A simple and high -performance immobilization technique of membrane protein from crude cell lysate sample for a membrane-based immunoassay application.

Membrane proteins are difficult to be extracted and to be coated on the substrate of the immunoassay reaction chamber because of their hydrophobicity. Traditional method to prepare membrane protein sample requires many steps of protein extraction and purification that may lead to protein structure deformation and protein dysfunction. This work proposes a simple technique to prepare and immobilize the membrane protein suspended in an unprocessed crude cell lysate sample. Membrane fractions in crude cell lysate were incorporated with the large unilamellar vesicle (LUV) that was mainly composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) before coating in the polystyrene plate by passive adsorption technique. Immunofluorescence staining and the Enzyme-Linked Immunosorbent Assay (ELISA) examination of a strictly conformation-dependent integral membrane protein, Myelin Oligodendrocyte Glycoprotein (MOG), demonstrate that LUV incorporated cell lysate sample obviously promotes MOG protein immobilization in the microplate well. With LUV incorporation, the dose-response curve of the MOG transfected cell lysate coating plate can be 2-9 times differentiated from that of the untransfected cell lysate coating plate. The LUV incorporated MOG transfected cell lysate can be efficiently coated in the microplate without carbonate/bicarbonate coating buffer assistance.

Hypercalcemia induces targeted autophagic degradation of aquaporin-2 at the onset of nephrogenic diabetes insipidus.

Hypercalcemia can cause renal dysfunction such as nephrogenic diabetes insipidus (NDI), but the mechanisms underlying hypercalcemia-induced NDI are not well understood. To elucidate the early molecular changes responsible for this disorder, we employed mass spectrometry-based proteomic analysis of inner medullary collecting ducts (IMCD) isolated from parathyroid hormone-treated rats at onset of hypercalcemia-induced NDI. Forty-one proteins, including the water channel aquaporin-2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the downregulated proteins were associated with cytoskeletal protein binding, regulation of actin filament polymerization, and cell-cell junctions. Targeted LC-MS/MS and immunoblot studies confirmed the downregulation of 16 proteins identified in the initial proteomic analysis and in additional experiments using a vitamin D treatment model of hypercalcemia-induced NDI. Evaluation of transcript levels and estimated half-life of the downregulated proteins suggested enhanced protein degradation as the possible regulatory mechanism. Electron microscopy showed defective intercellular junctions and autophagy in the IMCD cells from both vitamin D- and parathyroid hormone-treated rats. A significant increase in the number of autophagosomes was confirmed by immunofluorescence labeling of LC3. Colocalization of LC3 and Lamp1 with aquaporin-2 and other downregulated proteins was found in both models. Immunogold electron microscopy revealed aquaporin-2 in autophagosomes in IMCD cells from both hypercalcemia models. Finally, parathyroid hormone withdrawal reversed the NDI phenotype, accompanied by termination of aquaporin-2 autophagic degradation and normalization of both nonphoshorylated and S256-phosphorylated aquaporin-2 levels. Thus, enhanced autophagic degradation of proteins plays an important role in the initial mechanism of hypercalcemic-induced NDI.

Germinated brown rice protects against glutamate toxicity in HT22 hippocampal neurons through the jnk-mediated apoptotic pathway via the GABAA receptor.

The anti-apoptosis effect of germinated brown rice (GBR) focusing on differentiated HT22 cells results in improved nutritional values after the germination process of GBR which contains total phenolic compounds and γ-aminobutyric acid (GABA). Cell death induced by 5 mM glutamate was investigated for 24 h to determine whether GBR mediates cell death through GABA receptors by using antagonists. The results showed that GBR (100 µg/ml) suppressed glutamate-induced cytotoxicity and caused arrest at the G1/S phase of the cell cycle in differentiated HT22 cells. Furthermore, GBR significantly decreased the expression level of c-Jun, while its active form, p-c-Jun, is the downstream product of the JNK-mediated apoptotic pathway and causes subsequent cell death. In addition, bicuculline (12.5 nM), a GABAA antagonist, could eliminate GBR effects, but phaclofen (1 mM), a GABAB antagonist, could not. Surprisingly, GBR exhibited a better neuroprotective effect than a pure commercial GABA compound (0.115 µM). These results indicated that GBR possessed high anti-apoptotic activity and inhibited cell death in differentiated HT22 cells by perturbing re-entry of the cell cycle and apoptosis via the GABAA receptor. Hence, GBR could be further used as a valuable nutritional compound to prevent apoptosis-induced neurodegenerative diseases.

Systems-level analysis of cell-specific AQP2 gene expression in renal collecting duct.

We used a systems biology-based approach to investigate the basis of cell-specific expression of the water channel aquaporin-2 (AQP2) in the renal collecting duct. Computational analysis of the 5'-flanking region of the AQP2 gene (Genomatix) revealed 2 conserved clusters of putative transcriptional regulator (TR) binding elements (BEs) centered at -513 bp (corresponding to the SF1, NFAT, and FKHD TR families) and -224 bp (corresponding to the AP2, SRF, CREB, GATA, and HOX TR families). Three other conserved motifs corresponded to the ETS, EBOX, and RXR TR families. To identify TRs that potentially bind to these BEs, we carried out mRNA profiling (Affymetrix) in mouse mpkCCDc14 collecting duct cells, revealing expression of 25 TRs that are also expressed in native inner medullary collecting duct. One showed a significant positive correlation with AQP2 mRNA abundance among mpkCCD subclones (Ets1), and 2 showed a significant negative correlation (Elf1 and an orphan nuclear receptor Nr1h2). Transcriptomic profiling in native proximal tubules (PT), medullary thick ascending limbs (MTAL), and IMCDs from kidney identified 14 TRs (including Ets1 and HoxD3) expressed in the IMCD but not PT or MTAL (candidate AQP2 enhancer roles), and 5 TRs (including HoxA5, HoxA9 and HoxA10) expressed in PT and MTAL but not in IMCD (candidate AQP2 repressor roles). In luciferase reporter assays, overexpression of 3 ETS family TRs transactivated the mouse proximal AQP2 promoter. The results implicate ETS family TRs in cell-specific expression of AQP2 and point to HOX, RXR, CREB and GATA family TRs as playing likely additional roles.

Germinated Brown Rice Attenuates Cell Death in Vascular Cognitive Impaired Mice and Glutamate-Induced Toxicity In HT22 Cells.

Germinated brown rice (GBR) with unpolishing, soaking, and germinating processes can improve the texture, flavor, and nutritional value, including GABA and phenolic contents. The effect of GBR was first investigated in vascular cognitive impaired mice and glutamate-induced toxicity in HT22 cells with respect to standard pure GABA. Feeding mice with GBR for 5 weeks showed neuroprotection. In this study, the modified bilateral common carotid artery occlusion mice model was mild but a significant difference in cognitive impairment was still shown. Like pure GABA, GBR decreased cognitive deficits in memory behavioral tests and significantly attenuated hippocampal neuronal cell death at P < 0.001. Similarly to 0.125 µM of GABA, 100 µg/mL of GBR increased HT22 cell viability after glutamate toxicity. GBR affected less apoptotic cell death and less blocking by the GABAA antangonist bicuculline in comparison to GABA. When the results are taken together, the underlying mechanism of GBR protection may mediate though the GABAA receptor and its phenolic contents.

Transcriptional profiling of native inner medullary collecting duct cells from rat kidney.

Vasopressin acts on the inner medullary collecting duct (IMCD) in the kidney to regulate water and urea transport. To obtain a "parts list" of gene products expressed in the IMCD, we carried out mRNA profiling of freshly isolated rat IMCD cells using Affymetrix Rat 230 2.0 microarrays with approximately 31,000 features; 7,913 annotated transcripts were found to be expressed above background in the IMCD cells. We have created a new online database (the "IMCD Transcriptome Database;" http://dir.nhlbi.nih.gov/papers/lkem/imcdtr/) to make the results publicly accessible. Among the 30 transcripts with the greatest signals on the arrays were 3 water channels: aquaporin-2, aquaporin-3, and aquaporin-4, all of which have been reported to be targets for regulation by vasopressin. In addition, the transcript with the greatest signal among members of the solute carrier family of genes was the UT-A urea transporter (Slc14a2), which is also regulated by vasopressin. The V2 vasopressin receptor was strongly expressed, but the V1a and V1b vasopressin receptors did not produce signals above background. Among the 200 protein kinases expressed, the serum-glucocorticoid-regulated kinase (Sgk1) had the greatest signal intensity in the IMCD. WNK1 and WNK4 were also expressed in the IMCD with a relatively high signal intensity, as was protein kinase A (beta-catalytic subunit). In addition, a large number of transcripts corresponding to A kinase anchoring proteins and 14-3-3 proteins (phospho-S/T-binding proteins) were expressed. Altogether, the results combine with proteomics studies of the IMCD to provide a framework for modeling complex interaction networks responsible for vasopressin action in collecting duct cells.

Adipose Y5R mRNA is higher in obese than non-obese humans and is correlated with obesity parameters.

Neuropeptide Y is mainly expressed in the central nervous system to regulate food intake via its receptors, Y receptors, and in various peripheral tissues including adipose tissue. The objectives of this study were to compare Y5R mRNA and adipocyte parameters consisting of area, width, height, and perimeter either between obese and non-obese subjects or between subcutaneous and visceral fat as well as to compare between NPY, Y1R, Y2R, and Y5R mRNA expressions in subcutaneous and visceral adipose tissues. In subcutaneous and visceral adipose tissues, Y5R was greater in obese than in non-obese humans (both P < 0.05). Y1R mRNA expression was highest followed by Y5R, Y2R, and NPY mRNA expressions, respectively, in subcutaneous and visceral adipose tissues. Visceral Y5R mRNA had positive correlations with body weight, body mass index, waist circumference, hip circumference (R ≍ 0.4), and visceral Y1R mRNA (R = 0.773), but had a negative correlation with the quantitative insulin sensitivity check index (R=-0.421) (all P < 0.05). Subcutaneous and visceral adipocyte parameters were positively correlated with body weight, waist circumference, hip circumference, and waist-to-hip ratio, with greater values of correlation coefficient shown in visceral (R ≍ 0.5-0.8) than in subcutaneous adipocytes (R ≍ 0.4-0.6, all P < 0.05). The parameters of visceral adipocytes had positive correlations with serum NPY levels (R ≍ 0.4, all P < 0.05). Y5R mRNA in visceral adipose tissue is related to increased obesity and reduced insulin sensitivity. The dominant Y receptors in subcutaneous and visceral adipose tissue might be the Y1R and Y5R. Visceral adipocytes show higher correlations with obesity parameters than subcutaneous adipocytes, suggestive of an increased risk of metabolic syndrome in visceral obesity. Y1R and Y5R in visceral adipose tissue might be targets of drug development in prevention or treatment of adiposity. Impact statement Obesity, defined as excess fat accumulation, has been increasingly diagnosed worldwide causing adverse health consequences. The novel findings of this study were that Y5R mRNA expression in both subcutaneous and visceral fat was higher in obese than non-obese subjects. Furthermore, Y5R only in visceral fat, not subcutaneous fat, was positively correlated with visceral Y1R and obesity parameters but it was negatively correlated with the QUICKI. Moreover, we found that Y1R expression was highest followed by Y5R and Y2R, respectively, in both subcutaneous and visceral fat. Our results suggested that Y5R in visceral fat was associated with increased obesity and decreased insulin sensitivity. Y1R and Y5R might be the dominant receptors that mediate the effect of NPY-induced fat accumulation in both subcutaneous and visceral adipose tissues. Y1R and Y5R in visceral adipose tissue might be targets of drug development in prevention or treatment of obesity.

Autophagic degradation of aquaporin-2 is an early event in hypokalemia-induced nephrogenic diabetes insipidus.

Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.

Bacillus thuringiensis Cry4A and Cry4B mosquito-larvicidal proteins: homology-based 3D model and implications for toxin activity.

Three-dimensional (3D) models for the 65-kDa activated Cry4A and Cry4B delta-endotoxins from Bacillus thuringiensis subsp. israelensis that are specifically toxic to mosquito-larvae were constructed by homology modeling, based on atomic coordinates of the Cry1Aa and Cry3Aa crystal structures. They were structurally similar to the known structures, both derived 3D models displayed a three-domain organization: the N-terminal domain (I) is a seven-helix bundle, while the middle and C-terminal domains are primarily comprise of anti-parallel beta-sheets. Circular dichroism spectroscopy confirmed the secondary structural contents of the two homology-based Cry4 structures. A structural analysis of both Cry4 models revealed the following: (a) Residues Arg-235 and Arg-203 are located in the interhelical 5/6 loop within the domain I of Cry4A and Cry4B, respectively. Both are solvent exposed. This suggests that they are susceptible to tryptic cleavage. (b) The unique disulphide bond, together with a proline-rich region within the long loop connecting alpha4 and alpha5 of Cry4A, were identified. This implies their functional significance for membrane insertion. (c) Significant structural differences between both models were found within domain II that may reflect their different activity spectra. Structural insights from this molecular modeling study would therefore increase our understanding of the mechanic aspects of these two closely related mosquito-larvicidal proteins.

Ion channels formed in planar lipid bilayers by the dipteran-specific Cry4B Bacillus thuringiensis toxin and its alpha1-alpha5 fragment.

Trypsin activation of Cry4B, a 130-kDa Bacillus thuringiensis (Bt) protein, produces a 65-kDa toxin active against mosquito larvae. The active toxin is made of two protease resistant-products of ca. 45 kDa and ca. 20 kDa. The cloned 21-kDa fragment consisting of the N-terminal region of the toxin was previously shown to be capable of permeabilizing liposomes. The present study was designed to test the following hypotheses: (1) Cry4B, like several other Bt toxins, is a channel-forming toxin in plannar lipid bilayers; and (2) the 21-kDa N-terminal region, which maps for the first five helices (alpha1-alpha5) of domain 1 in other Cry toxins, and which putatively shares a similar tri-dimensional structure, is sufficient to account for the ion channel activity of the whole toxin. Using circular dichroism spectroscopy and planar lipid bilayers, we showed that the 21-kDa polypeptide existed as an alpha-helical structure and that both Cry4B and its alpha1-alpha5 fragment formed ion channels of 248 +/- 44 pS and 207 +/- 23 pS, respectively. The channels were cation-selective with a potassium-to-chloride permeability ratio of 6.7 for Cry4B and 4.5 for its fragment. However, contrary to the full-length toxin, the alpha1-alpha5 region formed channels at low dose; they tended to remain locked in their open state and displayed flickering activity bouts. Thus, like the full-length toxin, the alpha1-alpha5 region is a functional channel former. A pH-dependent, yet undefined region of the toxin may be involved in regulating the channel properties.