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PubMed2004

Differential DNases are selectively used in neuronal apoptosis depending on the differentiation state.

In this study, we investigate the roles of two apoptotic endonucleases, CAD and DNase gamma, in neuronal apoptosis. High expression of CAD, but not DNase gamma, is detected in proliferating N1E-115 neuroblastoma cells, and apoptotic DNA fragmentation induced by staurosporine under proliferating conditions is abolished by the expression of a caspase-resistant form of ICAD. After the induction of neuronal differentiation, CAD disappearance and the induction of DNase gamma occur simultaneously in N1E-115 cells. Apoptotic DNA fragmentation that occurs under differentiating conditions is suppressed by the downregulation of DNase gamma caused by its antisense RNA. The induction of DNase gamma is also observed during neuronal differentiation of PC12 cells, and apoptotic DNA fragmentation induced by NGF deprivation is inhibited by the antisense-mediated downregulation of DNase gamma. These observations suggest that DNA fragmentation in neuronal apoptosis is catalyzed by either CAD or DNase gamma depending on the differentiation state. Furthermore, DNase gamma is suggested to be involved in naturally occurring apoptosis in developing nervous systems.

Animals↗
PubMed2006

Molecular characterization of adenocarcinoma and squamous carcinoma of the uterine cervix using microarray analysis of gene expression.

In an attempt to understand the molecular mechanisms for the different clinical features between adenocarcinoma/adenosquamous carcinoma (AC/ASC) and squamous carcinoma (SC) of the uterine cervix, we analyzed gene expression profiles of different histological subtypes of cervical cancer. Cancer specimens and the surrounding normal tissue counterparts were separately collected from cervical cancer patients undergoing type III radical hysterectomy. Paired total RNA (cancer and normal tissues) was isolated and analyzed with cDNA microarrays containing duplicate spots of 7 334 sequence-verified human cDNA clones. Selected differentially expressed genes specific for AC or SC were further verified using real-time quantitative polymerase chain reaction (RTQ-PCR) and immunohistochemistry. Genes, including CEACAM5, TACSTD1, S100P and MSLN were upregulated in AC. Contrarily, genes involved in epidermal differentiation complex such as S100A9 and ANXA8 were upregulated in SC. Cross-validation of the results using an independent but comparable group of patients with known long-term outcomes (n = 63, median follow-up 70.3 months; range, 4-208 months) showed that the correlation between the selected 6 differentially expressed genes and histology was highly significant. CEACAM5 (p < 0.0001) and TACSTD1 (p = 0.009) were significant prognostic factors by multivariate Cox proportional hazards regression analysis. The combination of cDNA microarray, RTQ-PCR and immunohistochemical results of this study showed that it is possible to define different gene profiles for AC and SC. Moreover, TACSTD1 expression may be a novel poor prognostic factor.

Adenocarcinoma↗
PubMedDate not supplied

Confusing selective feeding with differential digestion in bacterivorous nanoflagellates.

Food selectivity and the mechanisms of food selection were analyzed by video microscopy for three species (Spumella, Ochromonas, Cafeteria) of interception-feeding heterotrophic nanoflagellates. The fate of individual prey particles, either live bacteria and/or inert particles, was recorded during the different stages of the particle-flagellate-interaction, which included capture, ingestion, digestion, and egestion. The experiments revealed species-specific differences and new insights into the underlying mechanisms of particle selection by bacterivorous flagellates. When beads and bacteria were offered simultaneously, both particles were ingested unselectively at similar rates. However, the chrysomonads Spumella and Ochromonas egested the inert beads after a vacuole passage time of only 2-3 min, which resulted in an increasing proportion of bacteria in the food vacuoles. Vacuole passage time for starved flagellates was significantly longer compared to that of exponential-phase flagellates for Spumella and Ochromonas. The bicosoecid Cafeteria stored all ingested particles, beads as well as bacteria, in food vacuoles for more then 30 min. Therefore "selective digestion" is one main mechanism responsible for differential processing of prey particles. This selection mechanism may explain some discrepancies of former experiments using inert particles as bacterial surrogates for measuring bacterivory.

Animals↗
PubMed2001

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.

Animals↗
PubMed2000

Nonrandom mating in Drosophila melanogaster laboratory populations derived from closely adjacent ecologically contrasting slopes at "Evolution Canyon".

Ecological differentiation of natural populations of Drosophila melanogaster, Drosophila simulans, and another drosophilid, Zaprionus tuberculatus, in "Evolution Canyon," Mount Carmel, Israel, is well established. The fitness complex of D. melanogaster includes oviposition temperature preferences, tolerance to high temperature, drought stress and starvation, and different longevity patterns. This remarkable differentiation has evolved despite small interslope distances (only 100-400 m), within easy dispersal distance. The differences between populations are those expected from genetic adaptation to local microclimates. How such differentiation could evolve and be maintained despite the likelihood of genetic exchange between populations is a challenging question. We hypothesized that interslope microclimatic differences caused strong differential selection for stress tolerance, accompanied by behavioral differentiation (habitat choice and reduced migration rate), reinforced by sexual isolation. Here we report highly significant mate choice by flies from different slopes of the canyon, with preference for sexual partners originating from the same slope. No preferences were found when the sexual partners belonged to different isofemale lines from the same slope.

Animals↗
PubMed1999

Involvement of type 4 cAMP-phosphodiesterase in the myogenic differentiation of L6 cells.

Myogenic cell differentiation is induced by Arg(8)-vasopressin, whereas high cAMP levels and protein kinase A (PKA) activity inhibit myogenesis. We investigated the role of type 4 phosphodiesterase (PDE4) during L6-C5 myoblast differentiation. Selective PDE4 inhibition resulted in suppression of differentiation induced by vasopressin. PDE4 inhibition prevented vasopressin-induced nuclear translocation of the muscle-specific transcription factor myogenin without affecting its overall expression level. The effects of PDE4 inhibition could be attributed to an increase of cAMP levels and PKA activity. RNase protection, reverse transcriptase PCR, immunoprecipitation, Western blot, and enzyme activity assays demonstrated that the PDE4D3 isoform is the major PDE4 expressed in L6-C5 myoblasts and myotubes, accounting for 75% of total cAMP-hydrolyzing activity. Vasopressin cell stimulation caused a biphasic increase of PDE4 activity, which peaked at 2 and 15 min and remained elevated for 48 h. In the continuous presence of vasopressin, cAMP levels and PKA activity were lowered. PDE4D3 overexpression increased spontaneous and vasopressin-dependent differentiation of L6-C5 cells. These results show that PDE4D3 plays a key role in the control of cAMP levels and differentiation of L6-C5 cells. Through the modulation of PDE4 activity, vasopressin inhibits the cAMP signal transduction pathway, which regulates myogenesis possibly by controlling the subcellular localization of myogenin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Embryonic Stem Cells in vitro - Prospects for Cell and Developmental Biology, Embryotoxicology and Cell Therapy.

Embryonic stem (ES) cells are able to differentiate in vitro via embryo-like aggregates, so-called "embryoid bodies", into derivatives of the endodermal, ectodermal and mesodermal lineage. We established standardised protocols for cardiogenesis, myogenesis, neurogenesis and vascular smooth muscle cell differentiation in vitro. The developmentally controlled expression of tissue-specific genes, proteins, ion channels and receptors during ES cell differentiation is the basis of several in vitro approaches: (1) "Loss of function" assays with ES cells containing homozygous mutations of specific genes, (2) "Gain of function" assays with ES cells overexpressing exogenous genes, (3) Developmental analysis of teratogenic/embryotoxic compounds in vitro, (4) Pharmacological assays and the establishment of model systems for pathological cell functions, and (5) The application of differentiation and growth factors for induction of selectively differentiated cells which, in the future, may be used as a source for tissue grafts. We propose the ES cell technology as valuable in vitro system to substitute and reduce the use of animals in basic and applied research.

Journal Article↗
PubMed2003

[Application of suppression subtractive hybridization technique in the investigation of heat adaptation mechanism].

OBJECTIVE: To understand whether there are genes differentially expressed in association with heat adaptation in resting heat-adapted rats at normal temperature, and to evaluate the feasibility of investigating heat adaptation mechanism in light of gene differential expression. METHODS: The mRNAs were extracted from rat liver tissues and reverse transcripted into double-strand cDNAs. Suppression subtractive hybridization (SSH) was applied to compare the gene expression profiles between heat adaptation group and normal temperature control group, and cDNA subtraction libraries of the differentially expressed genes were constructed. PCR-selective differential screening technique was used to identify the differentially expressed genes. RESULTS: Six cDNA fragments were identified in the preliminary experiment to be differentially expressed. CONCLUSIONS: There are genes differentially expressed in the liver of resting heat-adapted rats in association with heat adaptation at normal temperature, and these genes may be involved in maintaining heat adaptation. This study also indicates the feasibility of investigating heat adaptation mechanism by isolating differentially expressed genes associated with heat adaptation.

Adaptation, Physiological↗
PubMed2004

A theoretical analysis of gene selection.

A great deal of recent research has focused on the challenging task of selecting differentially expressed genes from microarray data ('gene selection'). Numerous gene selection algorithms have been proposed in the literature, but it is often unclear exactly how these algorithms respond to conditions like small sample-sizes or differing variances. Choosing an appropriate algorithm can therefore be difficult in many cases. In this paper we propose a theoretical analysis of gene selection, in which the probability of successfully selecting relevant genes, using a given gene ranking function, is explicitly calculated in terms of population parameters. The theory developed is applicable to any ranking function which has a known sampling distribution, or one which can be approximated analytically. In contrast to empirical methods, the analysis can easily be used to examine the behaviour of gene selection algorithms under a wide variety of conditions, even when the numbers of genes involved runs into the tens of thousands. The utility of our approach is illustrated by comparing three well-known gene ranking functions.

Algorithms↗
PubMed1996

Sex ratios in fetuses and liveborn infants with autosomal aneuploidy.

Ten data sources were used substantially to increase the available data for estimating fetal and livebirth sex ratios for Patau (trisomy 13), Edwards (trisomy 18), and Down (trisomy 21) syndromes and controls. The fetal sex ratio estimate was 0.88 (N = 584) for trisomy 13, 0.90 (N = 1702) for trisomy 18, and 1.16 (N = 3154) for trisomy 21. All were significantly different from prenatal controls (1.07). The estimated ratios in prenatal controls were 1.28 (N = 1409) for CVSs and 1.06 (N = 49427) for amniocenteses, indicating a clear differential selection against males, mostly during the first half of fetal development. By contrast, there were no sex ratio differences for any of the trisomies when comparing gestational ages < 16 and > 16 weeks. The livebirth sex ratio estimate was 0.90 (N = 293) for trisomy 13, 0.63 (N = 497) for trisomy 18, and 1.15 (N = 6424) for trisomy 21, the latter two being statistically different than controls (1.05) (N = 3660707). These ratios for trisomies 13 and 18 were also statistically different than the ratio for trisomy 21. Only in trisomy 18 did the sex ratios in fetuses and livebirths differ, indicating a prenatal selection against males > 16 weeks. No effects of maternal age or race were found on these estimates for any of the fetal or livebirth trisomies. Sex ratios for translocations and mosaics were also estimated for these aneuploids. Compared to previous estimates, these results are less extreme, most likely because of larger sample sizes and less sample bias. They support the hypothesis that these trisomy sex ratios are skewed at conception, or become so during embryonic development through differential intrauterine selection. The estimate for Down syndrome livebirths is also consistent with the hypothesis that its higher sex ratio is associated with paternal nondisjunction.

Adult↗
PubMedDate not supplied

New tetrachromic VOF stain (Type III-G.S) for normal and pathological fish tissues.

A new VOF Type III-G.S stain was applied to histological sections of different organs and tissues of healthy and pathological larvae, juvenile and adult fish species (Solea senegalensis; Sparus aurata; Diplodus sargo; Pagrus auriga; Argyrosomus regius and Halobatrachus didactylus). In comparison to the original Gutiérrez VOF stain, more acid dyes of contrasting colours and polychromatic/metachromatic properties were incorporated as essential constituents of the tetrachromic VOF stain. This facilitates the selective staining of different basic tissues and improves the morphological analysis of histochemical approaches of the cell components. The VOF Type III -6.5 stain is composed of a mixture of several dyes of varying size and molecular weight (Orange G<acid Fuchsin<Light green<Methyl Blue<Fast Green), which are used simultaneously, and it enables the individual tissues to be selectively differentiated and stained. Muscle fibers, collagen, reticulin and elastin fibers, erythrocytes, cartilage, bone, mucous cells, oocytes and larvae were selectively stained and differentiated. Dyes with small size and molecular weight (i.e Orange G), penetrate all tissue structures rapidly, but are only tightly retained in densely textured tissues (i.e erythrocytes). Methyl Blue is an interesting triarylmethane dye (large size and molecular weight), which is incorporated in this new VOF tetrachrome stain, and acquires histochemical significance when used at acid pH (2.8) because collagen and reticulin fibers, as well basophilic and metachromatic substances (strongly ionized sulphated glycoconjugates) can be identified. Muscle tissues show an evident green colour (Fast Green or Light Green affinities), even those isolated and/or diffuse muscle fibers present in the digestive submucosa layer. Connective tissues showed a specific and strong blue colour (Methyl Blue affinity) or mixed blue-red staining (Methyl Blue and Acid Fucshin affinities). Very noticeable is the staining of the mucous cells, as well as the hyaline capsule of the viral lymphocystic cells, which were stained blue-purple (carboxylated and/or strongly ionized sulphated groups). Cartilaginous tissues showed a blue or purple (Methyl Blue affinity) staining, and a specific red colour (Acid Fucshin affinity) was evident during calcification or in bone structures (i.e skeleton, fins, gills, teeth).

Animals↗
PubMed2002

Social stress in laying hens: differential dopamine and corticosterone responses after intermingling different genetic strains of chickens.

White Leghorn chickens were genetically selected for high (HGPS) or low (LGPS) group productivity and survivability. The selection resulted in two genetic lines with marked opposite changes in cannibalism and flightiness when housed in multiple-colony battery cages without beak trimming. The objective of the study was to examine whether the genetic selection differentially affected the neuroendocrine system of chickens from different strains in response to social stress. Based on the previous studies, social stress was induced by randomly pairing 17-wk-old hens from three genetic lines, i.e., HGPS, LGPS, and Dekalb XL (DXL), to form three mixed-line combinations. At 24 wk of age, the concentrations of plasma dopamine (DA) and corticosterone (CORT) showed no differences in DXL hens housed with HGPS or LGPS hens (P > 0.05). However, different regulations of DA and adrenal function were found between HGPS and LGPS hens when paired with DXL hens. Compared to HGPS hens, LGPS hens had greater levels of DA and CORT (P < 0.01 and P < 0.05, respectively). In addition, under the HGPS-LGPS social treatment, the concentrations of DA but not CORT were greater in LGPS hens than in HGPS hens (P < 0.05 and P > 0.05, respectively). The results indicated genetic selection for production and survivability differentially altered DA and CORT systems in response to social stress. The data suggested, compared to LGPS hens, HGPS hens had a better coping capability to social stress, which might have been responsible for their higher productivity and survivability.

Adrenal Glands↗
PubMed2003

Evidence for a ligand-mediated positive selection signal in differentiation to a mature B cell.

Positive selection is required for B cell differentiation, as indicated by the requirement for expression of the pre-B cell receptor (pre-BCR) and the BCR at the pre-B and immature B cell stages, respectively. Positive selection mediated by a tonic signal from these receptors is sufficient to drive B cell differentiation beyond the pre-B and immature B cell stages, but it is unclear whether additional positive selection signals are required for differentiation to a mature B-2 cell. We have identified a population of Ig transgenic B cells that differentiatively arrest at a transitional B cell stage in the spleen. They exhibit no evidence of Ag encounter or negative selection and can differentiate to mature B-2 cells in vivo upon weak BCR stimulation or adoptive transfer to irradiated hosts. These data are consistent with a requirement for a ligand-mediated BCR signal for differentiation to a mature B-2 cell.

Animals↗
PubMed1997

Symmetry breaking in Dictyostelium morphogenesis: evidence that a combination of cell cycle stage and positional information dictates cell fate.

The event that visibly breaks the symmetry of the Dictyostelium aggregate is the formation of a nipple-shaped tip at its apex, but there has been considerable debate as to the equivalence of cells entering development, particularly with regard to the effect of cell cycle position at the onset of starvation. We show that there is a strong correlation between cell cycle position at the time of starvation and the subsequent expression of a marker of tip cell differentiation. Cells starved in late-G2 phase selectively differentiate into tip cells. Previous evidence indicated that tip cells differentiate at the extreme periphery of the aggregate and then move to the apex. Taken in combination, these data suggest that cell cycle position at the onset of starvation affects differentiation fate by determining a cell's probable position within the aggregate, i.e., that both cell cycle and positional information are used to generate pattern.

Animals↗
PubMed1990

The use of selective media for the isolation of Pseudomonas pseudomallei in clinical practice.

Ashdown's selective-differential agar medium, with or without preenrichment in selective broth, was evaluated for the isolation of Pseudomonas pseudomallei from 1972 clinical specimens obtained from 643 subjects in Northeast Thailand; 226 patients proved to have meliodosis. The use of Ashdown's medium significantly increased the frequency of recovery of P. pseudomallei from sites or specimens with an extensive normal flora (throat, rectum, wounds and sputum) as compared to the recovery on blood and MacConkey agars (p less than 0.01). The isolation frequency from throat, rectal and wound swabs was further increased by the use of the broth pre-enrichment. The colonial morphology of P. pseudomallei on Ashdown's medium was sufficiently characteristic to allow presumptive identification. With the use of these selective media it was possible to culture P. pseudomallei from throat swabs taken from 87% of the patients from whom the organism could also be isolated from corresponding tracheal aspirates or sputum specimens. P. pseudomallei was isolated from rectal swabs taken from 51 patients, the first time that faecal excretion of the organism has been demonstrated in man. The diagnosis of melioidosis would not have been confirmed bacteriologically in eight patients (3.5%) without the use of the selective media. It is suggested that, in areas endemic for melioidosis, all sputum specimens should be cultured on selective media, such as Ashdown's. For the investigation of clinically suspected cases of melioidosis, and for follow-up during treatment of the disease, the use of broth pre-enrichment is recommended for specimens obtained from sites with an extensive normal flora.

Bacteriological Techniques↗
PubMed2004

Integrating phylogenetics and environmental niche models to explore speciation mechanisms in dendrobatid frogs.

We developed an approach that combines distribution data, environmental geographic information system layers, environmental niche models, and phylogenetic information to investigate speciation processes. We used Ecuadorian frogs of the family Dendrobatidae to illustrate our methodology. For dendrobatids there are several cases for which there is significant environmental divergence for allopatric and parapatric lineages. The consistent pattern that many related taxa or nodes exist in distinct environmental space reinforces Lynch and Duellman's hypothesis that differential selection likely played an important role in species differentiation of frogs in the Andes. There is also some evidence that the Río Esmeraldas basin is a geographic barrier to species distributed in low to middle elevations on the western side of the Andes. Another useful aspect of this approach is that it can point to common environmental parameters that correlate with speciation. For dendrobatids, sister clades generally segregate along temperature/elevational and/or seasonality axes. The joint analysis of environmental and geographic data for this group of dendrobatid frogs has identified potentially important speciation mechanisms and specific sister lineages that warrant intensive study to test hypotheses generated in this investigation. Further, the method outlined in this paper will be increasingly useful as knowledge of distribution and phylogeny of tropical species increases.

Analysis of Variance↗
PubMed2000

In vitro and in vivo inhibition of the 2 active sites of ACE by omapatrilat, a vasopeptidase inhibitor.

The vasopeptidase inhibitor omapatrilat inhibits both neutral endopeptidase and angiotensin-converting enzyme (ACE). The in vitro and in vivo inhibitory potency of omapatrilat and the specific ACE inhibitor fosinopril toward the 2 active sites of ACE (called N- and C-domains) was investigated with the use of 3 substrates: angiotensin I, which is equally cleaved by the 2 ACE domains; hippuryl-histidyl-leucine, specific synthetic substrate of the C-domain in high- salt conditions; and a newly synthesized specific substrate of the N-domain designed by acetylating the lysine residue of AcSDKP. In vitro, omapatrilat was 5 times more potent than fosinoprilat in inhibiting angiotensin I hydrolysis. Omapatrilat inhibited similarly both N- and C-domain hydrolysis, whereas fosinoprilat was slightly more specific for the N-domain. The in vivo selective inhibitory potency of single oral doses of 10 mg omapatrilat and 20 mg fosinopril were investigated in a double-blind, placebo-controlled, cross-over study in 9 mildly sodium-depleted normotensive subjects. In accordance with the in vitro results, fosinopril appeared to be more specific for the N-domain than the C-domain in vivo, since plasma and urine AcSDKP concentrations were significantly higher than those observed with omapatrilat. This study shows that it is possible to assess separately in vitro and in vivo the selectivity of ACE or ACE/neutral endopeptidase inhibitors. A differential selectivity may explain some peculiar properties observed with some ACE inhibitors.

Adolescent↗
PubMed1985

The influence of genetic increases in shank width on body weight, walking ability, and reproduction of turkeys.

Body weight and breast width have been greatly increased in the modern turkey. However, the relative amounts of leg muscles and leg bones have declined. A similar decline also occurs with age. It was theorized that this is an inherent weakness that magnifies other causes of leg problems. In an attempt to increase the relative amount of leg bone, a subline (FL) was developed by mass selection for increased shank width at 16 weeks of age from a long-term growth-selected line of turkeys (F). Shank width of FL was increased greatly by selection. The realized heritability of shank width, based on the regression of accumulated selection response on accumulated selection differential, was .33 +/- .05 over five generations of selection. Body weight of males from FL increased at a rate comparable to that of F at 16 weeks of age. However, there was no comparable improvement in body weight of FL females, resulting in a significant line X sex interaction. A similar interaction was observed for body weight at 8 and 20 weeks of age in these lines. The genetic increases in shank width in FL resulted in significantly improved walking ability of males at 16 weeks of age in comparison to F, even though body weight of males from the two lines was similar. The walking ability of FL did not change relative to the control line from which F originated. Thus, large genetic increases in body weight of males can be achieved without loss in walking ability by genetic increases in shank width.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗