Immunoblot Signals Research Articles

Optimizing renal transporter immunodetection: consequences of freeze-thaw during sample preparation.

Renal transporters (cotransporters, channels, and claudins) mediate homeostasis of fluids and electrolytes and are targets of hormonal and therapeutic regulators. Assessing renal transporter abundance with antibody probes by immunoblotting is an essential tool for mechanistic studies. Although journals require authors to demonstrate antibody specificity, there are no consensus guidelines for kidney sample preparation leading to lab-to-lab variability in immunoblot results. In this study, we determined the impact of sample preparation, specifically freeze-thawed (Frozen) versus freshly processed (Fresh) kidneys (female and male rats and mice) on immunoblot signal detection of 15 renal transporters and the impact of protease inhibitors during homogenization. In female Sprague-Dawley rat kidneys homogenized with aprotinin, Na2EDTA, PMSF, and phosphatase inhibitors, immunodetection signals were ∼50% lower in Frozen versus Fresh samples for most transporters. Inclusion of additional inhibitors (Roche cOmplete Protease Inhibitor, "+") only partially increased transporter immunoblot signals to near Fresh levels. In male Sprague-Dawley rats, immunoblot signal density was lower in Frozen+ versus Fresh+ despite additional inhibitors. In C57BL/6 male mice, immunoblot signals from proximal tubule transporters were lower in Frozen versus Fresh by ∼25-50% and greater in Frozen+. In contrast, immunodetection signal was equivalent in female Frozen+ versus female Fresh+ for claudin 2, villin, AQP1, NKCC2, NCC, ENaCβ, ENaCɣ, claudin 7, AQP2, NKAα1, and NKAβ1. Thus, kidney sample preparation variables, including freeze-thaw and protease inhibition, have substantial transporter-specific effects on quantification of renal transporter abundance by immunoblot. These findings underscore the critical importance of assessing and reporting the impact of sample preparation protocols on transporter recovery to ensure robust rigor and reproducibility. NEW & NOTEWORTHY Freeze-thawing kidneys before homogenization is widely accepted in renal research. This study demonstrates that if kidneys are freeze-thawed just once before homogenization, immunoblot signals are reduced in a transporter-specific manner in rats and mice dependent on sex and that immunoblot signals can be partially recovered by adding additional protease inhibitors. These findings underscore the critical importance of assessing the impact of sample preparation, including freeze-thaw versus fresh, to ensure robust rigor and reproducibility.

Factors Affecting Non-Enzymatic Protein Acylation by trans-3-Methylglutaconyl Coenzyme A.

The leucine catabolism pathway intermediate, trans-3-methylglutaconyl (3MGC) CoA, is considered to be the precursor of 3MGC acid, a urinary organic acid associated with specific inborn errors of metabolism (IEM). trans-3MGC CoA is an unstable molecule that can undergo a sequence of non-enzymatic chemical reactions that lead to either 3MGC acid or protein 3MGCylation. Herein, the susceptibility of trans-3MGC CoA to protein 3MGCylation was investigated. trans-3MGC CoA was generated through the activity of recombinant 3-methylcrotonyl CoA carboxylase (3MCCCase). Following enzyme incubations, reaction mixtures were spin-filtered to remove 3MCCCase. The recovered filtrates, containing trans-3MGC CoA, were then incubated in the presence of bovine serum albumin (BSA). Following this, sample aliquots were subjected to α-3MGC IgG immunoblot analysis to probe for 3MGCylated BSA. Experiments revealed a positive correlation between trans-3MGC CoA incubation temperature and 3MGCylated BSA immunoblot signal intensity. A similar correlation was observed between incubation time and 3MGCylated BSA immunoblot signal intensity. When trans-3MGC CoA hydratase (AUH) was included in incubations containing trans-3MGC CoA and BSA, 3MGCylated BSA immunoblot signal intensity decreased. Evidence that protein 3MGCylation occurs in vivo was obtained in studies with liver-specific 3-hydroxy-3-methylglutaryl (HMG) CoA lyase knockout mice. Therefore, trans-3MGC CoA is a reactive, potentially toxic metabolite, and under normal physiological conditions, lowering trans-3MGC CoA levels via AUH-mediated hydration to HMG CoA protects against aberrant non-enzymatic chemical reactions that lead to protein 3MGCylation and 3MGC acid production.

Allosteric activation of Tcell antigen receptor signaling by quaternary structure relaxation.

SummaryThe mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

Na+/K+-ATPase level and products of lipid peroxidation in live cells treated with therapeutic lithium for different periods in time (1, 7, and 28days); studies of Jurkat and HEK293 cells.

Regulation of Na+/K+-ATPase in bipolar disorder and lithium therapy has been investigated for more than 40years. Contradictory results in this area may be caused by the difference between acute and long-term Li effects on cell metabolism and variance in responsiveness of different cell types. We compared the time-course of Li action focusing on Na+/K+-ATPase and lipid peroxidation in two widely different cell models-Jurkat and HEK293. Na+/K+-ATPase expression level was determined in cells cultivated in the absence or presence of 1mM Li for different time spans (1, 7, and 28days) using [3H] ouabain binding and immunoblot assay of α-subunit. In parallel samples, the formation of malondialdehyde (MDA) was quantified by HPLC, and 4-hydroxy-2-nonenal (4-HNE) protein adducts were determined by immunoblot. Cultivation of Jurkat cells in 1mM Li medium resulted in downregulation of Na+/K+-ATPase (decrease of [3H] ouabain-biding sites and intensity of immunoblot signals) in all Li-groups. In HEK293 cells, the decrease of Na+/K+-ATPase was observed after the acute, 1-day exposure only. The long-term treatment with Li resulted in Na+/K+-ATPase upregulation. MDA and 4-HNE modified proteins were decreased in Jurkat cells in all Li-groups. On the other hand, in HEK293 cells, MDA concentration was decreased after the acute, 1-day Li exposure only; the long-term cultivations, for 7 or 28days, resulted in a significant increase of lipid peroxidation products. The Li-induced decrease of lipid peroxidation products was associated with the decrease of Na+/K+-ATPase level and vice versa.

Detection of annexin A8 antibodies in serum of patients with antiphospholipid syndrome.

IntroductionAntibodies specific for annexin A8 (AnxA8) have not been investigated in patients suffering from antiphospholipid syndrome (APS) yet. The aim of this study was to compare the presence of AnxA8 antibodies in serum of APS patients with that of age-matched healthy controls and to investigate whether AnxA8 antibodies are potential biomarkers for APS.Materials and methodsWe enrolled 22 APS patients and 22 healthy controls in this case-control study. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot to investigate the presence of AnxA8 antibodies, and we applied enzyme-linked immunosorbent assay to investigate the presence of cardiolipin (CL) and beta-2-glycoprotein I (ß2GPI) antibodies.ResultsThe serum of 9/22 APS patients showed AnxA8 IgG isotype antibody reactivity compared to serum of 2/22 healthy controls (P = 0.034). When we also included weak immunoblot signals, 12/22 APS patients exhibited AnxA8 IgG isotype antibody reactivity compared to 3/22 healthy controls (P = 0.005). We also investigated the presence of AnxA8 IgM isotype antibodies in the serum of APS patients but found no statistically significant difference between the APS patient group and healthy control group (P = 0.500). We further investigated the presence of ß2GPI and CL IgG and IgM isotype antibodies. AnxA8 IgG isotype antibodies were present in APS patients in a similar frequency as the APS “criteria” antibody against CL (P = 0.764).ConclusionWe demonstrated that AnxA8 IgG isotype antibodies are potential biomarkers for the diagnosis of APS.

Up-regulation of μ-, δ- and κ-opioid receptors in concanavalin A-stimulated rat spleen lymphocytes

Regulation of μ-, δ- and κ-opioid receptor protein level in spleen lymphocytes when stimulated by mitogen is not known. To answer the question whether these cells do express opioid receptor (OR) proteins, primary, fresh rat spleen lymphocytes were prepared and stimulated for 48 h with mitogenic dose of Con A. The unstimulated lymphocytes did not express μ- and δ-OR proteins in detectable amounts, however, stimulation with Con A resulted in appearance of clearly detectable immunoblot signals of both μ-OR and δ-OR. κ-OR were detected already in primary cells and increased 2.4-fold in Con A-stimulated cells. These results were supported by data obtained by flow cytometry analysis indicating a dramatic increase in number of μ-, δ- and κ-OR expressing cells after mitogen stimulation. The newly synthesized μ-, δ- and κ-OR in Con A-stimulated spleen lymphocytes were present in the cells interior and not functionally mature, at least in terms of their ability to enhance activity of trimeric G proteins determined by three different protocols of agonist-stimulated, high-affinity [35S]GTPγS binding assay. The up-regulation of μ-, δ- and κ-OR was associated with specific decrease of their cognate trimeric G proteins, Gi1α/Gi2α; the other Gα and Gβ subunits were unchanged. The level of β-arrestin-1/2 was also decreased in Con A-stimulated splenocytes. We conclude that up-regulation of OR expression level in spleen lymphocytes by Con A proceeds in conjunction with down-regulation of their intracellular signaling partners, Gi1α/Gi2α proteins and β-arrestin-1/2. These regulatory proteins are expressed in high amounts already in unstimulated cells and decreased by mitogen stimulation.

The Effect of Acute Temperature Change on the Localization of Aquaporin‐3 in the Erythrocytes of the Freeze‐tolerant Cope's Gray Treefrog

Cope's gray treefrogs (Dryophytes chrysoscelis) accumulate cryoprotective glycerol (>100 mM) throughout their body in response to falling temperatures, which enables them to tolerate freezing. The most likely pathway for the transmembrane transport of glycerol is through glycerol‐permeable aquaporins such as HC‐3, a homolog of Aquaporin‐3, which increases in both gene and protein expression for erythrocytes during cold acclimation. Much of the increase in immunoblot signal intensity occurs not at the nominal molecular weight of HC‐3 (~31.5 kDa), but at a higher molecular weight (~65–100 kDa) shown to be glycosylated HC‐3. We hypothesized that glycosylation of HC‐3 is involved in trafficking of the protein to the plasma membrane. To test this hypothesis, we established conditions in culture that enhance expression of HC‐3, and we assessed expression of glycosylated and un‐glycosylated HC‐3 in the plasma membrane via biotinylation and immunoblot.Erythrocytes were cultured at 20°C and 5°C over the course of 48 hours. Cell surface expression was assessed by biotinylation, which resulted in total and biotinylated (plasma membrane, PM) fractions assayed by immunoblot for HC‐3 and quantified with densitometry. Erythrocytes incubated at 20°C for 48 hours yielded total protein with 4‐fold greater glycosylated HC‐3 compared with 5°C‐incubated erythrocytes, but no change in the nominal molecular weight (unglycosylated) band. The biotinylated fraction of protein from 20°C‐incubated erythrocytes had another 4‐fold increase in glycosylated HC‐3 over the total, suggesting enrichment of glycosylated protein in HC‐3 localized to the plasma membrane. In 5°C‐incubated erythrocytes, total expression of HC‐3 was diminished compared with 20°C. In contrast to RBCs incubated at 20°C, cells at 5°C were enriched for glycosylated HC‐3 in the non‐biotinylated protein fraction compared with PM‐localized protein. Thus, while glycosylation is enhanced in the plasma membrane fraction of HC‐3 in 20°C‐incubated erythrocytes, the opposite occurs in cells incubated at 5°C, suggesting that while the glycosylation of HC‐3 isn't required for localization to the plasma membrane, its expression is variably regulated in response to environmental and humoral factors.We further hypothesized that epinephrine could induce changes in membrane localization of HC‐3 in cultured erythrocytes similar to those observed during cold acclimation. When erythrocytes incubated at 5°C for 48 hours were treated with epinephrine (1 μM) for 60 minutes, the expression of glycosylated protein in the plasma membrane fraction of HC‐3 increased >10‐fold compared with 5°C‐incubated erythrocytes without epinephrine treatment; the nominal MW band was not present in the plasma membrane fraction from cells incubated with epinephrine. Our data suggest that the enhanced expression of HC‐3 observed in erythrocytes from long‐term cold‐acclimated animals is not induced by a decrease in temperature, but changes to HC‐3 expression and subsequent trafficking to the plasma membrane can be activated by epinephrine.Support or Funding InformationNSF grant (IOS‐1121457)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cellulose-Affinity Isolation of Vaccine Candidate Antigens from Transgenic Plants

Apart from tradition, the biospecific affinity isolation has become one of the most rapidly growing concerns with cellulose binding domains (CBDs), a high-capacity tag for cost-effective purifications of fusion proteins. We report here a new strategy optimized for isolation of two fusion proteins, the FGF-1 (a human functional protein) and the H5N1 (a vaccine candidate antigen) tagged with CBD were grown in transient <i>Nicotiana benthamiana</i> and transgenic <i>Arabidopsis thaliana</i> respectively. A notable fraction of the recombinant proteins was lost through plant debris pelleted from the plant-slurry made. However, this issue was resolved by adjusting tissue-to-buffer ratios with 1:10 and 1:15 in those plants respectively. Washing efficiencies were improved by agitating column beds with acidic buffer (20mM NaAc. pH 4.0) in <i>Nicotiana</i> and alkaline buffer (10mM Tris-base pH 8.0) in <i>Arabidopsis</i>. Adsorption and coupling of tagged proteins on cellulose matrices were affected by the buffer-logged resins. The column-beds, after pumping the moisture out, showed efficient in binding of antigens with almost no losses detected by immunoblot signals. The bound antigens were released efficiently from the cellulose matrices by 1% Cellobiose and 2% Triethylamine respectively. The successive purifications of these antigenic proteins with identical tags likely indicate the efficiency of the proposed strategy in providing a generic and cost-effective method to purify fusion proteins propagated in transgenic plants.

Porcine milk induces a strengthening of barrier function in porcine jejunal epithelium in vitro.

Milk contains a variety of components that have been shown to affect the expression and localization of epithelial tight junction proteins and therefore the intestinal barrier. Thus, we hypothesized that milk would have an effect on intestinal barrier properties, owing to effects on the tight junction in an intraspecies porcine intestinal in vitro model. Jejunal samples of piglets derived from different age groups were analyzed. Transepithelial electrical resistance was recorded employing the Ussing chamber technique. Porcine milk or predigested milk in buffer solution was added to the apical side, and effects were compared to untreated controls. Unidirectional paracellular flux measurements were performed using sodium fluorescein. Tight junction protein expression and localization were analyzed by immunoblotting and immunofluorescence microscopy. Incubation with milk or predigested milk led to an increase in transepithelial electrical resistance, while paracellular permeability for sodium fluorescein did not result in significant changes. Densitometric analysis of immunoblot signals did not show significant alterations in claudin expression, but a reduction of claudin signals in apicolateral membrane compartments in both approaches became apparent via immunohistology. The functional effect might reflect a physiological protective mechanism, when offspring exclusively rely on their mother's milk and are exposed to a plethora of potentially barrier-perturbing factors.

Detection of a Perilipin‐5 Splice Variant

Alternative splicing occurs throughout the human genome, leading to multiple proteins from a single gene. The resulting proteins can be nearly identical or vastly different in how they function. The perilipins are a family of lipid droplet associated proteins that among other functions regulate interaction of lipases with their substrates. Alternative splicing within the perilipin family has been observed in perilipins 1 and 3, giving rise to proteins with varying functions. In the course of our studies on perilipin 5, an immunoblot signal was observed that corresponded to an approximately 35 kDa protein. Based on this data we hypothesized that perilipin 5 also undergoes alternative splicing. Western blot analysis of this shortened form of perilipin 5 has revealed its expression in C2C12 cultured myoblasts and in heart and liver mouse tissue. Further analysis of perilipin 5 cDNA through reverse transcriptase PCR provided evidence that this protein is a result of alternative splicing. Based on the current naming convention for the perilipin proteins, we have termed this protein perilipin 5b. The objective of the current study is to develop DNA based probes for use in northern blot analysis of perilipin 5. The genomic DNA sequence of perilipin 5 was analyzed to identify areas where splicing events could occur, and probes were designed accordingly to detect the different variants of perilipin 5. In the analysis of different mouse tissue samples, these probes have been used to detect which of the perilipin 5 splice variants are being expressed under different feeding conditions. Ongoing studies are seeking to determine the function of perilipin 5b.

Identification of Serologic Markers for School-Aged Children With Congenital Rubella Syndrome.

Congenital rubella syndrome (CRS) case identification is challenging in older children since laboratory markers of congenital rubella virus (RUBV) infection do not persist beyond age 12 months. We enrolled children with CRS born between 1998 and 2003 and compared their immune responses to RUBV with those of their mothers and a group of similarly aged children without CRS. Demographic data and sera were collected. Sera were tested for anti-RUBV immunoglobulin G (IgG), IgG avidity, and IgG response to the 3 viral structural proteins (E1, E2, and C), reflected by immunoblot fluorescent signals. We enrolled 32 children with CRS, 31 mothers, and 62 children without CRS. The immunoblot signal strength to C and the ratio of the C signal to the RUBV-specific IgG concentration were higher (P < .029 for both) and the ratio of the E1 signal to the RUBV-specific IgG concentration lower (P = .001) in children with CRS, compared with their mothers. Compared with children without CRS, children with CRS had more RUBV-specific IgG (P < .001), a stronger C signal (P < .001), and a stronger E2 signal (P ≤ .001). Two classification rules for children with versus children without CRS gave 100% specificity with >65% sensitivity. This study was the first to establish classification rules for identifying CRS in school-aged children, using laboratory biomarkers. These biomarkers should allow improved burden of disease estimates and monitoring of CRS control programs.

Stable expression and characterization of monomeric and dimeric recombinant hybrid-IgG/IgA immunoglobulins specific for Shiga toxin.

Antigen-specific immunoglobulin A (IgA) may be useful for preventing infectious diseases through passive immunization on the mucosal surface. We previously established mouse IgA and IgG monoclonal antibodies (mAbs) specific for the binding subunit of Shiga toxin 1 (Stx1B). We also developed a recombinant hybrid-IgG/IgA, in which variable regions from the IgG mAb were present. The binding activity of recombinant hybrid-IgG/IgA was verified by transient expression. Aiming at a constant supply, we established Chinese hamster ovary cells stably expressing monomeric or dimeric hybrid-IgG/IgA. The cDNAs encoding heavy and light chains were co-expressed for the monomeric hybrid-IgG/IgA, while those encoding heavy, light, and joining chains were co-expressed for the dimeric one. Serum-free culture supernatants of the cloned transfectants were subjected to size-exclusion chromatography. The elution patterns showed that the binding to immobilized Stx1B and the immunoblot signals of assembled immunoglobulins were correlated. In the transfectant for the dimeric hybrid-IgG/IgA, both monomers and dimers were observed. Size-exclusion chromatography enabled us to prepare a sample of the dimeric hybrid-IgG/IgA devoid of the monomeric one. The monomeric and dimeric forms of hybrid-IgG/IgA were prepared from the respective transfectants to examine the neutralization of Stx1. After pretreatment with monomeric or dimeric hybrid-IgG/IgA, the cytotoxicity of Stx1 toward Vero cells was abolished. Furthermore, the dimeric form was more than 10-fold more effective than the monomeric one in terms of toxin neutralization. These results suggest that the tetravalent feature of the binding sites of the dimeric hybrid-IgG/IgA contributes to the efficacy of toxin neutralization.

Humic substances interfere with detection of pathogenic prion protein

Studies examining the persistence of prions (the etiological agent of transmissible spongiform encephalopathies) in soil require accurate quantification of pathogenic prion protein (PrPTSE) extracted from or in the presence of soil particles. Here, we demonstrate that natural organic matter (NOM) in soil impacts PrPTSE detection by immunoblotting. Methods commonly used to extract PrPTSE from soils release substantial amounts of NOM, and NOM inhibited PrPTSE immunoblot signal. The degree of immunoblot interference increased with increasing NOM concentration and decreasing NOM polarity. Humic substances affected immunoblot detection of prion protein from both deer and hamsters. We also establish that after interaction with humic acid, PrPTSE remains infectious to hamsters inoculated intracerebrally, and humic acid appeared to slow disease progression. These results provide evidence for interactions between PrPTSE and humic substances that influence both accurate measurement of PrPTSE in soil and disease transmission.

The role of phosphatase and tensin homolog in drug-induced gingival overgrowth

We proposed that phosphatase and tensin homolog (PTEN) might be one of the signaling proteins that alter the balance between cell growth and cell death in drug-induced gingival overgrowth. The aim of this study was to investigate the expression of PTEN in subjects using cyclosporine A and to analyze the relationship between PTEN and cell proliferation marker, proliferating cell nuclear antigen (PCNA), in cyclosporine A-induced gingival overgrowth. In total, samples from 36 subjects, i.e. 24 cyclosporine A-mediated renal transplant patients with gingival overgrowth (n = 12) or without gingival overgrowth (n = 12) and 12 matched periodontally healthy subjects, were included in the study. PTEN and PCNA expressions in gingival tissues were analyzed using immunohistochemistry, PTEN expression was also analyzed by western blot. PTEN immunoreactivity was calculated with a histologic score (HSCORE) value and PCNA immunoreactivity was calculated with the PCNA-proliferative index. Phosphatase and tensin homolog HSCORE for the group with gingival overgrowth was found to be significantly lowest compared to the group without gingival overgrowth and the control group (p < 0.001) while the highest PTEN HSCORE was found in the control group. In addition, the PTEN HSCORE for the group without gingival overgrowth was significantly lower compared to controls (p < 0.001). The highest PCNA-proliferative index score was observed in the group with gingival overgrowth while the lowest score was observed in the control group (p < 0.001). The immunoblot signal for PTEN was significantly decreased in the group with gingival overgrowth compared to the group without gingival overgrowth and the control group (p < 0.001). Western blot results were different from immunohistochemistry and revealed there was no significant difference between the without gingival overgrowth and the control group (p > 0.05). Our results showing decreased PTEN levels in patients with gingival overgrowth supported with increased PCNA expression suggested that PTEN might take part in the imbalance between cell proliferation and death in drug-induced gingival overgrowth.

Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to VEGF receptor 2 (VEGFR2) on endothelial cells (ECs). Downstream activation of the extracellular related kinases 1/2 (ERK1/2) is important for angiogenesis to proceed. Receptor internalization has been implicated in VEGFR2 signaling, but its role in the activation of ERK1/2 is unclear. To explore this question we utilized pitstop and dynasore, two small molecule inhibitors of endocytosis. First, we confirmed that both inhibitors block the internalization of VEGFR2 in ECs. We then stimulated ECs with VEGF in the presence and absence of the inhibitors and examined VEGFR2 signaling to ERK1/2. Activation of VEGFR2 and C-Raf still occurred in the presence of the inhibitors, whereas the activation of MEK1/2 and ERK1/2 was abrogated. Therefore, although internalization is not required for activation of either VEGFR2 or C-Raf in ECs stimulated with VEGF, internalization is necessary to activate the more distal kinases in the cascade. Importantly, inhibition of internalization also prevented activation of ERK1/2 when ECs were stimulated with other pro-angiogenic growth factors, namely fibroblast growth factor 2 and hepatocyte growth factor. In contrast, the same inhibitors did not block ERK1/2 activation in fibroblasts or cancer cells stimulated with growth factors. Finally, we show that these small molecule inhibitors of endocytosis block angiogenesis in vitro and in vivo. Therefore, receptor internalization may be a generic requirement for pro-angiogenic growth factors to activate ERK1/2 signaling in human ECs, and targeting receptor trafficking may present a therapeutic opportunity to block tumor angiogenesis.

Photosynthesis in relation to D1, PsaA and Rubisco in marine and brackish water ecotypes of Fucus vesiculosus and Fucus radicans (Phaeophyceae)

The aim of this study was to investigate photosynthetic differences between the marine, Norwegian Sea ecotype and the brackish, Bothnian Sea ecotype of F. vesiculosus and F. radicans and to see whether photosynthetic differences could be connected with the relative amounts of D1 protein (PSII), PsaA (PSI) protein and/or Rubisco. For this purpose, we tested if a higher photosynthetic maximum (P max) in the Atlantic Ocean ecotype of F. vesiculosus relative to the Baltic Sea ecotype, and an increase of the P max in Baltic Sea ecotype of F. vesiculosus at higher salinity, could be due to an increase in the relative amounts of Rubisco. The proteins have been evaluated on a relative basis. Immunoblot signals showed that the amount of Rubisco was higher in both ecotypes of F. vesiculosus than in F. radicans, but no differences could be detected between the two ecotypes of F. vesiculosus. The results suggest an uneven photosystem protein stoichiometry in Fucus, with more of the PSI protein PsaA relative to the PSII protein D1. The difference in P max between the two ecotypes of F. vesiculosus might be related to the difficulties for the algae to adapt to the environment in Bothnian Sea.

Facilitation of μ-Opioid Receptor Activity by Preventing δ-Opioid Receptor-Mediated Codegradation

δ-opioid receptors (DORs) form heteromers with μ-opioid receptors (MORs) and negatively regulate MOR-mediated spinal analgesia. However, the underlying mechanism remains largely unclear. The present study shows that the activity of MORs can be enhanced by preventing MORs from DOR-mediated codegradation. Treatment with DOR-specific agonists led to endocytosis of both DORs and MORs. These receptors were further processed for ubiquitination and lysosomal degradation, resulting in a reduction of surface MORs. Such effects were attenuated by treatment with an interfering peptide containing the first transmembrane domain of MOR(MOR(TM1)), which interacted with DORs and disrupted the MOR/DOR interaction. Furthermore, the systemically applied fusion protein consisting of MOR(TM1) and TAT at the C terminus could disrupt the MOR/DOR interaction in the mouse spinal cord, enhance the morphine analgesia, and reduce the antinociceptive tolerance to morphine. Thus, dissociation of MORs from DORs in the cell membrane isa potential strategy to improve opioid analgesic therapies.

Rab5 Isoforms Differentially Regulate the Trafficking and Degradation of Epidermal Growth Factor Receptors

Ligand-mediated endocytosis is an intricate regulatory mechanism for epidermal growth factor receptor (EGFR) signal transduction. Coordinated trafficking of EGFR ensures its temporal and spatial communication with downstream signaling effectors. We focused our work on Rab5, a monomeric GTPase shown to participate in early stages of the endocytic pathway. Rab5 has three isoforms (A, B, and C) sharing more than 90% of sequence identity. We individually ablated endogenous isoforms in HeLa cells with short interfering RNAs and examined the loss-of-function phenotypes. We found that suppression of Rab5A or 5B hampered the degradation of EGFR, whereas Rab5C depletion had very little effect. The differential delay of EGFR degradation also corresponds with retarded progression of EGFR from early to late endosomes. We investigated the activators/effectors of Rab5A that can potentially separate its potency in EGFR degradation from other isoforms and found that Rin1, a Rab5 exchange factor, preferably associated with Rab5A. Moreover, Rab5A activation is sensitive to EGF stimulation, and suppression of Rin1 diminished this sensitivity. Based on our results together with previous work showing that Rin1 interacts with signal transducing adapter molecule to facilitate the degradation of EGFR (Kong, C., Su, X., Chen, P. I., and Stahl, P. D. (2007) J. Biol. Chem. 282, 15294-15301), we hypothesize that the selective association of Rab5A and Rin1 contributes to the dominance of Rab5A in EGFR trafficking, whereas the other isoforms may have major functions unrelated to the EGFR degradation pathway.

Distinct Pharmacological Effects of Inhibitors of Signal Peptide Peptidase and γ-Secretase

Signal peptide peptidase (SPP) and gamma-secretase are intramembrane aspartyl proteases that bear similar active site motifs but with opposite membrane topologies. Both proteases are inhibited by the same aspartyl protease transition-state analogue inhibitors, further evidence that these two enzymes have the same basic cleavage mechanism. Here we report that helical peptide inhibitors designed to mimic SPP substrates and interact with the SPP initial substrate-binding site (the "docking site") inhibit both SPP and gamma-secretase, but with submicromolar potency for SPP. SPP was labeled by helical peptide and transition-state analogue affinity probes but at distinct sites. Nonsteroidal anti-inflammatory drugs, which shift the site of proteolysis by SPP and gamma-secretase, did not affect the labeling of SPP or gamma-secretase by the helical peptide or transition-state analogue probes. On the other hand, another class of previously reported gamma-secretase modulators, naphthyl ketones, inhibited SPP activity as well as selective proteolysis by gamma-secretase. These naphthyl ketones significantly disrupted labeling of SPP by the helical peptide probe but did not block labeling of SPP by the transition-state analogue probe. With respect to gamma-secretase, the naphthyl ketone modulators allowed labeling by the transition-state analogue probe but not the helical peptide probe. Thus, the naphthyl ketones appear to alter the docking sites of both SPP and gamma-secretase. These results indicate that pharmacological effects of the four different classes of inhibitors (transition-state analogues, helical peptides, nonsteroidal anti-inflammatory drugs, and naphthyl ketones) are distinct from each other, and they reveal similarities and differences with how they affect SPP and gamma-secretase.