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Biomedical subjects

L Lu

Publications and source records attributed to L Lu.

At least 307 records · Page 17Linked to original sources
PubMed1998

[The measurement of baroreflex sensitivity in stress-induced hypertensive rats by spectral analysis].

Simultaneous spectral analysis of short-term systolic blood pressure variability (SBPV) and heart rate variability (HRV) was applied to determine the change of baroreflex sensitivity (BRS) in Sprague-Dawley (SD) rats. The modulus of the transfer function between fluctuations in systolic pressure and beat-to-beat interval would be an appropriate quantification of BRS. The animal experiment was performed on two groups of rats: normotensive rats and stress-induced hypertensive rats (SIHR). V1-receptor antagonist d(CH2)5Tye(Me) AVP of arginine vasopressin (AVP) was microinjected into intracerebroventricle. The results showed that, in base-line condition before administration, although the BRS in very low frequency band (0-0.035 Cycle/Beat, VLF), low frequency band (0.035-0.12 Cycle/Beat, LF), high frequency band (0.12-0.32 Cycle/Beat, HF) and total BRS(the sum of the three bands) were all decreased, the BRS in VLF(P < 0.05) and LF(P < 0.01) decreased significantly by statistics; and that, to the SIHR, the BRS in VLF was significantly lower after AVP-V1 administration than in base-line condition, while to normotensive rats, the BRS did not change. It indicates that the facilitating effect of AVP on the beroreflex in SIHR is mainly due to V1-receptor in central nerve system. In summary, the transfer function between SBPV and beat-to-beat interval fluctuation could be an index of BRS. This method can be developed for future clinical application.

Animals↗
PubMed1998

[The application of retinotomy and retinectomy for complicated retinal detachment].

OBJECTIVE: To investigate the techniques, therapeutic effects, indications, complications and control of retinotomy and retinectomy for complicated retinal detachment. METHODS: Based on the preliminary report of retinotomy and retinectomy, 36 eyes of 36 patients of complicated retinal detachment were operated on and followed-up for 3-12 months. The operative technique included buckling, vitrectomy, peeling, retinotomy and retinectomy, intraocular tamponade and laser treatment. RESULTS: Retina was reattached in 32 eyes in operation. Retinal detachment was recurrent in 8 eyes in followed-up in which retina was reattached again in 3 eyes by second operation. The last successful rate was 75% (in 27 eyes). The visual acuity was worse than funger conter in 33 eyes and worse than 0.05 in 3 eyes before operation. The visual acuity was better than 0.05 in 20 eyes (55.5%) after operation. The complications included bleeding, retinotomy expanding, complicated cataract, hypotony and so on. CONCLUSION: Retinotomy and retinectomy can improve the curative effect of complicated retinal detachment. But the complications of these operation were severe. The indications should be well controlled.

Follow-Up Studies↗
PubMed1998

In vitro and in vivo primate evaluation of carbon-11-etomidate and carbon-11-metomidate as potential tracers for PET imaging of the adrenal cortex and its tumors.

METHODS: With the purpose of developing a PET imaging agent for tumors of the adrenal cortex, we developed syntheses for 11C-etomidate and its methyl analog, 11C-metomidate. (R)-[O-ethyl-1-11C]Etomidate and (R)-[O-methyl-11C]metomidate were prepared by reaction of the appropriate respective 11C-labeled alkyl iodide and the tetrabutylammonium salt of the carboxylic acid derivative. The specificity of binding to the adrenal cortex was tested through the use of frozen section autoradiography of different tissues of the rat, pig and human. Inhibition of tracer binding was evaluated with etomidate, ketoconazole and metyrapone, well-known inhibitors of enzymes for steroid synthesis. Tracer binding to different human tumor samples was compared to immunohistochemical staining with antibodies for the steroid synthesis enzymes P450 11beta (11beta-hydroxylase), P450 scc (cholesterol side-chain cleavage enzyme), P450 C21 (21 -hydroxylase) and P450 17alpha (17alpha-hydroxylase). Three PET investigations, one with 11C-etomidate and two with 11C-metomidate, were performed in rhesus monkey sections, including the adrenals, liver and kidneys. Time-activity curves were generated from measured tracer uptake in these organs. RESULTS: In frozen section autoradiography of various tissues, high binding was seen in the adrenal cortex from all species, as well as in the tumors of adrenal cortical origin. The level of liver binding was about 50% of that in the adrenals, whereas that of all other organs was <10% of the adrenal binding. The adrenal binding was blocked by etomidate and ketoconazole at low doses but not by metyrapone. The binding in the adrenal tumor samples correlated with immunostaining for P450 11beta . PET studies in the monkey demonstrated high uptake in the adrenals with excellent visualization. The uptake increased with time without indication of washout. Slightly lower uptake was seen in the liver as compared to the adrenals, and in the late images, no organs other than adrenals and liver were seen. CONCLUSION: These investigations indicate that 11C-etomidate and 11C-metomidate have the potential to be useful specific agents for the visualization of the normal adrenal cortex and to provide positive identification of adrenal cortical tumors.

Adrenal Cortex↗
PubMed1998

In vitro and animal validation of bromine-76-bromodeoxyuridine as a proliferation marker.

UNLABELLED: The potential of 76Br-bromodeoxyuridine as a PET tracer for characterizing proliferation potential was investigated in multicellular tumor aggregates and in healthy rats and pigs. METHODS: Bromine-76-bromide was produced by proton irradiation of a 76Se-enriched target using a 17-MeV cyclotron and recovered by thermal diffusion. Bromine-76-BrdU was prepared from the corresponding trimethylstannate by an oxidative bromination. Multicellular aggregates from a carcinoid cell line and two bladder cancer cell lines were co-incubated with 76Br-BrdU and 3H-thymidine and the uptake and DNA incorporation analyzed. About 0.5 MBq 76Br-BrdU were injected in the tail vein of unanaesthetised Sprague-Dawley rats. Two to 36 hr later they were decapitated and the radioactivity concentration and fraction of radioactivity incorporated into DNA determined in five different organs and the blood. Parallel studies were performed in animals pretreated with hydroxyurea. In separate experiments, rats were given an injection of 76Br-bromide and organ uptake was evaluated after 20 hr. PET studies were performed in two pigs and the uptake in different organs was investigated after injection of 76Br-BrdU. In these studies, diuresis was induced by furosemide and mannitol and radioactivity in blood and organs was followed during 10 hr. RESULTS: In the cell aggregates, 30%-90% of the radioactivity was extracted in the DNA fraction. A good correlation was found between 76Br-BrdU and 3H-thymidine with respect to total uptake and DNA fraction. The DNA fraction increased from 2-10 hr after incubation. With in vivo injection in the rat, relatively high uptake of radioactivity was found in all organs, unrelated to the degree of DNA synthesis. However, inhibition by hydroxyurea occurred only in the spleen and intestines, organs which also showed a high degree of incorporation of 76Br-BrdU into DNA. In the pig, the highest in vivo uptake was observed in the red bone marrow and the intestines. In these organs, 70%-80% of the radioactivity was recovered in the DNA fraction. The concentration of radioactivity in the heart, liver and kidney was 3-10 times lower, and here the DNA fraction accounted for 10%-20% of the radioactivity. The decay-corrected radioactivity in blood and nonproliferating organs decreased with diuresis with a half-life of 13 and 16 hr, respectively. CONCLUSION: It is suggested that the radioactivity uptake as seen after the administration of 76Br-BrdU, is constituted by two parts: one relating to incorporation into DNA and one existing as free 76Br- or metabolites of 76Br-BrdU. If sufficient time has passed, 76Br- dominates other metabolites. A correct assessment of DNA-incorporated radioactivity using PET with 76Br-BrdU is not trivial and can only be made with due correction for 76Br-, using either a complementary investigation after hydroxyurea pretreatment (in animal studies) or a separate 76Br-bromide investigation. Alternatively, the free bromide can be eliminated partially through forced diuresis.

Animals↗
PubMed1998

Health and satisfaction among the elderly with chronic conditions: demographic differentials.

This study examines the state of health (physical and mental health) and avowed satisfaction (toward hospital services and towards life-as-a-whole), among 172 elderly subjects (108 outpatients from a family medicine clinic and 64 community residents) with chronic conditions living in Kaohsiung city. Results indicated that (a) the elderly did not perceive any great impact of illness(es) upon their lives and both their physical and mental health were satisfactory; (b) the elderly were generally satisfied with both the hospital services and life as a whole; (c) there were meaningful group differentials in health and satisfaction: older, male, single and financially-dependent elderly had poorer physical and mental health; female, single and financially dependent elderly avowed lower life satisfaction.

Aged↗
PubMed1997

Blockade of the CD40-CD40 ligand pathway potentiates the capacity of donor-derived dendritic cell progenitors to induce long-term cardiac allograft survival.

BACKGROUND: Failure of costimulatory molecule-deficient donor dendritic cells (DCs) to induce indefinite allograft acceptance may be a result of the 'late" up-regulation of these molecules on the DCs after interaction with host T cells. Ligation of CD40 on antigen-presenting cells by its cognate ligand CD40L is thought to induce expression of CD80 (B7-1) and CD86 (B7-2). We examined the influence of anti-CD40L monoclonal antibody (mAb) on the capacity of donor-derived DC progenitors to induce long-term allograft survival. METHODS: High purity DC progenitors were grown from B10 (H2b) mouse bone marrow in granulocyte-macrophage colony-stimulating factor and transforming growth factor beta1 (TGFbeta1). Mature DC were propagated in granulocyte-macrophage colony-stimulating factor and interleukin-4. Their phenotype was characterized by flow cytometric analysis and their function by mixed leukocyte reactivity. Anti-donor cytotoxic T lymphocyte activity in grafts and spleens of vascularized heart allograft recipients was also assessed. RESULTS: The TGFbeta3-cultured cells were (1) DEC 205-positive, MHC class II-positive, CD80dim, CD86dim, and CD40dim, (2) poor stimulators of naive allogeneic T-cell proliferation, and (3) able to prolong significantly B10 cardiac allograft survival in C3H (H2k) recipients when given (2 x 10[6] i.v.) 7 days before organ transplantation (median survival time [MST] 26 days vs. 12 days in controls, and 5 days in interleukin-4 DC-treated animals). Their allostimulatory activity was further diminished by addition of anti-CD40L mAb at the start of the mixed leukocyte cultures. Anti-CD40L mAb alone (250 microg/mouse, i.p.; day -7) did not prolong cardiac graft survival (MST 12 days). In contrast, TGFbeta-cultured DCs + anti-CD40L mAb extended graft survival to a MST of 77 days, and inhibited substantially the anti-donor cytotoxic T lymphocyte activity of graft-infiltrating cells and host spleen cells assessed 8 days after transplant. CONCLUSIONS: The CD40-CD40L pathway appears important in regulation of allogeneic DC-T-cell functional interaction in vivo; its blockade increases markedly the potential of costimulatory molecule-deficient DCs of donor origin to induce long-lasting allograft survival.

Animals↗
PubMed1997

Alpha2C-adrenoceptors mediate inhibition of forskolin-stimulated cAMP production in rat striatum.

The potential role of alpha2-adrenoceptors in modulating the activity of adenylyl cyclase in the rat striatum was examined. The selective alpha2-adrenoceptor agonist, UK14,304, produced a concentration-dependent inhibition of forskolin-stimulated accumulation of cAMP in striatal slices. The effect of UK14,304 was reversed by pre-incubation of striatal slices with the selective alpha2-adrenoceptor antagonist, RX821002. To determine whether alpha2C-adrenoceptors contribute to the alpha2-adrenoceptor-induced inhibition of forskolin-stimulated cAMP accumulation, an antisense oligodeoxynucleotide directed against alpha2C-adrenoceptor mRNA (alpha(2C)AS) or a random sequence (RS) was infused directly into the striatum. The ability of alpha(2C)AS to reduce the expression of alpha2C-adrenoceptors has been previously demonstrated. Alpha2C(AS) infusions did not reduce the ability of UK14,304 to inhibit forskolin-stimulated cAMP accumulation. Instead, alpha(2C)AS significantly enhanced forskolin-stimulated cAMP accumulation on the infusion side compared to the contralateral striatum. In contrast to the effects of alpha(2C)AS, infusions of RS had no effects on forskolin-stimulated cAMP accumulation or on the ability of UK14,304 to inhibit this effect. Incubation of striatal slices from untreated rats with RX821002 could mimic the ability of alpha(2C)AS infusion to enhance forskolin-stimulated cAMP accumulation, and did so in a concentration-dependent manner. Alpha2-adrenoceptors are negatively coupled to adenylyl cyclase in the rat striatum and alpha2C-adrenoceptors appear to be under tonic activation by an endogenous ligand in striatal slices.

Adenylyl Cyclases↗
PubMed1997

Augmentation of dendritic cells in murine organ donors by Flt3 ligand alters the balance between transplant tolerance and immunity.

Treatment of mice with the recently cloned hemopoietic growth factor Flt3 ligand (FL; 10 microg/day for 10 days) resulted in a large increase in myeloid lineage cells within the liver. While the number of nonparenchymal cells (NPC) harvested from liver increased about 9-fold, a 90-fold increase was observed in the proportion of CD11c+ dendritic cells (DC) recovered from NPC following overnight (18-h) culture in granulocyte-macrophage CSF. In contrast, only a 50% increase was seen in CD11c+ cells within heart single cell suspensions and in the number of DC obtained from hearts after 18-h culture. Liver NPC and heart cell suspensions freshly isolated from 10-day FL-treated animals exhibited increased T cell allostimulatory capacity compared with controls. Overnight cultured DC from livers of FL-treated animals expressed both higher levels of costimulatory molecules (CD80 and CD86) and allostimulatory activity than those from controls. Heart-derived DC also displayed enhanced stimulatory capacity. Pretreatment of organ donors with FL for either 5 or 10 days before transplant of organs to normal recipients abrogated the spontaneous liver allograft acceptance normally observed and resulted in delayed or acute graft rejection (median survival times, 40 and 12 days, respectively). Heart rejection was significantly accelerated by pretreatment of donors with FL for 5 or 10 days (median survival times, 8 and 7 days, respectively, vs 12 days in controls). These novel findings reveal the potent immunologic adjuvant properties of FL in vivo. They also show that substantial augmentation of the number of potential allostimulatory cells in donor organs before transplantation favors rejection rather than tolerance induction.

Animals↗
PubMed1997

Human Prk is a conserved protein serine/threonine kinase involved in regulating M phase functions.

Human prk encodes a novel protein serine/threonine kinase capable of strongly phosphorylating casein but not histone H1 in vitro. prk expression is tightly regulated at various levels during different stages of the cell cycle in lung fibroblasts. The Prk kinase activity is relatively low during mitosis, G1, and G1/S, and peaks during late S and G2 stages of the cell cycle. Recombinant human Prk expressed through the baculoviral vector system is capable of phosphorylating Cdc25C, a positive regulator for the G2/M transition. Human prk shares significant sequence homology with Saccharomyces cerevisiae CDC5 and Drosophila melanogaster polo, both of which are essential for mitosis and meiosis. Full-length prk transcripts greatly potentiate progesterone-induced meiotic maturation of Xenopus laevis oocytes. On the other hand, antisense prk transcripts significantly delay and reduce the rate of oocyte maturation. When expressed in a CDC5 mutant strain of S. cerevisiae, human Prk, but not a deletional mutant protein, fully rescues the temperature-sensitive phenotype of the budding yeast. Taken together, prk may represent a new protein kinase, playing an important role in regulating the onset and/or progression of mitosis in mammalian cells.

Amino Acid Sequence↗
PubMed1997

In vivo administration of flt3 ligand markedly stimulates generation of dendritic cell progenitors from mouse liver.

The study of liver dendritic cells (DC) and their progenitors is restricted by the small numbers that can be isolated or propagated from normal hepatic tissue. We examined the ex vivo growth, phenotype, and function of these cells after the administration to mice of the recently cloned hemopoietic growth factor flt3 ligand (FL), which is highly effective in mobilizing stem/progenitor cells. FL treatment (10 microg/day for 10 days) resulted in a mean 14-fold increase in the absolute number of nonparenchymal cells recovered from collagenase-digested livers compared with the control value. Culture of these nonparenchymal cells in granulocyte-macrophage CSF (GM-CSF; 1000 U/ml) resulted in the early formation of proliferating cell clusters and maximal release (within 4-5 days) of markedly increased numbers of nonadherent, low buoyant density cells per liver. Maximal release of low buoyant density cells propagated from control livers was at the later time of 6 to 8 days. Cells from both sources were DEC-205+, CD11c+, MHC class II+, CD80(low) (i.e., low level of CD80), CD86(low) and CD40(low). This immature phenotype was linked to poor T cell allostimulatory activity, indicative of DC progenitors. Propagation of cells from livers of FL-treated mice in GM-CSF and IL-4 resulted in a more mature DC phenotype and function. Maturational changes were also observed following exposure of the GM-CSF-stimulated progenitors to type 1 collagen for 3 additional days. The ability of FL to boost production of large numbers of liver DC progenitors provides opportunities for the further study of these important APC in normal liver immunobiology and in immune-mediated hepatic disorders.

Animals↗
PubMed1997

Suppression of cytochrome P450 Cyp2f2 mRNA levels in mice by the peroxisome proliferator diethylhexylphthalate.

Exposure to peroxisome proliferators, which are extensively used, causes a number of pleiotrophic effects. Prolonged exposure to the peroxisome proliferator, DEHP, causes hepatic hyperplasia and liver tumors in rats and mice. This exposure can also induce a number of enzymes. To identify additional genes that are regulated by DEHP, mRNA differential display was used. One of the genes affected is cytochrome 450 Cyp2f2, a naphthalene hydroxylase. Using northern analysis, RNase protection assay, and RT-PCR, we show that the Cyp2f2 mRNA levels are decreased in mouse liver following DEHP treatment. A smaller Cyp2f2 mRNA transcript was also detected in kidney and these transcript levels were also suppressed but to a lesser extent than that in the liver. The response to DEHP in mouse liver is both dose and time dependent.

Animals↗
PubMed1997

PLAB, a novel placental bone morphogenetic protein.

Bone morphogenetic proteins (BMP) constitute a sub-group of the large transforming growth factor-beta (TGF-beta) family. They play important roles in the embryonic development of multiple structures and in adult bone modeling. We have recently isolated a novel member of the BMP family from placenta, termed PLAB. PLAB is expressed highly in placenta, but can be found upon stringent analysis in low levels in most other tissues. At the amino acid level, PLAB is most closely related to BMP-8/OP-2, another member of the BMP family. Like TGF-beta, PLAB inhibits the proliferation of primitive hematopoietic progenitors. The high expression of PLAB by placenta raises the possibility that it may be a mediator of placental control of embryonic development.

3T3 Cells↗
PubMed1997

D-Glucose, forskolin and cytochalasin B affinities for the glucose transporter Glut1. Study of pH and reconstitution effects by biomembrane affinity chromatography.

The affinities of D-glucose and the transport inhibitors, forskolin and cytochalasin B (CB), for Glut1 were studied by frontal affinity chromatography at pH 5-10 on sterically immobilized proteoliposomes with reconstituted human red cell glucose transporter Glut1. The affinity of D-glucose for Glut1 became slightly weaker as the pH was increased. The inhibitor affinities decreased and became immeasurably weak above pH 9. At pH 7.4, the dissociation constants were 44 mM for glucose, 1.8 microM for forskolin and 72 nM for CB. The affinities of these solutes for Glut1 in red cell membrane vesicles and particularly for Glut1 in red cells were higher, as shown by chromatographic analyses.

Adult↗
PubMed1997

Fas ligand (CD95L) and B7 expression on dendritic cells provide counter-regulatory signals for T cell survival and proliferation.

Activation of T cells is induced efficiently by dendritic cells (DC), but little is known about the role of DC in the regulation of T cell death. In this study, highly purified DC (DEC-205+, MHC class II(high), B7-1+ [CD80+], B7-2high [CD86high], CD40+, CD11c+) grown from normal mouse bone marrow in granulocyte-macrophage CSF + IL-4 were found to express FasL (CD95L) mRNA by reverse transcriptase PCR and to uniformly express FasL by both flow cytometric and immunocytochemical analyses. These cells, but not DC propagated from FasL-deficient (B6.gld) mice, induced dose-dependent increases in DNA fragmentation in Fas+ Jurkat T cells over 18 h coculture. Addition of mouse Fas-Fc fusion protein at the start of the cultures diminished this effect. Even at high relative concentrations, however, B7-2high DC induced only low levels of DNA fragmentation in Con A or alloactivated splenic T cells, as determined by radio- or spectrofluorometric assays and by in situ nick-end labeling. However, in the presence of CTLA4Ig, a molecule that blocks the B7-CD28 costimulatory pathway, DC that failed to stimulate in primary MLR induced markedly augmented levels of apoptosis in alloactivated T cells. CTLA4Ig treatment also increased the level of DNA fragmentation induced by FasL-deficient DC, indicating the existence of additional potential (Fas-independent) pathways of DC-induced T cell death. These findings suggest that the costimulatory (B7-CD28) and T cell death-inducing pathways may play important counter-regulatory roles in dictating the outcome of (allogeneic) DC-T cell interactions.

Animals↗
PubMed1997

Apoptosis during B lymphopoiesis in mouse bone marrow.

To evaluate the magnitude of cell death and the critical stages at which it occurs during B lymphopoiesis in mouse bone marrow (BM), we have examined the kinetics of apoptosis at defined stages of B cell differentiation. FACS-sorted B220+ BM cells exhibited a low incidence of morphologically apoptotic cells by electron microscopy. In freshly prepared BM suspensions, the incidence of hypodiploid cells detected by multiparameter flow cytometry was greater among large dividing B220+ surface IgM- (sIgM-) precursor B cells and sIgM(low) immature B lymphocytes than among terminal deoxynucleotidyl transferase+ (TdT+) pro-B cells, small nondividing B220+ sIgM- precursors, and surface IgD+ mature B lymphocytes. During short-term culture, apoptotic cells, identified by both DNA content and in situ DNA strand break labeling, increased linearly with time without macrophage ingestion, providing an assay for the rate of entry into apoptosis. B220+ B lineage cells accumulated in apoptosis more rapidly than cells of other lineages. The apoptotic rate was greater among B220+ sIgM- precursor cells than sIgM+ B cells, and was highest among B220+ mu- pro-B cells. Coculture with stromal cells reduced the apoptotic rate of B220+ sIgM- precursors to a greater extent than that of sIgM+ B lymphocytes. The results lead to estimates of the actual number of B lineage cells undergoing apoptosis per unit time in successive differentiation compartments. The findings indicate that, although influenced by local microenvironmental factors, apoptotic cell death occurs most markedly at two developmental stages associated with Ig heavy chain gene rearrangement and Ag receptor expression, respectively.

Animals↗
PubMed1997

Apoptosis within spontaneously accepted mouse liver allografts: evidence for deletion of cytotoxic T cells and implications for tolerance induction.

MHC-mismatched liver grafts are accepted spontaneously between many mouse strains. The underlying mechanism(s) is unclear. In the B10 (H2(b)) to C3H (H2(k)) strain combination used in this study, donor T cells within the liver were rapidly replaced within 2 to 4 days of transplantation with those of the recipient. Freshly isolated liver graft-infiltrating cells harvested on days 4 and 7 exhibited strong CTL responses against donor alloantigens. CTL activity was reduced substantially, however, by day 14, although levels of CTL precursors in the spleen and liver remained high. Examination of the liver allografts by in situ terminal deoxynucleotidyltransferase-catalyzed dUTP-digoxigenin nick end labeling on days 4, 7, and 14 after transplantation revealed prominent apoptotic cells dispersed throughout the nonparenchymal cell population. When acute liver allograft rejection was induced by administration of IL-2 from days 0 to 4 post-transplant (median survival time, 5 days), apoptotic activity (day 4) was reduced substantially, whereas CTL activity was enhanced. Nonparenchymal cells isolated from allografts of unmodified recipients 4, 7, and 14 days after transplantation exhibited significantly higher DNA fragmentation after 18-h culture than cells from liver isografts. Moreover, the level was 4 to 5 times higher than that of cells from IL-2-treated mice (on day 4). These observations suggest that T cell deletion, not regulation, may be responsible for spontaneous liver allograft acceptance. The molecular recognition events that cause apoptosis of infiltrating T cells and why this occurs within liver grafts, but not heart or skin grafts, remain to be elucidated.

Animals↗
PubMed1997

Effect of substituting amino acids in extracellular disulfide loop on GABA rho1 subunit function.

Common features of GABA receptor/channels include a large extracellular NH2-terminal region that is the receptor domain, and four putative transmembrane segments that contribute to form the channel. A conserved Cys-Cys loop of the NH2-terminal domain in GABA receptor/channels is essential for maintaining the receptor and channel function. It has been suggested by other investigators that alteration of amino acids between these two cystines may affect the channel open kinetics. To investigate the mechanism underlying ligand binding and channel open, we have substituted several amino acids in the Cys-Cys loop of the GABA rho1 subunit and for the first time demonstrated the effect of mutations in the Cys-Cys loop on the single-channel kinetics of the rho1 subunit. For example, a single substitution of Arg198 in the extracellular Cys-Cys loop with Ala or Lys significantly reduced both the maximal amplitude of the whole-cell current and the single-channel open probability (Po). Single channel kinetics of mutants revealed no long open state, without changes in the short open state. There was a shift of apparent half-activation concentration (EC50) between the control and mutants in response to GABA. Our data further suggest that the Cys-Cys loop in the GABA rho1 subunit plays an important role in the receptor/channel function.

Amino Acid Sequence↗