Union City Research Articles

Integrin-Mediated Transforming Growth Factor-β Activation Regulates Homeostasis of the Pulmonary Epithelial-Mesenchymal Trophic Unit

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.

The Synthesis, Structural Characterization, and Receptor Specificity of the α-Conotoxin Vc1.1

The alpha-conotoxin Vc1.1 is a small disulfide-bonded peptide currently in development as a treatment for neuropathic pain. This study describes the synthesis, determination of the disulfide connectivity, and the determination of the three-dimensional structure of Vc1.1 using NMR spectroscopy. Vc1.1 was shown to inhibit nicotine-evoked membrane currents in isolated bovine chromaffin cells in a concentration-dependent manner and preferentially targets peripheral nicotinic acetylcholine receptor (nAChR) subtypes over central subtypes. Specifically, Vc1.1 is selective for alpha3-containing nAChR subtypes. The three-dimensional structure of Vc1.1 comprises a small alpha-helix spanning residues Pro6 to Asp11 and is braced by the I-III, II-IV disulfide connectivity seen in other alpha-conotoxins. A comparison of the structure of Vc1.1 with other alpha-conotoxins, taken together with nAChR selectivity data, suggests that the conserved proline at position 6 is important for binding, whereas a number of residues in the C-terminal portion of the peptide contribute toward the selectivity. The structure reported here should open new opportunities for further development of Vc1.1 or analogues as analgesic agents.

Different Domains of the AMPA Receptor Direct Stargazin-mediated Trafficking and Stargazin-mediated Modulation of Kinetics

Stargazin is an accessory protein of AMPA receptors that enhances surface expression and also affects the biophysical properties of the receptor. AMPA receptor domains necessary for either of these two processes have not yet been identified. Here, we used confocal imaging and electrophysiology of heterologously expressed, fluorophore-tagged GluR1, GluR2, and stargazin to study surface expression and desensitization kinetics. Stargazin-mediated trafficking was sensitive to the nature of the AMPA receptor cytoplasmic domain. The insertion of YFP after residue 15 of the truncated cytoplasmic tail of GluR1i perturbed stargazin-mediated trafficking of the receptor but not its modulation of desensitization kinetics. This construct also failed to permit fluorescence resonance energy transfer (FRET) with stargazin in the endoplasmic reticulum (ER), whereas FRET between fluorophore-tagged stargazin and non-truncated AMPA receptors demonstrated a specific interaction between these proteins, both in the ER and the plasma membrane. Rather than encoding a specific binding site, the fluorophore-tagged C terminus may restrict access to one or more ER retention sites. Although perturbations of the C terminus impeded stargazin-mediated trafficking to the plasma membrane, the effects of stargazin on the biophysical properties of AMPA receptors (i.e. modulation of desensitization) remained intact. These data provide strong evidence that the AMPA receptor domains required for stargazin modulation of gating and trafficking are separable.

Transient Meralgia Paresthetica After Pediatric Posterior Spine Fusion

Meralgia paresthetica (MP) rarely occurs during posterior spine surgery. The study goal was to examine risk factors associated with the incidence of MP. A review of 56 consecutive pediatric patients undergoing posterior spine fusion for scoliosis was performed. Patients with abnormal sensation in the lateral thigh preoperatively and prior spine surgery were excluded. All patients were positioned prone on the Jackson (Orthopaedic Systems, Inc., Union City, CA) spinal table with either (1) the lower leg support table and thigh supports or (2) the lower leg suspension sling. Data on patient weight, diagnosis, surgeon, duration of surgery, presence of MP, symptoms, and symptom duration were collected. A logistic regression analysis was performed between independent variables and presence of MP. There were 10/56 patients with MP (18%). Symptoms were anterolateral thigh numbness without pain or weakness. Symptoms in all cases were resolved, on average, before the 6-week postoperative visit (range 2-24 weeks). Patients with MP more often had idiopathic scoliosis (28% vs 7%; P < 0.05) were positioned with the lower leg sling instead of the flat table support (31% vs 13%; P < 0.05) and trended toward longer surgery times (451 vs 388 minutes; NS). Abnormal body mass index, age at surgery, surgeon, and sex did not correlate to MP. MP can occur after pediatric posterior spine surgery. Symptoms were minor, temporary, and did not require treatment. Shorter surgical times and use of thigh pads in conjunction with the lower leg support table may decrease the incidence of MP.

Amino Acid Residues Gln4020 and Lys4021 of the Ryanodine Receptor Type 1 Are Required for Activation by 4-Chloro-m-cresol

The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.

Evidence That DNA (Cytosine-5) Methyltransferase Regulates Synaptic Plasticity in the Hippocampus

DNA (cytosine-5) methylation represents one of the most widely used mechanisms of enduring cellular memory. Stable patterns of DNA methylation are established during development, resulting in creation of persisting cellular phenotypes. There is growing evidence that the nervous system has co-opted a number of cellular mechanisms used during development to subserve the formation of long term memory. In this study, we examined the role DNA (cytosine-5) methyltransferase (DNMT) activity might play in regulating the induction of synaptic plasticity. We found that the DNA within promoters for reelin and brain-derived neurotrophic factor, genes implicated in the induction of synaptic plasticity in the adult hippocampus, exhibited rapid and dramatic changes in cytosine methylation when DNMT activity was inhibited. Moreover, zebularine and 5-aza-2-deoxycytidine, inhibitors of DNMT activity, blocked the induction of long term potentiation at Schaffer collateral synapses. Activation of protein kinase C in the hippocampus decreased reelin promoter methylation and increased DNMT3A gene expression. Interestingly, DNMT activity is required for protein kinase C-induced increases in histone H3 acetylation. Considered together, these results suggest that DNMT activity is dynamically regulated in the adult nervous system and that DNMT may play a role in regulating the induction of synaptic plasticity in the mature CNS.

An Asymmetric Contribution to γ-Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2–3 of α1, β2, and γ2 Subunits

An Asymmetric Contribution to γ-Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2–3 of α1, β2, and γ2 Subunits

Effect of Prone Positioning Systems on Hemodynamic and Cardiac Function During Lumbar Spine Surgery: An Echocardiographic Study

Prospective randomized study of patients undergoing spine surgery. To compare changes in hemodynamic and cardiac function after prone positioning using different prone positioners. Prone positioning decreases blood pressure and cardiac function. Several studies have evaluated changes in cardiac function after prone positioning, and linked them to reduced venous return and ventricular compliance. This study compares different prone positioners using transesophageal echocardiography, and determines their effect on cardiac function and hemodynamics. After correction of fluid deficits with the patient under stable anesthesia, hemodynamic and cardiac performance was measured using transesophageal echocardiography. After prone positioning, repeat measurements were performed, and comparisons were made between prone and supine positions. No intergroup differences in demographics, fluid deficit, baseline hemodynamics, or differences from supine to prone position were noted. Cardiac output decreased with the Wilson (Union City, CA) and Siemens AG (Munich, Germany) frames, while cardiac index and stroke volume decreased with the Andrews (Hollywood, CA), Wilson, and Siemens systems. Cardiac preload decreased using the Andrews frame. The Jackson spine table (Hollywood, CA) and bolsters had the least effect on cardiac performance. Adequate fluid replacement reduced hypotension and hemodynamic instability after prone positioning. The Jackson spine table and longitudinal bolsters had minimal effects on cardiac function, and should be considered in patients with limited cardiac reserve.

Adherence with topical treatment is poor compared with adherence with oral agents: Implications for effective clinical use of topical agents

To the Editor: Adherence with oral and topical medications used in dermatology is not well characterized. In a randomized controlled study of moderate-to-severe hand dermatitis we compared adherence between an oral medication (prednisone) and a topical treatment (tacrolimus vs vehicle). Adherence to both medications was monitored using a medication cap that records the time and date each time the cap is removed from the bottle (MEMS caps; Aardex, Union City, Calif).

Purinergic Receptors Mediate Two Distinct Glutamate Release Pathways in Hippocampal Astrocytes

The purinergic P2X(7) receptor (P2X(7)R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X(7)Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X(7)R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca(2+)-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X(7)Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca(2+), and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X(7)R antagonists and amplified in low Ca(2+). In low Ca(2+) BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X(7)R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X(7)R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain.

283 TRANSCRIPTIONAL ANALYSIS OF BIOPSIES DERIVED FROM IN VITRO-PRODUCED BOVINE BLASTOCYSTS IN RELATION TO PREGNANCY SUCCESS AFTER TRANSFER TO RECIPIENTS

Early embryonic mortality is a recognized cause of reproductive failure in cattle leading to the loss of a large number of potential calves, retarded genetic progress, and significant loss of money and time in rebreeding cows. The purpose of this work was to address the relationship between the transcriptional profile of embryos, using the microarray technique, and pregnancy success based on blastocyst biopsies taken prior to transfer to recipients. Biopsies (3 to 40% of the intact embryo) were taken from IVP Day 7 blastocysts (n = 98) and 60 to 70% was transferred to recipients (2-year-old Simmental heifers) after re-expansion. Based on the success of pregnancy, biopsies were pooled in 3 groups: those resulting in no pregnancy (G1), resorption (G2), and calf delivery (G3). Gene expression analysis of these groups of biopsies was performed using a home-made bovine pre-implantation-specific array (with 219 clones) and bovine cDNA array (BlueChip) (with 2000 clones) (Sirard et al. 2005 Reprod. Fertil. Dev. 17, 47–57). Approximately 3 �g of amplified RNA from pooled biopsies (10 biopsies each) was used as a template in reverse transcription reactions incorporating amino-modified dUTPs into labeled cDNA using the CyScribeTM post-labelling kit (Amersham Bioscience, Freiburg, Germany). The synthesized cDNAs from all 3 groups were differentially labeled using Cy3 and Cy5 dyes. Slides were scanned using a GenePix 4000B scanner and images were analyzed using GenePix Pro Version 4.0 software (Axon Instruments, Union City, CA, USA). Data were analyzed using Significant Analysis for Microarray (SAM) software (http//www.stst.stanford.edu/tips/SAM). Quantitative real-time PCR was used to confirm the differentially expressed genes revealed by microarray experiments. Results revealed that 52 genes were differentially regulated between G1 and G3, and 58 genes were differentially regulated between G2 and G3. Biopsies resulting in calf delivery were enriched with genes necessary for implantation (COX-2 and CDX2), signal transduction (PLAU), polyamine biosynthesis (ODC1), response to oxidative stress (TXN), growth factor (BMP15), and placenta-specific 8 (PLAC8). Biopsies that ended up with resorption were enriched with transcripts involving protein phosphorylation (KRT8) plasma membrane (OCLN), and glucose metabolism (PGK1, AKR1B1). Biopsies resulting in no pregnancy were enriched with transcripts involving inflammatory cytokines (TNF), protein amino acid binding (EEF1A1), transcription factors (MSX1, PTTG1), glucose metabolism (PGK1, AKR1B1), and CD9, which is an inhibitor of implantation. In conclusion, we generated direct candidates of blastocyst-specific genes that determine the fate of the embryo after transfer.

250 TRANSCRIPTIONAL PROFILING OF BOVINE OOCYTES DERIVED FROM THE GROWTH AND DOMINANCE PHASES OF THE FIRST FOLLICULAR WAVE

Oocytes recovered at the growth phase have a higher blastocsyt rate (24.5%) than those recovered in the dominance phase (11.7%) (Machatkova et al. 2004 Theriogenology 61, 329–335). However, so far the intrinsic properties of these oocytes in relation to the gene expression patterns have not been investigated. Therefore, we aimed here to compare the transcriptional activity of bovine oocytes recovered at the growth and dominance phases of the first follicular wave. Sixty cyclic Simmental heifers (24 to 30 months old) were selected based on general clinical and ovarian normality, divided into 2 groups (30 heifers per group), and housed together. Estrous cycle was synchronized by 2 injections of PGF2α with an 11-day interval, and onset of estrus was considered as Day 0. Ultrasonography-guided ovum pickup (OPU) was performed to collect oocytes from small follicles (3–5 mm) at the growth (Day 3) and dominance (Day 7) phases in 2 sessions per phase. Triplicate pools of oocytes (each with 20 oocytes) from each developmental phase were used for transcriptional analysis using custom bovine cDNA array with 2000 clones (Sirard et al. 2005 Reprod. Fertil. Dev. 17, 47–57). Array images were analyzed using GenePix� Pro 4.0 (Axon Instruments, Inc., Union City, CA, USA), and then the data were LOESS normalized with GPRocessor 2.0a (http://bioinformatics.med.yale.edu/softwarelist.html). Microarray data were analyzed using Significance Analysis for Microarray (SAM) to obtain differentially regulated genes, followed by validation of 10 transcripts with real-time PCR. Data analysis revealed a total of 51 transcripts to be differentially regulated; out of these transcripts, 36 (70%) represented those with known function, 6 (12%) were those with unknown function, and 9 (18%) were novel transcripts. The quantitative real-time PCR validated the expression profile of 8 out of 10 to be in agreement with microarray results. Oocytes from the growth phase (Day 3) were found to be enriched with transcripts involved in protein biosynthesis (RPLP0, RPL8, RPL24, ARL6IP, RpS14, RpS15, RpS4x, and RPS3A), translation elongation (EF1A), ATP binding (ATP5A1), NADH dehydrogenase activity (FL396 and FL405), cytoskeleton (Actin, beta-Actin, H2AZ, and KRT8), calcium ion binding (S100A10 and ANXA2), signal transduction (G-beta like protein), and thiol-disulfide exchange intermediate (TXN). On the other hand, oocytes from the dominance phase (Day 7) were enriched with genes involved in cell cycle (CCNB1, CKS2, UBE2D3, and CDC31), transcription factors (MSX1, PTTG1, FANK1, and PWP1), aldehyde reductase activity (AKR1B1), nucleotide binding (TUBA6), growth factor (BMP15), and fertilization (ZP4). In conclusion, our results indicate that there are differences in transcriptional activity of oocytes derived from different stages of follicular development which may explain their reported differences in developmental competence. Transcripts identified in this study may be used as markers of oocyte competence.

Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4α

Hepatocyte nuclear factor 4alpha (HNF4alpha) regulates many genes that are preferentially expressed in liver. Mice lacking hepatic expression of HNF4alpha (HNF4alphaDeltaL) exhibited markedly increased levels of serum bile acids (BAs) compared with HNF4alpha-floxed (HNF4alphaF/F) mice. The expression of genes involved in the hydroxylation and side chain beta-oxidation of cholesterol, including oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase (CYP8B1), and sterol carrier protein x, was markedly decreased in HNF4alphaDeltaL mice. Cholesterol 7alpha-hydroxylase mRNA and protein were diminished only during the dark cycle in HNF4alphaDeltaL mice, whereas expression in the light cycle was not different between HNF4alphaDeltaL and HNF4alphaF/F mice. Because CYP8B1 expression was reduced in HNF4alphaDeltaL mice, it was studied in more detail. In agreement with the mRNA levels, CYP8B1 enzyme activity was absent in HNF4alphaDeltaL mice. An HNF4alpha binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4alpha-dependent transcription. Surprisingly, cholic acid-derived BAs, produced as a result of CYP8B1 activity, were still observed in the serum and gallbladder of these mice. These studies reveal that HNF4alpha plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis, including hydroxylation and side chain beta-oxidation of cholesterol in vivo.

Adherence to treatment in children and adolescent patients with cystic fibrosis

Purpose The purpose of this study was to monitor medication adherence in cystic fibrosis (CF) patients and its correlation with disease severity and patient age. Methods Children less than 12 years of age (group 1) and adolescents 12 years of age and older (group 2) were recruited from the University of Michigan CF Center. The study duration was 3 months. A total of 22 patients per group were enrolled. Adherence to ADEKs, an oral multivitamin, and dornase alfa, a nebulized mucolytic medication, was monitored. Adherence to ADEKs was monitored by using the Medication Event Monitoring System (MEMS) SmartCaps (APREX, AARDx, Inc., Union City, California). Dornase alfa adherence rate was monitored by counting empty medication vials. Results Thirty-three patients completed the study, 15 patients in group 1 and 18 patients in group 2. The overall mean adherence rates for ADEKs and dornase alfa were (± SD) 63.6% ± 24.0% and 66.5% ± 31.2%, respectively. The median ADEKs and dornase alfa adherence rate for group 1 was 84.6% and 79.1%, respectively ( p = .08); and for group 2 was 56.7% vs. 78.4%, respectively ( p = .07). There was a trend toward significance, suggesting that the adherence rate for ADEKs was higher than for dornase alfa ( p = .08) in group 1. Group 2 showed a trend toward adherence to dornase alfa than to ADEKs ( p = .07). There was a trend for ADEKs adherence between groups 1 and 2 ( p = .09), but not for dornase alfa ( p = .93). Conclusion Parental supervision and disease severity are likely to play a major role in adherence to medical management. Partnership with patients and families about the treatment plan might be important for improving adherence rate. The MEMS SmartCaps is an electronic monitoring technology that should be used to measure drug adherence objectively both in further larger clinical trials and in the outpatient setting.

Phosphatidylinositol 4,5-Bisphosphate Rescues TRPM4 Channels from Desensitization

TRPM4 is a Ca(2+)-activated nonselective cation channel that regulates membrane potential in response to intracellular Ca(2+) signaling. In lymphocytes it plays an essential role in shaping the pattern of intracellular Ca(2+) oscillations that lead to cytokine secretion. To better understand its role in this and other physiological processes, we investigated mechanisms by which TRPM4 is regulated. TRPM4 was expressed in ChoK1 cells, and currents were measured in excised patches. Under these conditions, TRPM4 currents were activated by micromolar concentrations of cytoplasmic Ca(2+) and progressively desensitized. Here we show that desensitization can be explained by a loss of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) from the channels. Poly-l-lysine, a PI(4,5)P(2) scavenger, caused rapid desensitization, whereas MgATP, at concentrations that activate lipid kinases, promoted recovery of TRPM4 currents. Application of exogenous PI(4,5)P(2) to the intracellular surface of the patch restored the properties of TRPM4 currents. Our results suggest that PI(4,5)P(2) acts to uncouple channel opening from changes in the transmembrane potential, allowing current activation at physiological voltages. These data argue that hydrolysis of PI(4,5)P(2) underlies desensitization of TRPM4 and support the idea that PI(4,5)P(2) is a general regulator for the gating of TRPM ion channels.

Regulation of Epithelial Na+ Channel Activity by Conserved Serine/Threonine Switches within Sorting Signals

The PY and YXXphi motifs are canonical sorting signals involved in trafficking. Nedd4-2 and the mu(2)-subunit of the AP-2 complex target these motifs to facilitate internalization. Epithelial Na(+) channel (ENaC) subunits contain both motifs in their cytosolic COOH termini where they overlap ((S/T)PPPXYX(S/T)phi). Just preceding the PY and embedded within the YXXphi motifs are conserved serine/threonine. We test here whether these conserved Ser/Thr modulate ENaC activity by influencing the function of the internalization domains. We find that co-expression of dominant-negative dynamin (K44A) with ENaC increases channel activity. Conversely, co-expression of Nedd4-2 and epsin with ENaC decrease activity. Alanine substitution of the conserved Thr(628) preceding the PY motif in gamma-mENaC had no effect on basal activity. Channels with this mutation, however, responded to K44A and epsin but not Nedd4-2. Similarly, mutation of the proline repeat in the PY motif of gamma-mENaC disrupted only Nedd4-2 regulation having no effect on regulation by K44A and epsin. Alanine substitution of the conserved Thr within the YXX motif of gamma-mENaC (T635A) increased basal activity. Channels containing this mutation responded to Nedd4-2 but not K44A and epsin. Channels containing the T635(D/E) substitution in gamma-mENaC did not have increased basal activity and responded to Nedd4-2 but not K44A. The double mutant T628A,T635A did not respond to Nedd4-2 or K44A. Mutation of Thr(628) and Thr(635) also disrupted ENaC precipitation with the mu(2)-subunit of the AP-2 complex. Moreover, the YXXphi motif, independent of the PY motif, was sufficient to target degradation with T635A disrupting this effect. These results demonstrate that the overlapping PY and YXXphi motifs in ENaC are, in some instances, capable of independent function and that the Ser/Thr just preceding and within these domains impact this function.

Spontaneous Thermal Motion of the GABAA Receptor M2 Channel-lining Segments

The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.

Decorin Evokes Protracted Internalization and Degradation of the Epidermal Growth Factor Receptor via Caveolar Endocytosis

Decorin inhibits the epidermal growth factor receptor (EGFR) by down-regulating its tyrosine kinase activity, thereby blocking the growth of a variety of transformed cells and tumor xenografts. In this study we provide evidence that decorin directly binds to the EGFR causing its dimerization, internalization, and ultimately its degradation. Using various pharmacological agents to disrupt clathrin-dependent and -independent endocytosis, we demonstrate that decorin evokes a protracted internalization of the EGFR primarily via caveolar-mediated endocytosis. In contrast to EGF, decorin targets the EGFR to caveolae, but not to early or recycling endosomes. Ultimately, however, both EGF- and decorin-induced pathways converge into late endosomes/lysosomes for final degradation. Thus, we have discovered a novel biological mechanism for decorin that could explain its anti-proliferative and anti-oncogenic mode of action.

Enhanced Shutoff of Phototransduction in Transgenic Mice Expressing Palmitoylation-deficient Rhodopsin*♦

Palmitoylation is a reversible, post-translational modification observed in a number of G-protein-coupled receptors. To gain a better understanding of its role in visual transduction, we produced transgenic knock-in mice that expressed a palmitoylation-deficient rhodopsin (Palm(-/-)). The mutant rhodopsin was expressed at wild-type levels and showed normal cellular localization to rod outer segments, indicating that neither rhodopsin stability nor its intracellular trafficking were compromised. But Palm(-/-) rods had briefer flash responses and reduced sensitivity to flashes and to steps of light. Upon exposure to light, rhodopsin became phosphorylated at a faster rate in mutant than in wild-type retinas. Since quench of rhodopsin begins with its phosphorylation, these results suggest that palmitoylation may modulate rod photoreceptor sensitivity by permitting rhodopsin to remain active for a longer period.

Scaling up success: lessons learned from technology-based educational improvement

Foreword (Ellen Condliffe Lagemann).Preface.1. Moving from Successful Local Practice to Effective State Policy: Lessons from Union City (Fred Carrigg, Margaret Honey, Ron Thorpe).2. Dewey Goes Digital: Scaling Up Constructivist Pedagogies and the Promise of New Technologies (Martha Stone Wiske, David Perkins).3. Adapting Innovations to Particular Contexts of Use: A Collaborative Framework (Barry J. Fishman).4. Designing for Scalable Educational Improvement: Processes of Inquiry in Practice (Susan R. Goldman).5. Scaling Up Professional Development in the United Kingdom, Singapore, and Chile (Laurence C. Peters).6. Technology as Proteus: Digital Infrastructures That Empower Scaling Up (Chris Dede, Robert Nelson).7. Scaling Up Data Use in Classrooms, Schools, and Districts (Sam Stringfield, Jeffrey C. Wayman, Mary E. Yakimowski-Srebnick).8. Foundations for Success in the Great City Schools: Lessons from Some Faster-Improving Districts (Michael Casserly, Jason C. Snipes).9. Scaling Up Technology-Based Educational Innovations (Barbara Means, William R. Penuel).10. Critiquing and Improving the Use of Data from High-Stakes Tests with the Aid of Dynamic Statistics Software (Jere Confrey, Katie M. Makar).11. Scaling Up Success: A Synthesis of Themes and Insights (Chris Dede, James P. Honan).The Contributors.Acknowledgments.Name Index.Subject Index.