Endogenous GUS Research Articles

Highly specific β‑glucuronidase light-up natural fluorescent probe with aggregation-induced emission and excited-state intramolecular proton transfer for inhibitors screening and in situ imaging in Escherichia coli

Expression of β-glucuronidase (GUS) in intestinal bacteria has attracted more and more attentions because of its relevant to drug-inducing enteropathy and enterohepatic circulation. Hence it is essential to develop a facile method for efficient inhibitors screening and in situ activity tracking of GUS. Here, a novel light-up natural fluorescent probe, quercetin-3-O-β-D-glucuronide (obtained from Bupleurum scorzonerifolium), for rapid, sensitive, and specific analysis of GUS activity was found. Hydrophilic quercetin-3-O-β-D-glucuronide was catalyzed by GUS to produce hydrophobic quercetin aggregates in situ. The occurrence of quercetin fluorescence at 540 nm (Stokes shift = 175 nm) with aggregation-induced emission and excited-state intramolecular proton transfer characteristics was light-up, which can be used to evaluate GUS activity. A good linear range (5.0–60.0 μg mL−1, R2 = 0.9904), high sensitivity (limit of detection at 0.7 μg mL−1), satisfying precision (relative standard deviation values at 1.2% and 3.0% for intra-day and inter-day variations) and superior selectivity to GUS were obtained. Then, a facile platform for screening of GUS inhibitors from natural compounds library was constructed successfully. Three natural compounds, resveratrol (IC50 = 0.45 ± 0.04 μM), hesperidin (IC50 = 1.46 ± 0.07 μM), and ursolic acid (IC50 = 7.85 ± 0.26 μM)) were screened out with stronger inhibitory efficiency. Furthermore, on account of the outstanding photostability and excellent biocompatibility, quercetin-3-O-β-D-glucuronide has been employed for in situ imaging of endogenous GUS in Escherichia coli noninvasively with low background noise. Results demonstrated the advantage of natural products for novel fluorescent probes and the great potential of quercetin-3-O-β-D-glucuronide in high-throughput inhibitors screening, GUS-related drug discovery, and disease diagnosis.

Highly sensitive light-up near-infrared fluorescent probe for detection and imaging of β-glucuronidase in human serum, living cells and tumor-bearing mice

β-Glucuronidase (GUS) plays a key role in tumor initiation, metastasis, and progression, and thus, has been proposed as a promising cancer biomarker. In this study, we designed an enzyme-activatable near-infrared (NIR) fluorescent probe (DCM-βGlcA) for the rapid and accurate detection of GUS activity in vitro, in vivo and ex vivo. The DCM-βGlcA was prepared by linking a glucuronic acid residue to dicyanomethylene-4H-pyran (DCM). This probe exhibited significant light-up NIR fluorescent signals at 680 nm after reacting with GUS and the Stokes shift could reach 150 nm. The DCM-βGlcA showed a high sensitivity toward GUS and an excellent linear relationship at concentrations ranging between 0 and 4 U L−1 (R2 = 0.9974) with the limit of detection as low as 0.19 U L−1. We used the DCM-βGlcA to identify GUS serum levels in both cancer patients and healthy individuals with a similar accuracy as that of an enzyme-linked immunosorbent assay (ELISA) while being easier and faster to perform. Moreover, the DCM-βGlcA was used for tracking endogenous GUS in living cells, thereby discriminating GUS-overexpressed liver cancer from normal cells. Additionally, the DCM-βGlcA was able to detect and image endogenous GUS in liver cancer tissue and tumor-bearing mouse models. These findings demonstrate the potential of the DCM-βGlcA as a promising tool for detecting and monitoring GUS activity in preclinical applications.

Off-on fluorogenic substrate harnessing ESIPT and AIE features for in situ and long-term tracking of β-glucuronidase in Escherichia coli

Detection and isolation of both O157:H7 and non-O157:H7 Escherichia coli are essential for the evaluation and surveillance of the associated foodborne disease risk. However, the development of a chromogenic/fluorogenic culture medium for detection and isolation is challenging yet urgent. β-Glucuronidase (GUS) serves as an important biomarker in E. coli. A novel and simple off-on fluorogenic substrate (BTBP-Gluc) was developed, based on a solid-state fluorophore (BTBP) with excited-state intramolecular proton transfer (ESIPT) and aggregation-induced emission (AIE) effects. BTBP-Gluc could be well-suited for visually detecting GUS activity under acidic conditions (3.0 ≤ pH＜7.0). It was successfully applied for detection of endogenous intracellular GUS activity in living E. coli and could produce the in situ and long-term localization of the target colonies and cell imaging of living E. coli. Additionally, it showed high selectivity, little toxicity, excellent cell permeability and sensitivity. BTBP-Gluc was combined with two tightly localizing chromogenic substrates, Magenta-Gal and X-Glu. These combinations were applied to the development of a new chromogenic-fluorogenic culture medium (C–F E. coli agar). On this new agar simultaneous visual detection and isolation of O157:H7 and non-O157:H7 E. coli with relatively high accuracy was achieved. C–F E. coli agar shows potential in serving as a simple sensitive tool for detecting and isolating O157:H7 and non-O157:H7 E. coli in food samples such as milk.

Activation of Astrocytic μ-Opioid Receptor Causes Conditioned Place Preference

The underlying mechanisms of how positive emotional valence (e.g., pleasure) causes preference of an associated context is poorly understood. Here, we show that activation of astrocytic μ-opioid receptor (MOR) drives conditioned place preference (CPP) by means of specific modulation of astrocytic MOR, an exemplar endogenous Gi protein-coupled receptor (Gi-GPCR), in the CA1 hippocampus. Long-term potentiation (LTP) induced by a subthreshold stimulation with the activation of astrocytic MOR at the Schaffer collateral pathway accounts for the memory acquisition to induce CPP. This astrocytic MOR-mediated LTP induction is dependent on astrocytic glutamate released upon activation of the astrocytic MOR and the consequent activation of the presynaptic mGluR1. The astrocytic MOR-dependent LTP and CPP were recapitulated by a chemogenetic activation of astrocyte-specifically expressed Gi-DREADD hM4Di. Our study reveals that the transduction of inhibitory Gi-signaling into augmented excitatory synaptic transmission through astrocytic glutamate is critical for the acquisition of contextual memory for CPP.

Acid Suppression Therapy Does Not Affect the Efficacy of Plecanatide: A Patient-Level Pooled Analysis of Two Large Randomized Controlled Trials

Introduction: Chronic Idiopathic Constipation (CIC) affects approximately 14% of the North American population. The most recently FDA-approved medication for the treatment of CIC is plecanatide, which is an analog of the endogenous GI peptide, uroguanylin. Like uroguanylin, it exhibits pH-sensitive activity and is most active in a slightly acidic environment. As many CIC patients may also be on concomitant acid suppressing medications such as proton pump inhibitors (PPIs) and histamine receptor antagonists (H2 blockers), it is important to determine whether plecanatide is efficacious if given with a concomitant acid suppressor. Methods: The efficacy and safety of plecanatide for the treatment of CIC has been established in two 12-week, double-blind, placebo-controlled trials. Enrolled patients met modified Rome III criteria for CIC. Patients were randomized 1:1 to plecanatide 3 mg or placebo (PBO). These phase 3 CIC studies permitted patients to enter the studies while on concomitant PPIs and/or H2 blockers if the dosing of the medication was not stopped or changed during the study. By combining data from the two phase 3 studies, there was a sufficiently large sample size to determine the efficacy of CIC patients being treated with plecanatide while on a concomitant acid suppressor. The primary endpoint for efficacy was the Efficacy Responder Rate, defined as a patient who had at least 3 complete spontaneous bowel movements (CSBMs) in a given week and an increase of at least 1 CSBM from baseline in the same week for at least 9 of the 12 treatment weeks and at least 3 of the last 4 weeks of the study. A post-hoc analysis was performed exploring the impact of concomitant acid suppressor use on efficacy parameters. Results: A total of 883 patients received plecanatide 3 mg across the two studies and 892 received PBO. Of these, approximately 10% also received concomitant treatment with acid suppressors (n=106 in the plecanatide group [96 on a PPI]; n=119 in the PBO group [102 on a PPI]). Significantly more patients who received plecanatide were Efficacy Responders as compared to PBO (23.6% plecanatide; 7.6% PBO [P=0.001]). Secondary endpoints were also significantly improved in the plecanatide group as compared to placebo (Table). Conclusion: In this subset analysis of two phase 3 trials, CIC patients treated with plecanatide, who were receiving a concomitant acid suppressing medication, demonstrated significant improvement in their constipation symptoms.444 Figure 1. Secondary Endpoint Outcome Measures

Effects of blockade of endogenous Gi signaling in Tie2-expressing cells on bone formation in a mouse model of heterotopic ossification.

Available evidence indicates that some Tie2-expressing (Tie2(+) ) cells serve as multipotent progenitors that have robust BMP-dependent osteogenic activity and mediate heterotopic ossification (HO). Since signaling through the G protein Gi is required for cell motility, we hypothesized that blockade of endogenous Gi signaling in Tie2(+) cell populations would prevent HO formation. Blockade of Gi signaling in Tie2(+) cells was accomplished in transgenic mice with expression of pertussis toxin (PTX) under the control of the Tie2 promoter (Tie2(+) /PTX(+) ). Bone formation within HOs was evaluated 2 weeks after BMP injection. Expression of PTX in Tie2(+) cells significantly reduced the bone volume (BV) of HOs in male and female mice. Orthotopic bones were assessed at the distal femur and expression of PTX significantly increased trabecular bone fractional volume and bone formation rate in females only. In adult Tie2(+) /GFP(+) mice, GFP(+) cells appeared both inside and at the surfaces of bone tissue within HOs and in orthotopic bones. In summary, blockade of Gi signaling in Tie2(+) cells reduced the accrual of HOs and stimulated osteogenesis in orthotopic bones. Targeting of Gi protein coupled receptors in Tie2(+) cells may be a novel therapeutic strategy in states of abnormal bone formation such as osteoporosis and HO.

English

We have chosen rice as a model crop to ascertain endogenous β-glucuronidase (family 79) activity and to differentiate the same from Escherichia coli based β-glucuronidase (family-2). The investigation dwells on characterizing endogenous β-glucuronidase (GUS) activity during the early stages of seed germination and from rice callus. Also, similar studies were made from homozygous transgenic rice line expressing E. coli GUS under the control of glyoxalase I promoter. Endogenous GUS activity was detected in plumules, shoots and calli of rice, nevertheless, showing differential response to pH. Further, endogenous GUS in rice was specifically inhibited by saccharic acid 1,4-lactone (SL) but, the E. coli β-glucuronidase remain unaffected, indicating distinct biochemical properties of family-2 and family-79 β-glucuronidase. Key words: Family 2, family 79, X-gluc, saccharic acid, 1,4-lactone.

Potential Repurposing of Known Drugs as Potent Bacterial β-Glucuronidase Inhibitors

The active metabolite of the chemotherapeutic irinotecan, SN-38, is detoxified through glucuronidation and then excreted into the gastrointestinal tract. Intestinal bacteria convert the glucuronidated metabolite back to the toxic SN-38 using β-glucuronidase (GUS), resulting in debilitating diarrhea. Inhibiting GUS activity may relieve this side effect of irinotecan. In this study, we sought to determine whether any known drugs have GUS inhibitory activity. We screened a library of Food and Drug Administration–approved drugs with a cell-free biochemical enzyme assay using purified bacterial GUS. After triage, five drugs were confirmed to inhibit purified bacterial GUS. Three of these were the monoamine oxidase inhibitors nialamide, isocarboxazid, and phenelzine with average IC50 values for inhibiting GUS of 71, 128, and 2300 nM, respectively. The tricyclic antidepressant amoxapine (IC50 = 388 nM) and the antimalarial mefloquine (IC50 = 1.2 µM) also had activity. Nialamide, isocarboxazid, and amoxapine had no significant activity against purified mammalian GUS but showed potent activity for inhibiting endogenous GUS activity in a cell-based assay using living intact Escherichia coli with average IC50 values of 17, 336, and 119 nM, respectively. Thus, nialamide, isocarboxazid, and amoxapine have potential to be repurposed as therapeutics to reduce diarrhea associated with irinotecan chemotherapy and warrant further investigation for this use.

Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: How to avoid interference with the Gus transgene in transformation studies

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed ( Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24 °C, 38 °C and 55 °C) were investigated. To avoid this problem in microspores, two incubation temperatures (38 °C and 55 °C) at different periods after GUS assay (4, 24 and 48 h) and in the presence of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4 h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55 °C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2 μM gibberellic acid.

Endogenous Gi signaling in osteoblasts participates in sexually dimorphic regulation of cancellous bone structure

Endogenous Gi signaling in osteoblasts participates in sexually dimorphic regulation of cancellous bone structure

Impact of ubiquitous inhibitors on the GUS gene reporter system: evidence from the model plants Arabidopsis, tobacco and rice and correction methods for quantitative assays of transgenic and endogenous GUS.

BackgroundThe β-glucuronidase (GUS) gene reporter system is one of the most effective and employed techniques in the study of gene regulation in plant molecular biology. Improving protocols for GUS assays have rendered the original method described by Jefferson amenable to various requirements and conditions, but the serious limitation caused by inhibitors of the enzyme activity in plant tissues has thus far been underestimated.ResultsWe report that inhibitors of GUS activity are ubiquitous in organ tissues of Arabidopsis, tobacco and rice, and significantly bias quantitative assessment of GUS activity in plant transformation experiments. Combined with previous literature reports on non-model species, our findings suggest that inhibitors may be common components of plant cells, with variable affinity towards the E. coli enzyme. The reduced inhibitory capacity towards the plant endogenous GUS discredits the hypothesis of a regulatory role of these compounds in plant cells, and their effect on the bacterial enzyme is better interpreted as a side effect due to their interaction with GUS during the assay. This is likely to have a bearing also on histochemical analyses, leading to inaccurate evaluations of GUS expression.ConclusionsIn order to achieve reliable results, inhibitor activity should be routinely tested during quantitative GUS assays. Two separate methods to correct the measured activity of the transgenic and endogenous GUS are presented.

A novel method for fluorometric continuous measurement of β-glucuronidase (GUS) activity using 4-methyl-umbelliferyl-β- d-glucuronide (MUG) as substrate

β-Glucuronidases (GUS) are histochemically and fluorometrically detectable enzymes that cleave the glycosidic bond of glucuronides in living organisms. Previously thought to be absent in plants, endogenous GUS activity has recently been demonstrated ubiquitous, and its function is being widely investigated in model plants. Further, the GUS gene from Escherichia coli is the most widely used reporter gene in plant transformation experiments. Fluorometric assay of GUS activity is universally performed using fluorogenic substrate 4-methyl-umbelliferyl-β- d-glucuronide (MUG) by discontinuous measurement, based on the general notion that basification of the solution following enzymatic cleavage is necessary to detect the fluorophore 4-methyl-umbelliferone (MU). In this report, we analyze MU and MUG spectroscopic characteristics at different pHs, and show that MU is a fluorescent compound also at pHs below its p K a, although with a different excitation spectrum compared to the ionized form. On the basis of this evidence, we show that MUG is a suitable substrate to perform continuous measurement of GUS activity at the pH optima of plant endogenous and E. coli GUS enzymes. An efficient and straightforward method which greatly improves the procedure to assess GUS activity is described, and insights are given on the principles leading to optimal setting of instrumental parameters.

Purification, Cloning and Functional Characterization of an Endogenous beta-Glucuronidase in Arabidopsis thaliana

Beta-glucuronidase (GUS) activities have been extensively characterized in bacteria, fungi, and animals, and the bacterial enzyme GUSA from Escherichia coli is commonly used as a reporter for gene expression studies in plants. Although endogenous GUS activity has been observed in plants, the nature and function of the enzymes involved remain elusive. Here we report on tissue-specific localization, partial purification and identification of AtGUS2, a GUS active under acidic conditions from Arabidopsis thaliana. This enzyme belongs to the GH79 family in the Carbohydrate-Active Enzymes database, which also includes mammalian heparanases that degrade the carbohydrate moieties of cell surface proteoglycans, and fungal enzymes active on arabinogalactan proteins (AGPs). We characterized a knockout insertion line (atgus2-1) and transgenic lines overexpressing AtGUS2 (Pro(35S):AtGUS2). Endogenous GUS activity assayed histochemically and biochemically was absent in atgus2-1 tissues and four times higher in Pro(35S):AtGUS2 lines. AGPs purified from atgus2-1 and Pro(35S):AtGUS2 seedlings showed higher and markedly lower glucuronic acid content, respectively. Our results suggest that endogenous GUS activity influences the sugar composition of the complex polysaccharide chains of AGPs. We also show that transgenics display hypocotyl and root growth defects compared to wild-type plants. Hypocotyl and root lengths are increased in Pro(35S):AtGUS2 seedlings, whereas hypocoyl length is reduced in atgus2-1 seedlings. These data are consistent with a role for the carbohydrate moieties of AGPs in cell growth.

Promoterless gus gene shows leaky β-glucuronidase activity during transformation of tomato with bspA gene for drought tolerance

Transformation of tomato (Lycopersicon esculentum Mill.) was carried out using disarmed Agrobacterium tumefaciens strain EHA 105 harboring a binary vector pBIG-HYG-bspA. The plasmid contains the bspA (boiling stable protein of aspen) gene under the control of a CaMV35S promoter and nopaline synthase (NOS) terminator, hygromycin phosphotransferase gene (hpt) driven by nopaline synthase promoter and polyadenylation signal of Agrobacterium gene7 as terminator and a promoterless gus gene. Very strong β-glucuronidase (GUS) expression was observed in transformed tomato plants but never in non-transformed (control). Since GUS expression was observed only in transformed plants, the possibility of the presence of endogenous GUS enzymes was ruled out. Possibility of false GUS positives was also ruled out because the GUS positive explants reacted positively to polymerase chain reaction (PCR) and PCR-Southern tests carried out for the presence of bspA gene, which indicated the integration of T-DNA in tomato genome. The promoterless GUS expression was hypothesized either due to leaky NOS termination signal of bspA gene or due to different cryptic promoters of plant origin. It was concluded that GUS expression was observed in the putative transgenics either due to the read through mechanism by the strong CaMV35S promoter or due to several cryptic promoters driving the gus gene in different transgenic lines.

Primary role for Gi protein signaling in the regulation of interleukin 12 production and the induction of T helper cell type 1 responses.

We explored the role of Gi protein signaling in the regulation of interleukin (IL)-12 production and T helper cell type 1 (Th1) T cell differentiation. In initial studies, we showed that treatment of normal mice with pertussis toxin (PT), which inhibits Gi protein signaling, enhanced the capacity of splenocytes to produce IL-12 in response to both microbial and nonmicrobial stimuli. In addition, PT treatment increased the production of tumor necrosis factor (TNF)-alpha and IL-10 by stimulated cells. These findings were corroborated by the fact that untreated Gi2alpha(2/-) mice exhibited enhanced production of IL-12 and TNF-alpha by splenocytes, and of IL-12 p40 by purified spleen CD8alpha(+) lymphoid dendritic cells. Finally, we showed that while normal BALB/c mice infected with Leishmania major exhibited a nonhealing phenotype, those treated with PT when infection was initiated exhibited a healing phenotype along with an enhancement of leishmania-specific Th1 responses in draining lymph nodes. Further, healing was prevented by coadministration of anti-IL-12 and PT. These data demonstrate that endogenous Gi protein signaling has a primary role in the regulation of IL-12 production and the induction of Th1 responses in vivo.

Efficiency of physical (light) or chemical (ABA, tetracycline, CuSO4 or 2-CBSU)-stimulus-dependent gus gene expression in tobacco cell suspensions.

In this study, the efficiency of inducible promoters to switch on gene expression in the presence of inducer or to switch it off in its absence was evaluated in tobacco cell suspensions transformed with the gus gene coding sequence. Either plant (pats1A, pSalT, pIn2-2) or microbial (pMre, pTet) inducible promoters were used to drive gus expression. The inducers were light, abscisic acid, 2-CBSU, CuSO4, tetracycline, respectively. For each construct (inducible promoter-gus coding sequence), the optimal induction conditions were determined (inducer concentration, induction time, and age of cells in culture cycle before induction). The efficiency of the inducible promoter was then evaluated under optimal induction conditions. GUS-expression levels obtained under non-inducing and inducing conditions were systematically compared. Thirty or forty percent of the clones transformed with the pSalT-gus or pTet-gus construct, respectively, showed high induction rates (>1000) and GUS activities of the same order as those obtained with a constitutive system. However, basal GUS levels were always high for the pTet-gus cell lines. Seventy or eighty-five percent of the cell lines transformed with the pMre-gus or pln2-2-gus construct, respectively, had induction rates of 1.5 to 1000. The pats1A-gus construct gave very low induction rates-55% of cell lines had induction rates less than 1.5. Only the pSalt-gus construct gave both the highest induction rates and basal GUS-levels equivalent to the endogenous GUS background.

Characterization of pedicel β‐glucuronidase activity in developing maize (Zea mays) kernels

A conspicuous endogenous maize (Zea mays L.) β‐glucuronidase (GUS) activity was observed in histochemical assays of non‐transformed maize kernels, confounding the use of Escherichia coli gusA as a reporter gene. Appearance of the endogenous activity was developmentally dependent and highly tissue‐specific, being localized to the upper pedicel (basal maternal kernel) tissues where the black layer forms in the latter stages of kernel development. Pedicel homogenates exhibited GUS activity using either p‐nitrophenyl‐β‐D‐glucuronide or 4‐methylumbelliferyl‐β‐D‐glucuronide (MUG) as substrates. Pedicel GUS was apparently not the result of endophyte contamination of enzyme isolates since no endophytes could be cultured. The MUG‐based activity had a pH optimum of 4 to 5 and was separable into two isoforms by anion exchange chromatography with Km values for MUG of 2.2 and 2.7 µM for the early‐ and late‐eluting forms, respectively. The pedicel GUS isoforms had very similar characteristics: native Mr of approximately 32000, stimulation by assay at 60°C, inhibition at high ionic strength or in the presence of EDTA and relative insensitivity to the E. coli GUS inhibitor saccharic acid‐1,4‐lactone. Only the early‐eluting form, however, was capable of hydrolyzing the histocbemical GUS substrate 5‐bromo‐4‐chloro‐3‐indoyl‐β‐D‐glucuronide. Neither isoform exhibited antifungal activity against Fusarium moniliforme. In contrast to the in vitro activity, pedicel endogenous GUS measured histochemically was completely inhibited by saccharic acid‐1,4‐lactone, unaffected by EDTA and greatly decreased by incubation at elevated assay temperature. A modification of the standard histochemical GUS assay allowed complete suppression of endogenous GUS activity while enhancing E. coli‐derived GUS activity in kernels transiently expressing the gusA gene. Possible roles of these endogenous GUS activities within the black layer region of the kernel pedicel are proposed.

Agonist Occupation of an α2A-Adrenoreceptor-Gi1α Fusion Protein Results in Activation of Both Receptor-linked and Endogenous Gi Proteins: COMPARISONS OF THEIR CONTRIBUTIONS TO GTPase ACTIVITY AND SIGNAL TRANSDUCTION AND ANALYSIS OF RECEPTOR-G PROTEIN ACTIVATION STOICHIOMETRY

A fusion protein between a pertussis toxin-resistant (C351G) mutant of the alpha subunit of the G protein Gi1 and the porcine alpha2A-adrenoreceptor was stably expressed in Rat 1 fibroblasts. Agonists caused stimulation of high affinity GTPase activity, which was partially prevented by pertussis toxin treatment, demonstrating that the toxin-resistant component of the GTPase activity was derived from the receptor-fused G protein and the remainder from endogenous Gialpha. Half-maximal stimulation of the GTPase activity of endogenous Gi was achieved with lower concentrations of agonist. Although the Km for GTP of the fusion protein-linked Gi was lower than for the endogenous G protein, Vmax measurements demonstrated that adrenaline activated some 5 mol of endogenous Gi/mol of fusion protein-linked Gi. The isolated alpha2A-adrenoreceptor could activate Gs; however, the fusion protein did not. Compared with adrenaline, the efficacy of a range of partial agonists to stimulate endogenous Gialpha was greater than for the fusion protein-constrained C351G Gi1alpha. alpha2A-Adrenoreceptor agonists could stimulate both p44 mitogen-activated protein kinase and p70 S6 kinase and inhibit forskolin-amplified adenylyl cyclase activity in untreated alpha2A-adrenoreceptor-C351G Gi1alpha fusion protein-expressing cells, but these signals were abolished following pertussis toxin treatment. These results demonstrate conclusively, and for the first time, that agonist occupancy of a receptor-G protein fusion protein can result in activation of G proteins other than that physically linked to the receptor. This was selective between G protein classes. Analysis of the contributions of fusion protein-linked and endogenous G proteins to agonist-stimulated GTPase activity provided a direct and original measure of receptor-G protein activation stoichiometry.

Pertussis Toxin-sensitive Activation of Phospholipase C by the C5a and fMet-Leu-Phe Receptors

Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PTx)-sensitive activation of phospholipase C (PLC) have been investigated using a cotransfection assay system in COS-7 cells. The abilities of the receptors for C5a and fMLP to activate PLC beta2 and PLC beta3 through the Gbetagamma subunits of endogenous Gi proteins in COS-7 cells were tested because both PLC beta2 and PLC beta3 were shown to be activated by the betagamma subunits of G proteins in in vitro reconstitution assays. Neither of the receptors can activate endogenous PLC beta3 or recombinant PLC beta3 in transfected COS-7 cells. However, both receptors can clearly activate PLC beta2 in a PTx-sensitive manner, suggesting that the receptors may interact with endogenous PTx-sensitive G proteins and activate PLC beta2 probably through the Gbetagamma subunits. These findings were further corroborated by the results that PLC beta3 could only be slightly activated by Gbeta1gamma1 or Gbeta1gamma5 in the cotransfection assay, whereas the Gbetagamma subunits strongly activated PLC beta2 under the same conditions. PLC beta3 can be activated by Galphaq, Galpha11, and Galpha16 in the cotransfection assay. In addition, the Ggamma2 and Ggamma3 mutants with substitution of the C-terminal Cys residue by a Ser residue, which can inhibit wild type Gbetagamma-mediated activation of PLC beta2, were able to inhibit C5a or fMLP-mediated activation of PLC beta2. These Ggamma mutants, however, showed little effect on m1-muscarinic receptor-mediated PLC activation, which is mediated by the Gq class of G proteins. These results all confirm that the Gbetagamma subunits are involved in PLC beta2 activation by the two chemoattractant receptors and suggest that in COS-7 cells activation of PLC beta3 by Gbetagamma may not be the primary pathway for the receptors.

β-Glucuronidase activity in transgenic and non-transgenic tobacco cells: specific elimination of plant inhibitors and minimization of endogenous GUS background

Bacterial β-glucuronidase is often introduced into plants as a reporter gene fused to constitutive or inducible promoters. However, the presence of both endogenous inhibitors of GUS activity and endogenous GUS enzymes in transgenic plants could lead to an underestimation of GUS. In this paper, a decrease of the V m values and a greater affinity ( K m) of the GUS enzyme for its substrate (p-NPG) has been recorded when increasing amounts of protein from untransformed tobacco cells has been added to the pure β-glucuronidase. The observed inhibition is not competitive and can be completely removed when the tobacco extracts are passed through Sephadex G-25 spin columns prior to the assays. After such a treatment, the activity of E. coli GUS in transgenic tobacco cells (constitutive or inducible systems) was stimulated by a factor 1.2 or 2 for p-NPG or 4-MUG substrates, respectively. This method was also effective in suppressing the endogenous GUS or GUS-like activity which can interfere with the activity originating from the introduced GUS gene.