GIRK4 confers appropriate processing and cell surface localization to G-protein-gated potassium channels

GIRK1 and GIRK4 subunits combine to form the heterotetrameric acetylcholine-activated potassium current (IKACh) channel in pacemaker cells of the heart. The channel is activated by direct binding of G-protein Gbetagamma subunits. The GIRK1 subunit is atypical in the GIRK family in having a unique ( approximately 125-amino acid) domain in its distal C terminus. GIRK1 cannot form functional channels by itself but must combine with another GIRK family member (GIRK2, GIRK3, or GIRK4), which are themselves capable of forming functional homotetramers. Here we show, using an extracellularly Flag-tagged GIRK1 subunit, that GIRK1 requires association with GIRK4 for cell surface localization. Furthermore, GIRK1 homomultimers reside in core-glycosylated and nonglycosylated states. Coexpression of GIRK4 caused the appearance of the mature glycosylated form of GIRK1. [35S]Methionine pulse-labeling experiments demonstrated that GIRK4 associates with GIRK1 either during or shortly after subunit synthesis. Mutant and chimeric channel subunits were utilized to identify domains responsible for GIRK1 localization. Truncation of the unique C-terminal domain of Delta374-501 resulted in an intracellular GIRK1 subunit that produced normal IKACh-like channels when coexpressed with GIRK4. Chimeras containing the C-terminal domain of GIRK1 from amino acid 194 to 501 were intracellularly localized, whereas chimeras containing the C terminus of GIRK4 localized to the cell surface. Deletion analysis of the GIRK4 C terminus identified a 25-amino acid region required for cell surface targeting of GIRK1/GIRK4 heterotetramers and a 25-amino acid region required for cell surface localization of GIRK4 homotetramers. GIRK1 appeared intracellular in atrial myocytes isolated from GIRK4 knockout mice and was not maturely glycosylated, supporting an essential role for GIRK4 in the processing and cell surface localization of IKACh in vivo.     

Remarkable preservation of biomolecules in ancient radish seeds

Desiccated seeds from a 6th century AD storage vessel recovered from Qasr Ibr?m, Egypt, were examined for the presence of lipids and nucleic acids. A remarkable degree of lipid preservation was discovered, the fatty acid and sterol profiles being very similar to those of modern radish seeds. The only significant differences were hydrolysis of triacylglycerols and depletion of the polyunsaturated fatty acids (C18:2 and C18:3). The delta 13 C values of the principal fatty acids were in the range -25.4 to -29.2/1000, which is congruent with modern radish (C3 seeds) taking account of isotopic shifts caused by recent changes in atmospheric CO2. Deoxyribonucleosides and nucleic acid bases were detected by direct chemical analysis, and polymerase chain reactions gave products with sequences comparable to those from modern radish. The degree of lipid preservation, which was much greater than that reported for other archaeological remains, suggests that the microenvironment within desiccated seeds retards biomolecular decay. The results illustrate the utility of combined lipid-nucleic acid analysis in chemotaxonomic and genotypic studies of archaeobotanical remains.     

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]i odo phenyl]2-ethoxy propanoic acid], which is specific for the gamma isoform of the peroxisomal proliferator activated receptor (PPARgamma), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARgamma1 and to a GST (glutathione S-transferase) fusion protein containing the ligand binding domain of human PPARgamma1 (KD = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARgamma1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an alpha-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPARgamma1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARgamma and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.     

Calcium regulation of nuclear pore permeability

The nuclear envelope is an integral part of the structural framework of the nucleus, and is involved in organizing intranuclear events. It serves as a selective barrier, actively transporting proteins required for normal nuclear function and exporting RNA. The movement of molecules across the nuclear envelope is critical for cellular homeostasis, and it allows cells to respond to external events. The only known pathway for direct communication between the cytoplasm and the nucleoplasm of a cell is through the nuclear pore complex. In the past decade, rapid advances have been made in elucidating the structure and function of the nuclear pore complex. Yet, researchers are just beginning to identify some of the regulatory mechanisms controlling transport through the pore complex. The nucleus is surrounded by a Ca2+ storage compartment, which sequesters and releases Ca2+ in response to intracellular second messengers, Recent evidence suggests that the nuclear Ca2+ store may indirectly regulate passive diffusion through the nuclear pore complex. The evidence for Ca2+ regulation of the nuclear pore complex will be discussed, along with the introduction of the simplest, testable model to describe the observations.     

Housing Theory,Housing Research and Housing Policy

Abstract

Jim Kemeny in 1992 criticized existing housing research for neglecting social theory and being overly positivist and policy focused. The result has been a strengthening of the conceptual basis of housing research in general, but also a growing schism between researchers who focus on policy relevancy and those that pursue more theoretical work. This paper challenges this schism and argues for theoretically based and policy-relevant research. First, the paper argues that the policy-making process is complex and can vary between different countries, situations and over time. Therefore, it is argued that many styles of research can influence policy in the right circumstances. Second, the paper challenges the idea that there can be theoretically free housing research arguing that all research has a theoretical foundation even though in many studies it is not explicit. Finally, the paper engages with the debate about where theory for housing research should come from and what it would look like.     

Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.     

The effect of defect introduction vs. load application sequencing on defect-induced stress distributions in steel samples

This study examines the effect of altering the sequence of defect introduction vs. load application on the defect-induced stress distributions in steel samples loaded under conditions of uniaxial tension. The stresses in the samples were measured using neutron diffraction and Magnetic Barkhausen Noise (MBN). In addition, the samples were scanned using Magnetic Flux Leakage (MFL) to determine the effect of sequencing on the MFL signal amplitude and trend with changing applied loads.At loads resulting in local plastic deformation at the defect edge, studies show distinct differences in the defect-induced stress distributions between through-holes milled prior to the application of load (pre-drilled), compared to those milled while the sample was held at-load (in situ). The consequences of these findings are discussed in light of MFL tool calibration and the subsequent determination of the Maximum Allowable Operating Pressure (MAOP) for in-service pipelines. The implications with regards to the hole-drilling technique for stress measurement are also discussed.     

Mechanical damage detection using magnetic flux leakage tools: modeling the effect of dent geometry and stresses

We have used three-dimensional (3D) magnetic finite element analysis (FEA) to simulate the MFL signal from a circular dent geometry with associated residual stresses. Strain distribution information around the dent was obtained from an earlier work using finite element structural modeling. In the magnetic FEA dent model, the localized residual stresses were simulated by assigning appropriate values of magnetic anisotropy to the relevant magnetic regions. The simulated flux leakage patterns were found to be in good agreement with the experimentally observed MFL patterns associated with dent shape as well as with the stress effects from the dent.     

Investigation of Optimum Field Amplitude for Stress Dependence of Magnetic Barkhausen Noise

An investigation of the sensitivity of magnetic Barkhausen noise (MBN) to changes in uniaxial tensile stress applied to mild steel under conditions of low, medium, and high excitation field amplitudes is presented. Field excitation in the lowest range that was tested produced the most sensitive response to change in stress for the squared-voltage-signal output integrated with respect to time (MBN_energy). The linear response extended to the highest applied stress of 220 MPa, which was 80% of yield strength. In this field range, the angle-dependent MBN_energy and distribution of pulse heights under applied stress conditions was observed to be consistent with previous measurements performed in the medium- to high-field amplitude ranges. However, the number of Barkhausen events was less, and increased linearly with applied stress up to 120 MPa. In contrast, at higher field amplitudes, the initial number of events was greater, but then decreased with increasing applied stress. The results at low field amplitudes are attributed to the additional 180deg domain wall activity, accompanied by increased magnetization level, which could be induced by tensile stress. The reduction in number of events at higher stresses and higher excitation field amplitudes, which corresponds to an increase in MBN_energy and broadened pulse-height distribution, is associated with increased collective Barkhausen behavior. These results demonstrate that improved sensitivity of Barkhausen signals to changes in stress can be achieved in mild steel if optimized field excitation is used.