Developmental Stage-dependent Expression Research Articles

RNAi targeting Nav and CPR via leaf delivery reduces adult emergence and increases the susceptibility to λ-cyholthin in Tuta absoluta (Meyrick)

The tomato leafminer, Tuta absoluta (Meyrick), one of the most economically destructive pests of tomato, causes severe yields losses of tomato production globally. Rapid evolution of insecticide resistance requires the development of alternative control strategy for this pest. RNA interference (RNAi) represents a promising, innovative control strategy against key agricultural insect pests, which has recently been licensed for Colorado Potato Beetle control. Here two essential genes, voltage-gated sodium channel (Nav) and NADPH-cytochrome P450 reductase (CPR) were evaluated as targets for RNAi using an ex vivo tomato leaf delivery system. Developmental stage-dependent expression profiles showed TaNav was most abundant in adult stages, whereas TaCPR was highly expressed in larval and adult stages. T. absoluta larvae feeding on tomato leaflets treated with dsRNA targeting TaNav and TaCPR showed significant knockdown of gene expression, leading to reduction in adult emergence. Additionally, tomato leaves treated with dsRNA targeting these two genes were significantly less damaged by larval feeding and mining. Furthermore, bioassay with LC30 doses of λ-cyholthin showed that silencing TaNav and TaCPR increased T. absoluta mortality about 32.2 and 17.4%, respectively, thus indicating that RNAi targeting TaNav and TaCPR could increase the susceptibility to λ-cyholthin in T. absoluta. This study demonstrates the potential of using RNAi targeting key genes, like TaNav and TaCPR, as an alternative technology for the control of this most destructive tomato pests in the future.

Molecular detection of maturation stages in the developing kidney

Recent advances in stem cell biology have enabled the generation of kidney organoids in vitro, and further maturation of these organoids is observed after experimental transplantation. However, the current organoids remain immature and their precise maturation stages are difficult to determine because of limited information on developmental stage-dependent gene expressions in the kidney in vivo. To establish relevant molecular coordinates, we performed single-cell RNA sequencing (scRNA-seq) on developing kidneys at different stages in the mouse. By selecting genes that exhibited upregulation at birth compared with embryonic day 15.5 as well as cell lineage-specific expression, we generated gene lists correlated with developmental stages in individual cell lineages. Application of these lists to transplanted embryonic kidneys revealed that most cell types, other than the collecting ducts, exhibited similar maturation to kidneys at the neonatal stage in vivo, revealing non-synchronous maturation across the cell lineages. Thus, our scRNA-seq data can serve as useful molecular coordinates to assess the maturation of developing kidneys and eventually of kidney organoids.

Molecular characterization of an NADPH cytochrome P450 reductase from Bemisia tabaci Q: Potential involvement in susceptibility to imidacloprid

NADPH cytochrome P450 reductase (CPR) is an integral component of cytochrome P450-mediated biological reactions, such as the metabolism of xenobiotics, including insecticides. CPR has been reported to be associated with insecticide tolerance in several insects. However, the biochemical characteristics and biological function of CPR in Bemisia tabaci Q (BtCPR) remain undefined. In this study, BtCPR was cloned, and bioinformatic analysis showed that BtCPR is a transmembrane protein with a molecular weight (MW) of 76.73 kDa and contains conserved binding domains (FMN, FAD, and NADPH). Tissue- and developmental stage-dependent expression indicated that the highest expression levels of BtCPR occurred in head tissue and in male adults. Transcripts of BtCPR in the field B. tabaci Q strain were 1.62-fold higher than those of the laboratory B. tabaci Q strain. In both field and laboratory adults, the susceptibility of BtCPR-knockdown B. tabaci Q to imidacloprid substantially increased compared to that of the B. tabaci Q control group. Furthermore, the heterologous expression of BtCPR in Sf9 cells exhibited catalytic activity for cytochrome c reduction, following Michaelis-Menten kinetics. Sf9 cells overexpressing BtCPR had greater viability than the control cells when treated with imidacloprid. The results suggest that BtCPR could affect the susceptibility of B. tabaci Q to imidacloprid and could also be considered a novel target for pest control.

Biochemical and Morphological Characterization of a Guanine Nucleotide Exchange Factor ARHGEF9 in Mouse Tissues.

ARHGEF9, also known as Collybistin, a guanine nucleotide exchange factor for Rho family GTPases, is thought to play an essential role in the mammalian brain. In this study, we prepared a specific polyclonal antibody against ARHGEF9, anti-ARHGEF9, and carried out expression analyses with mouse tissues especially brain. Western blotting analyses demonstrated tissue-dependent expression profiles of ARHGEF9 in the young adult mouse, and strongly suggested a role during brain development. Immunohistochemical analyses revealed developmental stage-dependent expression profiles of ARHGEF9 in cerebral cortex, hippocampus and cerebellum. ARHGEF9 exhibited partial localization at dendritic spines in cultured hippocampal neurons. From the obtained results, anti-ARHGEF9 was found to be a useful tool for biochemical and cell biological analyses of ARHGEF9.

Endometrial vascular development in heavy menstrual bleeding: altered spatio-temporal expression of endothelial cell markers and extracellular matrix components.

Are there any phenotypic and structural/architectural changes in the vessels of endometrium and superficial myometrium during the normal menstrual cycle in healthy women and those with heavy menstrual bleeding (HMB)? Spatial and temporal differences in protein levels of endothelial cell (EC) markers and components of the extracellular matrix (ECM) were detected across the menstrual cycle in healthy women and these are altered in HMB. HMB affects 30% of women of reproductive age with ~50% of cases being idiopathic. We have previously shown that the differentiation status of endometrial vascular smooth muscle cells (VSMCs) is altered in women with HMB, suggesting altered vessel maturation compared to controls. Endometrial arteriogenesis requires the co-ordinated maturation not only of the VSMCs but also the underlying ECs and surrounding ECM. We hypothesized that there are spatial and temporal patterns of protein expression of EC markers and vascular ECM components in the endometrium across the menstrual cycle, which are altered in women with HMB. Biopsies containing endometrium and superficial myometrium were taken from hysterectomy specimens from both healthy control women without endometrial pathology and women with subjective HMB in the proliferative (PP), early secretory (ESP), mid secretory (MSP) and late secretory (LSP) phases (N = 5 for each cycle phase and subject group). Samples were fixed in formalin and embedded in paraffin wax. Serial sections (3μm thick) were immunostained for EC markers (factor VIII related antigen (F8RA), CD34, CD31 and ulex europaeus-agglutinin I (UEA-1) lectin), structural ECM markers (osteopontin, laminin, fibronectin and collagen IV) and for Ki67 to assess proliferation. Immunoreactivity of vessels in superficial myometrium, endometrial stratum basalis, stratum functionalis and luminal region was scored using either a modified Quickscore or by counting the number of positive vessels. In control samples, all four EC markers showed a dynamic expression pattern according to the menstrual cycle phase, in both endometrial and myometrial vessels. EC protein marker expression was altered in women with HMB compared with controls, especially in the secretory phase in the endometrial luminal region and stratum functionalis. For example, in the LSP expression of UEA-1 and CD31 in the luminal region decreased in HMB (mean quickscore: 1 and 5, respectively) compared with controls (3.2 and 7.4, respectively) (both P = 0.008), while expression of F8RA and CD34 increased in HMB (1.4 and 8, respectively) compared with controls (0 and 5.8, respectively) (both P = 0.008). There was also a distinct pattern of expression of the vascular structural ECM protein components osteopontin, laminin, fibronectin and collagen IV in the superficial myometrium, stratum functionalis and stratum basalis during the menstrual cycle, which was altered in HMB. In particular, compared with controls, osteopontin expression in HMB was higher in stratum functionalis in the LSP (7.2 and 11.2, respectively P = 0.008), while collagen IV expression was reduced in stratum basalis in the MSP (4.6 and 2.8, respectively P = 0.002) and in stratum functionalis in the ESP (7 and 3.2, respectively P = 0.008). The protein expression of vascular EC markers and ECM components was assessed using a semi-quantitative approach in both straight and spiral arterioles. In our hospital, HMB is determined by subjective criteria and levels of blood loss were not assessed. Variation in the protein expression pattern between the four EC markers highlights the importance of choice of EC marker for investigation of endometrial vessels. Differences in expression of the different EC markers may reflect developmental stage dependent expression of EC markers in endometrial vessels, and their altered expression in HMB may reflect dysregulated vascular development. This hypothesis is supported by altered expression of ECM proteins within endometrial vessel walls, as well as our previous data showing a dysregulation in VSMC contractile protein expression in the endometrium of women with HMB. Taken together, these data support the suggestion that HMB symptoms are associated with weaker vascular structures, particularly in the LSP of the menstrual cycle, which may lead to increased and extended blood flow during menstruation. This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare. Not applicable.

Molecular Characterization of Six Small Heat Shock Proteins and Their Responses Under Cadmium Stress in Oxya chinensis (Orthoptera: Acridoidea).

Small heat shock proteins (sHSPs) have been implicated in many physiological processes and play important roles in the response to various stresses. In this study, the full-length sequences of six sHSPs: OcHSP19.1, 19.8, 20.4, 20.7, 21.1, and 23.8 were obtained from the rice grasshopper Oxya chinensis transcriptome database. The deduced amino acid sequences of the six OcsHSPs contain a typical α-crystallin domain, which consists of approximately 100 amino acid residues and five β-strands. The phylogenetic analysis suggested that OcHSP23.8 was orthologous to the sHSPs of other species and that OcHSP19.1, 20.4, 20.7, and 21.1 were species specific, whereas OcHSP19.8 did not cluster closely to Orthoptera but was placed on the basal end of the cluster. Developmental stage-dependent and tissue-specific expression patterns were evaluated using quantitative real-time polymerase chain reaction. The six genes were expressed in all developmental stages and showed clear tissue specificity. The cadmium acute experiment indicates that Cd(2+) can induce the six genes. However, various response patterns were observed among these genes under Cd(2+) stress conditions. OcHSP19.1, 19.8, 20.4, and 20.7 were highly induced by 2.61 mM Cd(2+) at 24 h. OcHSP23.8 was significantly upregulated by 2.61 mM Cd(2+) at 6 h. For OcHSP21.1, the highest expression levels were found after treatment with 0.87 mM Cd(2+) for 24 h, 1.74 mM Cd(2+) for 36 h, and 2.61 mM Cd(2+) for 12 h. These differential characteristics will facilitate future investigations into the physiological functions of sHSPs.

Peroxisome proliferator-activated receptor gamma (PPARγ) in yellow catfish Pelteobagrus fulvidraco: Molecular characterization, mRNA expression and transcriptional regulation by insulin in vivo and in vitro

Peroxisome proliferator-activated receptor gamma (PPARγ) is ligand-inducible transcription factor and has important roles in lipid metabolism, cell proliferation and inflammation. In the present study, yellow catfish Pelteobagrus fulvidraco PPARγ cDNA was isolated from liver by RT-PCR and RACE, and its molecular characterization and transcriptional regulation by insulin in vivo and in vitro were determined. The generation of PPARγ1 and PPARγ2 was due to alternative promoter of PPARγ gene. PPARγ1 and PPARγ2 mRNA covered 2426bp and 2537bp, respectively, with an open reading frame (ORF) of 1584bp encoding 527 amino acid residues. Yellow catfish PPARγ gene was organized in a manner similar to that of their mammalian homologs, implying a modular organization of the protein’s domains. A comparison between the yellow catfish PPARγ amino acid sequence and the correspondent sequences of several other species revealed the identity of 55–76.2%. Two PPARγ transcripts (PPARγ1 and PPARγ2) mRNAs were expressed in a wide range of tissues, but the abundance of each PPARγ mRNA showed the tissue- and developmental stage-dependent expression patterns. Intraperitoneal injection of insulin in vivo significantly stimulated the mRNA expression of total PPARγ and PPARγ1, but not PPARγ2 in the liver of yellow catfish. In contrast, incubation of hepatocytes with insulin in vitro increased the mRNA levels of PPARγ1, PPARγ2 and total PPARγ. To our knowledge, for the first time, the present study provides evidence that PPARγ1 and PPARγ2 are differentially expressed with and among tissues during different developmental stages and also regulated by insulin both in vivo and in vitro, which serves to increase our understanding on PPARγ physiological function in fish.

Hormone-sensitive lipase in yellow catfish Pelteobagrus fulvidraco: Molecular characterization, mRNA tissue expression and transcriptional regulation by leptin in vivo and in vitro

Hormone-sensitive lipase (hsl) plays a pivotal role in regulation of lipolysis in mammals, but information is very scarce about its gene structure and function in fish. In this study, two distinct hsl cDNAs, designated hsl1 and hsl2, were firstly isolated and characterized from yellow catfish Pelteobagrus fulvidraco. The validated cDNAs encoding for hsl1 and hsl2 were 2739 and 2629bp in length, encoding peptides of 679 and 813 amino acid residues, respectively, and shared 57.7% amino acid identity. The phylogenetic analysis revealed that hsl1 and hsl2 derived from paralogous genes that might have arisen during a teleost-specific genome duplication event. Both hsl mRNAs were expressed in a wide range of tissues, but the abundance of each hsl mRNA showed the tissue- and developmental stage-dependent expression patterns. Intraperitoneal injection in vivo and incubation in vitro of recombinant human leptin (rb-hLEP) stimulated the mRNA expression of hsl2, but not hsl1, in the liver and hepatocytes of P. fulvidraco, respectively, suggesting that two hsl isoforms might serve different roles in lipid metabolism. To our knowledge, for the first time, the present study provides evidence that two hsl mRNAs are differentially expressed with and among tissues during different developmental stages and also differentially regulated by leptin both in vivo and in vitro, which serves to increase our understanding on hsl physiological function in fish.

Genome-Wide Identification of Embryogenesis-Associated microRNAs in Radish (Raphanus sativus L.) by High-Throughput Sequencing

microRNAs (miRNAs), endogenous non-coding RNAs of approximately 21–24 nucleotides, are important regulators of transcriptional and post-transcriptional gene expression. These regulators play a key role during plant growth and development, including embryogenesis, which is crucial to the life cycle of most plant species. However, although embryogenesis-associated miRNAs have been isolated in a few species, the diversity of these regulatory miRNAs remains largely unexplored, especially in radish. In this study, two small RNA libraries were constructed from radish ovules before and after fertilization. Both libraries were sequenced by next generation sequencing (NGS) technology. This analysis identified 144 conserved and 34 non-conserved miRNAs (representing 43 known miRNA families) and 38 novel miRNAs (representing 28 miRNA families). Comparative analysis revealed that 29 known and 10 novel miRNA families were differentially expressed during embryogenesis. QRT-PCR analysis confirmed miRNA expression patterns and revealed tissue-specific and/or developmental stage-dependent expression for some miRNAs. Moreover, potential target predictions indicated that most of these targets were transcription factors involved in regulating plant growth, development and metabolism. Notably, target transcripts such as squamosa promoter-binding protein, auxin response factor and agamous-like MADS-box protein participated in radish embryogenesis.

Cloning, functional characterization, and expression profiles of NADPH-cytochrome P450 reductase gene from the Asiatic rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae)

NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems.

Genome-wide analysis of microRNAs in rubber tree (Hevea brasiliensis L.) using high-throughput sequencing

MicroRNAs (miRNAs) are short RNAs with essential roles in gene regulation in various organisms including higher plants. In contrast to the vast information on miRNAs from many economically important plants, almost nothing has been reported on the identification or analysis of miRNAs from rubber tree (Hevea brasiliensis L.), the most important natural rubber-producing crop. To identify miRNAs and their target genes in rubber tree, high-throughput sequencing combined with a computational approach was performed. Four small RNA libraries were constructed for deep sequencing from mature and young leaves of two rubber tree clones, PB 260 and PB 217, which provide high and low latex yield, respectively. 115 miRNAs belonging to 56 known miRNA families were identified, and northern hybridization validated miRNA expression and revealed developmental stage-dependent and clone-specific expression for some miRNAs. We took advantage of the newly released rubber tree genome assembly and predicted 20 novel miRNAs. Further, computational analysis uncovered potential targets of the known and novel miRNAs. Predicted target genes included not only transcription factors but also genes involved in various biological processes including stress responses, primary and secondary metabolism, and signal transduction. In particular, genes with roles in rubber biosynthesis are predicted targets of miRNAs. This study provides a basic catalog of miRNAs and their targets in rubber tree to facilitate future improvement and exploitation of rubber tree.

Crystal Structures of NAD(P)(H) Sensing NmrA-Like Proteins from Dictyostelium Discoideum

NAD(P)(H) functions as central coenzyme in catabolic and anabolic reaction. And recently, many results show that NAD(P)(H) is also involved in signaling pathways that regulate fundamental processes of cellular functions. NmrA in Aspergillus nidulans is known to discriminate between oxidized and reduced dinucleotides and suggested as redox sensor. There are several NmrA-like protein coding genes in Dictyostelium discoideum and they show developmental stage-dependent expression levels and especially DDB_G0291732 gene exhibits different expression pattern from the other NmrA-like protein coding genes. To study relation between structure and function, the crystal structures of DDB_G0291732 and DDB_G0286605 were determined. Crystal structures of DDB_G0291732 and DDB_G0286605 adopt the features of SDR-type NmrA-like proteins. DDB_G0291732 and DDB_G0286605 contain conserved N-terminal dinucleotide binding Rossmann fold with a central β sheet and the C-domain, containing a hydrophobic pocket connected to the N-domain. Structural comparisons with known 3D structures reveal that DDB_G0291732 and DDB_G0286605 are mostly matched with HSCARG; human NADP(H) sensor protein, NmrA; negative transcriptional regulator involved in nitrogen metabolite repression of Aspergillus nidulans and QOR2; new-type quinone oxidoreductase considered to function as transcriptional regulator of E. coli. No dehydrogenase activity could be prospected with DDB_G0291732 and DDB_G0286605 because the catalytic Tyr residue in the active site triad of Ser-Tyr-Lys is replaced by His and Asp in DDB_G0291732 and DDB_G0286605, respectively. Cofactor binding experiments reveal that DDB_G0291732 does not interact with pyridine dinucleotides and DDB_G0286605 has high affinity with NADP(H) but not with NAD(H). The mRNA expression levels of DDB_G0291732 and DDB_G0286605 genes after dehydroepiandrosterone (DHEA) or H2O2 treatment are monitored to examine how these proteins respond to redox states in D. discoideum.

Identification, characterization, and ontogenic study of a catechol O-methyltransferase from zebrafish

To establish the zebrafish as a model for investigating the methylation pathway of drug metabolism, we embarked on the molecular cloning of the zebrafish catechol O-methyltransferase (COMT). By searching the GenBank database, a zebrafish nucleotide sequence encoding a putative COMT was identified. Based on the sequence information, we designed and synthesized oligonucleotides corresponding to its 5′- and 3′-coding regions of this zebrafish COMT. Using the first-strand cDNA reverse-transcribed from the total RNA isolated from a 3-month-old adult female zebrafish as the template, the cDNA encoding the zebrafish COMT was PCR-amplified. The recombinant zebrafish COMT protein was subsequently expressed in and purified from BL21 (DE3) Escherichia coli cells transformed with the pGEX-2TK expression vector harboring the zebrafish COMT cDNA. Upon enzymatic characterization, purified COMT displayed methylating activity toward dopamine, dopa, and catecholestrogens, as well as three representative catechol drugs, methyldopa, dobutamine, and isoproterenol. A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of the zebrafish COMT during embryonic development and throughout the larval stage onto maturity. These results provide a foundation for investigating the involvement of COMT-mediated methylation in protection against the adverse effects of catechol drugs and other xenobiotic catechols during the developmental process.

Developmental toxicity of dextromethorphan in zebrafish embryos/larvae

Dextromethorphan is widely used in over-the-counter cough and cold medications. Its efficacy and safety for infants and young children remains to be clarified. The present study was designed to use zebrafish as a model to investigate the potential toxicity of dextromethorphan during embryonic and larval development. Three sets of zebrafish embryos/larvae were exposed to dextromethorphan at 24, 48 and 72 h post fertilization (hpf), respectively, during the embryonic/larval development. Compared with the 48 and 72 hpf exposure sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent manner. Bradycardia and reduced blood flow were observed for the embryos/larvae treated with increasing concentrations of dextromethorphan. Morphological effects of dextromethorphan exposure, including yolk sac and cardiac edema, craniofacial malformation, lordosis, non-inflated swim bladder and missing gill, were also more frequent and severe among zebrafish embryos/larvae exposed to dextromethorphan at 24 hpf. Whether the more frequent and severe developmental toxicity of dextromethorphan observed among the embryos/larvae in the 24 hpf exposure set, as compared with the 48 and 72 hpf exposure sets, is due to the developmental expression of the phase I and phase II enzymes involved in the metabolism of dextromethorphan remains to be clarified. A reverse transcription-polymerase chain reaction analysis, nevertheless, revealed developmental stage-dependent expression of mRNAs encoding SULT3 ST1 and SULT3 ST3, two enzymes previously shown to be capable of sulfating dextrorphan, an active metabolite of dextromethorphan.

Developmental stage-dependent expression of an 2,8-trisialic acid unit on glycoproteins in mouse brain

The monoclonal antibody mAb.A2B5 is a marker for the detection of oligodendrocyte progenitor cells that differentiate into type-2 astrocytes and oligodendrocytes. It is also a useful antibody for separating these cells from other lineage populations. The epitope of this antibody is considered to be the gangliosides GT3 and GQ1c. In this study, we sought to define more precisely the structure of the epitope. Accordingly, we chemically synthesized defined oligosialic acid structures linked to phosphatidylethanolamine and bovine serum albumin and used these to determine the antigenic specificity. mAb.A2B5 recognized the Neu5Acalpha2-->8Neu5Acalpha2-->8Neu5Acalpha--> structure on both glycolipids and glycoproteins. We then examined whether the mAb.A2B5 epitope exists on glycoproteins in developing mouse brains. Western blot analyses revealed the expression of four glycoproteins reactive with the mAb.A2B5, and their expression was dependent on the stage of neural development. All the immunoreactivity in these glycoproteins with mAb.A2B5 disappeared after sialidase treatment and were resistant to chloroform/methanol extraction. These epitopes were also detected in brain homogenates from both GD3 synthetase-null and GD3/GD2 synthetase double null mice. These findings show that the alpha2,8-trisialic acid (triSia) unit recognized by mAb.A2B5 resides not only on gangliosides but also on glycoproteins in developing mouse brain. We postulate that the triSia structure on glycoproteins may be involved in oligodendrocyte differentiation, similar to the case with the alpha2,8-triSia structure on gangliosides. Real time polymerase chain reaction analysis of the developmental expression of all known ST8Sia genes, which are responsible for the biosynthesis of alpha2,8-linked Sia residues, showed that ST8Sia III gene expression correlated with expression of the triSia epitope. We suggest that ST8Sia III is the principal sialyltransferase responsible for synthesis of the alpha2,8-triSia units on glycoproteins.

A Novel Hydroxysteroid-Sulfating Cytosolic Sulfotransferase, SULT3 ST3, from Zebrafish: Identification, Characterization, and Ontogenic Study

To establish the zebrafish as a model for investigating the drug metabolism through sulfation, we had embarked on establishing a complete repertoire of the zebrafish Phase II cytosolic sulfotransferases (SULTs). By searching the expressed sequence tag database, a zebrafish cDNA encoding a putative cytosolic sulfotransferase (SULT) was identified. Based on the sequence analysis, this zebrafish SULT was found to belong to the SULT3 gene family. The recombinant protein of the zebrafish SULT, designated SULT3 ST3, was expressed in and purified from BL21 (DE3) Escherichia coli cells transformed with the pGEX-2TK expression vector harboring SULT3 ST3 cDNA. Upon enzymatic characterization, purified SULT3 ST3 displayed sulfating activity toward hydroxysteroids, particularly pregnenolone and dehydroepiandrosterone (DHEA), as well as several drugs among various endogenous and xenobiotic compounds tested as substrates. The pH-dependence and kinetic constants of this enzyme with DHEA were determined. The regulatory effects of various divalent metal cations on the DHEA-sulfating activity of SULT3 ST3 were quantitatively evaluated. A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of SULT3 ST3 during embryonic development and throughout the larval stage onto maturity. Collectively, these results suggest a possible involvement of the newly discovered SULT3 ST3 in the metabolism of hydroxysteroids and xenobiotics including drugs in zebrafish.

The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2

MicroRNAs (miRNAs) are endogenous noncoding RNAs, about 22 nucleotides in length, that mediate post-transcriptional gene silencing by annealing to inexactly complementary sequences in the 3'-untranslated regions of target mRNAs. Our current understanding of the functions of miRNAs relies mainly on their tissue-specific or developmental stage-dependent expression and their evolutionary conservation, and therefore is primarily limited to their involvement in developmental regulation and oncogenesis. Of more than 300 miRNAs that have been identified, miR-1 and miR-133 are considered to be muscle specific. Here we show that miR-1 is overexpressed in individuals with coronary artery disease, and that when overexpressed in normal or infarcted rat hearts, it exacerbates arrhythmogenesis. Elimination of miR-1 by an antisense inhibitor in infarcted rat hearts relieved arrhythmogenesis. miR-1 overexpression slowed conduction and depolarized the cytoplasmic membrane by post-transcriptionally repressing KCNJ2 (which encodes the K(+) channel subunit Kir2.1) and GJA1 (which encodes connexin 43), and this likely accounts at least in part for its arrhythmogenic potential. Thus, miR-1 may have important pathophysiological functions in the heart, and is a potential antiarrhythmic target.

Molecular cloning, expression, characterization, and ontogenic study of novel zebrafish cytosolic SULTs

By searching the expressed sequence tag database, five novel zebrafish SULT cDNAs were identified. The recombinant proteins of these SULTs, designated SULT1 ST7 and ST8, SULT2 ST4, and SULT3 ST1 and ST2, were expressed using the pGEX-2TK GST gene fusion system and purified from transformed E. coli cells. The enzymatic characterization of the purified proteins was performed. Purified SULT1 ST7 and ST8 exhibited strong sulfating-activities toward n-propyl gallate and polychlorinated biphenylols, among various endogenous and xenobiotic compounds tested as substrates. SULT2 ST4 displayed a high substrate specificity toward dehydroepiandrosterone (DHEA) and pregnenolone; whereas SULT3 ST1 and ST2 exhibited strong activities toward 17-estradiol and DHEA. The pH-dependence and kinetic constants of these enzymes were determined. Some of the known environmental estrogens and divalent metal cations exerted inhibitory effects on the sulfating activities of these SULTs. Developmental stage-dependent expression experiments revealed distinct patterns of expression of these SULTs during embryonic development and throughout the larval stage onto maturity. These results provide essential information pertaining to the use of the zebrafish as a model for systematic investigation on fundamental issues regarding the cytosolic SULTs.

Identification of ten novel genes involved in human spermatogenesis by microarray analysis of testicular tissue

To identify novel genes that are down-regulated in the testicular tissue of infertile men. Prospective study. University-based reproductive clinics and genetics laboratory. Nine patients with normal spermatogenesis, and 15 patients with maturation arrest (MA) or Sertoli cell-only syndrome (SCOS). Testicular samples of patients with the same histology were pooled for complementary DNA (cDNA) microarray analysis. Novel, down-regulated genes. In total, 300 genes were significantly down-regulated in SCOS or MA samples, and 10 novel sterility-related genes were identified. Of the 10 novel genes, 6 genes (Hs.126780, Hs.553658, Hs.274135, Hs.268122, Hs.531701, and Hs.171130) encode proteins with predictable functional domains, and all these functional domains are believed to correlate with spermatogenesis and/or spermiogenesis. Conversely, the other 4 genes (Hs.351582, Hs.407480, Hs.552781, and Hs.355570) do not encompass known functional domains. Two genes (Hs.407480 and Hs.552781) lack mouse orthologues. Most novel genes showed a testis-specific expression pattern in both mice and humans. Reverse transcription-polymerase chain reaction (RT-PCR) showed three distinct types of developmental stage-dependent expressions of message ribonucleic acid (mRNA) for these novel genes in murine testes. These 10 novel genes provide targets to elucidate novel pathways involved in human spermatogenesis.

Identification of novel hydroxysteroid-sulfating cytosolic SULTs, SULT2 ST2 and SULT2 ST3, from zebrafish: Cloning, expression, characterization, and developmental expression

By searching the expressed sequence tag database, two zebrafish cDNAs encoding putative cytosolic sulfotransferases (SULTs) were identified. Sequence analysis indicated that these two zebrafish SULTs belong to the cytosolic SULT2 gene family. The recombinant form of these two novel zebrafish SULTs, designated SULT2 ST2 and SULT2 ST3, were expressed using the pGEX-2TK glutathione S-transferase (GST) gene fusion system and purified from transformed BL21 (DE3) Escherichia coli cells. Purified GST-fusion protein form of SULT2 ST2 and SULT2 ST3 exhibited strong sulfating activities toward dehydroepiandrosterone (DHEA) and corticosterone, respectively, among various endogenous compounds tested as substrates. Both enzymes displayed pH optima at ∼6.5. Kinetic constants of the two enzymes, as well as the GST-fusion protein form of the previously identified SULT2 ST1, with DHEA and corticosterone as substrates were determined. Developmental stage-dependent expression experiments revealed distinct patterns of expression of SULT2 ST2 and SULT2 ST3, as well as the previously identified SULT2 ST1, during embryonic development and throughout the larval stage onto maturity.