Biomedical subjects

Mei Zhang

Publications and source records attributed to Mei Zhang.

At least 19 recordsLinked to original sources

PubMed2026

High NKAP expression predicts poor prognosis of breast cancer patients.

NF-κB activating protein (NKAP) plays important roles in various cancers, including breast cancer. However, its expression and prognosis value in breast cancer remains uncertain. Gene expression profiling interactive analysis, Human protein atlas database, and University of Alabama at Birmingham Cancer data analysis portal database were used to predict the expression and prognostic value of NKAP in breast cancer. Immunohistochemistry, quantitative real time polymerase chain reaction (qRT-PCR) and western blot were performed to detect NKAP expression. The effects of NKAP on cell proliferation, migration and drug sensitivity were investigated in MDA-MB-231 and SK-BR-3 cells. NKAP protein expression differed in breast cancer tissues and paraneoplastic tissues based on the cancer genome atlas data. The high NKAP expression was significantly correlated with a poor prognosis in breast cancer patients. The results of immunohistochemistry, western blot assay, and qRT-PCR proved that NKAP was highly expressed in breast cancer tissues compared with paraneoplastic tissues. In addition, qRT-PCR results showed that high expression of NKAP was significantly correlated with the larger tumor size and higher TNM stage. Moreover, knockdown of NKAP significantly inhibited the proliferation, migration, and enhanced drug sensitivity of MDA-MB-231 and SK-BR-3 cells. NKAP is highly expressed in breast cancer tissues, and its high expression is closely associated with poor prognosis. NKAP also promotes proliferation, migration, and inhibits drug sensitivity of breast cancer cells.

Humans↗

PubMed2007

Protein binding of cefazolin is saturable in vivo both between and within patients.

AIMS: The aims of the study were a) to determine if there is evidence of saturable protein binding of cefazolin in plasma across the range of concentrations achieved clinically (between patient variability) and b) to investigate whether saturable protein binding is also evident from trough and peak concentrations in the same patient (within patient variability). METHODS: Unbound and total plasma concentrations were measured in patients who were treated with cefazolin intravenously by continuous infusion or intermittent injection. In study (i) single random samples were taken from one series of patients. In study (ii) paired samples (troughs and peaks) were taken from a second series of patients. RESULTS: Thirty-one patients were included in study (i). Linear regression analysis of the percentage unbound vs. unbound plasma concentrations revealed a slope significantly different from zero, suggesting saturable protein binding. Mean values for percentage unbound ranged from 9% at low concentrations (8.5 mg l(-1)) to 51% at high concentrations (140 mg l(-1)). Twelve patients were investigated in study (ii). Values for protein binding ranged from 85% at low concentrations (2.7 mg l(-1)) to 52% at high concentrations (200.3 mg l(-1)). The percentage unbound was significantly higher (P < 0.0001) at high (peak) concentrations than at lower (trough) concentrations, confirming saturable protein binding. CONCLUSIONS: The protein binding of cefazolin is saturable in vivo in humans, both between and within patients.

Adult↗

PubMed2007

A novel OPA1 mutation responsible for autosomal dominant optic atrophy with high frequency hearing loss in a Chinese family.

PURPOSE: To investigate the genetic findings and phenotypic characters of autosomal dominant optic atrophy (ADOA). DESIGN: Case report and experimental study. METHODS: Molecular genetic analysis and clinical examinations were performed in a Chinese family with ADOA. Mutations in OPA1 were detected by direct sequencing. Haplotypes were constructed and compared with the phenotypes in the family. RESULTS: Nine family members were diagnosed with ADOA and some of them were accompanied with hearing loss and/or high myopia. A novel heterozygous mutation, c.2848_2849delGA(p.Asp950CysfsX4), was detected in all ADOA patients. The mutation and the mutation bearing haplotype cosegregated with the nine affected members. One family member had high myopia without vision or hearing loss. This patient along with unaffected ones did not harbor the mutation. CONCLUSIONS: A novel mutation, c.2848_2849delGA in OPA1, was identified in a Chinese family with ADOA. This mutation is associated with hearing loss, but likely not high myopia.

Adolescent↗

PubMed2007

Myofibroblast upregulators are elevated in joint capsules in posttraumatic contractures.

We hypothesized specific growth factors are increased in the elbow capsules of patients with post traumatic elbow contractures. A model of surgically induced joint contracture in rabbit knees was developed to study the growth factor expression in joint contractures. This study demonstrates this model mimics the human condition and analyzes how the growth factor levels decrease with time in rabbit knees with contractures. Reverse transcription polymerase chain reaction was used to measure mRNA levels of transforming growth factor-beta1, connective tissue growth factor, ED-A of fibronectin, and alpha-smooth muscle actin normalized to a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. In the joint capsules of patients with elbow contractures, mRNA levels were increased for transforming growth factor- beta1, connective tissue growth factor, and alpha-smooth muscle actin. In the joint capsules of rabbit knees with contractures, mRNA levels were increased for transforming growth factor- beta1, connective tissue growth factor, ED-A of fibronectin, and alpha-smooth muscle actin. The mRNA levels for transforming growth factor-beta1, connective tissue growth factor, and alpha-smooth muscle actin decreased with time in rabbit knees. The elevated levels of these myofibroblast up-regulators and fibrogenic growth factors could explain the previously reported increase in myofibroblasts and collagen mRNA levels. The rabbit knee model correlated well with the human post traumatic elbow contractures.

Actins↗

PubMed2006

Involvement of integrins, MAPK, and NF-kappaB in regulation of the shear stress-induced MMP-9 expression in endothelial cells.

Variations in the matrix metalloproteinase (MMP)-9 gene are related to the presence and severity of atherosclerosis. The aim of this study was to determine the signaling pathways of MMP-9 in endothelial cells subjected to low fluid shear stress. We found that low fluid shear stress significantly increased MMP-9 expression, IkappaBalpha degradation, NF-kappaB DNA-binding activity and phosphorylation of MAPK in cultured human umbilical vein endothelial cells (HUVECs). Inhibition of NF-kappaB resulted in remarkable downregulation of stress-induced MMP-9 expression. Pretreatment of HUVECs with inhibitors of p38 mitogen-activating protein kinase (MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) also led to significant suppression of stress-induced MMP-9 expression and NF-kappaB DNA-binding activity. Similarly, addition of integrins inhibitor to HUVECs suppressed the stress-induced MMP-9 expression, IkappaBalpha degradation, NF-kappaB DNA-binding activity and the phosphorylation of p38 MAPK, ERK1/2. Our findings demonstrated that the shear stress-induced MMP-9 expression involved integrins-p38 MAPK or ERK1/2-NF-kappaB signaling pathways.

Cells, Cultured↗

PubMed2006

Factors associated with return for routine annual screening in an ovarian cancer screening program.

OBJECTIVE: To identify clinical, demographic, dispositional, and attitudinal variables associated with return for routine, annual transvaginal sonography (TVS) screening for ovarian cancer. METHODS: Asymptomatic, average to high risk, women (n=585) participating in a free university-based ovarian cancer screening program completed a baseline interview prior to undergoing an initial TVS screening test. During the baseline interview, demographic (age, education, partner status, race), clinical (family history of ovarian cancer), dispositional (optimism, health values), and attitudinal (perceptions of personal risk for ovarian cancer and effectiveness of screening, intentions to return for repeat routine screening, discomfort during screening, satisfaction with the screening process, ovarian cancer-specific distress) information was obtained. Return for repeat screening was documented from screening program records. RESULTS: Results from both multivariate proportional hazards and logistic regression analyses indicated that stated intentions to return for a repeat screening test within the next year was the strongest predictor of return for repeat screening. Possessing > or =12 years of education was also associated with a greater likelihood of repeat screening in both the proportional hazards and logistic regression analyses. CONCLUSIONS: Results provide further support for low education as a risk factor for suboptimal participation in cancer screening. Results also highlight the critical link between intentions to perform a health-protective behavior and subsequent performance of that behavior and suggest that repeat screening could be enhanced by eliciting both an intention to return for annual ovarian cancer screening as well as a specific plan for implementing this intention.

Adult↗

PubMed2006

CAP interacts with cytoskeletal proteins and regulates adhesion-mediated ERK activation and motility.

CAP/Ponsin belongs to the SoHo family of adaptor molecules that includes ArgBP2 and Vinexin. These proteins possess an N-terminal sorbin homology (SoHo) domain and three C-terminal SH3 domains that bind to diverse signaling molecules involved in a variety of cellular processes. Here, we show that CAP binds to the cytoskeletal proteins paxillin and vinculin. CAP localizes to cell-extracellular matrix (ECM) adhesion sites, and this process requires binding to vinculin. Overexpression of CAP induces the aggregation of paxillin, vinculin and actin at cell-ECM adhesion sites. Moreover, CAP inhibits adhesion-dependent processes such as cell spreading and focal adhesion turnover, whereas a CAP mutant that is unable to localize to cell-ECM adhesion sites is incapable of exerting these effects. Finally, depletion of CAP by siRNA-mediated knockdown leads to enhanced cell spreading, migration and the activation of the PAK/MEK/ERK pathway in REF52 cells. Taken together, these results indicate that CAP is a cytoskeletal adaptor protein involved in modulating adhesion-mediated signaling events that lead to cell migration.

Animals↗

PubMed2006

The occurrence and outcome of 39 pregnancies after 1621 cases of transcervical resection of endometrium.

BACKGROUND: To evaluate the outcomes and management of pregnancy after transcervical resection of the endometrium (TCRE). METHODS: Retrospective study of 39 pregnancies after 1621 procedures of TCRE. RESULTS: Among 1621 women who were successfully followed up after TCRE, there were 39 pregnancies in 32 women, including five ectopic pregnancies (12.8%) and 34 intrauterine pregnancies (87.2%). The majority of pregnancies (84.6%) occurred within the first 2 years. In the first year after TCRE, the incidence of pregnancy was 1.5%. In women who had amenorrhoea after TCRE, the chances of conception (2/676; 0.3%) were significantly (P < 0.001) lower than for those who continued to have period (30/945; 3.2%). Thirty-two cases with intrauterine pregnancy were terminated under ultrasound guidance with two difficult procedures. Only one pregnancy in our study resulted in spontaneous miscarriage which was managed by suction curettage. One term pregnancy had placenta increta resulting in Caesarean hysterectomy. CONCLUSIONS: Pregnancies after TCRE are associated with increased risk, and clinicians should be aware of the various complications of pregnancy that may occur after TCRE, including an increased risk of ectopic pregnancy. Surgical termination of pregnancy after TCRE is potentially a difficult procedure and should be carried out under ultrasound guidance.

Endometrium↗

PubMed2006

Effect of five triterpenoid compounds from the buds of Aralia elata on stimulus-induced superoxide generation, tyrosyl phosphorylation and translocation of cytosolic compounds to the cell membrane in human neutrophils.

The buds of Aralia elata (Miq.) Seem (Japanese angelica tree) have long been used as a tonic, antiarthritic and antidiabetic agent in China and Japan. We have isolated five triterpenoids, congmuyanosides A, C, D, echinocystic acid and 3-O-[beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl]-hederagenin from the buds of Aralia elata , and investigated their effects on stimulus-induced superoxide generation in human neutrophils. Congmuyanoside A, echinocystic acid and 3-O-[beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl]-hederagenin suppressed the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) in a concentration-dependent manner. Congmuyanosides C, D and echinocystic acid significantly suppressed the superoxide generation induced by phorbol 12-myristate 13-acetate (PMA) and arachidonic acid (AA). The compounds also suppressed fMLP- and AA-induced tyrosyl or PMA-induced serine/threonine phosphorylation and translocation of cytosolic compounds, p47 (phox), p67 (phox) and Rac to the cell membrane.

Aralia↗

PubMed2006

Effects of ceramide-1-phosphate on cultured cells: dependence on dodecane in the vehicle.

Ceramide-1-phosphate (C1P), the product of ceramide kinase, is a sphingophospholipid with recently recognized signaling properties. In particular, it was reported to be mitogenic and capable of direct stimulation of cytosolic phospholipase A(2alpha). Much of the present knowledge has relied on the use of C1P of various acyl chain lengths, together with diverse protocols to deliver it to cultured cells. A mixture of ethanol (or methanol) with dodecane, as the vehicle, has become popular. However, the contribution of this solvent to the observed effects of C1P has not been documented. Here, we show that addition of C1P in ethanol-dodecane to culture medium leads to irreversible cytotoxic effects. These culminate in mitochondrial swelling, vacuole formation, and cell death. Not only the toxicity of C1P, but also its ability to trigger prostaglandin E2 release, is fully dependent upon addition of a premade C1P-dodecane mixture. Furthermore, we show that these effects are not restricted to C1P. They result from the capacity of dodecane to interact with phospholipids; hence, they go undetected with a vehicle control. This study should raise awareness about the use of dodecane for phospholipid delivery and, in turn, help in unraveling C1P signaling, which is still poorly understood.

Alkanes↗

PubMed2006

KCNE2 is colocalized with KCNQ1 and KCNE1 in cardiac myocytes and may function as a negative modulator of I(Ks) current amplitude in the heart.

BACKGROUND: In heterologous expression systems, KCNE1 and KCNE2 each can associate with KCNQ1 and exert apparently opposite effects on its channel function. KCNQ1 and KCNE1 associate to form the slow delayed rectifier I(Ks) channels in the heart. Whether KCNE2 plays any role in I(Ks) function is not clear. OBJECTIVES: The purpose of this study was to study whether KCNE2 can associate with KCNQ1 in the presence of KCNE1 and modulate its function. METHODS: Voltage clamp methods were used to study channel function in cardiomyocytes and in oocytes or COS-7 cells and immunocytochemistry/coimmunoprecipitation was used to study protein colocalization/association. RESULTS: Adult rat ventricular myocytes express functional I(Ks), and KCNE2 is colocalized with KCNQ1 and KCNE1 at surface membrane and t-tubules. A detailed study of KCNQ1 modulation by KCNE2 at different KCNE2 expression levels reveals that, surprisingly, KCNE2 and KCNE1 share the major features in modulating KCNQ1 gating kinetics: slowing of activation, positive shift in the voltage range of activation, and suppression of inactivation. However, KCNE2 reduces KCNQ1 current amplitude whereas KCNE1 increases it, and KCNE2 induces a constitutively active KCNQ1 component whereas KCNE1 does not. Coimmunoprecipitation suggests that KCNQ1, KCNE1, and KCNE2 can form a tripartite complex, indicating that KCNE2 can bind to KCNQ1 in the presence of KCNE1. Coexpressing KCNE2 with KCNQ1 and KCNE1 leads to a decrease in the I(Ks) current amplitude without altering the gating kinetics. CONCLUSION: Our data suggest that KCNE2 is in close proximity to KCNQ1 and KCNE1 in cardiomyocytes and may participate in dynamic regulation of I(Ks) current amplitude in the heart.

Animals↗

PubMed2006

Electrical remodeling in a canine model of ischemic cardiomyopathy.

The nature of electrical remodeling in a canine model of ischemic cardiomyopathy (ICM; induced by repetitive intracoronary microembolizations) that exhibits spontaneous ventricular tachycardia is not entirely clear. We used the patch-clamp technique to record action potentials and ionic currents of left ventricular myocytes isolated from the region affected by microembolizations. We also used the immunoblot technique to examine channel subunit expression in adjacent affected tissue. Ventricular myocytes and tissue isolated from the corresponding region of normal hearts served as control. ICM myocytes had prolonged action potential duration (APD) and more pronounced APD dispersion. Slow delayed rectifier current (I(Ks)) was reduced at voltages positive to 0 mV, along with a negative shift in its voltage dependence of activation. Immunoblots showed that there was no change in KCNQ1.1 (I(Ks) pore-forming or alpha-subunit), but KCNE1 (I(Ks) auxiliary or beta-subunit) was reduced, and KCNQ1.2 (a truncated KCNQ1 splice variant with a dominant-negative effect on I(Ks)) was increased. Transient outward current (I(to)) was reduced, along with an acceleration of the slow phase of recovery from inactivation. Immunoblots showed that there was no change in Kv4.3 (alpha-subunit of fast-recovering I(to) component), but KChIP2 (beta-subunit of fast-recovering component) and Kv1.4 (alpha-subunit of slow-recovering component) were reduced. Inward rectifier current was reduced. L-type Ca current was unaltered. The immunoblot data provide mechanistic insights into the observed changes in current amplitude and gating kinetics of I(Ks) and I(to). We suggest that these changes, along with the decrease in inward rectifier current, contribute to APD prolongation in ICM hearts.

Action Potentials↗

PubMed2006

Identification of the A3 adenosine receptor in rat retinal ganglion cells.

PURPOSE: Adenosine can protect retinal ganglion cells from the death that accompanies a general ischemic challenge as well as excitotoxic death. In other tissues, both A1 and A3 adenosine receptor subtypes can mediate protection. While a role for the A1 adenosine receptor in ganglion cell protection has been established, a potential for the A3 receptor has only recently been proposed. Although the pharmacology is promising, the molecular identity of the responsible receptor is unclear as previous studies were unable to detect message for the A3 receptor in retinal ganglion cells. We combined laser capture microdisection (LCM) and immunopurification with traditional and real-time PCR to unequivocally demonstrate the presence of the A3 receptor message in rat retinal ganglion cells. METHODS: Retinal ganglion cells of Long-Evans rat pups were retrograde labeled with aminostilbamidine. Eyeballs were enucleated, embedded, frozen, sectioned, and fluorescent cells in the ganglion cell layer were collected with LCM. Purified ganglion cells were also isolated with a two-step panning procedure. cDNA for the A3 receptor obtained from the microdissected ganglion cell layer, immunopurified ganglion cells, whole retina and testis was amplified using RT-PCR, confirmed by DNA sequencing and compared with published sequences. A3 receptor message was also amplified using real-time PCR. Ca2+ levels in immunopanned ganglion cells were measured ratiometrically with fura-2. RESULTS: RNA from immunopurified ganglion cells and from dye-loaded cells in the ganglion cell layer contained message for the A3 receptor when amplified with either traditional RT-PCR or real-time PCR. The entire encoding region was sequenced and found to be 99% identical to the published code. The sequence closely resembled the consensus form of the gene, with other sequences deviating from this default code. Molecular identification was functionally confirmed in purified ganglion cells as the A3 receptor agonist Cl-IB-MECA prevented the excessive Ca2+ rise triggered by P2X7 agonist BzATP. CONCLUSIONS: Retinal ganglion cells express A3 adenosine receptor mRNA. Stimulation of this receptor can reduce the Ca2+ overload following excessive activation of P2X7 receptors.

Adenosine↗

PubMed2006

An unconventional nuclear localization motif is crucial for function of the Drosophila Wnt/wingless antagonist Naked cuticle.

Wnt/beta-catenin signals orchestrate cell fate and behavior throughout the animal kingdom. Aberrant Wnt signaling impacts nearly the entire spectrum of human disease, including birth defects, cancer, and osteoporosis. If Wnt signaling is to be effectively manipulated for therapeutic advantage, we first must understand how Wnt signals are normally controlled. Naked cuticle (Nkd) is a novel and evolutionarily conserved inducible antagonist of Wnt/beta-catenin signaling that is crucial for segmentation in the model genetic organism, the fruit fly Drosophila melanogaster. Nkd can bind and inhibit the Wnt signal transducer Dishevelled (Dsh), but the mechanism by which Nkd limits Wnt signaling in the fly embryo is not understood. Here we show that nkd mutants exhibit elevated levels of the beta-catenin homolog Armadillo but no alteration in Dsh abundance or distribution. In the fly embryo, Nkd and Dsh are predominantly cytoplasmic, although a recent report suggests that vertebrate Dsh requires nuclear localization for activity in gain-of-function assays. While Dsh-binding regions of Nkd contribute to its activity, we identify a conserved 30-amino-acid motif, separable from Dsh-binding regions, that is essential for Nkd function and nuclear localization. Replacement of the 30-aa motif with a conventional nuclear localization sequence rescued a small fraction of nkd mutant animals to adulthood. Our studies suggest that Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal.

Active Transport, Cell Nucleus↗

PubMed2006

Different effects of sulfur amino acids on prolidase and prolinase activity in normal and prolidase-deficient human erythrocytes.

BACKGROUND: Prolidase and prolinase activity is known to be enhanced significantly in some diseases. Recently, the effect of amino acids on prolidase and prolinase activity in normal and prolidase-deficient human erythrocytes was investigated. It was reported that both enzymes were enhanced by glycine and alanine in the presence of MnCl(2). METHODS: Erythrocytes were isolated from heparinized blood from normal human and a patient with prolidase deficiency. Effects of various sulfur amino acids on prolidase and prolinase activities against iminodipeptides in the presence of 1 or 0.1 mmol/l MnCl(2) were investigated. RESULTS: Prolinase activity against prolylglycine in normal and prolidase-deficient erythrocyte lysates was inhibited by L-methionine, NAc-L-methionine and D,L-methionine in a concentration-dependent manner, but D-methionine enhanced the activity in low concentrations (0-20 mmol/l). D,L-Homocysteine inhibited the activity more strongly than other sulfur amino acids tested in a concentration-dependent manner. On the other hand, prolidase activity against glycylproline was enhanced by L-methionine, D-methionine, D,L-methionine, D,L-homocysteine thiolactone and D,L-ethionine. The rates of enhancement by these sulfur amino acids were in the following order: D,L-ethionine>D,L-methionine, D-methionine, D,L-homocysteine thiolactone>L-methionine (10 mmol/l). CONCLUSION: The prolinase activity in normal and prolidase-deficient erythrocyte lysates was inhibited by L-methionine, D,L-ethionine and D,L-homocysteine. On the other hand, prolidase activity in their erythrocyte lysates was enhanced by D,L-ethionine, D-methionine and L-methionine. These results indicate the effects of these sulfur amino acids on prolidase and prolinase activities were different.

Amino Acids, Sulfur↗

PubMed2006

[Expression of alpha 1 anti-trypsin in proximal tubular epithelial cell line].

OBJECTIVE: To investigate the expression and regulation of alpha 1 anti-trypsin (AAT) in tubular epithelial cells. METHODS: The expression of AAT in tubular epithelial cell line HKC was detected with indirect immunofluorescence assay and confirmed by reverse transcription polymerase chain reaction (PCR) in transcription level respectively, and PCR product was sequenced. In order to observe the response of HCK to LPS, the different concentrations of LPS (0, 0.5, 1.0, 2.0 microg/ml) were used in the research and the regulation of AAT in HKC was semi-quantitatively detected with Western Blot and Real-Time PCR respectively. RESULTS: Indirect immunofluorescence staining showed that AAT was positive in HKC, but negative in renal interstitial fibroblast. By PCR, a significant messenger RNA band of AAT was found in HKC, but not in renal interstitial fibroblast. DNA sequencing indicated that the sequence of PCR product is consistent with AAT mRNA sequence in Gene Bank. Real-time PCR showed that the expression of AAT mRNA was up-regulated (fluorescence intensity ratio is 3.43 +/- 0.88 versus 1.22 +/- 0.20; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 4 h, and Western Blot showed that the synthesis of AAT significantly increased (band density ratio is 0.88 +/- 0.12 versus 0.59 +/- 0.05; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 8 h, as compared with unstimulated HKC. 0.5, 1.0 g/ml of LPS has no effect on the expression of AAT mRNA and AAT protein in HKC. CONCLUSIONS: HKC express AAT and the expression can be up-regulated by LPS.

Cell Line↗

PubMed2006

Characterization of an LQT5-related mutation in KCNE1, Y81C: implications for a role of KCNE1 cytoplasmic domain in IKs channel function.

BACKGROUND: Y81C is a new long QT-5 (LQT5)-related KCNE1 mutation, which is located in the post-transmembrane domain (post-TMD) region in close proximity to three other LQT5 mutations (S74L, D76N, and W87R). OBJECTIVE: We examine the effects of Y81C on the function and drug sensitivity of the slow delayed rectifier channel (I(Ks)) formed by KCNE1 with pore-forming KCNQ1 subunits. We also infer a structural basis for the detrimental effects of Y81C on I(Ks) function. METHODS: Wild-type (WT) and mutant (harboring Y81C) I(Ks) channels are expressed in oocytes or COS-7 cells. Channel function and KCNQ1 protein expression/subcellular distribution are studied by techniques of electrophysiology, biochemistry, and immunocytochemistry. Ab initio structure predictions of KCNE1 cytoplasmic domain are performed by the Robetta server. RESULTS: Relative to WT KCNE1, Y81C reduces I(Ks) current amplitude and shifts the voltage range of activation to a more positive range. Y81C does not reduce whole-cell KCNQ1 protein level or interfere with KCNQ1 trafficking to cell surface. Thus, its effects are mediated by altered KCNQ1/KCNE1 interactions in cell surface channels. Importantly, Y81C potentiates the effects of an I(Ks) activator. Preserving the aromatic or hydroxyl side chain at position 81 (Y81F or Y81T) does not prevent the detrimental effects of Y81C. Structure predictions suggest that the post-TMD region of KCNE1 may adopt a helical secondary structure. CONCLUSION: We propose that the post-TMD region of KCNE1 interacts with the KCNQ1 channel to modulate I(Ks) current amplitude and gating kinetics. Other LQT5 mutations in this region share the Y81C phenotype and probably affect the I(Ks) channel function by a similar mechanism.

Amino Acid Sequence↗

PubMed2006

Presenilin/gamma-secretase activity regulates protein clearance from the endocytic recycling compartment.

The presenilin (PS)/gamma-secretase complex proteolytically cleaves more than 20 different proteins in addition to the amyloid precursor protein (APP). These substrates are almost exclusively type I membrane proteins. Many undergo internalization from the cell surface followed by degradation or recycling back to the plasma membrane through the endocytic recycling compartment (ERC). Evidence shows that the PSs also regulate intracellular trafficking of APP and its C-terminal fragments (CTFs). To investigate whether PS/gamma-secretase activity is required for normal endosomal recycling, we performed live cell imaging experiments with fluorescently labeled transferrin, reported to specifically traffic through the ERC. By using pharmacological gamma-secretase inhibitors or cell lines lacking functional PS/gamma-secretase, here we show that PS/gamma-secretase activity is required for clearance of transferrin from the ERC. Interestingly, lack of PS/gamma-secretase function also resulted in the accumulation of APP and APP-CTFs in the ERC in addition to the cell surface. Familial Alzheimer's disease mutations in APP-CTFs did not affect endocytic recycling of these proteins. Our results suggest that PS/gamma-secretase activity is required for normal endosomal recycling of soluble and membrane-associated proteins through the ERC and propose a new mechanism by which impaired PS/gamma-secretase function may eventually contribute to neurodegeneration.

Amyloid Precursor Protein Secretases↗