ABSTRACT
Magnolia champaca Linn. has traditionally been used for medicinal activity in Asia for treating various chronic diseases as well as a source of food, medicines, and other commodities. Due to the long-used history of this plant, the present study was designed to explore the in vitro, in vivo and in silico anti-inflammatory and antineoplastic properties of the methanolic extract and fractions and the pure compound isolated from the most active chloroform fraction (CHF) of the stem bark of the plant. The isolated compound from the most active CHF was characterized and identified as a glycoside, trans-syringin, through chromatographic and spectroscopic (1H-NMR and 13C-NMR) analyses. In the in vitro anti-inflammatory assay, CHF was most effective in inhibiting inflammation and hemolysis of RBCs by 73.91 ± 1.70% and 75.92 ± 0.14%, respectively, induced by heat and hypotonicity compared to standard acetylsalicylic acid. In the egg albumin denaturation assay, CME and CHF showed the highest inhibition by 56.25 ± 0.82% and 65.82 ± 3.52%, respectively, contrasted with acetylsalicylic acid by 80.14 ± 2.44%. In an in vivo anti-inflammatory assay, statistically significant (p < 0.05) decreases in the parameters of inflammation, such as paw edema, leukocyte migration and vascular permeability, were recorded in a dose-dependent manner in the treated groups. In the antineoplastic assay, 45.26 ± 2.24% and 68.31 ± 3.26% inhibition of tumor cell growth for pure compound were observed compared to 73.26 ± 3.41% for standard vincristine. Apoptotic morphologic alterations, such as membrane and nuclear condensation and fragmentation, were also found in EAC cells after treatment with the isolated bioactive pure compound. Such treatment also reversed the increased WBC count and decreased RBC count to normal values compared to the untreated EAC cell-bearing mice and the standard vincristine-treated mice. Subsequently, in silico molecular docking studies substantiated the current findings, and the isolated pure compound and standard vincristine exhibited -6.4 kcal/mol and -7.3 kcal/mol binding affinities with topoisomerase-II. Additionally, isolated pure compound and standard diclofenac showed -8.2 kcal/mol and -7.6 kcal/mol binding affinities with the COX-2 enzyme, respectively. The analysis of this research suggests that the isolated bioactive pure compound possesses moderate to potent anti-inflammatory and antineoplastic activity and justifies the traditional uses of the stem bark of M. champaca. However, further investigations are necessary to analyze its bioactivity, proper mechanism of action and clinical trials for the revelation of new drug formulations.
ABSTRACT
BACKGROUND: Pithecellobium dulce (Roxb.), an evergreen medium-sized, spiny tree which have vast nutritional values and widely used in ayurvedic medicines and home remedies. The plant has also been a rich source of biologically active compounds. The present study was designed to isolate pure compound from ethyl acetate fraction of methanol extract of leaves and to know the efficacy as antioxidant as well as its anti-tumor activity on Ehrlich ascites carcinoma cell (EAC). METHODS: The leaves were extracted with methanol and fractionated with different solvents. The isolation of the compound was carried out by column chromatography from ethyl acetate fraction (EAF) and structure was revealed by 1H-NMR and 13C NMR. The antioxidant activity was investigated by the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals as well as the inhibition of oxidative damage of pUC19 plasmid DNA, hemolysis and lipid peroxidation induced by a water-soluble free radical initiator 2,2'-azo (2-asmidinopropane) dihydrochloride (AAPH) in human erythrocytes. In vivo anti-tumor activity of the compound was also evaluated by determining the viable tumor cell count, hematological profiles of experimental mice along with observing morphological changes of EAC cells by fluorescence microscope. RESULTS: The isolated compound kaempferol-3-O-alpha-L-rhamnoside effectively inhibited AAPH induced oxidation in DNA and human erythrocyte model and lipid per oxidation as well as a stronger DPPH radical scavenging activity. In anti-tumor assay, at a dose of 50 mg/kg body weight exhibit about 70.89 ± 6.62% EAC cell growth inhibition, whereas standard anticancer drug vincristine showed 77.84 ± 6.69% growth inhibition. CONCLUSION: The compound may have a great importance as a therapeutic agent in preventing oxidative damage of biomolecules and therapeutic use in chemotherapy.
Subject(s)
Antioxidants , Fabaceae , Animals , Antioxidants/chemistry , Mannosides , Methanol/analysis , Methanol/chemistry , Mice , Plant Extracts/chemistry , Plant Leaves/chemistry , ProanthocyanidinsABSTRACT
Among the major constituents of Leea rubra (Family Vitaceae) leaves, phenolic and flavonoind compounds are most important for therapeutic purposes and the plant parts have been used in traditional medicine to treat several diseases for long. Thus, in order to scientifically confirm the traditional uses of the L. rubra leaves, the present study was designed to investigate the efficacy of the isolated flavones against AAPH induced oxidative damage to pUC19 DNA by gel electrophoresis and antineoplastic activity was evaluated on Ehrlich ascites carcinoma (EAC) bearing Swiss albino mice by evaluating percentage inhibition of cell growth, morphological changes of EAC cells and hematological parameters of the mice. The isolation was carried out by column chromatography and structure was revealed by 1H-NMR and 13C NMR. The result shows that, the isolated compound was identified as myricetin 4'-methoxy-3-O-α-l-rhamnopyranoside based on previously reported data. The isolated flavone effectively inhibited AAPH-induced oxidative damage to DNA; because it could inhibit the formation of circular and linear forms of the DNA. In anti-proliferative assay, 76% growth inhibition of EAC cells was observed as compare to the control mice (p<0.05) at a dose 100 mg/kg body weight. Thus the isolated flavone showed great importance as a possible therapeutic agent in preventing oxidative damage to DNA and the chronic diseases caused by such DNA damage, and can also become important in cancer chemotherapy.
ABSTRACT
Medicinal plants are sources of antioxidant which may protect the body against oxidative stress related diseases and can be used as human food supplements. In this investigation, seeds of Hygrophila schulli (M. R. Almeida & S. M. Almeida) (Fam.-Acanthaceae), a herbaceous plant well known for its medicinal properties, has been examined for antioxidant activity of crude methanolic extract (CME) and its fraction using in vitro and in vivo assay as well as their protective activity against oxidative damage of DNA and RBC. Total phenolic and flavonoid content have also been estimated using the aluminum chloride colorimetric and Folin-Ciocalteu method. Among the different fractions of CME, Ethyl acetate fraction (EAF) had higher antioxidant activity in vitro assay and was selected for in vivo antioxidant activity in cadmium intoxicated mice. The EAF showed a significant (p < 0.05) increase in serum catalase and SOD activity compared to the control group. TBARS levels were restored to 17.42 and 19.19 nmol/mg protein, respectively, after treatment with EAF and standard ascorbic acid (AA); compared to the normal group (14.96 nmol/mg protein). Similarly, levels of albumin, bilirubin, uric acid, and alkaline phosphatase were also brought back to normal levels. EAF's protective role against oxidative damage of DNA has shown a significant reduction in destroying of nicked DNA. RBC as a target of oxidation by H2O2 and HOCl, EAF showed inhibition of oxidation in a concentration-dependent manner, compared to standard gallic acid. In this study, we confirmed that EAF could scavenge reactive oxygen species (ROS) thus preventing DNA strand scission and the extract can be used as a functional food or nutraceutical product for health benefits.
ABSTRACT
BACKGROUND: The various parts of Cresecentia cujete have some important biological activities. In folklore medicine leaves are used to treat hematomas, tumors and hypertension. Fruit decoction is used to treat diarrhea, stomachaches, cold, bronchitis, cough, asthma, and urethritis. The present study was designed to explore the anti-inflammatory and antibacterial potential of C. cujete leaves and stem bark. Anti-inflammatory activity was evaluated by in vitro human red blood cell (HRBC) membrane stabilization method and antibacterial activity by disc diffusion method. METHODS: In vitro anti-inflammatory activity was evaluated by human red blood cell (HRBC) membrane stabilization method while in vitro antibacterial activity was evaluated using cultures of Escherichia coli and Staphylococcus aureus by disc diffusion method. Total phenolic (TPC) and total flavonoid contents (TFC) of the crude extract and fractions were also determined by Folin-Ciocalteu's phenol reagent and by aluminium chloride method, respectively. RESULTS: The crude ethanol extract (CEE) of leaves and bark (concentration of each 1.0 mg/ml) demonstrated strong membrane stabilizing activity (53.86 and 61.85% protection, respectively), whereas their chloroform fractions (CHF) revealed moderate activity (48.74 ± 0.56 and 43.55 ± 6.20 %, respectively) compared with standard aspirin (concentration 0.10 mg/ml) which showed 75.81% protection in this test. All the samples showed a dose dependent anti-inflammatory activity in HRBC membrane stabilization test. Total phenolic (TPC) and total flavonoid contents (TFC) of the crude extract and fractions were also determined. Again, in in vitro antibacterial study, the extractives exhibited potent antibacterial activity. CONCLUSION: Results from this study showed that the leaves and bark of C. cujete possessed anti-inflammatory as well as antibacterial activities indicating that the plant extract has therapeutic potential against the bacterial infection and also have effect on disease processes by causing destabilization of biological membranes.
Subject(s)
Anti-Bacterial Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Bignoniaceae/chemistry , Plant Bark/chemistry , Plant Leaves/chemistry , Drug Evaluation, Preclinical , Erythrocyte Membrane/drug effects , Erythrocyte Membrane/physiology , Escherichia coli/drug effects , Flavonoids/analysis , Hemolysis/drug effects , Humans , Microbial Sensitivity Tests , Phenols/analysis , Plant Extracts/chemistry , Plant Extracts/pharmacology , Staphylococcus aureus/drug effectsABSTRACT
BACKGROUND: Tribolium castaneum (Herbst) is a harmful pest of stored grain and flour-based products in tropical and subtropical region. In the present study, rhizome of Drynaria quercifolia (J. Smith) was evaluated for pesticidal and pest repellency activities against T. castaneum, using surface film method and filter paper disc method, respectively. In addition, activity of the isolated compound 3,4-dihydroxybenzoic acid was evaluated against the pest. RESULTS: Chloroform soluble fraction of ethanol extract of rhizome of D. quercifolia showed significant pesticidal activity at doses 0.88 to 1.77 mg/cm(2) and significant pest repellency activity at doses 0.94 to 0.23 mg/cm(2). No pesticidal and pest repellency activity was found for petroleum ether, ethyl acetate and methanol soluble fractions of ethanol extract as well as for 3,4-dihydroxybenzoic acid. CONCLUSION: Considering our findings it can be concluded that chloroform soluble fraction of rhizome of D. quercifolia is useful in controlling T. castaneum of stored grain and flour-based products.
Subject(s)
Hydroxybenzoates/pharmacology , Insect Repellents/pharmacology , Pest Control/methods , Pesticides , Polypodiaceae/chemistry , Rhizome/chemistry , Tribolium/drug effects , Acetates , Alkanes , Animals , Chloroform , Ethanol , Hydroxybenzoates/isolation & purification , Lethal Dose 50 , Methanol , Plant Extracts/chemistry , Plant Extracts/isolation & purificationABSTRACT
OBJECTIVE: To ascertain analgesic, antibacterial and central nervous system (CNS) depressant activities of ethyl acetate, dichloromethane and carbon tetrachloride fractions of methanol extract of Albizia procera (A. procera) leaves. METHODS: Leaves extracts of A. procera were tested for analgesic activity by acetic acid induced and formalin test method in mice. The in vitro antibacterial activity was performed by agar well diffusion method. CNS depressant activity was evaluated by hole cross and open field tests. RESULTS: All the extracts at 200 mg/kg exhibited significant (P<0.01) analgesic activity in acetic acid induced and formalin tests method in mice. Analgesic activity of the ethyl acetate fraction was almost same like as standard drug indomethacin in acetic acid induced method. The CNS depressant activity of the extracts at 500 mg/kg was comparable to the positive control diazepam as determined by hole cross and open field test method. The extracts exhibited moderate antimicrobial activity against all the tested microorganisms (Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Esherichia coli, Shigella soneii, Shigella boydii) at concentration of 0.8 mg/disc. The measured diameter of zone of inhibition for the extracts was within the range of 7 to 12 mm which was less than the standard kanamycin (16-24 mm). CONCLUSIONS: It is concluded that all the extracts possess potential analgesic and CNS depressants activity. This study also showed that different fractions of methanol extract could be potential sources of new antimicrobial agents.
ABSTRACT
BACKGROUND: Antioxidant compounds like phenols and flavonoids scavenge free radicals and thus inhibit the oxidative mechanisms that lead to control degenerative and other diseases. The aim of this study was to investigate the antioxidant activity in vitro, total phenolic and flavonoid contents in ethanol extracts and fractions of Crescentia cujete leaves and stem bark. METHODS: Crescentia cujete leaves and bark crude ethanol extract (CEE) and their partitionates petroleum ether (PEF), chloroform (CHF), ethyl acetate (EAF) and aqueous (AQF) were firstly prepared. Different established testing methods, such as 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical, ferric reducing power (FRP), and total antioxidant capacity (TAC) assays were used to detect the antioxidant activity. Further, the total yield, total phenolic (TPC) and total flavonoid contents (TFC) of CEE and all the fractions were determined. Ethanol extracts of both leaves and stem bark were also subjected to preliminary phytochemical screening to detect the presence of secondary metabolites, using standard phytochemical methods (Thin layer chromatography and spray reagents). RESULTS: Phytochemical screening of crude ethanol extract of both leaves and stem bark revealed the presence of steroids, flavonoids, saponins, tannins, glycosides and terpenoids. All the fractions and CEE of leaves and bark exhibited antioxidant activities, however, EAF of leaves showing the highest antioxidant activity based on the results of DPPH, FRP and TAC assay tests. The above fraction has shown the significant DPPH scavenging activity (IC50 = 8.78 µg/ml) when compared with standard ascorbic acid (IC50 =7.68 µg/ml). The TAC and FRP activities increased with increasing crude extract/fractions content. The TPC (371.23 ± 15.77 mg GAE/g extract) and TFC (144.64 ± 5.82 mg QE/g extract) of EAF of leaves were found significantly higher as compared to other solvent fractions for both leaves and bark. TPC were highly correlated with the antioxidant activity (R2 = 0.9268 and 0.8515 in DPPH test for leaves and bark, respectively). CONCLUSION: The results of the study show that leaves of C. cujete possesses significant free radical scavenging properties compared with stem bark and a clear correlation exists between the antioxidant activity and phenolic content.
Subject(s)
Antioxidants/pharmacology , Bignoniaceae/chemistry , Flavonoids/pharmacology , Phenols/pharmacology , Plant Extracts/pharmacology , Antioxidants/analysis , Biphenyl Compounds/metabolism , Flavonoids/analysis , Free Radicals/metabolism , Humans , Oxidation-Reduction , Phenols/analysis , Picrates/metabolism , Plant Bark/chemistry , Plant Extracts/chemistry , Plant Leaves/chemistry , Plant Stems/chemistry , Tannins/analysis , Tannins/pharmacologyABSTRACT
BACKGROUND: Tribolium castaneum (Herbst) is a harmful pest of stored grain and flour-based products in tropical and subtropical region. In the present study, rhizome of Drynaria quercifolia (J. Smith) was evaluated for pesticidal and pest repellency activities against T. castaneum, using surface film method and filter paper disc method, respectively. In addition, activity of the isolated compound 3,4-dihydroxybenzoic acid was evaluated against the pest. RESULTS: Chloroform soluble fraction of ethanol extract of rhizome of D. quercifolia showed significant pesticidal activity at doses 0.88 to 1.77 mg/cm² and significant pest repellency activity at doses 0.94 to 0.23 mg/cm². No pesticidal and pest repellency activity was found for petroleum ether, ethyl acetate and methanol soluble fractions of ethanol extract as well as for 3,4-dihydroxybenzoic acid. CONCLUSION: Considering our findings it can be concluded that chloroform soluble fraction of rhizome of D. quercifoliais useful in controlling T. castaneum of stored grain and flour-based products.
Subject(s)
Animals , Pesticides , Tribolium/drug effects , Pest Control/methods , Polypodiaceae/chemistry , Rhizome/chemistry , Hydroxybenzoates/pharmacology , Insect Repellents/pharmacology , Plant Extracts/isolation & purification , Plant Extracts/chemistry , Chloroform , Ethanol , Methanol , Alkanes , Hydroxybenzoates/isolation & purification , Lethal Dose 50 , AcetatesABSTRACT
OBJECTIVE: To evaluate the pharmacological activity against diarrhea of methanol and petroleum ether extract of Desmodium puchellum (Family: Fabaceae) leaves. METHODS: The extract was evaluated for castor oil-induced diarrhea and enteropooling as well as intestinal motility in rats. Both of the extracts were given to the rats at 200 mg/kg orally. Loperamide was used as a standard drug for diarrhea. RESULTS: The diarrheal severity was reduced significantly (P<0.05) by methanol and petroleum ether extracts by 31.95% and 28.33%, respectively, whereas 54.75% inhibition was found for standard drug loperamide at 5 mg/kg. The two extracts also significantly (P<0.05) reduced the intestinal volume in case of castor oil induced enteropooling. CONCLUSIONS: It is concluded that both fractions contain some biologically active ingredients that are active for anti-diarrheal actions whereas methanol fraction has better potential.
Subject(s)
Antidiarrheals/pharmacology , Fabaceae/chemistry , Fibrinolytic Agents/pharmacology , Plant Extracts/chemistry , Plant Leaves/chemistry , Alkanes/chemistry , Animals , Castor Oil/adverse effects , Diarrhea/chemically induced , Diarrhea/drug therapy , Female , Gastrointestinal Motility/drug effects , Loperamide/pharmacology , Male , Methanol/chemistry , RatsABSTRACT
Zebrafish hoxb1b is expressed during epiboly in the posterior neural plate, with its anterior boundary at the prospective r4 region providing a positional cue for hindbrain formation. A similar function and expression is known for Hoxa1 in mice, suggesting a shared regulatory mechanism for hindbrain patterning in vertebrate embryos. To understand the evolution of the regulatory mechanisms of key genes in patterning of the central nervous system, we examined how hoxb1b transcription is regulated in zebrafish embryos and compared the regulatory mechanisms between mammals and teleosts that have undergone an additional genome duplication. By promoter analysis, we found that the expression of the reporter gene recapitulated hoxb1b expression when driven in transgenic embryos by a combination of the upstream 8.0-kb DNA and downstream 4.6-kb DNA. Furthermore, reporter expression expanded anteriorly when transgenic embryos were exposed to retinoic acid (RA) or LiCl, or injected with fgf3/8 mRNA, implicating the flanking DNA examined here in the responsiveness of hoxb1b to posteriorizing signals. We further identified at least two functional RA responsive elements in the downstream DNA that were shown to be major regulators of early hoxb1b expression during gastrulation, while the upstream DNA, which harbors repetitive sequences with apparent similarity to the autoregulatory sequence of mouse Hoxb1, contributed only to later hoxb1b expression, during somitogenesis. Possible implications in vertebrate evolution are discussed based on these findings.
Subject(s)
Body Patterning , Evolution, Molecular , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Rhombencephalon/embryology , Tretinoin/metabolism , Animals , Base Sequence , Conserved Sequence , Fibroblast Growth Factors/metabolism , Gastrulation , Mice , Molecular Sequence Data , Neural Plate/embryology , Neural Plate/metabolism , Promoter Regions, Genetic/drug effects , Rats , Receptors, Retinoic Acid/metabolism , Retinoid X Receptors/metabolism , Rhombencephalon/metabolism , Transcription, Genetic , Tretinoin/pharmacology , Wnt Proteins/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
Zebrafish pou5f1, also known as pou2, encodes a POU-family transcription factor that is transiently expressed in the prospective midbrain and anterior hindbrain during gastrulation, governing brain development. In the present study, we found that the main regulatory elements reside in the proximal upstream DNA sequence from -2.2 to -0.12 kb (the -2.2/-0.1 region). The electrophoretic gel mobility shift assay (EMSA) revealed four functional octamer sequences that can associate with zebrafish Pou2/Pou5f1. The expression of mutated reporter constructs, as well as EMSA, suggested that these four octamer sequences operate in a cooperative manner to drive expression in the mid/hindbrain. We also identified a retinoic acid (RA)-responsive element in this proximal region, which was required to repress transcription in the posterior part of the embryo. These data provide a scheme wherein pou2/pou5f1 expression in zebrafish embryos is regulated by both an autoregulatory loop and repression by RA emanating from the posterior mesoderm.
Subject(s)
Brain/embryology , Brain/metabolism , Gene Expression Regulation, Developmental , Octamer Transcription Factor-3/metabolism , Tretinoin/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Amino Acid Sequence , Animals , Base Sequence , Brain/cytology , Genes, Reporter , Mesoderm/cytology , Mesoderm/physiology , Molecular Sequence Data , Octamer Transcription Factor-3/genetics , Regulatory Sequences, Nucleic Acid , Sequence Alignment , Transcription, Genetic , Zebrafish/anatomy & histology , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
Fgf8 is expressed in the isthmic region of the developing brain, serving an organizing function in vertebrate embryos. We previously identified S4.2 downstream to the zebrafish fgf8 gene as a regulatory region that drives transcription in the anterior hindbrain. Here, we investigated the mechanism of fgf8 regulation by the S4.2 region during development. Reporter analyses in embryos revealed that S4.2 closely recapitulates fgf8 expression in the anteriormost hindbrain during somitogenesis. This region contains a sequence highly conserved in fgf8 of diverse vertebrates. Further analyses of S4.2 revealed a 342-bp core region composed of three subregions (#2, #3, and #4). Regions #3 and #4 drove expression broadly in the brain from the midbrain to r5 of the hindbrain, whereas a 28-bp sequence in #2 repressed ectopic expression in the midbrain and in r2 to r5. The enhancer function of S4.2 was absent in pax2a mutant embryos, while it was activated ectopically by pax2a misexpression in the hindbrain. We identified two sites in the core region that are bound by Pax2a in vitro and in vivo, the disruption of which abrogated the S4.2 activity. Thus, fgf8 expression in the anteriormost hindbrain involves activation and repression, with Pax2a as a pivotal regulator.
Subject(s)
Embryo, Nonmammalian/anatomy & histology , Fibroblast Growth Factors/genetics , Mesencephalon/embryology , Rhombencephalon/embryology , Transcription, Genetic , Zebrafish Proteins/genetics , Zebrafish/embryology , Animals , Base Sequence , DNA Primers , Gene Amplification , Mesencephalon/anatomy & histology , Regulatory Sequences, Nucleic Acid , Rhombencephalon/anatomy & histology , Zebrafish/anatomy & histologyABSTRACT
In vertebrate embryos, positioning of the boundary between the midbrain and hindbrain (MHB) and subsequent isthmus formation are dependent upon the interaction between the Otx2 and Gbx genes. In zebrafish, sequential expression of gbx1 and gbx2 in the anterior hindbrain contributes to this process, whereas in mouse embryos, a single Gbx gene (Gbx2) is responsible for MHB development. In the present study, to investigate the regulatory mechanism of gbx2 in the MHB/isthmic region of zebrafish embryos, we cloned the gene and showed that its organization is conserved among different vertebrates. Promoter analyses revealed three enhancers that direct reporter gene expression after the end of epiboly in the anterior-most hindbrain, which is a feature of the zebrafish gbx2 gene. One of the enhancers is located upstream of gbx2 (AMH1), while the other two enhancers are located downstream of gbx2 (AMH2 and AMH3). Detailed analysis of the AMH1 enhancer showed that it directs expression in the rhombomere 1 (r1) region and the dorsal thalamus, as has been shown for gbx2, whereas no expression was induced by the AMH1 enhancer in other embryonic regions in which gbx2 is expressed. The AMH1 enhancer is composed of multiple regulatory subregions that share the same spatial specificity. The most active of the regulatory subregions is a 291-bp region that contains at least two Pax2-binding sites, both of which are necessary for the function of the main component (PB1-A region) of the AMH1 enhancer. In accordance with these results, enhancer activity in the PB1-A region, as well as gbx2 expression in r1, was missing in no isthmus mutant embryos that lacked functional pax2a. In addition, we identified an upstream conserved sequence of 227bp that suppresses the enhancer activity of AMH1. Taken together, these findings suggest that gbx2 expression during the somitogenesis stage in zebrafish is regulated by a complex mechanism involving Pax2 as well as activators and suppressors in the regions flanking the gene.