Important Phenotypic Changes Research Articles

Phenoscape: Identifying Candidate Genes for Evolutionary Phenotypes.

Phenotypes resulting from mutations in genetic model organisms can help reveal candidate genes for evolutionarily important phenotypic changes in related taxa. Although testing candidate gene hypotheses experimentally in nonmodel organisms is typically difficult, ontology-driven information systems can help generate testable hypotheses about developmental processes in experimentally tractable organisms. Here, we tested candidate gene hypotheses suggested by expert use of the Phenoscape Knowledgebase, specifically looking for genes that are candidates responsible for evolutionarily interesting phenotypes in the ostariophysan fishes that bear resemblance to mutant phenotypes in zebrafish. For this, we searched ZFIN for genetic perturbations that result in either loss of basihyal element or loss of scales phenotypes, because these are the ancestral phenotypes observed in catfishes (Siluriformes). We tested the identified candidate genes by examining their endogenous expression patterns in the channel catfish, Ictalurus punctatus. The experimental results were consistent with the hypotheses that these features evolved through disruption in developmental pathways at, or upstream of, brpf1 and eda/edar for the ancestral losses of basihyal element and scales, respectively. These results demonstrate that ontological annotations of the phenotypic effects of genetic alterations in model organisms, when aggregated within a knowledgebase, can be used effectively to generate testable, and useful, hypotheses about evolutionary changes in morphology.

Visual and odour cues: plant responses to pollination and herbivory affect the behaviour of flower visitors

SummaryPlants evolved strategies to attract pollinators that are essential for reproduction. However, plant defence against herbivores may trade off with pollinator attraction.Here, we investigated the role of inducible plant secondary metabolites in such a trade‐off. Our objective was to reveal the mechanisms underlying the effects of induced plant responses to pollination and herbivory. We assessed how responses of plants to pollination and insect herbivory affect the behaviour of flower visitors. Subsequently, we investigated how the production of volatile and non‐volatile compounds changes after pollination and herbivory.Both herbivores and pollinators induced important phenotypic changes in flowers.Brassica nigraplants respond to pollination and herbivory with changes in the profile of volatiles and non‐volatiles of their flowers. Our results show that butterflies use different cues when searching for an oviposition site or a nectar source. Pollination status influenced the behaviour of butterflies, but not that of syrphid flies.We discuss the results in the context of the trade‐off between defence and reproduction in plants and suggest that systemic responses to herbivores can interfere with local responses to pollination. Therefore, these responses must be addressed in an integrated way because, in nature, plants are simultaneously exposed to herbivores and pollinators.

Speeding up the Analysis of Neuron Morphology using Parallel Processing

Researchers using High Content Screening systems generate thousands of fluorescently labeled cell images from which they measure subtle but important phenotypic changes summarized by dozens of parameters. Large image datasets and fast turnaround requirement have made the efficient High Content Analysis a challenging task. This paper studies multi-core based high performance image analysis and its application to data and compute-intensive High Content Analyses. A vertical parallelization strategy is employed and an automated parallelization framework is implemented to automatically dispatch image processing tasks. The strategy is based on allocation of different images to separate processors so that each image is analyzed sequentially on a single processor and multiple images are processed by separate processors in parallel. Experiments demonstrate that this approach, of a generic character, considerably increases throughput.

OsLG1 regulates a closed panicle trait in domesticated rice

Reduction in seed shattering was an important phenotypic change during cereal domestication. Here we show that a simple morphological change in rice panicle shape, controlled by the SPR3 locus, has a large impact on seed-shedding and pollinating behaviors. In the wild genetic background of rice, we found that plants with a cultivated-like type of closed panicle had significantly reduced seed shedding through seed retention. In addition, the long awns in closed panicles disturbed the free exposure of anthers and stigmas on the flowering spikelets, resulting in a significant reduction of the outcrossing rate. We localized the SPR3 locus to a 9.3-kb genomic region, and our complementation tests suggest that this region regulates the liguleless gene (OsLG1). Sequencing analysis identified reduced nucleotide diversity and a selective sweep at the SPR3 locus in cultivated rice. Our results suggest that a closed panicle was a selected trait during rice domestication.

Adenoviral dominant-negative soluble PDGFRβ improves hepatic collagen, systemic hemodynamics, and portal pressure in fibrotic rats

Platelet-derived growth factor (PDGF) is the most potent stimulus for proliferation and migration of stellate cells. PDGF receptor β (PDGFRβ) expression is an important phenotypic change in myofibroblastic cells that mediates proliferation and chemotaxis. Here we analyzed the relationship between PDGFRβ expression, hemodynamic deterioration, and fibrosis in CCl(4)-treated rats. Thereafter, we investigated the effects produced by an adenovirus encoding a dominant-negative soluble PDGFRβ (sPDGFRβ) on hemodynamic parameters, PDGFRβ signaling pathway, and fibrosis. Mean arterial pressure, portal pressure, PDGFRβ mRNA expression, and hepatic collagen were assessed in 6 controls and 21 rats induced to hepatic fibrosis/cirrhosis. Next, 30 fibrotic rats were randomized into three groups receiving iv saline and an adenovirus encoding for sPDGFRβ or β-galactosidase. After 7days, mean arterial pressure, portal pressure, serum sPDGFRβ, and hepatic collagen were measured. CCl(4)-treated animals for 18weeks showed a significantly higher increase in PDGFRβ mRNA compared to those treated for 13weeks and control rats. In CCl(4)-treated rats, the fibrous tissue area ranged from moderate to severe fibrosis. A direct relationship between the degree of fibrosis, hemodynamic changes, and PDGFRβ expression was observed. Fibrotic rats transduced with the adenovirus encoding sPDGFRβ showed increased mean arterial pressure, decreased portal pressure, lower activation of the PDGFRβ signaling pathway, and reduced hepatic collagen than fibrotic rats receiving β-galactosidase or saline. PDGFRβ activation closely correlates with hemodynamic disorders and increased fibrosis in CCl(4)-treated rats. Adenoviral dominant negative soluble PDGFRβ improved fibrosis. As a result, the hemodynamic abnormalities were ameliorated.

Relations between phenotypic changes of spores and biofilm production by Bacillus atrophaeus ATCC 9372 growing in solid-state fermentation

Bacillus spp. spores are usually obtained from strains cultivated in artificial media. However, in natural habitats, spores are predominantly formed from bacteria present in highly surface-associated communities of cells. Solid-state fermentation (SSF) is the culture method that best mimetizes the natural environment of many microorganisms that grow attached to the surface of solid particles. This study aims to confirm that sporulation through SSF of Bacillus atrophaeus occurs by biofilm formation and that this model of fermentation promotes important phenotypic changes in the spores. Sporulation on standard agar and by SSF with sand and sugarcane bagasse as support was followed by a comparative study of the formed spores. Growth characteristics, metabolic and enzymatic profiles confirmed that sporulation through SSF occurs by biofilm formation promoting important phenotypic changes. It was possible to demonstrate that spores coat had different structure and the presence of ridges only on SSF spores' surface. The sporulation conditions did not affect the dry-heat spore resistance. The type of support evaluated also influenced in the phenotypic alterations; however, the used substrates did not cause interference. This work provides novel information about B. atrophaeus response when submitted to different sporulation conditions and proposes a new concept about bacterial biofilm formation by SSF.

Fibroblast aging: intrinsic and extrinsic factors

Age-related changes and environmental stresses affect all of the cellular elements in the skin, but changes that occur in stromal fibroblasts are particularly important, because it is these cells that maintain the connective tissue of the skin. From the standpoint of maintaining connective tissue, the most important phenotypic changes include a progressive increase in connective tissue damage, mediated by matrix metalloproteinases (MMPs) and a reduction in new collagen synthesis. Intrinsic changes in the aged fibroblast as well as exposure to environmental insults (e.g. ultraviolet radiation) alter the intracellular signaling events that regulate both collagen synthesis and MMP elaboration. Furthermore, as the connective tissue becomes progressively damaged, it eventually becomes a stimulator of reduced fibroblast synthetic function. The result is a downward spiral that ends, if not checked, in the cosmetic and medical consequences that define aged skin.

Chymotrypsin-Like Protease (Chymase) Mediates Endothelial Activation by Factors Derived From Preeclamptic Placentas

Endothelial cells (EC) activation is an important inflammatory phenotypic change in the vascular system in women with preeclampsia (PE). In PE, maternal vessel chymotrypsin-like protease (CLP)/chymase expression was increased. Chymase is an inflammatory protease. In this study, we specifically examined whether placental-derived CLP could induce EC activation and whether EC activation is associated with increased cellular protease expression. Human uterine microvascular endothelial cells (UtMVECs) were used. Endothelial activation was determined by endothelial adhesion molecule P-selectin, E-selectin, inter-cellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM) expressions and by extracellular regulated kinase (ERK) activity. Activation of endogenous CLP/chymase associated with ERK phosphorylation was further examined by CLP/chymase short interfering RNA (siRNA). Our results showed that cells treated with PE placental conditioned medium revealed increased P-selectin, E-selectin, and VCAM-1 expressions and increased ERK phosphorylation. Increased endothelial adhesion molecule expression and phosphorylated ERK (pERK) induction could be attenuated or abolished by depletion of CLP in the conditioned medium or by transfecting ECs with CLP/chymase siRNA. These observations suggest that placental-derived CLP/chymase is responsible for inducing endothelial inflammatory phenotypic changes possibly by upregulation of cell adhesion molecule expressions, activation of cellular protease, and induction of ERK phosphorylation. We speculate that activation of endothelial CLP/chymase may directly relate to the increased inflammatory phenotypic changes in the vascular system in women with PE.

Constraints to Genetic Exchange Support Gene Coadaptation in a Tripartite RNA Virus

Genetic exchange by recombination, or reassortment of genomic segments, has been shown to be an important process in RNA virus evolution, resulting often in important phenotypic changes affecting host range and virulence. However, data from numerous systems indicate that reassortant or recombinant genotypes could be selected against in virus populations and suggest that there is coadaptation among viral genes. Little is known about the factors affecting the frequency of reassortants and recombinants along the virus life cycle. We have explored this issue by estimating the frequency of reassortant and recombinant genotypes in experimental populations of Cucumber mosaic virus derived from mixed infections with four different pairs of isolates that differed in about 12% of their nucleotide sequence. Genetic composition of progeny populations were analyzed at various steps of the virus life cycle during host colonization: infection of leaf cells, cell-to-cell movement within the inoculated leaf, encapsidation of progeny genomes, and systemic movement to upper noninoculated leaves. Results indicated that reassortant frequencies do not correspond to random expectations and that selection operates against reassortant genotypes. The intensity of selection, estimated through the use of log-linear models, increased as host colonization progressed. No recombinant was detected in any progeny. Hence, results showed the existence of constraints to genetic exchange linked to various steps of the virus life cycle, so that genotypes with heterologous gene combinations were less fit and disappeared from the population. These results contribute to explain the low frequency of recombinants and reassortants in natural populations of many viruses, in spite of high rates of genetic exchange. More generally, the present work supports the hypothesis of coadaptation of gene complexes within the viral genomes.

Proinflammatory cytokines released from microglia inhibit gap junctions in astrocytes: potentiation by β‐amyloid

Brain inflammation is characterized by a reactive gliosis involving the activation of astrocytes and microglia. This process, common to many brain injuries and diseases, underlies important phenotypic changes in these two glial cell types. One characteristic feature of astrocytes is their high level of intercellular communication mediated by gap junctions. Previously, we have reported that astrocyte gap junctional communication (AGJC) and the expression of connexin 43 (Cx43), the main constitutive protein of gap junctions, are inhibited in microglia (MG)-astrocyte cocultures. Here, we report that bacterial lipopolysaccharide activation of microglia increases their inhibitory effect on Cx43 expression and AGJC. This inhibition is mimicked by treating astrocyte cultures with conditioned medium harvested from activated microglia. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were identified as being the main factors responsible for this conditioned medium-mediated activity. Interestingly, an inflammatory response characterized by MG activation and reactive astrocytes occurs in Alzheimer's disease, at sites of beta-amyloid (Abeta) deposits. We found that this peptide potentiates the inhibitory effect of a conditioned medium diluted at a concentration that is not effective per se. This potentiation is prevented by treating astrocytes with specific blockers of IL-1beta and TNF-alpha activities. Thus, the suppression of communication between astrocytes, induced by activated MG could contribute to the proposed role of reactive gliosis in this neurodegenerative disease.

Triplex DNA-mediated downregulation of Ets2 expression results in growth inhibition and apoptosis in human prostate cancer cells.

Ets2 is a member of the Ets family of transcription factors that in humans comprise 25 distinct members. Various Ets-domain transcription factors have been implicated in cancer development. Ets2 is expressed in prostate and breast cancer cells and is thought to have a role in promoting growth and survival in these cell types. However, a definitive role and the mechanisms whereby Ets2 acts in cancer cells are still unclear. Structural and functional similarities as well as overlapping DNA binding specificities complicate the identification of the specific roles of the various Ets factors. In this study, we used a triplex-forming oligonucleotide (TFO) to selectively inhibit Ets2 transcription in prostate cancer cells. We had previously shown that the Ets2-targeting TFO, which was directed to a unique purine-rich sequence critical for Ets2 promoter activity, acted with a high degree of sequence-specificity and target selectivity. TFO-mediated downregulation of Ets2 in prostate cancer cells induced important phenotypic changes, including inhibition of anchorage-dependent and anchorage -independent growth, cell cycle alterations and induction of apoptotic cell death. Expression of Ets2 under the control of a heterologous promoter abolished the anti-proliferative effects of the TFO in both short- and long-term assays, suggesting that these effects were a direct result of downregulation of Ets2 transcription and confirming target selectivity of the TFO. Furthermore, normal human fibroblasts, which expressed low levels of Ets2, were not affected by the Ets2-targeting TFO. Downregulation of Ets2 in prostate cancer cells was associated with reduced levels of the anti-apoptotic protein bcl-x(L) and growth regulatory factors cyclin D1 and c-myc. These data revealed a specific role of this transcription factor in promoting growth and survival of prostate cancer cells. Furthermore, the activity and selectivity of the Ets2-targeting TFO suggest that it might represent a valid approach to prostate cancer therapy.

Expression of a retinol dehydrogenase (hRoDH-4), a member of the retinol/steroid dehydrogenase family implicated in retinoic acid biosynthesis, in normal and neoplastic endometria

Objective: Retinoic acid plays an essential role in epithelial differentiation, and retinoid homeostasis is disrupted in cancers of epithelial origin. The goal of this study was to determine whether hRoDH-4, an enzyme that can catalyze the first and rate-limiting step in retinoic acid biosynthesis, is expressed in normal endometrium and, if so, whether its expression is altered in endometrial cancer. Study design: Proliferative, secretory, hyperplastic, and neoplastic endometria were examined by immunocytochemistry for hRoDH-4 protein and by reverse transcriptase-polymerase chain reaction for the hRoDH-4 transcript. Results: In proliferative and secretory glandular epithelia, immunoreactive hRoDH-4 was uniformly pres-ent. In endometrial cancers, hRoDH-4 immunoreactivity was markedly reduced in many neoplastic epithelial cells. Expression of hRoDH-4 in normal and neoplastic endometrium was confirmed by findings on reverse transcriptase-polymerase chain reaction. Conclusion: These findings are consistent with the hypothesis that altered expression of enzymes essential for in situ retinoic acid biosynthesis is an important phenotypic change associated with the development of endometrial cancer. (Am J Obstet Gynecol 2002;186:675-83.)

Involvement of peroxynitrite on the early loss of p450 in short-term hepatocyte cultures.

The biological chemistry of nitric oxide (NO) in the oxygenated cellular environment is extremely complex. It involves the direct interaction of NO with specific biomolecules and the so-called indirect effects, due to secondary more potent oxidant species derived from NO which are also able to react with DNA, lipids, thiols and transition metals (Wink et al., 1996; Nathan, 1992). In addition to its regulatory role as a signalling molecule (Nathan, 1992; Moncada and Palmer, 1991) it has become evident that NO (or NO-derived species) is a critical factor involved in various toxicological mechanisms (Wink et al., 1996; Wang et al., 1998; Estevez et al., 1999; Wink et al., 1999). Some controversy exists however about the damaging vs. protective actions of NO on oxidative injury, whose biological significance in living cells and tissues remains still ill defined. Research in this laboratory (López-García, 1998; López-García and Sanz-Gonzalez, 2000) has shown that NO synthesis is significantly activated in hepatocytes from control rats following isolation by the classical collagenase-based procedure. NO overproduction appears to be due to the very early activation of liver constitutive Ca2+-dependent NO synthase (cNOS). Previous results have also provided first experimental evidence for the direct involvement of endogenously generated NO as a causal factor responsible for important phenotypic changes commonly observed in short-term cultured hepatocytes, which includes the early impairment of hepatocyte mitochondrial function--i.e., transient cell energy depletion--and glucose metabolism, and the well-known quick and irreversible loss of P450 content (López et al. 1987; López-García, 1998). This study aims to further characterise the mechanisms underlying this phenomenon. Results show that the hepatocyte isolation procedure (the commonly employed collagenase-based two step liver perfusion method) induces strong oxidative stress that lasts for at least 4 h in culture and involves both oxygen-derived (ROS) and nitrogen-derived (RNS) reactive species. On the basis of the combined use of dihydrorhodamine 123 (DHR) as a probe and L-NAME (N(G)-nitro-L-arginine methyl ester) to efficiently block NO synthesis, the analysis of the amount, the time-course pattern, and the nature of the species involved support the view that peroxynitrite* (PN) is readily formed within the early culture hours. Immunodetection of protein bound 3-nitrotyrosine provides direct evidence for PN generation upon hepatocyte isolation: several nitrated protein bands--most already present after only 30 min of liver perfusion and quantitatively increasing for the first 2 hours in culture--have been identified as preferential PN protein targets in the different cellular compartments. Since the early inhibition of NO synthesis is enough to provide full maintenance of the hepatocyte initial P450 content, results support the view that PN--while not affecting cell viability and monolayer development--is the main species likely responsible for the early loss of P450 in short-term cultured hepatocytes.

Phylogeny, speciation and species turnover. The case of the Mediterranean gastropods of genus Cyclope Risso, 1826

The genus Cyclope Risso, 1826 (family Nassariidae) has appeared in the fossil record since the Pliocene. Although it is still found today, the teleoconch morphology has never undergone modification, despite the fact that the protoconch morphologies of fossils (multispiral) and living forms (paucispiral) are different. They vary in their embryological and larval development and, hence, are two different species: C. migliorinii (Bevilacqua, 1928), the fossil species, and C. neritea (Linnaeus, 1758), the living species. We discuss the morphologic modifications in the evolution of this genus: the speciation that leads to its appearance and the speciation driving the Pliocene species to the living one. The order and the direction of these changes are based on phylogenetic analysis. No intermediate forms have been found showing a gradual morphological change that could have been worked by natural selection. Our analysis takes as the origin of the morphological novelties the genetic modifications in the ontogenetic processes which resulted in rapid and important phenotypic changes. Both speciation processes are sympatric cladogenetic. The changes that determine the appearance of the genus affect only the teleoconch, not the larval development. The modifications that lead from one species to the other, within the genus Cycope, affect the larval development exclusively. This points to a certain disconnection between the development of the embryo-larval phase and the young-adult formation, such that evolutionary processes could have occurred independently in different ontogenetic stages. The influence of larval ecology in relation to extinction of the ancestor and persistence of the derived species is also analysed. We hypothesize that climatic fluctuations may have affected the planktonic larvae of the fossil species, driving it to extinction. The living species, developing without the planktonic phase, would have resisted these climatic changes. We consider that the mechanisms described as drivers of the evolution of this genus can be of more general validity in prosobranch gastropods.

NEGATIVE REGULATION OF FIBRONECTIN GENE-TRANSCRIPTION BY EJ-HA-RAS AND C-SIS ONCOGENES IN BALB/C 3T3 CELLS

Down-regulation of fibronectin gene expression at cell surfaces by various oncogenes is an important phenotypic change of many transformed cells. Mechanisms effecting this loss of cell surface fibronectin remain poorly understood. An isogenic mouse fibroblast system, Balb/c 3T3, was used to investigate the effect of oncogene transformation on fibronectin gene transcription. Fibronectin mRNA levels were shown to decrease by >80% when 3T3 cells became stably transformed by EJ-Ha-ras or c-sis oncogenes. The mouse fibronectin gene promoter was subsequently cloned from a mouse genomic library and inserted into reporter gene constructs. Transient transfection assays with the 1.2 kb fibronectin promoter demonstrated that its activity was also decreased in the two transformed cell lines to a similar extent as fibronectin mRNA levels. Co-transfections into 3T3 cells with both the promoter construct and either Ha-ras or c-sis expression constructs provided further support for negative regulation of fibronectin gene transcription by oncogene expression. Potential mechanisms for such transcriptional regulation are proposed based on deletion analysis of the fibronectin promoter. These data suggest that down-regulation of fibronectin gene transcription can contribute directly to the loss of cell surface fibronectin in transformed murine fibroblasts.

Cell shape and increased free cytosolic calcium [Ca 2+] i induced by growth factors

Growth factors stimulate DNA synthesis of neoplastic cells but not of non-neoplastic cells in suspension cultures. Similarly, growth ceases in dense monolayers of non-neoplastic cells, while crowded neoplastic cells continue to grow. The mechanism of these important phenotypic changes in unknown; the block in growth stimulation could occur in early events of signal transduction at the plasma membrane or in a late step in the final steps of gene activation and induction of DNA synthesis. One particular early intracellular event, [Ca 2+] i increases, is in fact necessary for the induction of DNA synthesis in attached non-neoplastic Balb c 3T3 cell stimulated by platelet-derived growth factor (PDGF). We therefore used digital image analysis of intracellular Fura-2 fluorescence to determine whether PDGF can stimulate [Ca 2+] i transients in suspension or in dense monolayer cultures of Balb c 3T3 cells. In dense cells (>8 × 10 4 cells/cm 2) the basal [Ca 2+] i and [Ca 2+] i response to PDGF stimulation were both lower than those in sparser, more spread cells. PDGF also did not release internal stores of Ca 2+ or produce Ca 2+ influx in completely suspended cells. Remarkably, attachment alone, with minimal cell spreading, was enough to reinitiate the entire early signalling mechanism stimulated by PDGF. Thus, a block in PDGF-induced [Ca 2+] i increases may contribute to the inability of PDGF to stimulate DNA synthesis in suspended non-neoplastic cells. This early block in signal transduction must be abrogated in neoplastic cells growing in suspension and dense monolayer cultures.

Management of severe ovarian hyperstimulation.

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