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PubMed2000

Differential display analysis of gene expression during the induction of mucosal immunity.

One approach to understanding the physiologically relevant events during the induction of an immune response is to identify genes that are expressed when the immune system first encounters antigen. Such an investigation requires a naive but fully functional immune system, and the fetal lamb provides these conditions during the last trimester of gestation. 'Intestinal segments,' containing a jejunal Peyer's patch, were surgically prepared in fetal lambs (>120 days gestation) and individual 'intestinal segments' were injected with either culture medium or infectious bovine rotavirus. Peyer's patch tissue was collected 18 h postinfection. Histology and virus culture confirmed that bovine rotavirus had infected the mucosal epithelium. RNA was extracted from jejunal Peyer's patch tissue and mRNA differential display was used to identify genes expressed following rotavirus infection. Ten cDNAs were identified by differential display and these cDNAs were isolated, cloned, and sequenced. One of the cDNAs sequenced, displayed homology to the gene encoding the sperm surface protein Sp17. Differential expression of this gene in antigen-exposed jejunal Peyer's patches was confirmed by Northern blot and RT-PCR. The complete sequence for sheep Sp17 mRNA was obtained from a lambda cDNA library, prepared from the jejunal Peyer's patch of a young lamb. Sp17 expression was detected by RT-PCR in a variety of mucosa-associated lymphoid tissues but not in primary or other secondary lymphoid tissues. Thus, the fetal lamb model may be appropriate for identifying genes relevant to mucosal immunity.

Amino Acid Sequence↗
PubMed2000

The homogeneity assumption in differential prediction analysis: does it really matter?

In simulation studies, the F test for differences in regression slopes has tended to distort nominal Type I and II error rates when the 2 subgroup error variances exceeded a 1.50:1 ratio. This study examines the frequency and extent that this ratio is violated within data sets relevant to applied psychology. The General Aptitude Test Battery (GATB) validity study database contained ability data and overall job performance ratings. The Project A military database contained both ability and personality data, along with job performance factor scores and an overall job performance rating. Results suggest that subgroup (White-Black, male-female) error variances are often homogeneous enough to support F test results from past empirical work. Enough heterogeneity was found, however, to urge applied psychologists investigating differential prediction to explore their data and consider the possibility of alternative statistical tests.

Adult↗
PubMed2002

Acetylcholine-induced zymogen granule exocytosis: comparison between acini and single pancreatic acinar cells.

INTRODUCTION: Numerous studies have been carried out on the agonist-evoked calcium responses of single pancreatic acinar cells; however, several reports have shown that dissociation of the exocrine pancreas into predominantly single cells has an adverse effect on agonist-evoked amylase secretion. AIMS AND METHODOLOGY: To determine whether single acinar cells behave in an anomalous manner compared with cells within an intact acinus, we measured exocytosis in both single acinar cells and acini (2-5 cells) present in the same preparation. Exocytosis of individual zymogen granules was quantified in real-time by using the technique of continuous time-differential analysis of brightfield digital images. RESULTS: Basal rates of exocytosis were low in both single cells and intact acini. Application of 10 microM acetylcholine for 6 minutes stimulated a biphasic secretory response in acinar cells. Additionally, we found that exocytotic events occur repetitively in specific locations within the apical domain; i.e., there are exocytotic "hot spots." There were no statistically significant differences between the exocytotic rates, nor were there any differences in the characteristics of the exocytotic hot spots of single cells compared with those of acini. CONCLUSION: We conclude that time-differential analysis of brightfield images appears to be a useful tool for the investigation of the role of gap junctions in zymogen granule exocytosis and that single acinar cells provide a reasonable model for studies of acinar cell signaling and secretion.

Acetylcholine↗
PubMed1989

Analysis of differential growth of the right and the left leg.

In spite of well documented standards for length and annual growth rates of the femur and tibia, there is little information on short term longitudinal bone growth. We investigated differential growth dynamics of the lower leg in 10 children, aged 6:3 to 14:2 years, by knemometry, a novel and non-invasive technique of accurate lower leg length measurement with a technical error of 0.09 to 0.16 mm. Mini growth spurts were detectable in 7 of the children and occurred synchroneously in both legs. Approximately half of the variance of the weekly lower leg length increments could be attributed to synchrony of leg length increments, but a significant amount of residual variance remained which exceeded the technical error of the measurements. Run-analysis of the individual series of right vs. left differences of the weekly lower leg length increments provided evidence for alternating periods of overgrowth of one leg compared to the contralateral side in 5 out of the 10 children. We concluded that there is suggestive evidence of partial independence of lower leg growth in the short term.

Adolescent↗
PubMed2005

Using half-normal probability plot and regression analysis to differentiate complex traits: differentiating disease response of multigenic resistance and susceptibility in tomatoes to multiple pathogen isolates.

The need for a new analytical approach was encountered in the course of characterizing newly developed tomato lines resistant to late blight. Late blight resistant tomato lines were created in independent breeding programs using the accession Solanum pimpinellifolium L. (formerly Lycopersicon pimpinellifolium (L.) Miller) L3708 as the source of the resistance. However, initial field observation suggested that the late blight resistance in the lines produced by two independent breeding programs differed. Possible causes included a partial transfer of the late blight resistance derived from S. pimpinellifolium L3708 or the possibility of race specificity of this resistance. A crucial issue was determining the most appropriate and robust analytical method to use with data from laboratory analyses of the responses of nine tomato lines against five P. infestans isolates. Prior analysis by standard ANOVA revealed significant differences across tomato lines but could not determine whether the disease responses in the CLN-R lines were different from those of the heterozygous F(1) hybrids, created by crossing susceptible tomatoes with the fixed CU-R lines. A different analytical method was needed. Therefore, sporangia numbers/leaflet and diseased area data were analyzed using a half-normal probability plot and regression analysis. The results of this analysis show its utility for genetic or pathology studies. Considering only populations of the uniform tomato lines, this method confirms the results obtained by using a standard ANOVA, but provides a clearer demonstration of the distributions of the individuals within the populations and how this distribution impacts variance and the difference among the populations. This method also allows a joint analysis of the uniform lines with an additional population that is less uniform, because it is segregating. Such an analysis would be invalid using a standard ANOVA. The results of this joint analysis determined that the additional population was divergent from the fixed CU-R lines, and, against some isolates, against the CLN-R lines as well. Half-normal probability plot analysis method would be applicable more broadly beyond analysis of disease resistance data. It could be useful for data from populations that are not normally distributed, for traits which are affected by epistatic gene action, and could be useful for selection of extremes.

Genotype↗
PubMed2006

Analysis of differential detergent fractions of an AtT-20 cellular homogenate using one- and two-dimensional capillary electrophoresis.

Differential detergent fractionation was used to sequentially extract cytosolic, membrane, nuclear, and cytoskeletal fractions from AtT-20 cells. Extracted components were denatured by sodium dodecyl sulfate (SDS) and then labeled with the fluorogenic reagent 3-(2-furoyl) quinoline-1-carboxaldehyde. Both capillary sieving electrophoresis (CSE) and micellar electrokinetic capillary chromatography (MECC) were used to separate labeled components by one-dimensional (1D) electrophoresis. Labeled components were also separated by two-dimensional (2D) capillary electrophoresis; CSE was employed in the first dimension and MECC in the second dimension. Roughly 150 fractions were transferred from the first to the second capillary for this comprehensive analysis in 2.5 h.

Animals↗
PubMed2005

Analysis of differential gene expression patterns in colon cancer and cancer stroma using microdissected tissues.

BACKGROUND AND AIMS: The surrounding cancer stroma is increasingly recognized as playing an important role in cancer proliferation, invasion, and metastasis. Here, we analyzed patterns of gene expression in colon cancer cells, surrounding stroma, and normal mucosa and normal stroma using laser capture microdissection. METHODS: Tissues were fixed from 11 patients undergoing colorectal resection for cancer, and laser capture microdissection was performed. Samples (4 per patient) were extracted for RNA, which was then used for focused gene arrays. In addition, protein expression of selected genes was determined by Western blot or immunohistochemistry. RESULTS: We showed differential expression patterns in cancer cells and surrounding stroma compared with their normal counterparts. Genes contributing to angiogenesis, cell cycle regulation, and proliferation were significantly increased in cancer cells compared with the adjacent normal mucosa. Genes contributing to angiogenesis, cancer invasion, and metastasis were significantly increased in surrounding cancer stromal cells compared with normal stroma. CONCLUSIONS: Delineating the differential patterns of gene expression between colon cancers and the surrounding reactive stroma will better determine the role of the stromal components in the progression of colon cancers and may lead to chemopreventive therapy targeted specifically to the stroma.

Adenocarcinoma↗
PubMed1995

Statistical analysis of differential lissajous EMG from normal occlusion and Class III malocclusion.

The method of differential lissajous electromyography (DL-EMG) was applied to investigate the relationship among the integrated EMG activity, timing, and coordination of the bilateral superficial anterior temporal and masseter muscle activities in normal occlusion and Class III malocclusion subjects. In both Class III malocclusion and normal occlusion subjects, the working side muscles showed a higher mean cumulative voltage (MCV) and mean maximum peak voltage (MMPV) compared with the balancing side. In addition, a higher MCV and MMPV of the working side masseter was observed in the normal occlusion group compared with that seen in the Class III group during both right and left side chewing (p < 0.01). Discriminant analysis applied to examine the distribution, the size and the shape of DL-EMG pattern further indicated a statistical difference between subject groups (p < 0.01). Finally, there was a significantly higher percentage of clockwise DL-EMG pattern-generation in the normal group compared with that seen for Class III subjects (p < 0.01). These data indicate that, compared with normal subjects, patients with a Class III malocclusion have a demonstrably abnormal masticatory muscle balance which is well characterized by the DL-EMG method.

Adolescent↗
PubMed2006

Differential proteome analysis of colon carcinoma cell line SW480 after reconstitution of the tumour suppressor Smad4.

The tumour suppressor gene Smad4 is frequently inactivated in gastrointestinal carcinomas. Smad4 plays a pivotal role in transducing signals of the transforming growth factor-beta (TGF-beta) superfamily of proteins. Inactivation of Smad4 seems to occur late during tumour progression when tumours acquire invasive and metastatic properties. Identification of proteins directly or indirectly regulated by Smad4 would, therefore, ease the future design of new diagnostic and therapeutic strategies for gastrointestinal carcinoma. We have used human colon carcinoma cell line SW480 stably transfected with Smad4 as an in-vitro model system to identify Smad4-regulated proteins by applying two-dimensional gel electrophoresis (2DE) then MALDI-PMF/PFF-MS. We identified a total of 47 protein species with a Smad4-dependent expression. From the functions of the candidate proteins we obtained new insights into Smad4's participation in processes, for example apoptosis, differentiation, and proliferation.

Amino Acid Sequence↗
PubMed2006

Analysis of differentially expressed genes in fetal skin of scarless and scar-forming periods of gestational rats.

OBJECTIVE: To study the differences of gene expression between earlier gestational skin and later gestational skin of rats with the aids of single primer amplification (SPA) and high-density oligonucleotide DNA array to understand the molecular mechanism of scarless healing. METHODS: Total RNAs were isolated from fetal rat skin of the scarless (E15) and scar-forming (E18) periods of gestation (term = 21.5 days). The RNAs from earlier gestational skin (EGS) and later gestational skin (LGS) were both reversely transcribed to cDNAs, then labeled with the incorporation of fluorescent dCTP for preparing the hybridization probes by SPA method. The mixed probes were then hybridized to the oligonucleotide DNA arrays which contained 5,705 probes representing 5,705 rat genes. After highly stringent washing, these DNA arrays were scanned for fluorescent signals to display the differentially expressed genes between the 2 groups of skin. RESULTS: Among 5,705 rat genes, there were 53 genes (0.93 percent) with differentially expressed levels between EGS and LGS groups, 27 genes, including fibroblast growth factor 2 (FGF2) and follistatin were up-regulated (0.47%) and 26 genes were down-regulated (0.46%) in fetal skin during scarless period versus scar-forming period. Higher expressions of FGF2 and follistatin in EGS than those in LGS were also revealed by RT-PCR method. CONCLUSIONS: High-density oligonucleotide DNA array provided a powerful tool for investigating differential gene expression in earlier and later gestational fetal skins. This technology validates that the mechanism of fetal scarless healing is very complicate and the change of many gene expressions is associated with fetal scarless healing.

Animals↗
PubMed2004

Microarray analysis identified differentially expressed genes in keratocytes from keratoconus patients.

PURPOSE: To identify differentially expressed genes in human keratoconus keratocyte by cDNA microarray. METHODS: Normal and keratoconic cornea were cultured for keratocytes. RNA was extracted. cDNA probe labeled with fluorescence dye was made from Poly A+ RNA, hybridized with microarray slide, containing 164 human apoptosis genes. Signal intensity was measured. Expression ratio between keratoconus and normal was determined using ImaGene Ver.3.0. Identified genes were further evaluated by RT-PCR and real-time PCR. RESULTS: Five over-expressed and four under-expressed genes were identified. Of these, differential expression of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), human insulin-like growth factor binding protein 5 (IGFBP5), and IGFBP3 were verified and confirmed by RT-PCR. Real-time PCR showed TNFAIP6 increased by 3.3 folds, while IGFBP5, IGFBP3 decreased by 14 and 11 folds respectively. CONCLUSIONS: The identified genes could be important and deserve further investigation. Significant differential expression of TNFAIP6, IGFBP5, and IGFBP3 may indicate an important role of these genes in the mechanism underlying stromal thinning.

Adult↗
PubMed2000

Real-time studies of zymogen granule exocytosis in intact rat pancreatic acinar cells.

An adequate understanding of secretion requires the measurement of exocytosis on the same time scale as that used for second messenger dynamics. To investigate the kinetics of ACh-evoked secretion in pancreatic acinar cells, exocytosis of zymogen granules was quantified by continuous, time-differential analysis of digital images. The validity of this method was confirmed by simultaneous fluorescence imaging of quinacrine-loaded zymogen granules. Basal rates of exocytosis were low (0.2 events min(-1)). ACh stimulated a biphasic increase in secretory activity, maximal rates exceeding 20 events min(-1) after 10 s of ACh application (10 microM). Over the next 15 s the rate of exocytosis fell to less than 4 events min(-1); then began a second phase of secretion that peaked 15 s later at approximately 11 events min(-1), but subsequently declined in the continued presence of agonist. Measurements of fura-2 fluorescence demonstrated a biphasic increase in intracellular [Ca2+] ([Ca2+]i). Comparison of the [Ca2+]i records and time-differential analysis revealed that the fall in exocytotic rate following the initial burst occurred despite the fact that [Ca2+]i remained high. The second phase of secretion depended on both [Ca2+]i and [ACh]. At 10 microM ACh there was a decrease in the steepness of the relationship between [Ca2+]i and exocytosis that led to an enhancement of the slow secretory phase. We propose that acinar cells contain two pools of secretory vesicles: a small pool of granules that is exocytosed rapidly, but is quickly depleted; and a reserve pool of granules that can be recruited by ACh in a process that is modulated by second messengers other than calcium.

Acetylcholine↗
PubMed2003

Analysis of differential immune responses induced by innate and adaptive immunity following transplantation.

The roles of innate and adaptive immunity in allograft rejection remain incompletely understood. Previous studies analysing lymphocyte deficient or syngeneic graft recipients have identified subsets of inflammatory chemokines and cytokines induced by antigen independent mechanisms. In the current study, we analysed a panel of 60 inflammatory parameters including serum cytokines, intragraft chemokines and cytokines, receptors, and cellular markers. Our results confirmed the up-regulation of a subset of markers by innate mechanisms and also identified a subset of parameters up-regulated only in the context of an adaptive response. Thus, we successfully differentiated markers of the innate and adaptive phases of rejection. Current paradigms emphasize that innate signals can promote a subsequent adaptive response. Interestingly, in our studies, expression of the markers induced by innate mechanisms was markedly amplified in the allogeneic, but not syngeneic or lymphocyte deficient, recipients. These results suggest that inflammatory mediators can have functional overlap between the innate and adaptive responses, and that the adaptive component of the rejection process amplifies the innate response by positive feedback regulation.

Analysis of Variance↗
PubMed1991

Water deficit modulates gene expression in growing zones of soybean seedlings. Analysis of differentially expressed cDNAs, a new beta-tubulin gene, and expression of genes encoding cell wall proteins.

Transfer of soybean seedlings to low-water-potential vermiculite (psi w = -0.3 MPa) results in a reversible decrease in hypocotyl growth and modulation of several polysomal mRNAs (Plant Physiol 92: 205-214). We report here the isolation of two cDNA clones (pGE16 and pGE95) which correspond to genes whose mRNA levels are increased, and one cDNA clone (pGE23) which corresponds to a gene whose mRNA level is decreased in the hypocotyl zone of cell elongation by water deficit. In well-watered seedlings mRNAs hybridizing to pGE16 and pGE95 are most abundant in mature regions of the seedling, but in water-deficient seedlings mRNA levels are reduced in mature regions and enhanced in elongating regions. RNA corresponding to soybean proline-rich protein 1 (sbPRP1) shows a similar tissue distribution and response to water deficit. In contrast, in well-watered seedlings, the gene corresponding to pGE23 was highly expressed in the hypocotyl and root growing zones. Transfer of seedlings to low-water-potential vermiculite caused a rapid decrease in mRNA hybridizing to pGE23. Sequence analysis revealed that pGE23 has high homology with beta-tubulin. Water deficit also reduced the level of mRNA hybridizing to JCW1, an auxin-modulated gene, although with different kinetics. Furthermore, mRNA encoding actin, glycine-rich proteins (GRPs), and hydroxyproline-rich glycoproteins (HRGPs) were down-regulated in the hypocotyl zone of elongation of seedlings exposed to water deficit. No effect of water deficit was observed on the expression of chalcone synthase. Decreased expression of beta-tubulin, actin, JCW1, HRGP and GRP and increased expression of sbPRP1, pGE95 and pGE16 in the hypocotyl zone of cell elongation could participate in the reversible growth inhibition observed in water-deficient soybean seedlings.

Actins↗
PubMed2006

Microarray analysis reveals differential gene expression patterns in tumors of the pineal region.

Several types of tumors are known to originate from the pineal region, among them pineal parenchymal tumors (PPTs) and papillary tumors of the pineal region (PTPRs), probably derived from the subcommissural organ. As a result of their rarity, their histologic diagnosis remains difficult. To identify molecular markers, using CodeLink oligonucleotide arrays, gene expression was studied in 3 PPTs (2 pineocytomas and one pineoblastoma), 2 PTPRs, and one chordoid glioma, another rare tumor of the third ventricle. Because PTPR and chordoid glioma may present ependymal differentiation, gene expression was also analyzed in 4 ependymomas. The gene patterns of the 3 PPTs fell in the same cluster. The pineocytomas showed high expression of TPH, HIOMT, and genes related to phototransduction in the retina (OPN4, RGS16, and CRB3), whereas the pineoblastoma showed high expression of UBEC2, SOX4, TERT, TEP1, PRAME, CD24, POU4F2, and HOXD13. Using reverse transcriptase-polymerase chain reaction on 13 PPTs, we demonstrated that PRAME, CD24, POU4F2, and HOXD13 might be candidates for grading PPT with intermediate differentiation. PTPRs, classified with chordoid glioma and separately from ependymomas, showed high expression of SPEDF, KRT18, and genes encoding proteins reported to be expressed in the subcommissural organ, namely ZFH4, RFX3, TTR, and CGRP. Our results highlight the usefulness of gene expression profiling for classify tumors of the pineal region and identify genes with potential use as diagnostic markers.

Adolescent↗
PubMed2006

Microarray analysis reveals differential gene expression in hybrid sunflower species.

This paper describes the creation of a cDNA microarray for annual sunflowers and its use to elucidate patterns of gene expression in Helianthus annuus, Helianthus petiolaris, and the homoploid hybrid species Helianthus deserticola. The array comprises 3743 ESTs (expressed sequence tags) representing approximately 2897 unique genes. It has an average clone/EST identity rate of 91%, is applicable across species boundaries within the annual sunflowers, and shows patterns of gene expression that are highly reproducible according to real-time RT-PCR (reverse transcription-polymerase chain reaction) results. Overall, 12.8% of genes on the array showed statistically significant differential expression across the three species. Helianthus deserticola displayed transgressive, or extreme, expression for 58 genes, with roughly equal numbers exhibiting up- or down-regulation relative to both parental species. Transport-related proteins were strongly over-represented among the transgressively expressed genes, which makes functional sense given the extreme desert floor habitat of H. deserticola. The potential adaptive value of differential gene expression was evaluated for five genes in two populations of early generation (BC2) hybrids between the parental species grown in the H. deserticola habitat. One gene (a G protein-coupled receptor) had a significant association with fitness and maps close to a QTL controlling traits that may be adaptive in the desert habitat.

Base Sequence↗
PubMed2002

Global analysis of differentially expressed genes during progression of calcium oxalate nephrolithiasis.

The process of nephrolithiasis development is poorly understood at the molecular level. Here, we constructed a cDNA microarray from a rat kidney normalized cDNA library, and investigated the pattern of gene expression in rat kidneys from a calcium oxalate (CaOx) nephrolithiasis model. One hundred and seventy-three genes were found to be at least 2-fold regulated at one or more time points during progression of nephrolithiasis. RT-PCR and immunohistochemical analyses confirmed differential expression at both transcriptional and translational levels of genes identified by cDNA microarray screening. The differentially regulated genes were grouped into six clusters based on their expression profiles; the magnitude and the temporal patterns of gene expression identified known and novel molecular components involved in inflammation and matrix expansion in the CaOx nephrolithiasis kidney. This microarray study is the first report on gene expression programs underlying the process of nephrolithiasis.

Animals↗
PubMed2002

Identification and comparative analysis of differentially expressed proteins in rat striatum following 6-hydroxydopamine lesions of the nigrostriatal pathway: up-regulation of amyloid precursor-like protein 2 expression.

During neurodegenerative processes, cascades of degeneration and subsequent regeneration are triggered. However, the molecular nature of the factors involved in the neurodegeneration of the CNS remains largely unknown. In this study, the variations of protein expression in the striatum of adult Sprague-Dawley rats following 6-hydroxydopamine lesions were investigated, in order to better understand the molecular events occurring in the denervated target tissue. The rat striatum, ipsilateral to the lesion was analysed by two-dimensional gel electrophoresis followed by matrix assisted laser desorption/ionization-time of flight mass spectrometry. Seven proteins were up-regulated (188.1-750% compared to control) in response to the lesion: amyloid precursor-like protein 2 (APLP2), kininogen, glucokinase, tropomyosin alpha chain, type brain-1 and calpactin I light chain; whilst four proteins, neural epidermal growth factor-like 2, minichromosome maintenance 6, and thyroid hormone receptor beta-2, were down-regulated (to between 36% and 59% of levels in sham-operated controls). Three proteins that did not match with available data in the SWISS-PROT protein database were also determined. Immunohistochemical analysis demonstrated colocalization of APLP2 and tyrosine hydroxylase in the nigral neurons. Moreover, reduction of APLP2-positive neurons in the substantia nigra pars compacta as well as the increases in the substantia nigra pars reticulata and in the striatum were observed. Furthermore, the conditioned medium of the Chinese hamster ovary cells over-expressing APLP2-751 (chondroitin sulphate-modified), but not APLP2-763 (nonchondroitin sulphate-modified), was able to increase the number of the tyrosine hydroxylase-positive neurons in fetal mesencephalic cultures. These results suggest that the expression of APLP2, a protein that has been thought to be associated with Alzheimer's disease, is up-regulated in the striatum following dopaminergic denervation. They also support the view that chondroitin sulphate-modified APLP2 protein may play an important role in the dopaminergic nigrostriatal system.

Adrenergic Agents↗