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PubMedDate not supplied

Searching for excystment-regulated genes in Sterkiella histriomuscorum (Ciliophora, Oxytrichidae): a mRNA differential display analysis of gene expression in excysting cells.

In the absence of food, the oxytrichid Sterkiella histriomuscorum transforms like many ciliates into resting cysts. When transferred back into feeding medium, the cyst re-transforms into a vegetative cell. The entry into and exit from the dormant cyst stage are complex developmental processes still poorly investigated at the molecular level. Assuming that these changes in state could involve changes in gene expression, we have used the technique of mRNA differential display to detect differentially expressed genes in cysts and two different stages of excysting cell. Variation in the temporal expression pattern of transcripts could be detected and, in using an inverse-PCR strategy on circularized macronuclear DNA, we have sequenced the macronuclear genes of three of the isolated cDNAs. which correspond to 1) a nucleotide-binding domain-encoding gene, 2) a DHHC-domain-carrying gene, and 3) a phosphatase type 2C-encoding gene. For the first two genes, Northern blot analyses supported an excystment-associated regulated gene expression. We discuss their possible role during excystment and we show that the combination of differential display and inverse PCR constitutes a powerful approach to isolate excystment-regulated genes in hypotrichs.

Amino Acid Sequence↗
PubMed2001

Differential display analysis of gene expression altered by ras oncogene.

The goal of the signal transduction pathways, such as those controlled by Ras, is in large part to ensure highly stringent regulation of the target genes in the nucleus, which are collectively responsible for the signal output, or phenotypes, of the cell. Understanding of the Ras effect ultimately requires the identification of these downstream target genes. Reverse genetic approaches would trace back the pathways by which they are regulated by Ras. While newer methods such as DNA microarray are emerging, differential display has allowed the identification of a greater number of differentially expressed genes than have been cloned by all the other methods combined, based on Medline search. Much of this success has been attributed to its simplicity (RT-PCR and DNA-sequencing gel) and versatility (compare more than two RNAs for both up- and downregulated genes). It has become obvious that finding the genes by either differential display or DNA microarray is only the first step toward the understanding of biological problems under investigation. It is hoped that finding the right genes through careful experimental designs, such as outlined here, will narrow down the number of relevant genes and increase the odds for solving the puzzles of nature, such as ras.

Animals↗
PubMed1999

Cloning, molecular analysis and differential cell localisation of the p36 RACK analogue antigen from the parasite protozoon Crithidia fasciculata.

The family of the RACK molecules (receptors for activated C kinases) are present in all the species studied so far. In the genus Leishmania, these molecules also induce a strong immune reaction against the infection. We have cloned and characterised the gene that encodes the RACK analogue from the parasite trypanosomatid Crithidia fasciculata (CACK). The molecule seems to be encoded by two genes. The sequence analysis of the cloned open reading frame indicates the existence of a high degree of conservation not only with other members of the Trypanosomatidae but also with mammalians. The study of the protein kinase C phosphorylation sites shows the presence of three of them, shared with the mammalian species, additional to those present in the other protozoa suggesting a certain phylogenetic distance between the protozoon Crithidia fasciculata and the rest of the Trypanosomatidae. The CACK-encoded polypeptide shows an additional sequence of four amino acids at the carboxy-terminal end, which produces a different folding of the fragment with the presence of an alpha-helix instead of the beta-sheet usual in all the other species studied. A similar result is elicited at the amino-terminal end by the change of three amino acid residues. The immunolocalisation experiments show that the CACK displays a pattern with a distribution mainly at the plasma membrane, different from that of the related Leishmania species used as control, that displays a distribution close to the nucleus. Altogether, the data suggest that the existence of the structural differences found may have functional consequences.

Amino Acid Sequence↗
PubMed1991

Are malignant melanoma patients at higher risk for a second cancer?

This study tests whether malignant melanoma (MM) patients are at higher risk of having an unrelated second cancer by comparing the observed incidence of a second cancer in a given population of MM patients with the expected number in an age-matched and sex-matched group of healthy people followed for a similar period. The analysis was based on the person-years method in which the main consideration is the follow-up period after the diagnosis of MM. Of 370 patients with histologically confirmed MM, 27 (7.3%) had a second noncutaneous invasive cancer, diagnosed either simultaneously (within 6 months, five patients) or after the diagnosis of MM (22 patients). The follow-up period for the entire MM group was 1253 person-years, a period during which the expected number of cancer cases in the normal population, according to the Israel Cancer Registry, was 6.6. The observed-expected ratio or the relative risk (RR) was 4.1 (P less than 0.01). After excluding the five patients with simultaneous diagnosis of MM and a second cancer, analysis of the remaining 22 patients in whom MM definitely preceded the second cancer showed an RR of 3.3 (P less than 0.01). For the entire group, there were nine patients with breast cancer, five with head and neck cancer (two with thyroid and three with oral cavity cancer), five with gynecologic cancer (one with uterine and four with ovarian cancer), five myeloproliferative malignancies (one with lymphoma, three with chronic lymphocytic leukemia, and one with myeloma), three gastrointestinal carcinomas (two with colon and one with stomach cancer), and two soft tissue sarcomas. When the differential analysis according to gender and age was done, it was found that the RR was higher for women (5.5, P less than 0.01) than for men where the RR was 2.2 (P less than 0.05). Differential analysis for various age groups showed that the trend for second cancer was consistent in all age groups, with a slight increase in the younger ones. None of the variables of MM, such as location of the primary tumor, level of invasion, or stage, were predictive for a second cancer. Furthermore, the RR for a second cancer did not relate significantly with the treatment given to the MM patient. Concerning the type of second cancer, it was found that the RR was especially high for breast cancer--6.6. These data indicate that MM patients may be at higher risk for having a noncutaneous invasive cancer compared with the general population.

Adolescent↗
PubMed1999

Prion protein and neuronal differentiation: quantitative analysis of prnp gene expression in a murine inducible neuroectodermal progenitor.

The biological function of the cellular prion protein, PrP(c), is currently unknown. The presence of PrP(c) transcripts in the developing neural tube from embryonic day 13.5 and the predominant expression of PrP(c) in the adult brain is suggestive of a role in the onset and/or modulation of neuronal functions. We took advantage of the bipotential neuroectodermal 1C11 cell line to monitor PrP(c) expression during its bioaminergic differentiations. The F9-derived 1C11 precursor cell line displays a stable and immature phenotype in the absence of extracellular signal and, upon induction, has the capacity to acquire a complete serotonergic or noradrenergic phenotype, the two pathways being mutually exclusive. A real-time quantitative PCR assay was developed to assess PrP(c) gene expression at definite times of the two programs that correspond to sequential acquisition of neurotransmitter-specific functions. 1C11 cells and their differentiated progenies express significant amounts of PrP transcripts and of the corresponding protein. A unique decrease in prnp gene expression is observed upon entry into the serotonergic pathway, correlating with a downregulation at the protein level. Moreover, nerve growth factor (NGF) is shown to induce a decrease in the level of prnp gene expression along the serotonergic - but not the noradrenergic - pathway. Our study accurately establishes that prnp gene expression (i) is strongly upregulated concomitantly with cell fate restriction of multipotential cells towards the neural lineage; (ii) is differentially regulated along the serotonergic versus noradrenergic differentiation program of a unique neuroectodermal progenitor. The 1C11 cell line may provide a new tool for studying prion infectivity in a well-defined neuronal context.

Amyloid↗
PubMed2000

[Application of DNA analysis for differential diagnosis of familial hypercholesterolemia and familial defect of apolipoprotein b-100].

AIM: To determine occurrence of apolipoprotein B-100 mutation in codon 3500 (apoB3500) in patients with primary hypercholesterolemia in Russia. MATERIALS AND METHODS: The study included 71 patients with clinical diagnosis of familial hypercholesterolemia (FH) and 24 relatives. All the subjects were tested for the presence of apoB3500 mutation using polymerase chain reaction and cleavage with restriction enzyme HhaII. Samples demonstrating anomalous pattern were further analysed by automatic DNA sequencing. RESULTS: Apob3500 mutation was detected in two (2.8%) female patients. In both cases cholesterol levels were severely increased although clinical features were different. CONCLUSION: Some cases of primary hypercholesterolemia in Russia may be due to familial defective apoB-100. Further screening of FH patients is required for a precise estimation of the incidence rate of familial defective apoB-100 in this country.

Adult↗
PubMed2005

Differential display analysis reveals the expression of glutathione S-transferase omega and novel genes through an ITAM-containing receptor in ascidian immunocytes.

The immunoreceptor tyrosine-based activation motif (ITAM) plays an important role in signal transduction through antigen receptors in mammalian lymphocytes. We previously reported that an ITAM-containing receptor, ascidian hemocyte ITAM-containing receptor 1 (AhITAMR1), exists on the hemocyte surfaces of the ascidian Halocynthia roretzi, and is involved in both phagocytosis and hemocyte aggregation. In this study, we carried out differential display screening of upregulated genes during H. roretzi hemocyte aggregation and found that at least three genes are upregulated. One encodes glutathione S-transferase omega (GSTomega), while the other two encode novel proteins. The expression of all three genes was induced by treatment with a specific monoclonal antibody against AhITAMR1, while their expression was inhibited by wortmannin, BAPTA-AM, and cyclosporin A. We also found that the expression of GSTomega was induced by treatment with anti-T cell receptor antibody in mouse peripheral T cells. We propose that signal transduction pathways mediated by ITAM-containing receptors are conserved from ascidian hemocytes to mammalian T cells.

Animals↗
PubMed1986

Exophthalmos caused by excessive fat: CT volumetric analysis and differential diagnosis.

CT has proven to be excellent in identifying orbital pathology responsible for proptosis. Occasionally, no discrete mass or extraocular muscle enlargement to explain the exophthalmos is found, only an appearance suggestive of an abnormal increase in orbital fat volume. Fifteen patients were studied with proptosis apparently resulting from increased orbital fat. Clinical follow-up revealed that four of them had Graves orbitopathy, unilateral in one; two had Cushing disease/syndrome; and nine were obese without endocrinopathy. The orbital volume and percentage orbital fat volume were measured by CT software analysis in these patients and in a control group of 16 patients without proptosis. Measurements of proptosis and thickness of the scalp fat pad at the inion level were also performed. Significantly greater values for orbital fat volume, percentage fat volume, and proptosis were found in the proptosis group compared with the control group. There was excellent correlation between proptosis and percentage fat volume, supporting the contention that increased orbital fat is responsible for the proptosis. The thickness of the scalp fat pad at the inion level was significantly greater in obese and Cushing patients than in control subjects, but the thickness was not significantly greater in Graves patients than in controls. Proptosis and inion fat were well correlated (r = 0.74) in the control and obese patients, which suggests a relation between general body fat and orbital fat volume.

Adipose Tissue↗
PubMed2006

Novel differential neuroproteomics analysis of traumatic brain injury in rats.

Approximately two million traumatic brain injury (TBI) incidents occur annually in the United States, yet there are no specific therapeutic treatments. The absence of brain injury diagnostic endpoints was identified as a significant roadblock to TBI therapeutic development. To this end, our laboratory has studied mechanisms of cellular injury for biomarker discovery and possible therapeutic strategies. In this study, pooled naïve and injured cortical samples (48 h postinjury; rat controlled cortical impact model) were processed and analyzed using a differential neuroproteomics platform. Protein separation was performed using combined cation/anion exchange chromatography-PAGE. Differential proteins were then trypsinized and analyzed with reversed-phase LC-MSMS for protein identification and quantitative confirmation. The results included 59 differential protein components of which 21 decreased and 38 increased in abundance after TBI. Proteins with decreased abundance included collapsin response mediator protein 2 (CRMP-2), glyceraldehyde-3-phosphate dehydrogenase, microtubule-associated proteins MAP2A/2B, and hexokinase. Conversely C-reactive protein, transferrin, and breakdown products of CRMP-2, synaptotagmin, and alphaII-spectrin were found to be elevated after TBI. Differential changes in the above mentioned proteins were confirmed by quantitative immunoblotting. Results from this work provide insight into mechanisms of traumatic brain injury and yield putative biochemical markers to potentially facilitate patient management by monitoring the severity, progression, and treatment of injury.

Animals↗
PubMed1996

A model phage display subtraction method with potential for analysis of differential gene expression.

In order to establish a subtractive procedure that makes it possible to enrich selectively phage displayed antibodies directed against proteins constituting a difference between two populations of cells, a competitive selection strategy utilising two solid phases was developed and tested. Antibodies recognising a defined difference between two otherwise identical protein mixtures were isolated and their specificity confirmed. To test further the efficacy of selection inhibition during the competitive selections, selections towards a total cell extract were performed with and without competition from the same extract. An analysis of the resulting phage antibodies confirmed the subtractive nature of the system described.

Animals↗
PubMed2002

Ultrastructural analysis of differentiation in Legionella pneumophila.

Legionella pneumophila is an adaptive pathogen that replicates in the intracellular environment of fundamentally divergent hosts (freshwater protozoa and mammalian cells) and is capable of surviving long periods of starvation in water when between hosts. Physiological adaptation to these quite diverse environments seems to be accompanied by morphological changes (Garduño et al., p. 82-85, in Marre et al., ed., Legionella, 2001) and conceivably involves developmental differentiation. In following the fine-structural pathway of L. pneumophila through both in vitro and in vivo growth cycles, we have now discovered that this bacterium displays an unprecedented number of morphological forms, as revealed in ultrathin sections and freeze-fracture replicas for transmission electron microscopy. Many of the forms were identified by the obvious ultrastructural properties of their cell envelope, which included changes in the relative opaqueness of membrane leaflets, vesiculation, and/or profuse invagination of the inner membrane. These changes were best documented with image analysis software to obtain intensity tracings of the envelope in cross sections. Also prominent were changes in the distribution of intramembranous particles (clearly revealed in replicas of freeze-fractured specimens) and the formation of cytoplasmic inclusions. Our results confirm that L. pneumophila is a highly pleomorphic bacterium and clarify some early observations suggesting sporogenic differentiation in L. pneumophila. Since morphological changes occurred in a conserved sequence within the growth cycle, our results also provide strong evidence for the existence of a developmental cycle in L. pneumophila that is likely accompanied by profound physiological alterations and stage-specific patterns of gene expression.

Cell Membrane↗
PubMed2005

Contrast-enhanced dynamic MR imaging parameters and histological types of invasive ductal carcinoma of breast.

The diagnosis of breast cancer has progressed owing to the improvement of both MRI equipments and MR techniques. CE-MRI is expected to be more useful for diagnosis of the existence and characteristics of breast cancer than mammography and ultrasound. Forty-three breast cancer patients, who underwent breast MRI examination before surgery and had histologically confirmed invasive ductal carcinomas, were included in this study. In 19, seven and 17 of them, carcinomas were histologically classified as papillotubular, solid-tubular, and scirrhous type, respectively. In dynamic MRI studies, the MR parameters were calculated from the signal intensity determined before and at five time-points after a bolus of 0.1 mmol Gd-DTPA/kg in the chosen region of interest (ROI). The differential analysis of MR parameters with reference to histopathology was performed by one-way analysis of variance (ANOVA) in dynamic studies. Four of 15 parameters analyzed, ENH1-2, ENH1, MSLP, and SAT3, were significantly different between papillotubular and scirrhous carcinoma. The dynamic curve of papillotubular carcinoma reached a maximum in the very early phase and then decreased slowly (linear pattern), while that of scirrhous carcinoma peaked in the slightly later phase and then decreased (parabolic pattern). In the differential analysis by Kruskal-Wallis test, although no significant differences were found in any morphological parameters among three histological types, spicula formation and rim enhancement tended to appear comparatively frequently in scirrhous and solid-tubular carcinoma. CE-MRI might reveal the differences of growth characteristics among different histological types of breast carcinomas and provide a diagnostic method useful for estimation of the prognosis of breast cancer.

Adult↗
PubMed2006

Microarray analysis of differentially expressed fetal genes in placental tissue derived from early and late onset severe pre-eclampsia.

Although it has been well documented that pre-eclampsia is caused by a combination of maternal and fetal susceptibility genes, little is known about the precise etiology of this complicated disorder. To investigate how the expression of fetal genes contributes to the mechanisms underlying the progression of this disease, we have analyzed differentially expressed genes using placentas from 13 normal pregnancies and 14 pregnancies with severe pre-eclampsia. We performed genome-wide expression profiling using high-density oligonucleotide microarrays, followed by validation using real-time PCR. Among the 47,000 genes that were screened in the microarray, 137 genes were found to be differentially expressed between normal and pre-eclamptic tissues. Among these candidates, 70 were up-regulated and 67 were down-regulated. The up-regulated genes included leptin and inhibin A, which are well-known biological markers for pre-eclampsia, as well as FLT1, which was recently proved to be tightly linked with the etiology of this disease. Gene ontology analysis further revealed several biological processes that could be associated with the development of pre-eclampsia, including response to stress, host-pathogen interactions, lipid metabolism, and carbohydrate metabolism. Analyses of biological mechanisms highlighted some important pathways that may be involved in this disorder, such as the TGF-beta and CEBPA-related pathways. Furthermore, when our present subjects were classified as either severe cases of early onset or late onset pre-eclampsia, the expression of 11 genes could be correlated with the severity of this disorder. These genes may therefore prove to be novel biological markers by which the severity of this condition could be predicted. Our data are likely to be a useful future resource in the elucidation of the disease-process and in the identification of novel markers for pre-eclampsia.

Adult↗
PubMed2006

Differential expression analysis of proteins from neutrophils in the periparturient period and neutrophils from dexamethasone-treated dairy cows.

Neutrophils play an important role in the host immune system's defense against pathogens. It has been established that neutrophil functionality is suppressed in dairy cows at parturition. The periparturient immunosuppression seen in dairy cattle is associated with an increase in the incidence of mastitis. Using amine-reactive isobaric tagging reagents we have measured relative protein expression from normal prepartum neutrophils and neutrophils isolated during immunosuppression at parturition. We found over 40 proteins that are differentially expressed at parturition compared to prepartum. In addition, we measured relative protein expression from normal neutrophils and neutrophils obtained from cows treated with an immunosuppressive dose of dexamethasone. We found over 70 proteins are differentially expressed during dexamethasone treatment. We then compared protein expression changes in dexamethasone-induced immunosuppression to periparturient immunosuppression. A number of proteins underwent similar expression changes in both dexamethasone and periparturient immunosuppressed neutrophils. Most significantly, we found a significant number of proteins whose relative expression was not the same for these two different conditions that cause neutrophil dysfunction. The data demonstrates that there are both similarities and differences in neutrophil protein expression in the naturally occurring immunosuppression observed at parturition compared to dexamethasone-induced immunosuppression in the bovine neutrophil.

Animals↗
PubMed2003

A differential pharmacokinetic analysis of the erythropoietin receptor population in newborn and adult sheep.

Strong evidence indicates that erythropoietin (Epo) is eliminated via Epo receptors (EpoR). Epo receptors may be classified as erythropoietic receptors that are largely located on erythroid progenitor cells in the bone marrow (BM) and nonerythropoietic receptors present in most tissues. Epo's elimination kinetics was studied using a very sensitive tracer interaction method (TIM) before and after chemical ablation of BM as an indirect way of evaluating the EpoR through an assortment of pharmacokinetic parameters (VM, KM, K, and CL) used in differentiating the EpoR population in newborn and adult sheep. TIM identified a parallel nonlinear Michaelis-Menten (VM and KM), and linear (K) elimination pathway and found the latter pathway to be significantly (p < 0.01) more dominant in lamb: K/(VM/KM + K) = 0.309 (25.3) versus 0.0895 (18.4) mean (CV%) lambs versus adult sheep. The significantly (p < 0.01) larger total clearance found for lambs indicates a larger nonhematopoietic tissue clearance of Epo (CL = 118 (10.9) ml/h/kg versus 67.8 (19.3) lamb versus adult sheep). The VM/KM ratio for the nonlinear pathway was not found to be significantly different (p > 0.05) between newborn and adults with values of 1.10 (15.8) and 1.30 (3.81) h-1, respectively. We proposed the hypothesis that the linear pathway is via nonhematopoietic EpoR. Assuming that Epo's elimination largely depends not only on erythropoietic EpoR but also on nonhematopoietic EpoR, this work shows a significant difference in the relative proportions of the two EpoR populations in lamb and adult sheep. The larger dominance of the nonhematopoietic EpoR in lamb supports the hypothesis that these receptors are more needed in early life, e.g., providing neuroprotection from perinatal hypoxemic-ischemic episodes.

Alkylating Agents↗
PubMed2003

[Differential protein analysis in rat renal proximal tubule epithelial cells in response to acetazolamide and its relation with the inhibition of AQP1].

AIM: To study the endogenous mechanism for the inhibition of aquaporin-1 expression in rat renal proximal tubule epithelial cells in response to acetazolamide. METHODS: Primary cultured rat renal proximal tubule epithelia cells were divided into two groups: one was subjected to 1 x 10(-5) mol.L-1 acetazolamide, the other served as normal control. When grown to sub-confluency, the cells were disintegrated to perform isoelectrofocusing electrophoresis in order to find the differential proteins induced by the acetazolamide treatment. The differential proteins were defined by peptide mass fingerprinting technology. RESULTS: Two differential proteins were found in the cell disintegrant. The pI 3.8 protein was reduced after treatment, which showed 21.4% similarity with the brush border membrane myosin from rat brain and testis, and 27% with glycogen phosphorylase; The pI 5.5 protein was increased on the contrary, with 20% similarity to phosphatidylinositol transfer protein alpha isoform. CONCLUSION: Acetazolamide inhibited AQP1 expression probably by affecting the expression of pI 3.8 and pI 5.5 proteins.

Acetazolamide↗
PubMed2001

Global analysis of differential gene expression after transformation with the v-H-ras oncogene in a murine tumor model.

Mouse PB-3c mast cells stably transfected with the v-H-ras oncogene induce tumor formation in vivo when implanted into mice. Such tumor cells are characterized by an autocrine IL-3 loop. DNA microarrays allow simultaneous transcript imaging of several thousand genes and the technique was applied in this tumor model to analyse gene expression following malignant transformation. Using three independent tumor lines derived from the same precursor the expression of about 400 out of 11 000 genes was modulated in each tumor. A subset of only 75 genes (0.68%) is shared and up- or downregulated in all three lines. A significant portion of this gene pool possesses functions related to tumorigenesis such as cell adhesion, signaling or transcriptional regulation. Apart from a number of expressed sequence tags (EST's) we find downregulation of four interferon-inducible genes in the tumor lines. Finally, when we extrapolate our data to the complete mouse genome, we estimate that about 500 genes are differentially expressed in tumor cells compared to the precursor cell PB-3c.

Animals↗
PubMed2005

Oligonucleotide microarray analysis of differential transporter regulation in the regenerating rat liver.

AIMS: The aim of this study was to investigate the regulation of hepatic transport systems during liver regeneration. METHODS: A DNA oligonucleotide microarray was developed with probes for 400 transcripts. Data were confirmed using real-time PCR and on a functional level in the perfused rat liver. Liver homogenates were taken 3-48 h following 2/3-hepatectomy in rats and compared with sham-operated and non-operated controls. RESULTS: A more than two-fold increase or decrease of expression was obtained in 183 genes following partial hepatectomy and in 16 genes in sham-operated rats. A strong induction during liver regeneration was detected for the amino acid transporters LAT4, SN2 and sodium-dependent neutral amino acid transporter (ASCT)2, whereas amino acid transport system (ATA)2 and ATA3 expressions remained unchanged. The upregulation of ASCT2 may be responsible for the increase in sodium-dependent neutral amino acid influx important for liver cell proliferation. Expression of the osmolyte transporters Smit, TauT and Bgt1 was almost unchanged indicating that osmolytes are not involved in the cell volume increase during liver regeneration. The basolateral bile salt transporter Ntcp messenger RNA (mRNA) was significantly downregulated, whereas bile salt export pump (Bsep) and multidrug resistance protein (Mrp)2 expressions remained almost unchanged. An increased mRNA expression following partial hepatectomy was detected for organic anion transporting polypeptide (Oatp)5, Octn1, Octn2 and SGLT2. In contrast, Mrp6, Oatp 2, Oatp 3, Oatp 4 and Oatp 7 were downregulated. A five-fold upregulation at the protein level was shown for the Na(+)-K(+)-2Cl- cotransporter sodium-potassium-2-chloride cotransporter (NKCC1). CONCLUSIONS: The data show a differential regulation of hepatic transport systems during liver regeneration.

Amino Acid Transport Systems↗