Assay Of Beta-galactosidase Activity Research Articles

O35 THE SYNERGISM BETWEEN HIGH GLUCOSE AND INTERLEUKIN-1beta PROMOTES INFLAMMASOME ACTIVATION AND THE RELEASE OF SMALL EXTRACELLULAR VESICLES IN HUMAN AORTIC SMOOTH MUSCLE CELLS

Background and objective: Hyperglycemia and inflammatory cytokines like interleukin (IL)-1beta play a key role in the vascular complications associated to diabetes melllitus (DM), such as atherosclerosis and hypertension. Understanding the pathophysiological mechanisms involved, including altered intercellular communication, is essential to identify potential therapeutical targets. We investigated the synergism of IL-1beta and high glucose on: (1) the activation of nucleotide-binding-oligomerization-domain, leucine-rich-repeat, and pyrin-domain-containing-3 (NLRP3)-inflammasome, an innate immunity component linked to vascular disease, and (2) the production and cargo of small extracellular vesicles (sEVs) as key players in vascular intercellular communication. Methods: Human aortic smooth muscle cells (HASMC) were challenged for 18 h with IL-1beta (10 ng/ml) in the presence of normal (5,5 mmol/L) or high glucose (22 mmol/L). Protein levels were quantified by Western blot, while the formation of ASC-like specks, reflecting NLRP3 activation, was assessed by indirect immunofluorescence. sEVs were determined by nanotracking analysis and endothelial senescence by the beta-galactosidase activity assay. Results: In HASMC cultures, IL-1beta favored NLRP3-inflammasome priming, as shown by NF-kappaB (phospho-p65) activation while NLRP3 and pro-IL-1beta levels were increased. The cytokine equally promoted NLRP3-inflammasome complex assembly, caspase-1 activation, and a subsequent release of mature IL-1beta in an auto-inflammatory loop. High glucose alone did not promote these effects, but it exacerbated some actions of IL-1beta and potentiated the release of HASMC-derived sEVs induced by IL-1beta, increasing their cargo content in NLRP3-inflammasome components. Moreover, these sEVs induce autocrine NLRP3-inflammasome activation in naïve HASMC and paracrine senescence in human endothelial cells. The IL-1 receptor blocker anakinra (1 mg/ml) blunted the IL-1beta effect, as well as their exacerbation by high glucose. Conclusion: High glucose exacerbates the activation of NLRP3-inflammasome by IL-1beta and alters intercellular communication by modifying the number and content of sEVs, which can expand inflammation in neighboring tissues. By preventing such synergy, biological drugs blocking IL-1beta receptors arise as potential pharmacological tools to attenuate DM associated vascular abnormalities.

Abstract 1850: Characterizing senescence response to PARP inhibition may provide opportunities for enhanced efficacy through combinations with senolytic agents

Abstract Background: Inhibition of poly (ADP-ribose) polymerase (PARP) is an exciting treatment strategy recently approved for prostate cancer patients with homologous recombination repair defects. Despite this advance in the field, it remains unclear how PARP inhibitor (PARPi) sensitive cells respond to treatment. We previously demonstrated that treatment with the PARPi olaparib induces not only cell death, but also G2/M arrested senescence characterized by activation of the p53 signaling pathway. We hypothesize that targeting PARPi induced senescence may provide a means to enhance the efficacy of PARPi treatment. In our current work, we sought to 1) understand whether senescence induction is a generalized response to all PARPi’s and 2) characterize senescence induction to guide the development of novel treatment strategies combining a PARPi with a senolytic drug. Methods: PARPi sensitive LNCaP and C4-2B prostate tumor cells were treated with olaparib, rucaparib, niraparib, or talazoparib for 5 days to induce senescence. Both vehicle treated and quiescent cells (LNCaP and C4-2B cultured in FBS-low (0.2%) conditions) were used as controls. Cell viability, flow cytometry, and beta-galactosidase activity assays tested response to PARPi’s. Western blot was used to detect PARP activity, apoptosis, and DNA damage response. RNA-sequencing was performed to characterize senescence induced signaling alterations. Results: We found that exposure to rucaparib, niraparib, and talazoparib all induce a robust G2/M arrested senescence response in LNCaP and C4-2B cells, suggesting that senescence induction is a class effect of PARPi’s. PARPi induced senescence is characterized by activation of the p53 signaling pathway and significantly increased expression of the cyclin-dependent kinase inhibitor p21. Furthermore, PARPi induced senescence is distinct from quiescence, suggesting that response to PARP inhibition is phenotypically different from a more general growth arrest. RNA-sequencing revealed several signaling changes associated with senescence which may provide novel treatment opportunities. Conclusions: Senolytics are a class of drugs thought to specifically target senescent cells. Our results demonstrate that PARPi’s induce senescence. Future work will be directed at further characterizing PARPi induced senescence, leading to rationally selected senolytic drug combinations which may enhance the efficacy of PARPi therapy. Citation Format: Alan P. Lombard, Wei Lou, Christopher P. Evans, Allen C. Gao. Characterizing senescence response to PARP inhibition may provide opportunities for enhanced efficacy through combinations with senolytic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1850.

In-situ SERS readout strategy to improve the reliability of beta-galactosidase activity assay based on X-gal staining in shortening incubation times

Beta-galactosidase (β-gal) activity is closed related with senescence cells and aging-associated diseases, however, the traditional readout of β-gal activity based on X-gal staining was limited to low sensitivity in short incubation times and false positives in long incubation times. Here, we expose the potential role of insoluble X-gal hydrolysates in causing false positives by diffusion pollution depending on organic medium and then propose the in-situ Surface-enhanced Raman spectroscopy (SERS) readout strategy to identify and locate β-gal positive cells. By building the blue-white screening model and fabricating SERS-active needle sensor, the sensitive detection of β-gal has been realized with the detection limit of less than 1 nmol L−1. The in-situ SERS readout strategy is proved to be necessary and feasible to improve the reliability of X-gal staining assay through shortening the time to a few hours. Moreover, its application was also preliminarily evaluated to analyse individual cells and tissues, which showed the well consistency for judgement of β-gal activity cells at different times. Consequently, by improving reliability and reducing time consumption, this SERS readout strategy may be of great significance to promote the application of X-gal staining assay in biology and biomedicine.

An Optimized Protocol for Histochemical Detection of Senescence-associated Beta-galactosidase Activity in Cryopreserved Liver Tissue

Senescence-associated beta-galactosidase (SA-β-gal) activity assay is commonly used to evaluate the increased beta-galactosidase (β-gal) activity in senescent cells related to enhanced lysosomal activity. Although the optimal pH for β-gal is 4.0, this enzymatic activity has been most commonly investigated at a suboptimal pH by using histochemical reaction on fresh tissue material. In the current study, we optimized a SA-β-gal activity histochemistry protocol that can also be applied on cryopreserved hepatic tissue. This protocol was developed on livers obtained from control rats and after bile duct resection (BDR). A significant increase in β-gal liver activity was observed in BDR rats vs controls after 2 hr of staining at physiological pH 4.0 (6.98 ± 1.19% of stained/total area vs 0.38 ± 0.22; p<0.01) and after overnight staining at pH 5.8 (24.09 ± 6.88 vs 0.12 ± 0.08; p<0.01). Although we noticed that β-gal activity staining decreased with cryopreservation time (from 4 to 12 months of storage at -80C; p<0.05), the enhanced staining observed in BDR compared with controls remained detectable up to 12 months after cryopreservation (p<0.01). In conclusion, we provide an optimized protocol for SA-β-gal activity histochemical detection at physiological pH 4.0 on long-term cryopreserved liver tissue.

Synergistic Effect of Mitochondrial and Lysosomal Dysfunction in Parkinson's Disease.

Crosstalk between lysosomes and mitochondria plays a central role in Parkinson’s Disease (PD). Lysosomal function may be influenced by mitochondrial quality control, dynamics and/or respiration, but whether dysfunction of endocytic or autophagic pathway is associated with mitochondrial impairment determining accumulation of defective mitochondria, is not yet understood. Here, we performed live imaging, western blotting analysis, sequencing of mitochondrial DNA (mtDNA) and senescence-associated beta-galactosidase activity assay on primary fibroblasts from a young patient affected by PD, her mother and a healthy control to analyze the occurrence of mtDNA mutations, lysosomal abundance, acidification and function, mitochondrial biogenesis activation and senescence. We showed synergistic alterations in lysosomal functions and mitochondrial biogenesis, likely associated with a mitochondrial genetic defect, with a consequent block of mitochondrial turnover and occurrence of premature cellular senescence in PARK2-PD fibroblasts, suggesting that these alterations represent potential mechanisms contributing to the loss of dopaminergic neurons.

Beta-galactosidase activity assay for yeast

Beta-galactosidase activity assay for yeast

Selective and Efficient Elimination of Vibrio cholerae with a Chemical Modulator that Targets Glucose Metabolism.

Vibrio cholerae, a Gram-negative bacterium, is the causative agent of pandemic cholera. Previous studies have shown that the survival of the seventh pandemic El Tor biotype V. cholerae strain N16961 requires production of acetoin in a glucose-rich environment. The production of acetoin, a neutral fermentation end-product, allows V. cholerae to metabolize glucose without a pH drop, which is mediated by the production of organic acid. This finding suggests that inhibition of acetoin fermentation can result in V. cholerae elimination by causing a pH imbalance under glucose-rich conditions. Here, we developed a simple high-throughput screening method and identified an inducer of medium acidification (iMAC). Of 8364 compounds screened, we identified one chemical, 5-(4-chloro-2-nitrobenzoyl)-6-hydroxy-1,3-dimethylpyrimidine-2,4(1H,3H)-dione, that successfully killed glucose-metabolizing N16961 by inducing acidic stress. When N16961 was grown with abundant glucose in the presence of iMAC, acetoin production was completely suppressed and concomitant accumulation of lactate and acetate was observed. Using a beta-galactosidase activity assay with a single-copy palsD::lacZ reporter fusion, we show that that iMAC likely inhibits acetoin production at the transcriptional level. Thin-layer chromatography revealed that iMAC causes a significantly reduced accumulation of intracellular (p)ppGpp, a bacterial stringent response alarmone known to positively regulate acetoin production. In vivo bacterial colonization and fluid accumulation were also markedly decreased after iMAC treatment. Finally, we demonstrate iMAC-induced bacterial killing for 22 different V. cholerae strains belonging to diverse serotypes. Together, our results suggest that iMAC, acting as a metabolic modulator, has strong potential as a novel antibacterial agent for treatment against cholera.

Scanning assay of β-galactosidase activity

Beta-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-beta-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of beta-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, beta-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the beta-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular beta-galactosidase activity in E. coli Tuner (DE3) expressing -galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of beta-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.

Quantifying the antiestrogen activity of wastewater treatment plant effluent using the yeast estrogen screen

The yeast estrogen screen (YES) assay was used to measure both estrogenic and antiestrogenic activity of wastewater treatment plant (WWTP) effluent for the purpose of developing a method to quantify antiestrogenic activity. Wastewater treatment plant effluent samples were concentrated by solid-phase extraction (SPE) and serially diluted. Five microliters of each dilution plus 195 microl of assay medium was placed in well plates and tested for estrogenic substances. Antiestrogen activity in WWTP effluent samples was indirectly measured by an effluent-volume-dependent suppression of the beta-galactosidase activity induced by an estradiol (E2) standard. Antiestrogens and estrogens were quantified by median inhibition concentration (IC50) and median effective concentration (EC50) statistics, respectively, and were expressed in terms of effluent volume (prior to concentration by SPE). Antiestrogen IC50 and estrogen EC50 values, calculated by standard linear regression methods, averaged 25.6 microl and 22.1 microl effluent, respectively. Taken together, these values suggest that antiestrogens were responsible for approximately a 50% reduction in estrogen-induced activity in WWTP effluent. Therefore, measurements of estrogenicity by the YES assay in WWTP effluent that typically contains a mixture of estrogenic and antiestrogenic substances should be considered net estrogenic activity. The potential for false-positive antiestrogen activity was addressed by assays of beta-galactosidase activity in effluent, by measurements of yeast cell turbidity, and by stirred cell ultrafiltration for removal of solid-phase coextracted organic substances.

The transcription factor ZBP‐89 suppresses p16 expression through a histone modification mechanism to affect cell senescence

The transcription factor ZBP-89 has been implicated in the induction of growth arrest and apoptosis. In this article, we demonstrate that ZBP-89 was able to restrain senescence in NCI-H460 human lung cancer cells, through epigenetically regulating p(16INK4a) expression. Specifically, our results indicate that knockdown of ZBP-89 by RNA interference stimulated cellular senescence in NCI-H460 cells, as judged by the senescence-associated beta-galactosidase activity assay and senescence-associated heterochromatin foci assay, and this process could be reversed by RNA interference-mediated p16(INK4a) silencing. We also show that histone deacetylase (HDAC) 3 and HDAC4 inhibited p16(INK4a) promoter activity in a dose-dependent manner. Furthermore, chromatin immunoprecipitation assays verified that HDAC3 was recruited to the p16(INK4a) promoter by ZBP-89 through an epigenetic mechanism involving histone acetylation modification. Moreover, immunofluorescence and coimmunoprecipitation assays revealed that ZBP-89 and HDAC3 formed a complex. These data suggest that ZBP-89 and HDAC3, but not HDAC4, can work coordinately to restrain cell senescence by downregulating p16(INK4a) expression through an epigenetic modification of histones.

MhbR, a LysR-type regulator involved in 3-hydroxybenzoate catabolism via gentisate in Klebsiella pneumoniae M5a1

In Klebsiella pneumoniae M5a1, mhbTDHIM genes are involved in 3-hydroxybenzoate catabolism via the gentisate pathway. mhbR, which encodes a LysR-type transcriptional regulator, is divergently transcribed from the mhb structural genes. MhbR was found to be necessary for the expression of catabolic genes. Transcriptional studies demonstrated that the mhb structural genes are transcribed as an operon. The promoters of mhbR and the mhb operon are sigma(70)-type and overlap with each other. 5' Deletion analysis of the promoter transcription activity showed that a 233bp fragment (position -144 to +89 according to the transcriptional start site of mhb operon) contained the element necessary for induction. beta-Galactosidase activity assays and electrophoretic mobility shift assays showed that an inverted repeat sequence site 1 (ATAACCTCCAGGTTAT, position -70 to -55) within this fragment was critical for regulation.

A bifunctional chimeric protein consisting of MutS and beta-galactosidase

A bifunctional protein consisting of MutS, a mismatch binding protein and a beta-galactosidase reporter domain has been constructed. The fusion of beta-galactosidase to the MutS C-terminus was obtained by cloning the Escherichia coli lacZ gene encoding beta-galactosidase into a plasmid vector carrying the Thermus thermophilus mutS gene. Milligram amounts of this huge chimeric protein (217 kDa monomer) were purified from 1l of overexpressing E. coli cells using metal-chelate affinity chromatography. The mismatch binding properties of the fusion protein were confirmed by DNA mobility shift assay in polyacrylamide gels. Binding to biotinylated mismatched DNA immobilized on streptavidin microplates followed by colorimetric reaction with X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), demonstrated both mismatch recognition and beta-galactosidase activity of the chimeric protein. The activity of beta-galactosidase domain of the fusion was similar to that of the native enzyme. A colorimetric assay for beta-galactosidase activity using X-Gal supplemented with NBT (nitro blue tetrazolium) allowed detection of 50 and 500 fmol of the chimeric protein with naked eye in 45 microl volumes after 120 and 15 min incubation, respectively.

Analysis of the Interaction between hPFTAIRE1 and PLZF in a Yeast Two-hybrid System

hPFTAIRE1 is a Cdc2-related kinase family member. To search its substrates and regulatory proteins, hPFTAIRE1 was fused to LexA and used as a bait to screen a human brain LexA two-hybrid library. In this screening, seven hPFTAIRE1 interacting proteins, including promyelocytic leukemia zinc finger (PLZF), were obtained. The interaction between PLZF and hPFTAIRE1 was confirmed by beta-galactosidase assay and Leu growth activity. PLZF encodes a transcription factor belonging to the POZ/BTB domain and Krüpel zinc finger (POK) family. The highly conserved POZ/BTB domain plays a critical role in protein-protein interaction. We deleted the POZ/BTB and Krüpel zinc finger domains, respectively, and observed the interaction between hPFTAIRE1 and truncated PLZFs by liquid beta-galactosidase activity assay. A weak interaction was detected between hPFTAIRE1 and PLZF. We also observed the interaction between PLZF and another Cdc2-related kinase, PCTAIRE1. A similar result was observed. The interaction between PLZF and hPFTAIRE1 or PCTAIRE1 was confirmed by co-immunoprecipitation assay in a yeast system. PLZF is a phosphoprotein and plays multiple roles during cell growth. Our results suggest that hPFTAIRE1 and PCTAIRE1 may play important roles in the functional regulation of PLZF.

Alcohol suppresses IL-2-induced CC chemokine production by natural killer cells.

Natural killer (NK) cells are a critical component of the host innate immune system. We investigated whether alcohol impairs NK cell function, particularly production of CC chemokines induced by interleukin (IL)-2, the natural ligands for CCR5 receptor. Primary NK cells and NK cell line (YTS) were cultured with or without alcohol (10 to 80 mM) for three hours. The culture supernatants were then harvested and used to treat human peripheral blood monocyte-derived macrophages and a HeLa cell line, which expresses CD4, CCR5, and CXCR4 receptors (MAGI cells). CC chemokine expression by YTS and primary NK cells treated with or without alcohol was analyzed with the real-time RT-PCR and ELISA. [Ca(2)(+)]i and Western blot assays were used to determine calcium-mediated intracellular signaling pathway and NF-kappaB p65 expression. HIV strains (Bal and UG024) were used to infect macrophages and MAGI cells. In addition, ADA (macrophage-tropic strain) and murine leukemia virus (MLV) envelope-pseudotyped HIV infection was carried out in macrophages. HIV infectivity was determined by HIV reverse transcriptase (RT) and beta-galactosidase activity assays. Alcohol inhibited IL-2-induced CC chemokine (CCL3 and CCL4) expression by NK cells. Functional tests demonstrated that this reduced expression of CC chemokines was associated with diminished anti-HIV ability of NK cells. Alcohol also reduced the ability of NK cells to response to CCL3-mediated chemotaxis. Alcohol inhibited IL-2-induced NF-kappaB p65 protein expression and calcium mobilization by NK cells. Alcohol, through the inhibition of IL-2-induced NF-kappaB p65 protein expression and intracellular calcium mobilization, suppressed NK cell production of CC chemokines. This suppression of CC chemokine production was associated with diminished anti-HIV activity of NK cells. Thus, by inhibiting NK cell-mediated innate immunity against HIV, alcohol consumption may have a cofactor role in the immunopathogenesis of HIV disease.

Regulation of RcsA by the ClpYQ (HslUV) protease in Escherichia coli.

Escherichia coli ClpYQ protease and Lon protease possess a redundant function for degradation of SulA, a cell division inhibitor. An experimental cue implied that the capsule synthesis activator RcsA, a known substrate of Lon, is probably a specific substrate for the ClpYQ protease. This paper shows that overexpression of ClpQ and ClpY suppresses the mucoid phenotype of a lon mutant. Since the cpsB (wcaB) gene, involved in capsule synthesis, is activated by RcsA, the reporter construct cpsB-lacZ was used to assay for beta-galactosidase activity and thus follow RcsA stability. The expression of cpsB-lacZ was increased in double mutants of lon in combination with clpQ or/and clpY mutation(s) compared with the wild-type or lon single mutants. Overproduction of ClpYQ or ClpQ decreased cpsB-lacZ expression. Additionally, a P(BAD)-rcsA fusion construct showed quantitatively that an inducible RcsA activates cpsB-lacZ expression. The effect of RcsA on cpsB-lacZ expression was shown to be influenced by the ClpYQ activities. Moreover, a rcsA(Red)-lacZ translational fusion construct showed higher activity of RcsA(Red)-LacZ in a clpQ clpY strain than in the wild-type. By contrast, overproduction of cellular ClpYQ resulted in decreased beta-galactosidase levels of RcsA(Red)-LacZ. Taken together, the data indicate that ClpYQ acts as a secondary protease in degrading the Lon substrate RcsA.

Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse.

We studied the gene therapy efficacy of diabetes-associated wound healing disorder with an adeno-associated virus (AAV) vector expressing the 165-amino acid isoform of human vascular endothelial growth factor-A (VEGF-A) by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db+/db+ mice and their normal littermates ( db+/+m). Animals were randomized to receive intradermally into the wound edges either rAAV-LacZ (a control gene), or rAAV-VEGF165. Animals were killed on different days (7 and 14 days after skin injury) and wounded skin tissues were used for gene marker studies, histological evaluation and immunohistochemistry, and wound breaking strength analysis. Furthermore we studied the VEGF mature protein in the wounds. We found that AAV vectors are highly efficient for gene transfer to the mouse skin, displaying an exquisite tropism for the panniculus carnosus by using the beta-galactosidase activity assay. We confirmed the increased expression of the angiogenic factor at day 7 by measuring the wound content of the mature protein. Delivery of VEGF165 to incisional skin wounds of diabetic mice resulted in a remarkable induction of new vessel formation with consequent improvement in the wound healing process. The rAAV-VEGF165 gene improved wound healing in diabetic mice through the stimulation of angiogenesis, reepithelization, synthesis and maturation of extracellular matrix. Moreover the recombinant AAV encoding the human VEGF165 increased the breaking strength of the wound and enhanced the wound content of VEGF. Our study suggests that VEGF gene transfer might represent a new approach to treat wound healing disorders associated with diabetes.

The Neisseria meningitidis adhesion regulatory protein CrgA acts through oligomerization and interaction with RNA polymerase.

CrgA is a LysR-type transcriptional regulator involved in the intimate adhesion of Neisseria meningitidis to target human epithelial cells. It is negatively autoregulated, and its expression is transiently induced upon contact with target cells. We analysed the functional organization of CrgA using in frame deleted proteins. Four truncated proteins were constructed and purified. They were deleted between residues 20 and 40, 121 and 154, 111 and 181 and 268 and 291. Meningococcal mutants harbouring the corresponding deleted crgA alleles were also constructed. All mutants showed a reduced ability to adhere to epithelial cells. beta-Galactosidase activity assays using a crgA-lacZ transcriptional fusion showed that all the mutations except the 268-291 deletion resulted in loss of induction upon contact with target cells. Gel mobility shift assays and cross-linking assays showed that the oligomerization of CrgA is required for DNA binding and that the N-terminal part of CrgA is directly involved in DNA binding through a helix-turn-helix motif. The C-terminal region is also involved in DNA binding, probably by permitting the oligomerization of CrgA. The C-terminal region also seemed to interact with RNA polymerase. Therefore, the binding of CrgA and its interaction with RNA polymerase may inhibit the clearance of meningococcal promoters that are repressed by CrgA during the intimate adhesion of N. meningitidis to target cells.

Enhancement of the adenoviral sensitivity of human ovarian cancer cells by transient expression of coxsackievirus and adenovirus receptor (CAR).

The coxsackievirus and adenovirus receptor (CAR) is known to be a primary receptor for attachment during adenovirus infection of target cells and thus is closely related to adenoviral infection efficiency. To extend this notion to human ovarian cancer, we investigated the difference in expression levels of CAR in human ovarian cancer cell lines and whether their adenoviral sensitivities are enhanced by transient transfection of the CAR gene. Adenoviral infection efficiency was examined by flow cytometry analysis, beta-galactosidase staining, and beta-galactosidase activity assay. Expression of the CAR-specific mRNA and protein was analyzed by reverse transcription polymerase chain reaction and flow cytometry, respectively. Expression of CAR in human ovarian cancer cell lines (SKOV3, 2774, PA-1, and OVCAR3) appeared to be correlated with their susceptibilities to adenovirus-mediated gene delivery. The 2774 and PA-1 cells expressing an easily detectable level of CAR on the cell surface showed a higher susceptibility to infection with both AdCMVGFP and AdRSVbetagal than SKOV3 and OVCAR3 cells, both of which had hardly detectable levels of CAR. Ectopic expression of the CAR gene by transient transfection of these ovarian cancer cells resulted in a dramatic increase in their adenoviral sensitivities. These data show that the expression of CAR is closely related to susceptibility to adenovirus infection in human ovarian cancer cells. These results indicate that the CAR gene can be a useful tool in boosting the efficiency of adenoviral vector-mediated gene therapy against human ovarian cancer.

Molecular characterization of a chromosomal locus in Staphylococcus aureus that contributes to oxidative defence and is highly induced by the cell-wall-active antibiotic oxacillin.

Previous studies employing two-dimensional gel electrophoresis and N-terminal protein sequencing have shown elevated synthesis of the enzyme methionine sulfoxide reductase (MsrA) in Staphylococcus aureus in response to cell-wall-active antibiotics. In the present study, the S. aureus msrA gene was cloned, overexpressed, purified as His-tagged MsrA and shown to have methionine sulfoxide reductase activity. The transcription of msrA was studied by assaying beta-galactosidase activity in an msrA promoter::lacZ fusion strain and by Northern blot analysis. Transcription of msrA was increased by oxacillin; but not by a variety of other stresses including H2O2. Northern blot analysis revealed that the size of the msrA transcript was 2.3 kb, considerably larger than the 531 nt msrA ORF. The msrA transcription start site was mapped 25 nt upstream of the msrA start codon. Computer analysis from database sequences indicated at least three additional ORFs downstream of msrA. The deduced amino acid sequences of two of these three ORFs showed significant sequence homologies to PilB, and enzyme IIA of the phosphotransferase system, respectively. The third ORF could not be identified by homology searches. Northern blot hybridization with probes specific to the msrA downstream region indicated that the S. aureus msrA was transcribed as part of a polycistronic message. Interestingly, purified S. aureus PilB was shown to possess approximately approximately 28-fold higher methionine sulfoxide reductase activity than the MsrA. An insertional knockout mutation in the first gene of this operon resulted in increased susceptibility of the mutant to H2O2 compared to the parent strain, but not to oxacillin.

Neurotensin induces mating in Saccharomyces cerevisiae cells that express human neurotensin receptor type 1 in place of the endogenous pheromone receptor.

Heterologous expression of the human neurotensin receptor type I (hNT1-R) has been achieved in the yeast Saccharomyces cerevisiae. Immunoanalysis of membranes prepared from cells expressing a c-myc-tagged version of hNT1-R revealed multiple c-myc cross-reacting polypeptides of high molecular mass, suggesting that hNT1-R was glycosylated in yeast. High-affinity binding sites for 125I-labeled-[monoiodo-Tyr3]neurotensin ([125I-Tyr3]NT) were detected on hNT1-R-expressing cells with Kd and Bmax values of 3.2 nM and of 500 receptors per cell, respectively. Competition binding studies of neurotensin with SR142948 and SR48692, two nonpeptidic antagonists of hNT1-R, indicated that the yeast-produced recombinant receptor displayed the same pharmacological properties as hNT1-R expressed in mammalian cells. Interestingly, neurotensin activated the pheromone pathway in hNT1-R-expressing cells in a dose-dependent fashion, as revealed by a beta-galactosidase activity assay with a pheromone-responsive Fus1:lacZ construct. Mutational inactivation of the SST2 and STE2 genes increased the level of beta-galactosidase activity in response to neurotensin by twofold. Recombinant hNT1-R-producing cells, which lacked the endogenous G-protein-coupled receptor for the alpha pheromone, mated with wild-type MATalpha haploid cells in response to neurotensin, leading to bona fide diploid zygote formation. This is the first report of a mammalian receptor that can replace the endogenous pheromone receptor when produced in yeast, by signaling a fully effective, agonist-induced, mating process.