Altered gene expression in the host brain caused by a trematode parasite: Neuropeptide genes are preferentially affected during parasitosis

Schistosome parasites adjust the physiology and behavior of their intermediate molluscan hosts to their own benefit. Previous studies demonstrated effects of the avian-schistosome Trichobilharzia ocellata on peptidergic centers in the brain of the intermediate snail host Lymnaea stagnalis. In particular, electrophysiological properties and peptide release of growth- and reproduction-controlling neuroendocrine neurons were affected. We now have examined the possibility that the expression of genes that control physiology and behavior of the host might be altered during parasitosis. A cDNA library of the brain of parasitized Lymnaea was constructed and differentially screened by using mRNA from the brain of both parasitized and nonparasitized snails. This screening yielded a number of clones, including previously identified cDNAs as well as novel neuronal transcripts, which appear to be differentially regulated. The majority of these transcripts encode neuropeptides. Reverse Northern blot analysis confirmed that neuropeptide gene expression is indeed affected in parasitized animals. Moreover, the expression profiles of 10 transcripts tested showed a differential, parasitic stage-specific regulation. Changes in expression could in many cases already be observed between 1.5 and 5 hr postinfection, suggesting that changes in gene expression are a direct effect of parasitosis. We suggest that direct regulation of neuropeptide gene expression is a strategy of parasites to induce physiological and behavioral changes in the host.

Molecular cloning of a cytochrome P450 taxane 10β-hydroxylase cDNA from Taxus and functional expression in yeast

The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5α-acetoxy-10β-ol. Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations. The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences. Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates. This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10β-hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10β-hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means. The 10β-hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway.

High-sensitivity array analysis of gene expression for the early detection of disseminated breast tumor cells in peripheral blood

Early detection is an effective means of reducing cancer mortality. Here, we describe a highly sensitive high-throughput screen that can identify panels of markers for the early detection of solid tumor cells disseminated in peripheral blood. The method is a two-step combination of differential display and high-sensitivity cDNA arrays. In a primary screen, differential display identified 170 candidate marker genes differentially expressed between breast tumor cells and normal breast epithelial cells. In a secondary screen, high-sensitivity arrays assessed expression levels of these genes in 48 blood samples, 22 from healthy volunteers and 26 from breast cancer patients. Cluster analysis identified a group of 12 genes that were elevated in the blood of cancer patients. Permutation analysis of individual genes defined five core genes (P ≤ 0.05, permax test). As a group, the 12 genes generally distinguished accurately between healthy volunteers and patients with breast cancer. Mean expression levels of the 12 genes were elevated in 77% (10 of 13) untreated invasive cancer patients, whereas cluster analysis correctly classified volunteers and patients (P = 0.0022, Fisher's exact test). Quantitative real-time PCR confirmed array results and indicated that the sensitivity of the assay (1:2 × 108 transcripts) was sufficient to detect disseminated solid tumor cells in blood. Expression-based blood assays developed with the screening approach described here have the potential to detect and classify solid tumor cells originating from virtually any primary site in the body.

GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP

The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power of linking cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-AFLP, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. We have developed a computer program (GenEST) that predicts the sizes of virtual transcript-derived fragments (TDFs) of in silico-digested cDNA sequences retrieved from databases. The vast majority of the resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. Sequencing of the corresponding bands excised from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostochiensis. Various novel pathogenicity factors were identified by combining ESTs from the infective stage juveniles with expression profiles of ∼4000 genes in five developmental stages produced by cDNA-AFLP.

Regulation of neuroblast mitosis is determined by PACAP receptor isoform expression

Although neurogenesis in the embryo proceeds in a region- or lineage-specific fashion coincident with neuropeptide expression, a regulatory role for G protein-coupled receptors (GPCR) remains undefined. Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates sympathetic neuroblast proliferation, whereas the peptide inhibits embryonic cortical precursor mitosis. Here, by using ectopic expression strategies, we show that the opposing mitogenic effects of PACAP are determined by expression of PACAP receptor splice isoforms and differential coupling to the phospholipase C (PLC) pathway, as opposed to differences in cellular context. In embryonic day 14 (E14) cortical precursors transfected with the hop receptor variant, but not cells transfected with the short variant, PACAP activates the PLC pathway, increasing intracellular calcium and eliciting translocation of protein kinase C. Ectopic expression of the hop variant in cortical neuroblasts transforms the antimitotic effect of PACAP into a promitogenic signal. Furthermore, PACAP promitogenic effects required PLC pathway function indicated by antagonist U-73122 studies in hop-transfected cortical cells and native sympathetic neuroblasts. These observations highlight the critical role of lineage-specific expression of GPCR variants in determining mitogenic signaling in neural precursors.

NPH4, a Conditional Modulator of Auxin-Dependent Differential Growth Responses in Arabidopsis1

Although sessile in nature, plants are able to use a number of mechanisms to modify their morphology in response to changing environmental conditions. Differential growth is one such mechanism. Despite its importance in plant development, little is known about the molecular events regulating the establishment of differential growth. Here we report analyses of the nph4 (nonphototropic hypocotyl) mutants of Arabidopsis that suggest that the NPH4 protein plays a central role in the modulation of auxin-dependent differential growth. Results from physiological studies demonstrate that NPH4 activity is conditionally required for a number of differential growth responses, including phototropism, gravitropism, phytochrome-dependent hypocotyl curvature, apical hook maintenance, and abaxial/adaxial leaf-blade expansion. The nph4 mutants exhibited auxin resistance and severely impaired auxin-dependent gene expression, indicating that the defects associated with differential growth likely arise because of altered auxin responsiveness. Moreover, the auxin signaling events mediating phototropism are genetically correlated with the abundance of the NPH4 protein.

Differential Regulation of Enolase during Anaerobiosis in Maize1

It was reported previously that enolase enzyme activity and ENO1 transcript levels are induced by anaerobic stress in maize (Zea mays). Here we show that not all isoforms of maize enolase are anaerobically induced. We cloned and sequenced a second enolase cDNA clone (pENO2) from maize. Sequence analysis showed that pENO2 shares 75.6% nucleotide and 89.5% deduced amino acid sequence identity with pENO1 and is encoded by a distinct gene. Expression of ENO2 is constitutive under aerobic conditions, whereas ENO1 levels are induced 10-fold in maize roots after 24 h of anaerobic treatment. Western-blot analysis and N-terminal sequencing of in vivo-labeled maize roots identified two major proteins selectively synthesized upon anaerobic stress as isozymes of enolase. We describe the expression of enolase in maize roots under anaerobic stress.

Differential Control of Xanthophylls and Light-Induced Stress Proteins, as Opposed to Light-Harvesting Chlorophyll a/b Proteins, during Photosynthetic Acclimation of Barley Leaves to Light Irradiance

Barley (Hordeum vulgare L.) plants were grown at different photon flux densities ranging from 100 to 1800 μmol m−2 s−1 in air and/or in atmospheres with reduced levels of O2 and CO2. Low O2 and CO2 partial pressures allowed plants to grow under high photosystem II (PSII) excitation pressure, estimated in vivo by chlorophyll fluorescence measurements, at moderate photon flux densities. The xanthophyll-cycle pigments, the early light-inducible proteins, and their mRNA accumulated with increasing PSII excitation pressure irrespective of the way high excitation pressure was obtained (high-light irradiance or decreased CO2 and O2 availability). These findings indicate that the reduction state of electron transport chain components could be involved in light sensing for the regulation of nuclear-encoded chloroplast gene expression. In contrast, no correlation was found between the reduction state of PSII and various indicators of the PSII light-harvesting system, such as the chlorophyll a-to-b ratio, the abundance of the major pigment-protein complex of PSII (LHCII), the mRNA level of LHCII, the light-saturation curve of O2 evolution, and the induced chlorophyll-fluorescence rise. We conclude that the chlorophyll antenna size of PSII is not governed by the redox state of PSII in higher plants and, consequently, regulation of early light-inducible protein synthesis is different from that of LHCII.

Effects of Short- and Long-Term Elevated CO2 on the Expression of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Genes and Carbohydrate Accumulation in Leaves of Arabidopsis thaliana (L.) Heynh.1

To investigate the proposed molecular characteristics of sugar-mediated repression of photosynthetic genes during plant acclimation to elevated CO2, we examined the relationship between the accumulation and metabolism of nonstructural carbohydrates and changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) gene expression in leaves of Arabidopsis thaliana exposed to elevated CO2. Long-term growth of Arabidopsis at high CO2 (1000 μL L−1) resulted in a 2-fold increase in nonstructural carbohydrates, a large decrease in the expression of Rubisco protein and in the transcript of rbcL, the gene encoding the large subunit of Rubisco (approximately 35–40%), and an even greater decline in mRNA of rbcS, the gene encoding the small subunit (approximately 60%). This differential response of protein and mRNAs suggests that transcriptional/posttranscriptional processes and protein turnover may determine the final amount of leaf Rubisco protein at high CO2. Analysis of mRNA levels of individual rbcS genes indicated that reduction in total rbcS transcripts was caused by decreased expression of all four rbcS genes. Short-term transfer of Arabidopsis plants grown at ambient CO2 to high CO2 resulted in a decrease in total rbcS mRNA by d 6, whereas Rubisco content and rbcL mRNA decreased by d 9. Transfer to high CO2 reduced the maximum expression level of the primary rbcS genes (1A and, particularly, 3B) by limiting their normal pattern of accumulation through the night period. The decreased nighttime levels of rbcS mRNA were associated with a nocturnal increase in leaf hexoses. We suggest that prolonged nighttime hexose metabolism resulting from exposure to elevated CO2 affects rbcS transcript accumulation and, ultimately, the level of Rubisco protein.

Organization and expression of canine olfactory receptor genes.

Four members of the canine olfactory receptor gene family were characterized. The predicted proteins shared 40-64% identity with previously identified olfactory receptors. The four subfamilies identified in Southern hybridization experiments had as few as 2 and as many as 20 members. All four genes were expressed exclusively in olfactory epithelium. Expression of multiple members of the larger subfamilies was detected, suggesting that most if not all of the cross-hybridizing bands in genomic Southern blots represented actively transcribed olfactory receptor genes. Analysis of large DNA fragments using Southern blots of pulsed-field gels indicated that subfamily members were clustered together, and that two of the subfamilies were closely linked in the dog genome. Analysis of the four olfactory receptor gene subfamilies in 26 breeds of dog provided evidence that the number of genes per subfamily was stable in spite of differential selection on the basis of olfactory acuity in scent hounds, sight hounds, and toy breeds.

Differential regulation of beta 1 and beta 2 integrin avidity by chemoattractants in eosinophils.

The CC chemokines regulated on activation normal T expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), and the anaphylatoxin C5a, induce activation, degranulation, chemotaxis, and transendothelial migration of eosinophils. Adhesion assays on purified ligands showed differential regulation of beta 1 and beta 2 integrin avidity in eosinophils. Adhesiveness of VLA-4 (alpha 4 beta 1, CD29/CD49d) for vascular cell adhesion molecule 1 or fibronectin was rapidly increased but subsequently reduced by RANTES, MCP-3, or C5a. The deactivation of VLA-4 lead to cell detachment, whereas phorbol 12-myristate 13-acetate induced sustained activation of VLA-4. In contrast, chemoattractants stimulated a prolonged increase in the adhesiveness of Mac-1 (alpha M beta 2, CD11b/CD18) for intercellular adhesion molecule 1. Inhibition by pertussis toxin confirmed signaling via G protein-coupled receptors. Chemoattractants induced transient, while phorbol 12-myristate 13-acetate induced sustained actin polymerization. Disruption of actin filaments by cytochalasins inhibited increases in avidity of VLA-4 but not of Mac-1. Chemoattractants did not upregulate a Mn2+-inducible beta 1 neoepitope defined by the mAb 9EG7, but induced prolonged expression of a Mac-1 activation epitope recognized by the mAb CBRM1/5. This mAb inhibited chemoattractant-stimulated adhesion of eosinophils to intercellular adhesion molecule 1. Thus, regulation of VLA-4 was dependent on the actin cytoskeleton, whereas conformational changes appeared to be crucial for activation of Mac-1. To our knowledge, this is the first demonstration that physiological agonists, such as chemoattractants, can differentially regulate the avidity of a beta 1 and a beta 2 integrin expressed on the same leukocyte.

Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules.

To replicate, HIV-1 must integrate a cDNA copy of the viral RNA genome into a chromosome of the host. The integration system is a promising target for antiretroviral agents, but to date no clinically useful integration inhibitors have been identified. Previous screens for integrase inhibitors have assayed inhibition of reactions containing HIV-1 integrase purified from an Escherichia coli expression system. Here we compare action of inhibitors in vitro on purified integrase and on subviral preintegration complexes (PICs) isolated from lymphoid cells infected with HIV-1. We find that many inhibitors active against purified integrase are inactive against PICs. Using PIC assays as a primary screen, we have identified three new anthraquinone inhibitors active against PICs and also against purified integrase. We propose that PIC assays are the closest in vitro match to integration in vivo and, as such, are particularly appropriate for identifying promising integration inhibitors.

Differential display of intestinal mRNAs regulated by dietary zinc.

Regulation of gene expression by zinc is well established, especially through the metal response elements of the metallothionein genes; however, most other aspects of the functions of zinc in gene expression remain unknown. We have looked for intestinal mRNAs that are regulated by dietary zinc status. Using the reverse transcriptase-PCR method of mRNA differential display, we compared intestinal mRNA from rats that were maintained for 18 days in one of three dietary groups: zinc-deficient, zinc-adequate, and pair-fed zinc-adequate. At the end of this period, total RNA was prepared from the intestine and analyzed by mRNA differential display. Under these conditions, only differentially displayed cDNA bands that varied in the zinc-deficient group, relative to the zinc-adequate groups, were selected. Utilizing two anchored oligo-dT3' PCR primers and a total of 27 arbitrary decamers as 5' PCR primers, our results yielded 47 differentially displayed cDNA bands from intestinal RNA. Thirty were increased in zinc deficiency, and 17 were decreased. Nineteen bands were subcloned and sequenced. Eleven of these were detectable on Northern blots, of which four were confirmed as regulated. Three of these have homology to known genes: cholecystokinin, uroguanylin, and ubiquinone oxidoreductase. The fourth is a novel sequence as it has no significant homology in GenBank. The remainder of those cloned included novel sequences, as well as matches to reported expressed sequence tags, and functionally identified genes. Further characterization of the regulated sequences identified here will show whether they are primary or secondary effects of zinc deficiency.

Differential effects of staphylococcal toxic shock syndrome toxin-1 on B cell apoptosis.

Superantigens, such as toxic shock syndrome toxin 1 (TSST-1), have been implicated in the pathogenesis of several autoimmune and allergic diseases associated with polyclonal B cell activation. In this report, we studied the in vitro effects of TSST-1 on B cell activation. We show herein that TSST-1 produced antagonistic effects on Ig synthesis by peripheral blood mononuclear cells (PBMC) from normal subjects, depending on the concentration used; Ig production was inhibited at 1000 pg/ml (P < 0.01) and enhanced at 1 and 0.01 pg/ml (P < 0.01) of toxin. Cultures of PBMC were then examined for morphologic features and DNA fragmentation characteristic for apoptosis. B cells exhibited a significantly higher (P < 0.01) incidence of apoptosis after stimulation with 1000 pg/ml of TSST-1 compared with 1 or 0.01 pg/ml of toxin or medium alone. Abundant expression of Fas, a cell surface protein that mediates apoptosis, was detected on B cells after stimulation with 1000 pg/ml of TSST-1 and was significantly higher on B cells undergoing apoptosis than on live cells (P = 0.01). Additionally, increased Fas expression and B cell death occurred at concentrations of TSST-1 inducing the production of high amounts of gamma interferon (IFN-gamma), and both events could be blocked by neutralizing anti-IFN-gamma antibody. These findings suggest that high concentrations of TSST-1 can induce IFN-gamma-dependent B cell apoptosis, whereas at low concentrations it stimulates Ig synthesis by PBMC from normal subjects. These findings support the concept that staphylococcal toxins have a role in B cell hyperactivity in autoimmunity and allergy.

Adenylyl cyclase inhibition and altered G protein subunit expression and ADP-ribosylation patterns in tissues and cells from Gi2 alpha-/-mice.

The inhibition of alpha i2-/- mouse cardiac isoproterenol-stimulated adenylyl cyclase (AC; EC 4.6.1.1) activity by carbachol and that of alpha i2-/- adipocyte AC by phenylisopropyladenosine (PIA), prostaglandin E2, and nicotinic acid were partially, but not completely, inhibited. While the inhibition of cardiac AC was affected in all alpha i2-/- animals tested, only 50% of the alpha i2-/- animals showed an impaired inhibition of adipocyte AC, indicative of a partial penetrance of this phenotype. In agreement with previous results, the data show that Gi2 mediates hormonal inhibition of AC and that Gi3 and/or Gi1 is capable of doing the same but with a lower efficacy. Disruption of the alpha i2 gene affected about equally the actions of all the receptors studied, indicating that none of them exhibits a striking specificity for one type of Gi over another and that receptors are likely to he selective rather than specific in their interaction with functionally homologous G proteins (e.g., Gi1, Gi2, Gi3). Western analysis of G protein subunit levels in simian virus 40-transformed primary embryonic fibroblasts from alpha i2+/+ and alpha i2-/- animals showed that alpha i2 accounts for about 50% of the immunopositive G protein alpha subunits and that loss of the alpha i2 is accompanied by a parallel reduction in G beta 35 and G beta 36 subunits and by a 30-50% increase in alpha i3. This suggests that G beta-gamma levels may be regulated passively through differential rates of turnover in their free vs. trimeric states. The existence of compensatory increase(s) in alpha i subunit expression raises the possibility that the lack of effect of a missing alpha i2 on AC inhibition in adipocytes of some alpha i2-/- animals may be the reflection of a more pronounced compensatory expression of alpha i3 and/or alpha i1.