA; n = 28), adenosquamous carcinoma (ASCA; n = 8), neuroendocrine carcinoma (NECA; n = 15), and other variant or mixed types (n = 21) were studied. Ninety-seven percent of SCCA, 0% of ADCA, and 0% of SCUC showed strong (>75% v <30%) positivity for p63 (P<.001). p63 sharply distinguished SCCA (p63+) from ADCA (p63-), Large-cell, poorly differentiated carcinomas were distinguished as putative glandular (glassy cell) or squamous (lymphoepithelial-like or spindle cell) types based on p63 staining. Eight (73%) of 11 neuroendocrine tumors tested were chromogranin positive; all showed no or low (<30%) levels of p63 immunostaining. Absence of p63 was also associated with a subset of nonneuroendocrine undifferentiated carcinomas. Transitions from squamous to columnar or undifferentiated morphology coincided with loss of p63 expression. A strong association between HPV 16 and p63 positivity was identified because of the colocalization of both within tumors of squamous phenotype. p63 is a powerful marker for squamous differentiation and, when diffusely expressed, excludes a glandular or neuroendocrine differentiation. p63 may be useful for differentiating pure squamous or glandular from adenosquamous carcinomas, tracking shifts in differentiation within tumors, supporting (by its absence) the diagnosis of neuroendocrine carcinomas, and clarifying the spectrum of poorly differentiated carcinomas lacking either squamous or neuroendocrine differentiation.

Adenocarcinoma↗
PubMed2001

Epileptogenesis induces long-term alterations in intracellular calcium release and sequestration mechanisms in the hippocampal neuronal culture model of epilepsy.

Calcium and calcium-dependent processes have been hypothesized to be involved in the induction of epilepsy. It has been shown that epileptic neurons have altered calcium homeostatic mechanisms following epileptogenesis in the hippocampal neuronal culture (HNC) and pilocarpine models of epilepsy. To investigate the mechanisms causing these alterations in [Ca2+]i homeostatic processes following epileptogenesis, we utilized the HNC model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs). Using [Ca2+]i imaging, studies were initiated to evaluate the mechanisms mediating these changes in [Ca2+]i homeostasis. 'Epileptic' neurons required much longer to restore a glutamate induced [Ca2+]i load to baseline levels than control neurons. Inhibition of Ca2+ entry through voltage and receptor gated Ca2+ channels and stretch activated Ca2+ channels had no effect on the prolonged glutamate induced increase in [Ca2+]i in epileptic neurons. Employing thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), it was shown that thapsigargin inhibited sequestration of [Ca2+]i by SERCA was significantly decreased in 'epileptic' neurons. Using Ca2+ induced Ca2+ release (CICR) cell permeable inhibitors for the ryanodine receptor (dantrolene) and the IP3 receptor (2-amino-ethoxydiphenylborate, 2APB) mediated CICR, we demonstrated that CICR was significantly augmented in the 'epileptic' neurons, and determined that the IP3 receptor mediated CICR was the major release mechanism altered in epileptogenesis. These data indicate that both inhibition of SERCA and augmentation of CICR activity contribute to the alterations accounting for the impaired calcium homeostatic processes observed in 'epileptic' neurons. The results suggest that persistent changes in [Ca2+]i levels following epileptogenesis may contribute to the long-term plasticity changes manifested in epilepsy and that understanding the basic mechanisms mediating these changes may provide an insight into the development of novel therapeutic approaches to treat epilepsy and prevent or reverse epileptogenesis.

Animals↗
PubMed2001

Expression, purification, characterization and clinical relevance of rAed a 1--a 68-kDa recombinant mosquito Aedes aegypti salivary allergen.

Accurate diagnosis of mosquito allergy has been precluded by the difficulty of obtaining salivary allergens. In this study, we expressed, purified, characterized and investigated the clinical relevance of a recombinant Aedes aegypti salivary allergen, rAed a 1. Two cDNA segments were ligated together to form the full-length Aed a 1 gene. rAed a 1 was expressed using a baculovirus/insect cell system, and purified using a combination of anion-exchange and gel-filtration chromatography. The purified rAed a 1 bound to human IgE, as detected by ELISA, ELISA inhibition tests and immunoblot analyses. Epicutaneous tests with rAed a 1 and a commercial whole-body AE: aegypti extract, and AE: aegypti bite tests were performed in 48 subjects. Nine of 31 (29%) of the subjects with positive immediate bite tests also had a positive rAed a 1 immediate skin reaction and 32% had an positive immediate test to the commercial extract. Six of 33 (18%) of the subjects with positive delayed bite tests also had a positive rAed a 1 delayed skin reaction and 6% had a positive delayed test to the commercial extract. Furthermore, rAed a 1-induced flare sizes significantly correlated with mosquito bite-induced flare sizes. None of the subjects with negative bite tests had a positive skin test to rAed a 1 or to commercial extract. We conclude that the rAed a 1 has identical antigenicity and biological activity to native Aed a 1, can be used in the in vitro and in vivo diagnosis of mosquito allergy, and is more sensitive than mosquito whole-body extract for detecting delayed skin reactions.

Aedes↗
PubMed2001

Extracellular calmodulin stimulates light-independent RbcS-GUS expression in suspension-cultured cells of transgenic tobacco.

The RbcS genes encode the small subunits of rubisco; the expression of these genes is controlled in a light-dependent and independent manner. It has been reported that intracellular calmodulin (CaM) is involved in light-dependent RbcS expression. In this report, the role of extracellular CaM in regulating expression of RbcS in darkness was examined. The time course of expression of RbcS-GUS and that of the secretion of CaM in the suspended transgenic tobacco cells in darkness were very similar. Both showed initial increase followed by decline with maximum CaM secretion preceding maximum GUS expression by 24 h. The concentration of CaM in the culture medium is regulated light independently. Purified CaM alone added to the media enhanced RbcS-GUS expression in darkness. The addition of membrane-impermeable CaM inhibitors, such as anti-CaM antiserum or W7-agarose, repressed the expression of RbcS-GUS in darkness, but this inhibitory effect was completely reversed by adding exogenous purified CaM. These results provide the first evidence that extracellular CaM is involved in the regulation of light-independent RbcS gene expression.

Calmodulin↗