Enhancement of recombinant gamma-aminobutyric acid type A receptor currents by chronic activation of cAMP-dependent protein kinase

alpha 1, beta 1, and gamma 2S gamma-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNAs were transiently expressed in derivative cell lines of mouse L929 fibroblasts, which possessed different levels of the catalytic subunit of cAMP-dependent protein kinase (PKA). These cell lines included L929 (intermediate levels of kinase), C alpha 12 (elevated levels of kinase), and RAB10 (low levels of kinase) cells. Pharmacological analysis of GABA-evoked whole-cell currents revealed that, compared with expression in L929 and RAB10 cells, expression of alpha 1 beta 1 gamma 2S GABARs in C alpha 12 cells produced a selective enhancement of single whole-cell current amplitudes. No other pharmacological properties (Hill slope, EC50, or diazepam sensitivity) of the expressed alpha 1 beta 1 gamma 2S GABARs were modified. The GABAR current enhancement in C alpha 12 cells was blocked by substitution of a beta 1 subunit mutated at the PKA consensus phosphorylation site, Ser409 [beta 1(S409A)], for the wild-type beta subunit. Interestingly, enhancement was specific for GABARs containing all three subunits, because it was not seen after expression of alpha 1 beta 1 or alpha 1 beta 1 (S409A) GABAR subunit combinations. Single-channel conductance and gating properties were not different for alpha 1 beta 1 gamma 2S or alpha 1 beta 1 (S409A) gamma 2S GABARs expressed in each cell line, suggesting that PKA did not enhance whole-cell currents by altering these properties of GABARs. These results suggested that unlike acute application of PKA, which has been shown to produce a decrease in GABAR current, chronic elevation of PKA activity can result in enhancement of GABAR currents. More importantly, this effect occurred only with GABARs composed of alpha 1 beta 1 gamma 2S subunits and not alpha 1 beta 1 subunits and was mediated by a single amino acid residue (Ser409) of the beta 1 subunit.     

Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdelta1

Structural studies of phospholipase C delta1 (PLCdelta1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCdelta1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His311, Asn312, Glu341, Asp343, His356, Glu390, Lys438, Lys440, Ser522, Arg549, and Tyr551) and residues from the hydrophobic ridge (Leu320, Phe360, and Trp555). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4, 5-bis-phosphate (PIP2) identified Ser522, Lys438, and Arg549 as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys440 selectively affected only hydrolysis of PIP2. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn312, Glu390, Glu341, and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed the importance of His311 ( approximately 20,000-fold reduction) and His356 ( approximately 6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2 hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.     

Identification and characterization of two distinct truncated forms of gp130 and a soluble form of leukemia inhibitory factor receptor alpha-chain in normal human urine and plasma

Leukemia inhibitory factor (LIF) is a polyfunctional cytokine known to require at least two distinct receptor components (LIF receptor alpha-chain and gp130) in order to form a high affinity, functional receptor complex. In this report, we present evidence that there are two distinct truncated forms of gp130 in normal human urine and plasma: a large form with a molecular weight of approximately 100, 000, which is similar to a previously described form of soluble gp130 in human serum, and a previously undescribed small form with a molecular weight of approximately 50,000. Using a panel of monoclonal antibodies raised against the extracellular domain of human gp130, we were able to show that the small form of the urinary gp130 probably contained only the hemopoietin domain. Both forms of gp130 bound LIF specifically and were capable of forming heterotrimeric complexes with soluble human LIF receptor alpha-chain in the presence of human LIF. In addition to the soluble forms of gp130, a soluble form of LIF receptor alpha-chain was also detected in human urine and plasma.     

Two-dimensional mapping and microsequencing of lysosomal proteins from human placenta

Lysosomes degrade a wide range of macromolecules to yield monomer products which are exported out of the lysosome by a series of transporters. In addition, lysosomes perform a range of other functions which are cell or tissue specific. In order to gain insight into the tissue specific role of lysosomes, carrier-ampholyte two-dimensional electrophoresis (2-DE) was used in combination with N-terminal sequencing to identify the major proteins present in both the membrane and luminal space of placental lysosomes. From the 45 N-terminal peptide sequences generated, 14 luminal and five membrane proteins were identified while three other sequences were novel. The sequenced proteins were a mixture of lysosomal and non-lysosomal proteins. The lysosomal proteins consisted of gamma-interferon-inducible protein (IP-30), Saposin D, cathepsins B and D, beta-hexosaminidase, palmitoyl protein thioesterase, alpha-glucosidase, and LAMP-1. The non-lysosomal proteins were serum albumin, serotransferrin, haemoglobin gamma G chain, alpha-1-antitrypsin, placental lactogen, endoplasmin, peptide binding protein 74, p60 lymphocyte protein, p450 side chain cleavage enzyme and placental alkaline phosphatase. The 2-DE maps obtained in this study are the first to identify the major proteins in both the lumen and membrane of placental lysosomes through sequence analysis, and thus provide the basis upon which to build a complete 2-DE database of the lysosome. Furthermore, the identities of the proteins sequenced from the placental lysosomes suggest a role for lysosomes in the transport of nutrients across the trophoblastic layer.     

Monoclonal antibodies to mitochondrial E2 components define autoepitopes in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial Abs (AMA). The autoantigens recognized by AMA are the E2 components of the pyruvate dehydrogenase complex (PDC-E2), the branched chain 2-oxoacid dehydrogenase complex E (BCOADC-E2), and the 2-oxoglutarate dehydrogenase complex E (OGDC-E2). Previous studies using murine monoclonal and human combinatorial Abs to PDC-E2 have demonstrated an intense linear staining pattern in the apical region of biliary epithelial cells (BEC) in PBC but not control liver. We therefore examined whether mAbs to the other mitochondrial autoantigens BCOADC-E2 and OGDC-E2 demonstrated disease-specific patterns of reactivity. Using an expressed recombinant "trihybrid" protein containing the lipoyl domains of PDC-E2, OGDC-E2, and BCOADC-E2, we immunized BALB/c mice to produce 35 mAbs specific for one or more of the above mitochondrial autoantigens. Seven of these mAbs uniquely stained the apical region of BEC in PBC. Of these seven, one was reactive to PDC-E2, two recognized BCOADC-E2, three were reactive to OGDC-E2, and one recognized all three Ags. Our current data demonstrate that, similar to our previous studies regarding PDC-E2, mAbs to BCOADC-E2 and OGDC-E2, or a molecule that cross-reacts with the inner lipoyl domain of all three enzymes, also show a uniquely intense staining pattern in the apical region of BEC in patients with PBC when compared with diseased controls. The abundance of such disease-specific determinants in the target cells of PBC raises interesting possibilities regarding the role of these autoantigens in the pathogenesis of this disease.     

Effect of aging on the sensitivity of growth hormone secretion to insulin-like growth factor-I negative feedback

To determine the effect of aging on the suppression of GH secretion by insulin-like growth factor (IGF)-I, we studied 11 healthy young adults (6 men, 5 women, mean +/- SD: 25.2 +/- 4.6 yr old; body mass index 23.7 +/- 1.8 kg/m2) and 11 older adults (6 men, 5 women, 69.5 +/- 5.8 yr old; body mass index 24.2 +/- 2.5 kg/m2). Saline (control) or recombinant human IGF-I (rhIGF-I) (2 h baseline then, in sequence, 2.5 h each of 1, 3, and 10 micrograms/kg.h) was infused iv during the last 9.5 h of a 40.5-h fast; serum glucose was clamped within 15% of baseline. Baseline serum GH concentrations (mean +/- SE: 3.3 +/- 0.7 vs. 1.9 +/- 0.5 micrograms/L, P = 0.02) and total IGF-I concentrations (219 +/- 15 vs. 103 +/- 19 micrograms/L, P < 0.01) were higher in the younger subjects. In both age groups, GH concentrations were significantly decreased by 3 and 10 micrograms/kg.h, but not by 1 microgram/kg.h rhIGF-I. The absolute decrease in GH concentrations was greater in young than in older subjects during the 3 and 10 micrograms/kg.h rhIGF-I infusion periods, but both young and older subjects suppressed to a similar GH level during the last hour of the rhIGF-I infusion (0.78 +/- 0.24 microgram/L and 0.61 +/- 0.16 microgram/L, respectively). The older subjects had a greater increase above baseline in serum concentrations of both total (306 +/- 24 vs. 244 +/- 14 micrograms/L, P = 0.04) and free IGF-I (8.5 +/- 1.4 vs. 4.2 +/- 0.6 micrograms/L, P = 0.01) than the young subjects during rhIGF-I infusion, and their GH suppression expressed in relation to increases in both total and free serum IGF-I concentrations was significantly less than in the young subjects. We conclude that the ability of exogenous rhIGF-I to suppress serum GH concentrations declines with increasing age. This suggests that increased sensitivity to endogenous IGF-I negative feedback is not a cause of the decline in GH secretion that occurs with aging.     

Metaphors in the talk of "depressed" women in psychotherapy.

Variations in how we describe, explain, and treat "depression" and in who is labeled "depressed" attest to the significance of the social-historical contexts in which our understandings and practices are based. A prevalent, modern-day (Western) conception of "depression" is as a gendered (primarily female), devalued condition characterized by a discourse of the deficient self. In an effort to learn more about the particular forms of this discourse, the metaphors used by 10 depressed women (aged 28–62 yrs) to talk to their psychotherapists about their "selves" in relation to their perceived difficulties were studied. Two broad cultural imperatives were constructed from these metaphors: (1) Don't be too mothering and (2) Don't be too child-like. At the core of these 2 imperatives were messages about the importance of autonomy, for both oneself and others. This valuing of autonomy can be clearly located at the heart of 20th-century Western ideals. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

An experimental stationary quadrotor with variable DOF

Unmanned air vehicles (UAV) and especially quadrotors have drawn great attention in recent years because of their maneuverability, ease of design and control. Most of the works concentrate mostly on control; yet, design and communication are also some sub-topics. In this research, a stationary, four rotor air vehicle with limited/controlled degree of freedom is constructed so that various control algorithms and their changing effects with varying vehicle dynamics can be studied on the ground for safety purposes. Ascending/descending, pitch/yaw/roll motions can be limited/controlled by setting the vehicle’s degree of freedom mechanically, resulting better net observability of the control algorithms on the vehicle’s dynamic performance. Design, in terms of mechanics, mechatronics and software is presented and the usability of the system is shown. Parallel self tuning fuzzy PD + PD control is applied to the system for preliminary studies and results are discussed. Inspite of the sensor noise, satisfactory pitch/roll/yaw control is achieved.