Reverse Transcription-polymerase Chain Reaction Experiments Research Articles

Exclusive overproduction of recombinant versatile peroxidase MnP2 by genetically modified white rot fungus, Pleurotus ostreatus

By combining a homologous recombinant gene expression system and optimization of the culture conditions, hyper overproduction of Pleurtous ostreatus MnP2 was achieved. Genetically modified P. ostreatus strains with the recombinant mnp2 sequence under the control of sdi1 expression signals, were subjected to agitated culture using media supplemented with wheat bran or its hot-water extract. The best result, whereby 7300 U/l of MnP was produced by a recombinant strain TM2-18, indicated that more than 30-fold overproduction of the recombinant MnP2 compared to the previous result was achieved. On the other hand, no MnP activity was detected for the wild-type strain under the same conditions. Accumulation of the recombinant, but not endogenous, mnp2 transcripts was demonstrated in reverse-transcription PCR experiments. These results indicated that the recombinant MnP2 was exclusively expressed by the recombinant strain. Purified recombinant MnP2 showed almost identical properties to native MnP2 in electrophoresis, spectroscopic and kinetic analyses, including determination of K(m) and V(max) values for Mn(II), H(2)O(2) and veratryl alcohol. Moreover, the recombinant MnP2 directly oxidized a high-molecularweight substrate RNase A in the absence of redox mediators, as does native MnP2. The homologous overproduction system will provide a plat form for exclusive production of mutant or variant peroxidases with a desired property in basidiomycete.

Grape Seed Extract Is an Aromatase Inhibitor and a Suppressor of Aromatase Expression

Aromatase is the enzyme that converts androgen to estrogen. It is expressed at higher levels in breast cancer tissues than normal breast tissues. Grape seed extract (GSE) contains high levels of procyanidin dimers that have been shown in our laboratory to be potent inhibitors of aromatase. In this study, GSE was found to inhibit aromatase activity in a dose-dependent manner and reduce androgen-dependent tumor growth in an aromatase-transfected MCF-7 (MCF-7aro) breast cancer xenograft model, agreeing with our previous findings. We have also examined the effect of GSE on aromatase expression. Reverse transcription-PCR experiments showed that treatment with 60 mug/mL of GSE suppressed the levels of exon I.3-, exon PII-, and exon I.6-containing aromatase mRNAs in MCF-7 and SK-BR-3 cells. The levels of exon I.1-containing mRNA, however, did not change with GSE treatment. Transient transfection experiments with luciferase-aromatase promoter I.3/II or I.4 reporter vectors showed the suppression of the promoter activity in a dose-dependent manner. The GSE treatment also led to the down-regulation of two transcription factors, cyclic AMP-responsive element binding protein-1 (CREB-1) and glucocorticoid receptor (GR). CREB-1 and GR are known to up-regulate aromatase gene expression through promoters I.3/II and I.4, respectively. We believe that these results are exciting in that they show GSE to be potentially useful in the prevention/treatment of hormone-dependent breast cancer through the inhibition of aromatase activity as well as its expression.

Lepidopteran Ortholog of Drosophila Breathless Is a Receptor for the Baculovirus Fibroblast Growth Factor

The Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a gene homologous to the mammalian fibroblast growth factor (FGF) family. We report the cloning of B. mori and Spodoptera frugiperda orthologous genes (Bmbtl and Sfbtl, respectively) of Drosophila melanogaster breathless (btl) encoding a receptor for Branchless/FGF and show that these genes encode the receptor for a baculovirus-encoded FGF (vFGF). Sequence analysis showed that BmBtl is composed of 856 amino acid residues, which potentially encodes a 97.3-kDa polypeptide and shares structural features and sequence similarities with the FGF receptor family. Reverse transcription-PCR experiments showed that Bmbtl was abundantly expressed in the trachea and midgut in B. mori larvae, with moderate expression observed in the hemocytes and the B. mori cultured cell line BmN. We generated Sf-9 cells that stably expressed His-tagged BmBtl. Western blot analysis revealed that BmBtl was an approximately 110-kDa protein. Immunoprecipitation experiments showed that BmNPV vFGF markedly phosphorylated BmBtl in Sf-9 cells. In addition, we found that BmBtl overexpression enhanced the migration activity for BmNPV vFGF. Furthermore, we generated Sf-9 cells in which Sfbtl was knocked down by transfection with double-strand RNA-expressing plasmids. In these cells, cell motility triggered by vFGF was markedly reduced. These results strongly suggest that the Btl orthologs, BmBtl and SfBtl, are the receptors for vFGF, which mediate vFGF-induced host cell chemotaxis.

Fluoxetine and Cocaine Induce the Epigenetic Factors MeCP2 and MBD1 in Adult Rat Brain

Once bound to methylated CpG sites, methyl-CpG-binding protein 2 (MeCP2) is thought to silence transcription of downstream genes by recruiting a histone deacetylase (HDAC). Mutations within the MeCP2 gene have been found to cause Rett syndrome, a disorder of arrested neuronal development. Using immunohistochemistry, we found that Mecp2, as well as the methyl-CpG-binding protein MBD1, were significantly induced in normal adult rat brain after repeated injections of fluoxetine or cocaine for 10 days (one injection per day). Mecp2 was not induced by repeated injections of 1-(2-bis(4-fluorphenyl)-methoxy)-ethyl)-4-(3-phenyl-propyl)piperazine (GBR-12909) or nortriptyline. Together, the data indicate that the serotonergic system is predominantly involved. Using real-time reverse transcription-polymerase chain reaction experiments, MBD1 mRNA and both Mecp2_e1 and Mecp2_e2 transcripts were found to be induced by fluoxetine. Induction of the methylbinding proteins was accompanied with enhanced HDAC2 labeling intensity and mRNA synthesis in response to fluoxetine. In tandem, acetylated forms of histone H3 were found to be decreased. The effect was characterized in three serotonin projection areas, the caudate-putamen, the frontal cortex, and the dentate gyrus subregion of hippocampus. Our data highlight GABAergic neurons as major target cells expressing Mecp2 in response to the serotonin-elevating agents and suggest that serotonin signaling enhances gene silencing in postmitotic neurons.

Characterization of novel carbazole catabolism genes from gram-positive carbazole degrader Nocardioides aromaticivorans IC177.

Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation (car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta-2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa, carAc, and carAd, respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. Escherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo-p-dioxin but less activity for dibenzofuran and biphenyl.

Mg 2+ On-Off Switch

Mg 2+ , a ubiquitous divalent cation, is required for the activity of many enzymes and stabilizes RNA structures and membranes. Because of its importance, organisms have mechanisms for controlling the intracellular concentration of Mg 2+ ([Mg 2+ ] i ). In Salmonella enterica serovar Typhimurium, decreased periplasmic Mg 2+ concentrations are sensed by the PhoQ protein, which triggers the phosphorylation of the PhoP transcription factor, thereby promoting the expression of genes encoding Mg 2+ transporters, mtgA and mtgB . However, expression of mtgA is also stimulated by low [Mg 2+ ] i in strains defective in the PhoP/PhoQ system. Cromie et al. show that Mg 2+ triggers changes in the structure of the 5' untranslated region (5′-UTR) of the mtgA transcript, which in high [Mg 2+ ] block transcription elongation and in low [Mg 2+ ] allow transcription through the coding region. Using quantitative RT-PCR (reverse transcription polymerase chain reaction) experiments, the authors showed that the 5′-UTR was detectable at higher [Mg 2+ ] than was the coding region. Substitution of 100 base pairs of the 5′-UTR with an irrelevant sequence resulted in constitutive mtgA expression in a strain in which PhoP was constitutively active. Transfer of the 5′-UTR from mtgA to a LacZ reporter resulted in stimulation of β-galactosidase activity in low [Mg 2+ ]. Secondary structure modeling suggested that a two-loop structure (stem loops A and B) was adopted under high [Mg 2+ ] conditions and a single-loop structure (stem loop C) was adopted under low [Mg 2+ ] conditions. These structures were supported by RNase and chemical adduct experiments, which showed differential accessibility to residues depending on [Mg 2+ ]. Further disruption of specific stem loop structures by placement of a derivative of the lac promoter at different positions in the 5′-UTR of mtgA in front of LacZ showed that when the formation of stem loop C was blocked all expression was blocked, and when the formation of stem loop A or all three stem loops was blocked, then expression was independent of [Mg 2+ ]. Point mutations in the 5′-UTR to disrupt specific stem loops also supported the model that stem loop A formed the Mg 2+ sensor such that when [Mg 2+ ] was high, stem loops A and B were present (and transcription was blocked) and when [Mg 2+ ] was low, stem loop C was present (and transcription proceeded). These proposed structures were also predicted for mtgA 5′-UTR from six other Gram-negative bacteria, suggesting that this Mg 2+ switch may be a common mechanism for regulating the abundance of these Mg 2+ transporters. M. J. Cromie, Y. Shi, T. Latifi, E. A. Groisman, An RNA sensor for intracellular Mg 2+ . Cell 125 , 71-84 (2006). [Online Journal]

Mutation analysis of the paraplegin gene ( SPG7 ) in patients with hereditary spastic paraplegia

Mutations in the SPG7 gene, which encodes paraplegin, are responsible for an autosomal recessive hereditary spastic paraplegia (HSP). To screen the SPG7 gene in a large population of HSP families compatible with autosomal recessive transmission. The authors analyzed 136 probands with pure or complex HSP for mutations in the SPG7 using denaturation high-performance liquid chromatography and direct sequencing. The authors identified 47 variants including 6 mutations, 27 polymorphisms, and 14 changes with unknown effects. In one family from Morocco, compound c.850_851delTTinsC and c.1742_1744delTGG heterozygous mutations were shown to be causative. This family had complex HSP with cerebellar impairment. Progression of the disease was rapid, resulting in a severe disease after 8 years of duration. Also detected were 20 families with one heterozygous mutation that was not found in a large control population. The mutations produced highly defective proteins in four of these families, suggesting that they were probably causative. Direct sequencing of all exons and reverse transcription PCR experiments demonstrated the absence of a second mutation. However, the p.Ala510Val missense substitution previously described as a polymorphism was shown to be significantly associated with HSP, suggesting that it had a functional effect. SPG7 mutations account for less than 5% of hereditary spastic paraplegia (HSP) families compatible with autosomal recessive inheritance. Cerebellar signs or cerebellar atrophy on brain imaging were the most frequent additional features in patients with SPG7 HSP. Rare nucleotide variants in SPG7 are frequent, complicating routine diagnosis.

Sodium and Epithelial Sodium Channels Participate in the Regulation of the Capacitation-associated Hyperpolarization in Mouse Sperm

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E(m)) hyperpolarizes. However, the mechanisms that regulate sperm E(m) are not well understood. Here we show that sperm hyperpolarize when external Na(+) is replaced by N-methyl-glucamine. Readdition of external Na(+) restores a more depolarized E(m) by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na(+) transporter, possibly involving an amiloride sensitive Na(+) channel, may contribute to the sperm resting E(m). Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na(+) current whose characteristics match those of the epithelial Na(+) channel (ENaC) family of epithelial Na(+) channels. Indeed, ENaC-alpha and -delta mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-alpha and -delta proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-alpha to the flagellar midpiece and of ENaC-delta to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na(+). These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane.

Acid-sensing ion channels in taste buds

Taste receptor cells detect gustatory stimuli using a complex arrangement of ion channels, G protein-coupled receptors, and signaling cascades. Sour and salty tastes are detected by ion channels in the rat. Using a combination of homology screening and functional expression approaches, we screened a rat circumvallate papilla cDNA library and identified acid-sensing ion channel-2a (ASIC2a) and ASIC2b as candidates for the rat sour-sensing channels. In situ hybridization and reverse transcription-polymerase chain reaction experiments revealed that ASIC2a and ASIC2b transcripts were localized in taste bud cells. Immunohistochemistry and immunoprecipitation also revealed that both subunits were expressed in a subset of taste cells and that some of the cells expressed ASIC2a/ASIC2b heteromeric assemblies. Electrophysiological studies demonstrated that stimulation of acetic acid produced larger ASIC2 currents than did hydrochloric acid at the same pH. ASIC2a/ ASIC2b channels generated maximal inward currents at pH <or= 2.0, which agrees well with the in vivo pH-sensitivity of rat taste cells. The amiloride-sensitivity of ASIC2a/ ASIC2b heteromer lessened with decreasing pH and almost completely disappeared at pH 2.0. These data suggest that ASIC2a and ASIC2b may play roles in sour taste transduction.

Pax3 transcripts in melanoblast development

The transcription factor encoded by PAX3 is among the first expressed in the embryo, with a key role in development of the melanocytic lineage. Re-expression of PAX3, consistently observed in cutaneous malignant melanoma (CMM) as compared to normal melanocytes, appears linked to progression of CMM. Previous research has identified PAX3d (encoded by exons 1-9) as the predominant isoform present in CMM, together the with an alternate isoform PAX3c (encoded by exons 1-8). We investigated the expression of Pax3c and Pax3d transcripts during mouse development. The reverse transcription-polymerase chain reaction and immunohistochemistry experiments presented here implicate these transcripts in melanoblast development and demonstrate significant spatial and temporal differences in their expression. Differences in expression were also noted during active hair regrowth in adult skin, which is accompanied by proliferation and migration of melanoblasts into the hair cortex to color new hair. Results indicate that the defined spatial and temporal expression of Pax3d may be linked to either melanoblast proliferation or migration during melanogenesis.

Chlamydia trachomatis enters a viable but non-cultivable (persistent) state within herpes simplex virus type 2 (HSV-2) co-infected host cells

Epidemiological and clinical studies have shown that double infection with herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occurs in vivo. We hypothesized that co-infection would alter replication of these agents. To test this hypothesis, HeLa cells were infected with C. trachomatis serovar E, followed 24 h later by HSV-2 strain 333. Transmission electron microscopic (TEM) analyses indicated that, by 10 h after HSV addition, reticulate bodies (RBs) in co-infected cells were swollen, aberrantly shaped and electron-lucent. In infectious titre assays, HSV-2 co-infection abrogated production of infectious chlamydial progeny. Western blot analyses indicated that accumulation of chlamydial major outer membrane protein (MOMP) was decreased by HSV co-infection while accumulation of chlamydial heat-shock protein 60-1 (HSP60-1) was increased. Polymerase chain reaction (PCR) experiments indicated that chlamydial genome copy number was unaltered by HSV-2 superinfection. Semi-quantitative, reverse transcription PCR (RT-PCR) experiments demonstrated that levels of chlamydial groEL, ftsK, ftsW, dnaA and unprocessed 16S rRNA transcripts were not changed by HSV-2 super-infection. These data indicate that HSV-2 superinfection drives chlamydia into a viable but non-cultivable state, which is the hallmark of persistence. Because chlamydial HSP60-1 has been associated with immunopathology in vivo, these results also suggest that disease severity might be increased in co-infected individuals.

Investigation of Oscillatoria spongeliae-dominated bacterial communities in four dictyoceratid sponges.

Certain species of marine sponges in the order Dictyoceratida harbor large populations of the cyanobacterial symbiont Oscillatoria spongeliae in the mesohyl (interior) of the sponge. We show that in four of these sponge species (Lamellodysidea herbacea, Lamellodysidea chlorea, Lendenfeldia chondrodes, and Phyllospongia papyracea) from Palau there is a consistent community of alpha-proteobacteria in addition to O. spongeliae that fall within the Rhodobacter group based on 16S rRNA gene analysis. Some of the alpha-proteobacteria in Lendenfeldia chondrodes and P. papyracea but not in the Lamellodysidea spp. contained site-specific insertions in the 16S rRNA gene. Reverse transcription-PCR experiments demonstrated that the largest insertion found in this study (63 bp) is present in the mature rRNA. Lendenfeldia chondrodes was the only sponge found to have another cyanobacterium in the tissue, a Synechocystis sp. We found that the Synechocystis sp. was present in both the pinacoderm (surface epithelial tissue) and mesohyl, in contrast to O. spongeliae, which was only found in the mesohyl through the use of specific fluorescence in situ hybridization experiments. Of the four sponge species, only P. papyracea was found to contain a significant number of gamma-proteobacteria. These results demonstrate that O. spongeliae-dominated bacterial communities in different sponge species can vary considerably and increase our understanding of the bacterial communities found in marine invertebrates.

Differential Expression of TASK Channels between Horizontal Interneurons and Pyramidal Cells of Rat Hippocampus

Among the electrophysiological properties differentiating stratum oriens horizontal interneurons from pyramidal neurons of the CA1 hippocampal subfield are the more depolarized resting potential and the higher input resistance; additionally, these interneurons are also less sensitive to ischemic damage than pyramidal cells. A differential expression of pH-sensitive leakage potassium channels (TASK) could contribute to all of these differences. To test this hypothesis, we studied the expression and properties of TASK channels in the two cell types. Electrophysiological recordings from acute slices showed that barium- and bupivacaine-sensitive TASK currents were detectable in pyramidal cells but not in interneurons and that extracellular acidification caused a much stronger depolarization in pyramidal cells than in interneurons. This pyramidal cell depolarization was paralleled by an increase of the input resistance, suggesting the blockade of a background conductance. Single-cell reverse transcription-PCR experiments showed that the expression profile of TASK channels differ between the two cell types and suggested that these channels mediate an important share of the leakage current of pyramidal cells. We suggest that the different expression of TASK channels in these cell types contribute to their electrophysiological differences and may result in cell-specific sensitivity to extracellular acidification in conditions such as epilepsy and ischemia.

Cell-specific localization of the sulphydryl oxidase QSOX in rat peripheral tissues

Rat quiescin/sulphydryl oxidase (rQSOX) introduces disulphide bridges into peptides and proteins with the reduction of molecular oxygen to hydrogen peroxide. Its occurrence has been previously highlighted in a wide range of organs by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses, methods that have provided information concerning its expression in whole organs but that do not reveal the cell types expressing this enzyme. In this report, in addition to RT-PCR and Western blot experiments, the cell-specific localization of rQSOX has been investigated in a wide range of male and female adult rat tissues by using in situ hybridization and immunohistochemistry. Labelling was detected in most organs and systems including the immune, endocrine and reproductive systems, the respiratory, digestive and urinary tracts and the skin. No labelling was observed in the heart, blood vessel endothelium, liver or smooth and skeletal muscles. rQSOX expression was mainly localized in epithelial cells specialized in secretion, strengthening the hypothesis that QSOX enzymes play an important role in the mechanism of secretion, notably in the folding of secreted proteins. The intracellular patterns of immunolabelling indicate that the protein usually follows the secretory pathway, which is in accordance with its secreted nature and its presumed involvement in the elaboration of the extracellular matrix. In seminiferous tubules, where a high level of expression was noticed, QSOX might play an important physiological role in sperm function and serve as a marker for the diagnosis of male infertility.

Expression of Estrogen Receptor ESR1 and Its 46-kDa Variant in the Gubernaculum Testis1

Testicular descent corresponds to migration of the testis from the abdominal cavity to the scrotum and is essential for proper functioning of the testis. Recent advances in the characterization of estrogen receptor (ESR) subtypes and isoforms in various tissues prompted us to study ESRs within the gubernaculum testis, a structure involved in testicular descent. In the rat gubernaculum, we searched for ESR alpha (Esr1) and beta (Esr2) and for the androgen receptor (Ar), androgens being known to regulate testicular descent. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that Esr1, Esr2, and Ar mRNAs were all expressed in the gubernaculum. Using PEETA (Primer extension, Electrophoresis, Elution, Tailing, and Amplification), we established that all Esr1 leader exons, previously identified in other organs, such as the uterus and pituitary, were transcribed in the gubernaculum, with the major form being O/B. The RNA protection assays, RT-PCR, and Western blot experiments revealed that isoform-specific mRNA transcripts generated by alternative splicing of the C-leader sequence on coding exons 1 and 2 of the Esr1 gene gave the 46- and 66-kDa ESR1 proteins. The ESR1 and AR proteins were found to colocalize in the parenchymal cells of the gubernaculum early in development, whereas AR also was strongly expressed in the muscular cells, both during fetal and postnatal life. The ESR2 protein was weakly expressed, principally in the muscular cells, but only once testicular descent had occurred. The levels of the 46-kDa ESR1 variant (ER46) exceeded those of the 66-kDa ESR1 form (ER66) at periods when the gubernaculum developed. Conversely, the 66-kDa form appears to predominate clearly when the gubernaculum growth was low or completed. The possible role of estrogens on the modulation of the androgen-dependent growth of the gubernaculum and, more widely, on testicular descent is discussed.

Pituitary adenylate cyclase-activating peptide/vasoactive intestinal peptide receptors in human normal mammary gland and breast cancer tissue

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) bind similarly to VPAC1 and VPAC2 receptors, whereas PACAP binds with higher affinity than VIP to PAC1 receptors. Here we demonstrate by different approaches the expression of the three subclasses of PACAP/VIP receptors in human normal and malignant breast tissue. At the mRNA level, reverse transcription–polymerase chain reaction experiments showed VPAC1 and VPAC2 receptors as well as various isoforms (null, hip/hop) of PAC1 receptors due to alternative splicing. At the protein level, Western blot experiments revealed the three subclasses of receptor although no conclusive differences could be established when comparing control, peritumoral and tumoral tissue samples. Immunohistochemistry showed the distribution of these receptors: they were located at epithelial cells in normal and cancer conditions but also in leukocytes at the stromal level in carcinomatous tissue. A weaker immunostaining of PAC1 receptors in normal tissue and a strong density of the three PACAP/VIP receptor subclasses in cancer tissue may be related to differential expression patterns during breast tumor progression but more samples need to be studied to validate this hypothesis. PAC1, VPAC1 and VPAC2 receptors were functional, as shown by their coupling to adenylate cyclase stimulation: VIP, PACAP-27 and PACAP-38 behaved similarly at this level, whereas both VPAC receptors acted alike as shown by means of specific peptide agonists and antagonists. The present results together with the known presence of PACAP and VIP in the mammary gland support a paracrine/autocrine involvement of both peptides at this level in physiological and pathological conditions, i.e. during malignant transformation.

The xylem and phloem transcriptomes from secondary tissues of the Arabidopsis root-hypocotyl.

The growth of secondary xylem and phloem depends on the division of cells in the vascular cambium and results in an increase in the diameter of the root and stem. Very little is known about the genetic mechanisms that control cambial activity and the differentiation of secondary xylem and phloem cell types. To begin to identify new genes required for vascular cell differentiation and function, we performed genome-wide expression profiling of xylem and phloem-cambium isolated from the root-hypocotyl of Arabidopsis (Arabidopsis thaliana). Gene expression in the remaining nonvascular tissue was also profiled. From these transcript profiles, we assembled three sets of genes with expression significantly biased toward xylem, phloem-cambium, or nonvascular tissue. We also assembled three two-tissue sets of genes with expression significantly biased toward xylem/phloem-cambium, xylem/nonvascular, or phloem-cambium/nonvascular tissues. Localizations predicted by transcript profiles were supported by results from promoter-reporter and reverse transcription-polymerase chain reaction experiments with nine xylem- or phloem-cambium-biased genes. An analysis of the members of the phloem-cambium gene set suggested that some genes involved in regulating primary meristems are also regulators of the cambium. Secondary phloem was implicated in the synthesis of auxin, glucosinolates, cytokinin, and gibberellic acid. Transcript profiles also supported the importance of class III HD ZIP and KANADI transcription factors as regulators of radial patterning during secondary growth, and identified several members of the G2-like, NAC, AP2, MADS, and MYB transcription factor families that may play roles as regulators of xylem or phloem cell differentiation and activity.

The role of nitric oxide synthase in reduced vasocontractile responsiveness induced by prolonged alpha1-adrenergic receptor stimulation in rat thoracic aorta.

1 Prolonged exposure (6-12 h) of rat aorta to alpha1-adrenergic receptor (alpha(1)AR) agonist phenylephrine (Phe) leads to a decrease in alpha(1)AR-mediated vasoconstriction. This reduced responsiveness to alpha(1)AR stimulation was strongly dependent on the intactness of the endothelium. 2 We examined the effect of Phe on nitric oxide synthase (NOS) activity by measuring the conversion of [(3)H]L-arginine to [(3)H]L-citrulline in rat aorta or in endothelial cells isolated from rat aorta. Phe stimulation increased NOS activity in control aortas. This response was antagonized by prazosin. However, Phe increased neither the activity of NOS nor intracellular Ca(2+) in the isolated endothelial cells from the control aortas, whereas acetylcholine (Ach) was able to stimulate both responses in these cells. This result suggests that Phe stimulates alpha(1)AR on vascular smooth muscle cells and has an indirect influence on endothelial cells to increase NOS activity. 3 In Phe-exposed aortic rings, basal NOS activity was found to have increased compared to vehicle-exposed control rings. Stimulation with Phe or Ach caused a small increase over basal NOS activity in these preparations. Prolonged exposure to Phe also caused an enhancement of Ach-mediated vasorelaxation in rat aorta. 4 Immunoblot and reverse transcription-polymerase chain reaction experiments showed that prolonged exposure of rat aorta to Phe resulted in an increased expression of eNOS, but not iNOS. This increase was antagonized by nonselective antagonist prazosin. Immunohistochemical staining experiments also showed that expression of eNOS increased in endothelial cells after Phe exposure of the aortas. 5 These results, all together, showed that prolonged exposure of rat aorta to alpha(l)AR agonist Phe enhanced the expression of eNOS and basal NOS activity, which probably causes a decreased vasocontractile response to Phe or to other agonists such as 5HT (5-hydroxytryptamine) in rat aorta. 6 This phenomenon can be considered more as a functional antagonism of vasocontractile response to agonists mediated by endothelium than a specific desensitization of alpha(1)AR-mediated signalling in vascular smooth muscle cells.

High-Salt Diet Inhibits Expression of Angiotensin Type 2 Receptor in Resistance Arteries

Recent studies suggested that type 2 angiotensin receptor (AT2R) could contribute to regulation of blood pressure and/or vascular remodeling. A key question relates to the effects of potential modulators of vascular AT2R expression. In the present work, we evaluated if high salt intake (70 mmol/L NaCl in drinking water) could modulate rat mesenteric artery AT2R function and expression. Angiotensin II dose-response curves were studied in rat perfused pressurized small-diameter arteries in the presence of losartan (AT1R antagonist). Arteries were precontracted with phenylephrine, yielding approximately 30% decrease in resting diameter. AT2R activation by angiotensin-induced dose-dependent relaxation of precontracted arteries (60.1+/-9.1% of phenylephrine-induced contraction, P<0.05). In contrast, AT2R-dependent relaxation was not observed in arteries obtained from rats on high-salt diet. Semi-quantitative reverse-transcription polymerase chain reaction experiments demonstrated reduced amount of AT2R mRNA in arteries of rats on high-salt diet (65.5+/-7.5% of control levels, P<0.05). Western blot studies demonstrated decreased AT2R in mesenteric artery protein fractions of high-salt diet rats (60.0+/-18.0 of control levels, P<0.05). In a second set of experiments, adrenalectomy (4 days) blunted AT2R-mediated vasorelaxation and decreased AT2R mRNA (72.0+/-11.0% of control levels, P<0.05). AT2R abundance in protein fractions of mesenteric arteries of ADX rats was also diminished (64.0+/-13% of control levels, P<0.05). Both, AT2R mRNA and protein downregulation were prevented by mineralocorticoid replacement therapy. Finally, physiological concentrations of aldosterone caused a dose-dependent increase in AT2R mRNA of small diameter mesenteric artery explants. The results are consistent with aldosterone-mediated upregulation AT2R.

SarA Is an Essential Positive Regulator of Staphylococcus epidermidis Biofilm Development

Staphylococcus epidermidis biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA)--poly-N-acetylglucosamine polysaccharide (PNAG) by the products of the icaADBC operon. Recent evidence indicates that SarA, a central regulatory element that controls the production of Staphylococcus aureus virulence factors, is essential for the synthesis of PIA/PNAG and the ensuing biofilm development in this species. Based on the presence of a sarA homolog, we hypothesized that SarA could also be involved in the regulation of the biofilm formation process in S. epidermidis. To investigate this, we constructed nonpolar sarA deletions in two genetically unrelated S. epidermidis clinical strains, O-47 and CH845. The SarA mutants were completely defective in biofilm formation, both in the steady-state conditions of a microtiter dish assay and in the flow conditions of microfermentors. Reverse transcription-PCR experiments showed that the mutation in the sarA gene resulted in downregulation of the icaADBC operon transcription in an IcaR-independent manner. Purified SarA protein showed high-affinity binding to the icaA promoter region by electrophoretic mobility shift assays. Consequently, mutation in sarA provoked a significant decrease in the amount of PIA/PNAG on the cell surface. Furthermore, heterologous complementation of S. aureus sarA mutants with the sarA gene of S. epidermidis completely restored biofilm formation. In summary, SarA appeared to be a positive regulator of transcription of the ica locus, and in its absence, PIA/PNAG production and biofilm formation were diminished. Additionally, we present experimental evidence showing that SarA may be an important regulatory element that controls S. epidermidis virulence factors other than biofilm formation.