Biomedical subjects

X Liu

Publications and source records attributed to X Liu.

At least 1,027 records · Page 57Linked to original sources

PubMed1998

Differential expression of Nur77 family members in human T-lymphotropic virus type 1-infected cells: transactivation of the TR3/nur77 gene by Tax protein.

We analyzed the differential expression and regulation of three members of the Nur77 transcription factor family by the human T-lymphotropic virus type 1 (HTLV-1) Tax protein. We have demonstrated that in both HTLV-1-infected cells and Tax-expressing JPX-9 cells, TR3/nur77 is highly expressed, whereas neither NOR-1 nor NOT expression is detectable. Transient transfection analysis further confirmed the Tax transactivation of the TR3/nur77 promoter but not the NOR-1 promoter in different cell types. Furthermore, expression of a luciferase reporter gene driven by the NGFI-B (rat homolog of TR3/Nur77) response element (NBRE) provided evidence that Tax-mediated transactivation resulted in the induction of a functional protein. Cotransfection assays with the TR3/nur77 promoter sequence or the NBRE binding motif together with a series of Tax mutants have shown that Tax-induced TR3/nur77 expression is mediated by CREB/ATF-related transcription factors.

Activating Transcription Factor 1↗

PubMed1998

Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia.

To study and define the early time-dependent response (< or = 6 h) of blocker-sensitive epithelial Na+ channels (ENaCs) to stimulation of Na+ transport by aldosterone, we used a new modified method of blocker-induced noise analysis to determine the changes of single-channel current (iNa) channel open probability (Po), and channel density (NT) under transient conditions of transport as measured by macroscopic short-circuit currents (Isc). In three groups of experiments in which spontaneous baseline rates of transport averaged 1.06, 5.40, and 15.14 microA/cm2, stimulation of transport occurred due to increase of blocker-sensitive channels. NT varied linearly over a 70-fold range of transport (0.5-35 microA/cm2). Relatively small and slow time-dependent but aldosterone-independent decreases of Po occurred during control (10-20% over 2 h) and aldosterone experimental periods (10-30% over 6 h). When the Po of control and aldosterone-treated tissues was examined over the 70-fold extended range of Na+ transport, Po was observed to vary inversely with Isc, falling from approximately 0.5 to approximately 0.15 at the highest rates of Na+ transport or approximately 25% per 3-fold increase of transport. Because decreases of Po from any source cannot explain stimulation of transport by aldosterone, it is concluded that the early time-dependent stimulation of Na+ transport in A6 epithelia is due exclusively to increase of apical membrane NT.

Aldosterone↗

PubMed1998

Hormonal regulation of ENaCs: insulin and aldosterone.

Although a variety of hormones and other agents modulate renal Na+ transport acting by way of the epithelial Na+ channel (ENaC), the mode(s), pathways, and their interrelationships in regulation of the channel remain largely unknown. It is likely that several hormones may be present concurrently in vivo, and it is, therefore, important to understand potential interactions among the various regulatory factors as they interact with the Na+ transport pathway to effect modulation of Na+ reabsorption in distal tubules and other native tissues. This study represents specifically a determination of the interaction between two hormones, namely, aldosterone and insulin, which stimulate Na+ transport by entirely different mechanisms. We have used a noninvasive pulse protocol of blocker-induced noise analysis to determine changes in single-channel current (iNa), channel open probability (Po), and functional channel density (NT) of amiloride-sensitive ENaCs at various time points following treatment with insulin for 3 h of unstimulated control and aldosterone-pretreated A6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10-30 min the density of the pool of apical membrane ENaCs (NT) involved in transport. The very early (10 min) increases of channel density were accompanied by relatively small decreases of iNa (10-20%) and decreases of p.o. (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes of iNa, p.o., and NT were transient, returning very slowly over 3 h toward their respective control values at the time of addition of insulin. We conclude that aldosterone and insulin act independently to stimulate apical Na+ entry into the cells of A6 epithelia by increase of channel density.

Aldosterone↗

PubMed1998

Differential effects of phorbol ester (PMA) on blocker-sensitive ENaCs of frog skin and A6 epithelia.

Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) caused complex transient perturbations of amiloride-sensitive short-circuit Na+ currents (INa) in A6 epithelia and frog skins that were tissue and concentration dependent. A noninvasive channel blocker pulse method of noise analysis (18) was used to investigate how PMA caused time-dependent changes of apical membrane epithelial Na+ channel (ENaC) single-channel currents, channel open probabilities (Po), and channel densities (NT). In A6 epithelia, 5 and 50 nM PMA caused within 7 min concentration-dependent sustained decreases of Po (approximately 55% below control, 50 nM) and rapid compensatory transient increases of NT within 7 min ( approximately 220% above control, 50 nM), resulting in either small transient increases of INa at 5 nM PMA or small biphasic decreases of INa at 50 nM PMA. In contrast to A6 epithelia, 50 and 500 nM PMA in frog skin caused after a delay of at least 10 min transient increases of NT to approximately 60-70% above control at 30-60 min. Unlike A6 epithelia, Po was increased approximately 15% above control within 7 min and remained within +/-10-15% of control for the duration of the 2-h experiments. Despite differences in the time courses of secondary inhibition of transport in A6 epithelia and frog skin, the delayed downregulation of transport was due to time-dependent decreases of NT from their preelevated levels in both tissues. Whereas Po is decreased within minutes in A6 epithelia as measured by noise analysis or by patch clamp (8), the discrepancy in regulation of NT in A6 epithelia as measured by noise analysis and patch clamp is most likely explained by the inability of on-cell patches formed before treatment of tissues with PMA to respond to regulation of their channel densities.

Amiloride↗

PubMed1998

Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells.

This study examines the Ca2+ influx-dependent regulation of the Ca2+-activated K+ channel (KCa) in human submandibular gland (HSG) cells. Carbachol (CCh) induced sustained increases in the KCa current and cytosolic Ca2+ concentration ([Ca2+]i), which were prevented by loading cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA). Removal of extracellular Ca2+ and addition of La3+ or Gd3+, but not Zn2+, inhibited the increases in KCa current and [Ca2+]i. Ca2+ influx during refill (i.e., addition of Ca2+ to cells treated with CCh and then atropine in Ca2+-free medium) failed to evoke increases in the KCa current but achieved internal Ca2+ store refill. When refill was prevented by thapsigargin, Ca2+ readdition induced rapid activation of KCa. These data provide further evidence that intracellular Ca2+ accumulation provides tight buffering of [Ca2+]i at the site of Ca2+ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggest that the Ca2+ influx-dependent regulation of the sustained KCa current in CCh-stimulated HSG cells is mediated by the uptake of Ca2+ into the internal Ca2+ store and release via the inositol 1,4,5-trisphosphate-sensitive channel.

Atropine↗

PubMed1998

Distribution of heme oxygenase and effects of exogenous carbon monoxide in canine jejunum.

Carbon monoxide (CO) has been postulated to be a messenger in the gastrointestinal tract. The aims of this study were to determine the distribution of heme oxygenase (HO), the source for endogenous CO in the canine jejunum, and to determine the effects of CO on jejunal circular smooth muscle cells. HO-2 isoform was present in a population of myenteric and submucosal neuronal cell bodies, in nerve fibers innervating the muscle layers, and in smooth muscle cells. HO-1 isozyme was not detected in the canine jejunum. Exogenous CO increased whole cell current by 285 +/- 86%, hyperpolarized the membrane potential by 8.5 +/- 2.9 mV, and increased guanosine 3',5'-cyclic monophosphate (cGMP) levels in smooth muscle cells. 8-Bromo-cGMP also increased the whole cell current. The data suggest that endogenous activity of HO-2 may be a source of CO in the canine jejunum and that exogenously applied CO can modulate intestinal smooth muscle electrical activity. It is therefore reasonable to suggest a role for endogenously produced CO as a messenger in the canine jejunum.

Alkaloids↗

PubMed1998

Role of Na+ and Ca2+ in stretch-induced Na(+)-K(+)-ATPase alpha-subunit regulation in aortic smooth muscle cells.

This study was designed to test the role of Na+ and Ca2+ entry in the stretch-induced Na(+)-K(+)-ATPase alpha 1- and alpha 2-isoform upregulation observed in our previous studies. We measured intracellular Na+ in cyclically stretched rat aortic smooth muscle cells, with or without gadolinium treatment, for various durations and performed Western blotting to analyze the effects of stretch and the calcium channel blocker isradipine on the expression of alpha-isoforms. Intracellular Na+ was elevated significantly after 1- and 2-h stretch, but returned to baseline after 1-, 2-, and 4-day stretch. This increase in intracellular Na+ was blocked by gadolinium. Both alpha 1- and alpha 2-isoforms were upregulated after either 2 or 4 days of cyclical stretch. Isradipine had no apparent effect on stretch-induced upregulation on either alpha-isoform, thus suggesting that Ca2+ entry through L-type channels is not involved in the stretch-induced upregulation. We therefore conclude that a transient intracellular Na+ elevation during stretch may serve as a signal to mediate the alpha 1- and alpha 2-isoform upregulation.

Animals↗

PubMed1998

Human bronchial epithelial cells can contract type I collagen gels.

Fibroblasts can contract collagen gels, a process thought to be related to tissue remodeling. Because epithelial cells are also involved in repair responses, we postulated that human bronchial epithelial cells (HBECs) could cause contraction of collagen gels. To evaluate this, HBECs were plated on the top of native type I collagen gels and were incubated for 48 h. After this, the gels were released and floated in LHC-9-RPMI 1640 for varying times, and gel size was measured with an image analyzer. HBECs caused a marked contraction of the gels within 24 h; the area was reduced by 88 +/- 4% (P < 0.01). The degree of gel contraction was dependent on cell density; 12,500 cells/cm2 resulted in maximal contraction, and half-maximal contraction occurred at 7,500 cells/cm2. Contraction varied inversely with the collagen concentration (91 +/- 1% with 0.5 mg/ml collagen vs. 43 +/- 5% with 1.5 mg/ml collagen). In contrast to fibroblasts that contract gels most efficiently when cast into the gel, HBEC-mediated contraction was significantly (P < 0.01) more efficient when cells were on top of the gels rather than when cast into the gels. Anti-beta 1-integrin antibody blocked HBEC-mediated contraction by > 50%, whereas anti-alpha 2-, anti-alpha 3-, anti-alpha v-, anti-alpha v beta 5-, anti-beta 2-, or anti-beta 4-integrin antibody was without effect. The combination of anti-beta 1-integrin antibody and an anti-alpha-subfamily antibody completely blocked gel contraction induced by HBECs. In contrast, anti-cellular fibronectin antibody did not block HBEC-induced gel contraction, whereas it did block fibroblast-mediated gel contraction. In summary, human airway epithelial cells can contract type I collagen gels, a process that appears to require integrins but may not require fibronectin. This process may contribute to airway remodeling.

Animals↗

PubMed1998

Cigarette smoke extract inhibits fibroblast-mediated collagen gel contraction.

Cigarette smoking, the major cause of pulmonary emphysema, is characterized by destruction of alveolar walls. Because tissue destruction represents a balance between injury and repair, we hypothesized that cigarette smoke exposure may contribute to the development of emphysema through the inhibition of tissue contraction during the repair process. To partially evaluate this hypothesis, we investigated the effects of cigarette smoke extract (CSE) on the ability of cultured fibroblasts to mediate collagen gel contraction in vitro: CSE inhibited fibroblast-mediated gel contraction in a concentration-dependent manner (P < 0.01). Production of prostaglandin E2, a known inhibitor of fibroblast contraction, was unchanged by CSE as was cell surface integrin expression. In contrast, fibronectin production by fibroblasts was inhibited (P < 0.01), and addition of exogenous fibronectin partially restored the contractile activity, thus suggesting at least one mechanism to explain inhibition of gel contraction by CSE. When CSE was treated to remove volatile components, it showed less inhibitory activity on fibroblast-mediated gel contraction. Therefore, we also examined the effects of acrolein and acetaldehyde, two volatile components of cigarette smoke. Inhibition of contraction was observed at 5 microM acrolein and at 0.5 mM acetaldehyde. In conclusion, cigarette smoke inhibited fibroblast-mediated gel contraction, and this inhibition was due, at least in part, to the volatile components of cigarette smoke and may be mediated, at least in part, by a decrease in fibroblast fibronectin production. By inhibition of repair, these smoke components may contribute to the development of pulmonary emphysema.

Cell Survival↗

PubMed1998

Mechanisms of the antidiabetic effects of the beta 3-adrenergic agonist CL-316243 in obese Zucker-ZDF rats.

Previous studies have demonstrated that chronic cold exposure activates the sympathetic nervous system, increases energy expenditure, improves glucose uptake in peripheral tissues [brown and white adipose tissues (BAT and WAT) and muscles] of normal rats. The goal of the present studies was to test whether the selective beta 3-adrenergic agonist CL-316243 (CL) would mimic the beneficial beneficial effects of cold exposure in lean and obese ZDF/Gmi-fa male (ZDF) rats, a new model of type II diabetes. In obese ZDF rats, chronic infusion of CL (1 mg.kg-1.day-1 for 14 days) significantly decreased body weight gain, food intake, and WAT weight. It also increased total tissue cytochrome oxidase activity, not only in BAT (15 times), but also in WAT (2-4) times, suggesting that it progressively enhanced mitochondriogenesis in adipose tissues. CL treatment normalized hyperglycemia and reduced hyperinsulinemia and circulating free fatty acid (FFA) levels. It also improved glucose tolerance and reduced insulin response during an intravenous glucose tolerance test. In general, the beneficial effects of CL were more pronounced in obese than in lean rats. Hyperinsulinemic-euglycemic glucose clamps combined with the [2-3H]deoxyglucose method revealed that CL markedly improved insulin responsiveness in obese rats (3-4 times) and increased glucose uptake in BAT (21 times), WAT (3 times), skeletal muscles (2-3 times), and in the diaphragm (2.8 times), but not in the heart. It is concluded that chronic CL treatment improves glucose tolerance and insulin responsiveness in obese ZDF rats by a mechanism similar to that induced by chronic cold exposure, i.e., by stimulating facultative thermaogenesis, mitochondriogenesis, and glucose utilization in BAT and WAT. In addition to this mechanism, the reduction in plasma FFA levels induced by chronic CL treatment may further contribute to enhance glucose uptake in skeletal muscles (a tissue that does not express typical beta 3-adrenoceptors) via the "glucose-fatty acid" cycle. The antiobesity and antidiabetic properties of CL suggest that selective beta 3-adrenergic agonists may represent useful agents for the treatment of type II diabetes.

Adipose Tissue, Brown↗

PubMed1998

PMA and staurosporine affect expression of the PCK gene in LLC-PK1-F+ cells.

The addition of phorbol 12-myristate 13-acetate (PMA) to renal LLC-PK1-F+ cells caused a rapid decrease in the level of phosphoenolpyruvate carboxykinase (PCK) mRNA and reversed the stimulatory effects of exposure to acidic medium (pH 6.9, 10 mM HCO-3) or cAMP. In contrast, prolonged treatment with PMA increased the levels of PCK mRNA. The two effects correlated with the membrane translocation and downregulation of the alpha-isozyme of protein kinase C and were blocked by pretreatment with specific inhibitors of protein kinase C. The rapid decrease in PCK mRNA caused by PMA occurred with a half-life (t1/2 = 1 h) that is significantly faster than that measured during recovery from acid medium or following inhibition of transcription (t1/2 = 4 h). The effect of PMA was reversed by staurosporine, which apparently acts by inhibiting a signaling pathway other than protein kinase C. Staurosporine had no effect on the half-life of the PCK mRNA, but it stimulated the activity of a chloramphenicol acetyltransferase gene that was driven by the initial 490 base pairs of the PCK promoter and transiently transfected into LLC-PK1-F+ cells. This effect was additive to that of cAMP, and neither stimulation was reversed by PMA. The stimulatory effect of staurosporine was mapped to the cAMP response element (CRE-1) and P3(II) element of the PCK promoter. The data indicate that, in LLC-PK1-F+ cells, activation of protein kinase C decreases the stability of the PCK mRNA, whereas transcription of the PCK gene may be suppressed by a kinase that is inhibited by staurosporine.

Animals↗

PubMed1998

Regulatory effects of soluble growth factors on choriocapillaris endothelial growth and survival.

The effects of soluble growth factors on regulating the survival and growth of choriocapillaris endothelial (CCE) cells were investigated in vitro. CCE cells were cultured in a serum-free medium in the presence or absence of various soluble growth factors. Cell growth and cell viability were assessed by counting viable cells. Results showed that acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) significantly stimulated CCE growth in a dose-dependent manner. Platelet-derived growth factor BB (PDGF, 0.4-10 ng/ml), insulin-like growth factor (IGF, 0.4-10 ng/ml) or insulin (0.4-10 micrograms/ml) alone did not affect the growth of CCE cells. In the presence of insulin (10 micrograms/ml), however, PDGF stimulated CCE growth in a dose-dependent manner. By contrast, transforming growth factor beta 1 (TGF-beta 1) induced CCE death in the absence of other growth factors and inhibited the CCE growth induced by aFGF, bFGF or VEGF. When CCE cells had been cultured in media containing aFGF, bFGF, VEGF or the combination of PDGF and insulin for 48 h, withdrawal of aFGF, bFGF or VEGF, but not PDGF, from the media resulted in CCE death. The CCE death induced by either an introduction of TGF-beta 1 or withdrawal of aFGF, bFGF or VEGF was defined as apoptosis based on morphologic characteristics (condensation and fragmentation of nuclei, shrinkage of cells in size) and DNA fragmentation in multiples of approximately 180 base pairs. Phorbol 12-myristate 13-acetate (PMA, 2 or 5 nM) protected CCE cells against apoptosis induced by the introduction of TGF-beta 1 and withdrawal of aFGF, bFGF or VEGF, while H7 (50 microM), but not HA1004 (50 microM), abrogated the protective effect of PMA on CCE apoptosis. However, cycloheximide (0.1 microM), a protein synthesis inhibitor, was only effective to protect CCE cells from apoptosis induced by aFGF, bFGF or VEGF withdrawal, but not that induced by the introduction of TGF-beta 1. Moreover, cycloheximide and a low concentration of PMA (2 nM) showed an additive effect on protecting CCE cells from apoptosis as the result of a growth factor withdrawal. These data may suggest that some growth factors, such as aFGF, bFGF, VEGF and TGF-beta 1, play a more critical role than the others, such as PDGF and IGF, in the regulation of CCE growth and survival. CCE apoptosis as a result of withdrawal of a survival factor or of receiving a death factor such as TGF-beta 1 may involve different mechanisms, which merits further study.

Animals↗

PubMed1998

Managed behavioral health services for children under carve-out contracts.

OBJECTIVE: Service costs and utilization patterns of children in carved-out behavioral health care plans were examined and compared with those of adults. METHODS: Twelve-month data on utilization and costs of behavioral health care from one managed behavioral health care carve-out organization, United Behavioral Health, were examined for three age groups of children--birth to five years, six to 12 years, and 13 to 17 years-and for adults. More than 600,000 enrollees in 108 different plans were included in the data. Rates of use and intensity of use were examined separately by type of service-inpatient, outpatient, and partial hospitalization. RESULTS: Only a small number of all enrollees used any behavioral health care services--4.2 percent used outpatient services, .3 percent used inpatient services, and .2 percent used partial hospitalization services. Adolescents were more than twice as likely as adults and about seven times as likely as children aged 6 to 12 to use inpatient services. Adolescents also had a slightly higher probability of using outpatient care than adults, while younger children had lower rates of outpatient use than adolescents or adults. Adolescents were also more likely than adults and other children to have very high costs of inpatient care (mean costs=$8,975 for adolescents and $4,750 for adults). Adults were more likely than other groups to have higher outpatient costs ($640 for adults and $513 for all children). CONCLUSIONS: The finding that children, and adolescents in particular, are more likely to have very high inpatient costs compared with adults implies that they may benefit most from the elimination of caps on mental health care costs covered by insurance. This profile of children's behavioral health care utilization patterns can be useful to policy makers in considering expansions in children's health insurance coverage.

Adolescent↗

PubMed1998

Effects of growth hormone, activin, and follistatin on the development of preantral follicle from immature female mice.

The aim of this study was to investigate whether GH and insulin-like growth factor I (IGF-I) are involved in preantral folliculogenesis and, if so, to clarify the relationship between GH/IGF-I and activin/follistatin (FS) systems in immature female mice. Ovaries were obtained from 11-day-old mice, and preantral follicles, 100-105 microm in diameter, were mechanically isolated and selected for culture. Ten preantral follicles per well were cultured with different quantities and combinations of additives as follows: no additives (control), recombinant human FSH (rhFSH), IGF-I, recombinant human GH (rhGH), activin A, and recombinant human FS (rhFS). Mean diameters of the follicles were measured daily, and estradiol and immunoreactive inhibin levels in the cultured medium were assayed by RIA on day 4. rhGH showed stimulatory effects on the follicular diameter and the secretion of estradiol and immunoreactive inhibin. These effects were augmented by the presence of IGF-I and activin A. IGF-I alone did not show any stimulatory effect. The addition of rhFSH to activin A or to rhGH and activin A promoted preantral follicular growth and hormone production. On the other hand, GH- or activin-stimulated follicular growth was suppressed by rhFS in a dose-dependent manner. These results indicate that activin A and rhGH may play an important role in controlling earlier phases of follicular development during the infantile period, which is considered to be gonadotropin independent.

Activins↗

PubMed1998

A unique constitutively activating mutation in third transmembrane helix of luteinizing hormone receptor causes sporadic male gonadotropin-independent precocious puberty.

Several constitutively activating mutations have been demonstrated in the sixth transmembrane helix of the human LH receptor (hLHR) in boys with gonadotropin-independent precocious puberty. In the current study, we examined two unrelated Brazilian boys with gonadotropin-independent precocious puberty caused by two different heterozygous activating mutations of the hLHR. Direct sequencing of the entire exon 11 of the hLHR revealed a heterozygous substitution of T for G at nucleotide 1370, that converts Leu 457 to Arg in the third transmembrane helix of the hLHR in one affected boy. His biological parents had a normal hLHR gene sequence, establishing the sporadic nature of this novel Leu457Arg mutation. Human embryonic 293 cells expressing hLHR mutant (L457R) or hLHR wild-type bound CG with high affinity. However, cells expressing hLHR(L457R) exhibited significantly higher basal levels of cAMP (7- to 14-fold) than cells expressing the wild-type receptor, indicating constitutive activation of hLHR(L457R). Basal levels of cAMP in hLHR(L457R)-expressing cells were, nonetheless, not as great as the levels of cAMP produced by hLHR wild-type-expressing cells incubated with a saturating concentration of CG. Furthermore, cells expressing hLHR(L457R) were unresponsive to further stimulation by CG. This finding was confirmed in the patient by lack of an increase in serum testosterone after CG stimulation. These results suggest that the conformation of hLHR(L457R) mutant represents a different activated receptor state (R*) than the agonist-occupied wild-type receptor. We also identified the previously described Ala568Val mutation in the third intracellular loop of the LHR in the other affected African-Brazilian boy and his normal prepubertal sister, suggesting the inherited form of precocious puberty in this boy. We conclude that the third transmembrane helix is a potential area for activating mutations of the hLHR that cause male precocious puberty.

Amino Acid Sequence↗

PubMed1998

A homozygous microdeletion in helix 7 of the luteinizing hormone receptor associated with familial testicular and ovarian resistance is due to both decreased cell surface expression and impaired effector activation by the cell surface receptor.

In this report, the genomic DNA was examined from two siblings with gonadal LH resistance. A 46,XY pseudohermaphrodite presented with female external genitalia and his 46,XX sister exhibited menstrual irregularities (oligoamenorrhea) and infertility. Exons 1-11 of the LH receptor (LHR) gene were amplified by the PCR using different sets of intronic primers and were directly sequenced. Sequencing revealed that both individuals carried a deletion of nucleotides 1822-1827, resulting in the deletion of Leu-608 and Val-609 within the seventh transmembrane helix. This mutation was introduced into a recombinant human (h) LHR cDNA. Transfections of 293 cells with hLHR(wt) vs. hLHR(deltaL608,V609) revealed that very little of the mutant receptor was expressed at the cell surface. This was due to both a decrease in the total amount of receptor expressed as well as to an increased intracellular retention of the mutant receptor. In spite of the decreased cell surface expression of the mutant, sufficient amounts were present to allow for assessment of its functions. Equilibrium binding assays showed that the cell surface hLHR(deltaL608,V609) binds hCG with an affinity comparable to that of the wild-type receptor. However, the cells expressing the hLHR(deltaL608,V609) exhibit only a 1.5- to 2.4-fold stimulation of cAMP production in response to hCG. In contrast, cells expressing comparably low levels of hLHR(wt) responded to hCG with 11- to 30-fold increases of cAMP levels. Therefore, the testicular and ovarian unresponsiveness to LH in these patients appears to be due to a mutation of the hLHR gene in which Leu-608 and Val-609 are deleted. As a consequence, the majority of the mutant receptor is retained intracellularly. The small percentage of mutant receptor that is expressed at the cell surface binds hormone normally but is unable to activate Gs.

Adult↗

PubMed1998

Opl: a zinc finger protein that regulates neural determination and patterning in Xenopus.

In order to study the mechanism of neural patterning in Xenopus, we used subtractive cloning to isolate genes activated early during this process. One gene isolated was opl, (odd-paired-like) that resembles the Drosophila pair-rule gene odd-paired and encodes a zinc finger protein that is a member of the Zic gene family. At the onset of gastrulation, opl is expressed throughout the presumptive neural plate, indicating that neural determination has begun at this stage while, by neurula, opl expression is restricted to the dorsal neural tube and neural crest. opl encodes a transcriptional activator, with a carboxy terminal regulatory domain, which when removed increases opl activity. opl both sensitizes animal cap ectoderm to the neural inducer noggin and alters the spectrum of genes induced by noggin, allowing activation of the midbrain marker engrailed. Consistent with the later dorsal neural expression of opl, the activated form of opl is able to induce neural crest and dorsal neural tube markers both in animal caps and whole embryos. In ventral ectoderm, opl induces formation of loose cell aggregates that may indicate neural crest precursor cells. Aggregates do not express an epidermal marker, indicating that opl suppresses ventral fates. Together, these data suggest that opl may mediate neural competence and may be involved in activation of midbrain, dorsal neural and neural crest fates.

Amino Acid Sequence↗

PubMed1998

Medea is a Drosophila Smad4 homolog that is differentially required to potentiate DPP responses.

Mothers against dpp (Mad) mediates Decapentaplegic (DPP) signaling throughout Drosophila development. Here we demonstrate that Medea encodes a MAD-related protein that functions in DPP signaling. MEDEA is most similar to mammalian Smad4 and forms heteromeric complexes with MAD. Like dpp, Medea is essential for embryonic dorsal/ventral patterning. However, Mad is essential in the germline for oogenesis whereas Medea is dispensable. In the wing primordium, loss of Medea most severely affects regions receiving low DPP signal. MEDEA is localized in the cytoplasm, is not regulated by phosphorylation, and requires physical association with MAD for nuclear translocation. Furthermore, inactivating MEDEA mutations prevent nuclear translocation either by preventing interaction with MAD or by trapping MAD/MEDEA complexes in the cytosol. Thus MAD-mediated nuclear translocation is essential for MEDEA function. Together these data show that, while MAD is essential for mediating all DPP signals, heteromeric MAD/MEDEA complexes function to modify or enhance DPP responses. We propose that this provides a general model for Smad4/MEDEA function in signaling by the TGF-beta family.

Amino Acid Sequence↗