Functional characterization of glucosamine-6-phosphate synthase (GlmS) in Salmonella enterica serovar Enteritidis.

Salmonella is a threat to public health due to consumption of contaminated food. Screening of a transposon library identified a unique mutant that was growth and host cell binding deficient. The objective of this study was to determine the functional role of glucosamine-6-phosphate synthase (GlmS) in the biology and pathogenesis of Salmonella. To examine this, we created a glmS mutant (ΔglmS) of Salmonella and examined the effect on cell envelope integrity, growth, metabolism, and pathogenesis. Our data indicated ΔglmS was defective in growth unless media were supplemented with D-glucosamine (D-GlcN). Examination of the bacterial cell envelope revealed that ΔglmS was highly sensitive to detergents, hydrophobic antibiotics, and bile salts compared to the wild type (WT). A release assay indicated that ΔglmS secreted higher amounts of ß-lactamase than the WT in culture supernatant fractions. Furthermore, ΔglmS was attenuated in cell culture models of Salmonella infection. Taken together, this study determined an important role for GlmS in the pathogenesis and biology of Salmonella.

RNA modification enzymes encoded by the gid operon: Implications in biology and virulence of bacteria.

Ribonucleic acid (RNA) molecules consist of numerous chemically modified nucleosides that are highly conserved in eukarya, archeae, and bacteria, while others are unique to each domain of life. In bacteria, hundreds of RNA modification enzymes have been identified and implicated in biological pathways associated with many cell processes. The glucose-inhibited division (gid) operon encodes genes for two RNA modification enzymes named GidA and GidB. Studies have shown GidA is essential for the proper biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) of bacterial transfer RNA (tRNA) with GidB responsible for the methylation of the 16S ribosomal RNA (rRNA). Furthermore, deletion of gidA and gidB has shown to alter numerous bacterial properties like virulence, stress response, morphology, growth, antibiotic susceptibility, and others. In this review, we discuss the present knowledge of the RNA modification enzymes GidA and GidB, and their potential role in the biology and virulence of bacteria.

Role of the flagellar basal-body protein, FlgC, in the binding of Salmonella enterica serovar Enteritidis to host cells.

Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Binding of the bacterium to the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified flgC as a potential binding mutant of SE. Therefore, we hypothesize FlgC which plays a significant role in the binding ability of SE to the intestinal mucosa of poultry. To test our hypothesis, we created a mutant of SE in which flgC was deleted. We then tested the in vitro and in vivo binding ability of ∆flgC when compared to the wild-type SE strain. Our data showed a significant decrease in the binding ability of ∆flgC to intestinal epithelial cells as well as in the small intestine and cecum of poultry. Furthermore, the decrease in binding correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. Overall, these studies indicate FlgC is a major factor in the binding ability of Salmonella to the intestinal mucosa of poultry.

tRNA modification enzymes GidA and MnmE: potential role in virulence of bacterial pathogens.

Transfer RNA (tRNA) is an RNA molecule that carries amino acids to the ribosomes for protein synthesis. These tRNAs function at the peptidyl (P) and aminoacyl (A) binding sites of the ribosome during translation, with each codon being recognized by a specific tRNA. Due to this specificity, tRNA modification is essential for translational efficiency. Many enzymes have been implicated in the modification of bacterial tRNAs, and these enzymes may complex with one another or interact individually with the tRNA. Approximately, 100 tRNA modification enzymes have been identified with glucose-inhibited division (GidA) protein and MnmE being two of the enzymes studied. In Escherichia coli and Salmonella, GidA and MnmE bind together to form a functional complex responsible for the proper biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm5s²U34) of tRNAs. Studies have implicated this pathway in a major pathogenic regulatory mechanism as deletion of gidA and/or mnmE has attenuated several bacterial pathogens like Salmonella enterica serovar Typhimurium, Pseudomonas syringae, Aeromonas hydrophila, and many others. In this review, we summarize the potential role of the GidA/MnmE tRNA modification pathway in bacterial virulence, interactions with the host, and potential therapeutic strategies resulting from a greater understanding of this regulatory mechanism.

Virulence characteristics of Salmonella following deletion of genes encoding the tRNA modification enzymes GidA and MnmE.

Salmonella is an important foodborne pathogen causing major public health problems throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division (gidA) gene significantly altered Salmonella virulence in both in vitro and in vivo models of infection. In Escherichia coli, GidA and MnmE have been shown to modify several bacterial factors by a post-transcriptional mechanism to modify tRNA. Therefore, we hypothesize that GidA and MnmE complex together to modulate virulence genes in Salmonella using a similar mechanism. To test our hypothesis, and to examine the relative contribution of GidA and MnmE in modulation of Salmonella virulence, we constructed gidA and mnmE single mutants as well as a gidA mnmE double mutant strain of Salmonella. Results from the in vitro data displayed a reduction in growth, motility, intracellular replication, and invasion of T84 intestinal epithelial cells in the mutant strains compared to the wild-type Salmonella strain. The in vivo data showed a significant attenuation of the mutant strains as indicated by the induction of inflammatory cytokines and chemokines, as well as in the severity of histopathological lesions in the liver and spleen, compared to mice infected with the wild-type strain. Also, a significant increase in the LD50 was observed in mice infected with the mutant strains, and mice immunized with the mutants were protected against a lethal dose of wild-type Salmonella. A pull-down assay indicated that Salmonella GidA and MnmE bind together, and HPLC analysis revealed that deletion of gidA and/or mnmE altered Salmonella tRNA modification. Overall, the data suggest MnmE and GidA bind together and use a post-transcriptional mechanism to modify tRNA to regulate Salmonella pathogenesis.

GidA expression in Salmonella is modulated under certain environmental conditions.

Glucose-inhibited division (GidA) protein is widely distributed in nature, and is highly conserved among bacteria and eukarya. In our previous study, a gidA mutant was attenuated in both in vitro and in vivo models of Salmonella infection. Furthermore, deletion of gidA resulted in a marked reduction in the expression of many virulence genes and proteins, suggesting a role for GidA in the regulation of Salmonella virulence. In this study, the effect of different environmental conditions (glucose, EDTA, and pH 5) on GidA expression in Salmonella was examined. Transcriptional analysis using real-time RT-PCR and a ß-galactosidase assay, displayed no differences in gidA transcription and promoter activity in different environmental conditions. Conversely, semiquantitative Western blot analysis revealed a significant increase in the GidA expression in Salmonella when grown under different environmental conditions. Salmonella in vitro virulence assays showed an increased virulence potential in the environmental conditions correlating to the increase in GidA expression. Together, our data indicate that GidA expression is modulated under different environmental conditions which correlate to increased Salmonella in vitro virulence.

Immunological characterization of a gidA mutant strain of Salmonella for potential use in a live-attenuated vaccine.

BACKGROUND: Salmonella is often associated with gastrointestinal disease outbreaks in humans throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division gene (gidA) significantly attenuated Salmonella enterica serovar Typhimurium (STM) virulence in both in vitro and in vivo models of infection. Most importantly, immunization with the gidA mutant protected mice from a lethal dose challenge of wild-type STM. In this study, we further characterize the gidA mutant STM strain for potential use in a live-attenuated vaccine. RESULTS: The protective efficacy of immunization with the gidA mutant was evaluated by challenging immunized mice with a lethal dose of wild-type STM. Sera levels of IgG2a and IgG1, passive transfer of sera and cells, and cytokine profiling were performed to study the induction of humoral and cellular immune responses induced by immunization with the gidA mutant strain. Additionally, a lymphocyte proliferation assay was performed to gauge the splenocyte survival in response to treatment with STM cell lysate. Mice immunized with the gidA mutant strain were fully protected from a lethal dose challenge of wild-type STM. Naïve mice receiving either cells or sera from immunized mice were partially protected from a lethal dose challenge of wild-type STM. The lymphocyte proliferation assay displayed a significant response of splenocytes from immunized mice when compared to splenocytes from non-immunized control mice. Furthermore, the immunized mice displayed significantly higher levels of IgG1 and IgG2a with a marked increase in IgG1. Additionally, immunization with the gidA mutant strain evoked higher levels of IL-2, IFN-γ, and IL-10 cytokines in splenocytes induced with STM cell lysate. CONCLUSIONS: Together, the results demonstrate that immunization with the gidA mutant strain protects mice by inducing humoral and cellular immune responses with the humoral immune response potentially being the main mechanism of protection.

Deletion of glucose-inhibited division (gidA) gene alters the morphological and replication characteristics of Salmonella enterica Serovar typhimurium.

Salmonella is an important food-borne pathogen that continues to plague the United States food industry. Characterization of bacterial factors involved in food-borne illnesses could help develop new ways to control salmonellosis. We have previously shown that deletion of glucose-inhibited division gene (gidA) significantly altered the virulence potential of Salmonella in both in vitro and in vivo models of infection. Most importantly, the gidA mutant cells displayed a filamentous morphology compared to the wild-type Salmonella cells. In our current study, we investigated the role of GidA in Salmonella cell division using fluorescence and electron microscopy, transcriptional, and proteomic assays. Scanning electron microscopy data indicated a filamentous morphology with few constrictions in the gidA mutant cells. The filamentation of the gidA mutant cells is most likely due to the defect in chromosome segregation, with little to no sign of septa formation observed using fluorescence and transmission electron microscopy. Furthermore, deletion of gidA altered the expression of many genes and proteins responsible for cell division and chromosome segregation as indicated by global transcriptional profiling and semi-quantitative western blot analysis. Taken together, our data indicate GidA as a potential regulator of Salmonella cell division genes.

Biological and virulence characteristics of Salmonella enterica serovar Typhimurium following deletion of glucose-inhibited division (gidA) gene.

Salmonella enterica serovar Typhimurium is a frequent cause of enteric disease due to the consumption of contaminated food. Identification and characterization of bacterial factors involved in Salmonella pathogenesis would help develop effective strategies for controlling salmonellosis. To investigate the role of glucose-inhibited division gene (gidA) in Salmonella virulence, we constructed a Salmonella mutant strain in which gidA was deleted. Deletion of gidA rendered Salmonella deficient in the invasion of intestinal epithelial cells, bacterial motility, intracellular survival, and induction of cytotoxicity in host cells. Deletion of gidA rendered the organism to display a filamentous morphology compared to the normal rod-shaped nature of Salmonella. Furthermore, a significant attenuation in the induction of inflammatory cytokines and chemokines, histopathological lesions, and systemic infection was observed in mice infected with the gidA mutant. Most importantly, a significant increase in LD(50) was observed in mice infected with the gidA mutant, and mice immunized with the gidA mutant were able to survive a lethal dose of wild-type Salmonella. Additionally, deletion of gidA significantly altered the expression of several bacterial factors associated with pathogenesis as indicated by global transcriptional and proteomic profiling. Taken together, our data indicate GidA as a potential regulator of Salmonella virulence genes.

Biological and virulence characteristics of the YqhC mutant of Salmonella.

Previous work by the present authors indicated a murein lipoprotein mutant of Salmonella shows a marked down-regulation in expression of yqhC. Because YqhC is a putative DNA-binding protein, it is likely involved in modulation of Salmonella genes. Deletion of yqhC renders Salmonella defective in invasion of intestinal epithelial cells, motility, and induction of cytotoxicity. In the present study, further attenuation in induction of inflammatory cytokines/chemokines and histopathological lesions was seen in mice infected with the yqhC mutant. On the other hand, deletion of yqhC did not significantly affect the LD(50) in mice or the ability of Salmonella to survive and replicate in vivo. To better understand how YqhC affects Salmonella virulence and to identify factors potentially modulated by YqhC, comparative transcriptome and proteome analysis of the yqhC mutant and the WT Salmonella was performed. Data from these experiments indicate that deletion of yqhC significantly alters the transcription of several genes associated with the SPI-1 encoded T3SS and flagellar regulons, correlating with the yqhC mutant phenotype. Overall, this study indicates that deletion of the yqhC gene causes a number of virulence-related defects in vitro, but has a modest effect in vivo, despite affecting induction of inflammatory cytokines and histopathology.

Distribution of virulence factors and molecular fingerprinting of Aeromonas species isolates from water and clinical samples: suggestive evidence of water-to-human transmission.

A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.

Cold shock exoribonuclease R (VacB) is involved in Aeromonas hydrophila pathogenesis.

In this study, we cloned and sequenced a virulence-associated gene (vacB) from a clinical isolate SSU of Aeromonas hydrophila. We identified this gene based on our recently annotated genome sequence of the environmental isolate ATCC 7966(T) of A. hydrophila and the vacB gene of Shigella flexneri. The A. hydrophila VacB protein contained 798 amino acid residues, had a molecular mass of 90.5 kDa, and exhibited an exoribonuclease (RNase R) activity. The RNase R of A. hydrophila was a cold-shock protein and was required for bacterial growth at low temperature. The vacB isogenic mutant, which we developed by homologous recombination using marker exchange mutagenesis, was unable to grow at 4 degrees C. In contrast, the wild-type (WT) A. hydrophila exhibited significant growth at this low temperature. Importantly, the vacB mutant was not defective in growth at 37 degrees C. The vacB mutant also exhibited reduced motility, and these growth and motility phenotype defects were restored after complementation of the vacB mutant. The A. hydrophila RNase R-lacking strain was found to be less virulent in a mouse lethality model (70% survival) when given by the intraperitoneal route at as two 50% lethal doses (LD(50)). On the other hand, the WT and complemented strains of A. hydrophila caused 80 to 90% of the mice to succumb to infection at the same LD(50) dose. Overall, this is the first report demonstrating the role of RNase R in modulating the expression of A. hydrophila virulence.

Braun lipoprotein (Lpp) contributes to virulence of yersiniae: potential role of Lpp in inducing bubonic and pneumonic plague.

Yersinia pestis evolved from Y. pseudotuberculosis to become the causative agent of bubonic and pneumonic plague. We identified a homolog of the Salmonella enterica serovar Typhimurium lipoprotein (lpp) gene in Yersinia species and prepared lpp gene deletion mutants of Y. pseudotuberculosis YPIII, Y. pestis KIM/D27 (pigmentation locus minus), and Y. pestis CO92 with reduced virulence. Mice injected via the intraperitoneal route with 5 x 10(7) CFU of the Deltalpp KIM/D27 mutant survived a month, even though this would have constituted a lethal dose for the parental KIM/D27 strain. Subsequently, these Deltalpp KIM/D27-injected mice were solidly protected against an intranasally administered, highly virulent Y. pestis CO92 strain when it was given as five 50% lethal doses (LD(50)). In a parallel study with the pneumonic plague mouse model, after 72 h postinfection, the lungs of animals infected with wild-type (WT) Y. pestis CO92 and given a subinhibitory dose of levofloxacin had acute inflammation, edema, and masses of bacteria, while the lung tissue appeared essentially normal in mice inoculated with the Deltalpp mutant of CO92 and given the same dose of levofloxacin. Importantly, while WT Y. pestis CO92 could be detected in the bloodstreams and spleens of infected mice at 72 h postinfection, the Deltalpp mutant of CO92 could not be detected in those organs. Furthermore, the levels of cytokines/chemokines detected in the sera were significantly lower in animals infected with the Deltalpp mutant than in those infected with WT CO92. Additionally, the Deltalpp mutant was more rapidly killed by macrophages than was the WT CO92 strain. These data provided evidence that the Deltalpp mutants of yersiniae were significantly attenuated and could be useful tools in the development of new vaccines.

Identification of a new hemolysin from diarrheal isolate SSU of Aeromonas hydrophila.

A clinical strain SSU of Aeromonas hydrophila produces a potent cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. A new gene, which encoded a hemolysin of 439-amino acid residues with a molecular mass of 49 kDa, was identified. This hemolysin (HlyA) was detected based on the observation that the act gene minus mutant of A. hydrophila SSU still had residual hemolytic activity. The new hemolysin gene (hlyA) was cloned, sequenced, and overexpressed in Escherichia coli. The hlyA gene exhibited 96% identity with its homolog found in a recently annotated genome sequence of an environmental isolate, namely the type strain ATCC 7966 of A. hydrophila subspecies hydrophila. The hlyA gene did not exhibit any homology with other known hemolysins and aerolysin genes detected in Aeromonas isolates. However, this hemolysin exhibited significant homology with hemolysin of Vibrio vulnificus as well as with the cystathionine beta synthase domain protein of Shewanella oneidensis. The HlyA protein was activated only after treatment with trypsin and the resulting hemolytic activity was not neutralizable with antibodies to Act. The presence of the hlyA gene in clinical and water Aeromonas isolates was investigated and DNA fingerprint analysis was performed to demonstrate its possible role in Aeromonas virulence.

Evaluation of knowledge and behavior of workers in Prince Mohammed International Airport in Western Saudi Arabia regarding public health emergency measures applied during Hajj season 2014.

OBJECTIVES: To evaluate the knowledge and behavior of workers at a Saudi airport regarding public health emergency measures applied during Hajj season. METHODS: This study is a cross-sectional study conducted at the Prince Mohammed International Airport in Al-Madinah Al-Munawwarah, Saudi Arabia between August and September 2014. Data were collected by semi-structured questionnaires during personal interviews. Non-random purposive sampling was conducted to target workers at higher risk of acquiring infection from travellers. RESULTS: One hundred and eighty-six participants were recruited of whom 92.5% were males. The study participants were workers in 8 different sectors. Twenty-six percent of the participants were health workers. Non-health workers were more likely to be concerned on acquiring infection while working at the airport compared with health workers (p=0.023). The most commonly feared disease was Ebola viral disease (EBV) among 30% of health workers, and 47% of non-health workers. Approximately 47% of non-health workers reported no knowledge of the procedures implemented during public health emergencies. The proportion of participants who received public health related training among non-health workers was significantly lower compared with health workers (p less than 0.00001). CONCLUSION: More emphasis should be given to educating airport workers on the potential health threats at the airport. Specific guidelines for public health emergencies at the airport should be established and communicated with airport sectors.

Evaluation of applied public health emergency system at Prince Mohammed International Airport in Almedinah during Hajj season 2014: a qualitative case study.

BACKGROUND: During the Hajj season 2014, several public health measures were applied by the Ministry of Health at Prince Mohammed International Airport in Almedinah. However, several operational defects affected the provision of preventive health services for passengers and airport workers. This study aims to evaluate the applied public health emergency system at the airport, detect any potential gaps and to provide appropriate operational solutions. METHODS: This is a qualitative case study conducted at Prince Mohammed International Airport in Almedinah during the 2014 Hajj season, September 2014. Data were collected via semi-structured interviews, focus groups and policy document reviews. Interviews were conducted with the 14 individuals of the airport's decision makers and relevant health practitioners. Data were recorded via taking notes during interviews and data coding was performed to produce the main themes and subthemes of the study. RESULTS: The main findings of the study revealed three main defects affecting the applied public health emergency system at the airport. The main themes were mainly related to shortage in logistics related to public health emergency systems, shortage in proper documentation of policies and lack of documented protocols of communications among airport stakeholders. CONCLUSIONS: The study highlighted the main factors hindering the application of public health emergency measures at the airport. A Public Health Emergency Contingency Plan was proposed as a method to regulate the process of providing logistics for public health preventive services, the method of producing documented policies and methods of producing Memoranda of Understandings as communication regulators.

Role of StdA in adhesion of Salmonella enterica serovar Enteritidis phage type 8 to host intestinal epithelial cells.

BACKGROUND: Salmonella is often implicated in foodborne outbreaks, and is a major public health concern in the United States and throughout the world. Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Adhesion to epithelial cells in the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified stdA as a potential adhesion mutant of SE. Therefore, we hypothesize StdA plays a significant role in adhesion of SE to the intestinal mucosa of poultry. METHODS AND RESULTS: To test our hypothesis, we created a mutant of SE in which stdA was deleted. Growth and motility were assayed along with the in vitro and in vivo adhesion ability of the ∆stdA when compared to the wild-type SE strain. Our data showed a significant decrease in motility in ∆stdA when compared to the wild-type and complemented strain. A decrease in adhesion to intestinal epithelial cells as well as in the small intestine and cecum of poultry was observed in ∆stdA. Furthermore, the lack of adhesion correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. CONCLUSIONS: These studies suggest StdA is a major contributor to the adhesion of Salmonella to the intestinal mucosa of poultry.

Deletion of gene encoding methyltransferase (gidB) confers high-level antimicrobial resistance in Salmonella.

The glucose-inhibited division gene (gid)B, which resides in the gid operon, was thought to have a role in the modulation of genes similar to that of gidA. Recent studies have indicated that GidB is a methyltransferase enzyme that is involved in the methylation of the 16S ribosomal RNA (rRNA) in Escherichia coli. In this study, we investigated the role of GidB in susceptibility to antibiotics and the overall biology of Salmonella. A gidB isogenic mutant of Salmonella was constructed and subsequently characterized under different conditions. Our data indicated that growth and invasion characteristics of the gidB mutant were similar to those of the wild type (WT). The gidB mutant was outgrown by the WT in a competitive growth assay, indicating a compromised overall bacterial fitness. Under the stress of nalidixic acid, the gidB mutant's motility was significantly reduced. Similarly, the mutant showed a filamentous morphology and smaller colony size compared with the rod-shaped and large colonies of the WT in the presence of nalidixic acid. Most importantly, deletion of gidB conferred high-level resistance to the aminoglycoside antibiotics streptomycin and neomycin. A primer extension assay determined the methylation site for the WT to be at G527 of the 16S rRNA. A lack of methylation in the mutant indicated that GidB is required for this methylation. Taken together, these data indicate that the GidB enzyme has a significant role in the alteration of antibiotic susceptibility and the modulation of growth and morphology under stress conditions in Salmonella.

Regulation of catechol O-methyltransferase expression in granulosa cells: a potential role for follicular arrest in polycystic ovary syndrome.

OBJECTIVE: To investigate the regulation of catechol O-methyltransferase (COMT) expression in granulosa cells and assess potential effects of 2-methoxyestradiol (2-ME2) and COMT inhibitors on granulosa cell steroidogenesis and proliferation. DESIGN AND SETTING: Controlled experimental study in an academic research laboratory. INTERVENTION(S): JC410 porcine and HGL5 human granulosa cell lines were used for in vitro experiments. Effects of 2-ME2 and COMT inhibitor treatment on DNA proliferation and steroidogenesis were assessed by using Hoechst dye and p450SCC-luciferase reporter assays. Effects of dihydrotestosterone (DHT), insulin, and all-trans retinoic acid (ATRA) on COMT messenger RNA expression were investigated by using COMTP1 promoter-luciferase reporter and Northern blot. MAIN OUTCOME MEASURE(S): Granulosa cell steroidogenesis and proliferation following COMP inhibitor and 2-ME2 treatment. Regulation of COMT expression with DHT, insulin, and ATRA. RESULT(S): 2-Methoxyestradiol had a dual effect on granulosa cell proliferation and p450SCC- luciferase activity; low doses were stimulatory and high doses were inhibitory. Catechol O-methyltransferase inhibitor was associated with up to a 65% increase in JC410 cell number and a maximal 5.6-fold increase in p450SCC-luciferase activity at 20 micromol/L. Dihydrotestosterone, insulin, and ATRA all induced a dose-dependent increase in COMTP1-luciferase transactivation, as well as up-regulated COMT messenger RNA expression in granulosa cells. CONCLUSION(S): Catechol O-methyltransferase expression in granulosa cells was up-regulated by insulin, DHT, and ATRA. Catechol O-methyltransferase product, 2-ME2, decreased, whereas COMT inhibitor increased granulosa cell proliferation and steroidogenesis. These data suggest that COMT overexpression with subsequent increased level of 2-ME2 may lead to ovulatory dysfunction.