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Biomedical subjects

U Marti

Publications and source records attributed to U Marti.

At least 19 recordsLinked to original sources
PubMed2002

Differential expression of IgG Fc binding protein (FcgammaBP) in human normal thyroid tissue, thyroid adenomas and thyroid carcinomas.

The genetic events involved in thyroid carcinogenesis are still incompletely understood. Several rearrangements and mutations of oncogenes have been implicated in the development of thyroid papillary carcinomas, follicular adenomas and carcinomas. However, none of these molecular alterations is suitable either as a general marker for the diagnosis of thyroid carcinomas or to differentiate between thyroid follicular adenomas and carcinomas. In order to identify new genes with altered expression which could serve as such markers, we analyzed RNA from thyroid tumor and normal tissue using a novel technique called restriction-mediated differential display. Several differentially expressed genes were identified, including the gene for IgG Fc binding protein (FcgammaBP). Differential expression of FcgammaBP was confirmed by quantitative real-time RT-PCR. Our experiments showed that IgG Fc binding protein (FcgammaBP) is differentially expressed in normal thyroid tissue, thyroid adenomas and thyroid carcinomas. While the FcgammaBP gene is constitutively expressed in normal thyroid tissue, its expression is significantly increased in follicular thyroid adenomas and significantly decreased in papillary and follicular thyroid carcinomas. Thus, measurement of the expression levels of FcgammaBP in thyroid biopsies might help to make the otherwise difficult distinction between a thyroid follicular adenoma and a follicular carcinoma.

Adenoma↗
PubMed2001

Advances ofcapillary electrophoresis in clinical and forensic analysis (1999-2000).

In this paper, capillary electrophoresis in clinical and forensic analysis is reviewed on the basis of the literature of 1999, 2000 and the first papers in 2001. An overview of progress relevant examples for each major field of application, namely (i) analysis of drug seizures, explosives residues, gunshot residues and inks, (ii) monitoring of drugs, endogenous small molecules and ions in biofluids and tissues, (iii) general screening for serum proteins and analysis of specific proteins (carbohydrate deficient transferrin, alpha1-antitrypsin, lipoproteins and hemoglobins) in biological fluids, and (iv) analysis of nucleic acids and oligonucleotides in biological samples, including oligonucleotide therapeutics, are presented.

Clinical Chemistry Tests↗
PubMed2001

Nuclear localization of epidermal growth factor and epidermal growth factor receptors in human thyroid tissues.

Epidermal growth factor (EGF) has widespread growth effects, and in some tissues proliferation is associated with the nuclear localization of EGF and epidermal growth factor receptor (EGFR). In the thyroid, EGF promotes growth but differs from thyrotropin (TSH) in inhibiting rather than stimulating functional parameters. We have therefore studied the occurrence and cellular distribution of EGF and EGFR in normal thyroid, in Graves' disease, where growth is mediated through the thyrotropin receptor (TSHR), and in a variety of human thyroid tumors. In the normal gland the staining was variable, but largely cytoplasmic, for both EGF and EGFR. In Graves' disease there was strong cytoplasmic staining for both EGF and EGFR, with frequent positive nuclei. Nuclear positivity for EGF and particularly for EGFR was also a feature of both follicular adenomas and follicular carcinomas. Interestingly, nuclear staining was almost absent in papillary carcinomas. These findings document for the first time the presence of nuclear EGF and EGFR in thyroid. Their predominant occurrence in tissues with increased growth (Graves' disease, follicular adenoma, and carcinoma) may indicate that nuclear EGF and EGFR play a role in growth regulation in these conditions. The absence of nuclear EGF and EGFR in papillary carcinomas would suggest that the role played by EGF in growth control differs between papillary carcinoma and follicular adenomas/carcinomas of the thyroid.

Adenoma↗
PubMed2000

Growth hormone regulates growth hormone receptor gene transcription in primary human thyroid cells.

In this study the regulation of GH-receptor gene (GHR/GHBP) transcription by different concentrations of GH (0, 12.5, 25, 50, 150, 500 ng/ml) with and without variable TSH concentrations (0.5, 2, 20 mU/l) in primary human thyroid cells cultured in serum-free hormonally-defined medium was studied. The incubation time was 6 h and GHR/GHBP mRNA expression was quantitatively assessed by using PCR amplification at hourly intervals. Correlating with the GH-concentrations added a constant and significant increase of GHR/GHBP gene transcription was found. After the addition of 12.5 ng/ml GH, GHR/GHBP mRNA concentration remained constant over the incubation period of 6 h but in comparison with the experiments where no GH was added there was a significant change of GHR/GHBP mRNA expression. Following the addition of 25 ng/ml GH a slight but further increase of GHR/GHBP transcription products was seen which increased even more in the experiments where higher GH concentrations were used. These data focusing on GHR/GHBP gene transcription derived from cDNA synthesis and quantitative PCR amplification were confirmed by run-on experiments. Furthermore, cycloheximide did not affect these changes supporting the notion that GH stimulates GHR/GHBP gene transcription directly. In a second set of experiments, in combination with variable TSH levels, identical GH concentrations were used and no difference in either GHR/GHBP mRNA levels or in transcription rate (run-on experiments) could be found. In conclusion, we report data showing that primary thyroid cells express functional GH-receptors in which GH has a direct and dose dependent effect on the GHR/GHBP gene transcription. Furthermore, TSH does not a have a major impact on GHR/GHBP gene regulation.

Base Sequence↗
PubMed2000

The nuclear accumulation of a variant epidermal growth factor receptor (EGFR) lacking the transmembrane domain requires coexpression of a full-length EGFR.

Both the epidermal growth factor (EGF) and its receptor (EGFR) accumulate in the nucleoplasm during liver regeneration. This localization in a nonmembraneous compartment presents a challenge in that the standard form of EGFR is a transmembrane protein and suggests the existence of a variant, soluble form of EGFR. To investigate the localization of such a putative EGFR splice variant, we generated a transmembrane-devoid form of EGFR. We placed this transmembrane-negative [TM(-)] EGFR construct and full-length wild-type (wt) EGFR either in a retroviral transfection vector or in an inducible expression vector. Mouse 3T3 cells, which express endogenous EGFR, were transfected with the TM(-) EGFR construct. The expression of these TM(-) EGFR, detected with a specific antibody against human EGFR using a confocal laser-scanning microscope, was predominantly found in the cytoplasm with no nuclear localization. After an overnight incubation with EGF the TM(-) EGFR accumulated in the nucleus. In mouse NR6 cells, which lack endogenous EGFR, transfected TM(-) EGFR were found in the cytoplasm, but incubation with EGF did not result in a nuclear accumulation of TM(-) EGFR. However, NR6 cells transfected with both TM(-) EGFR and wt EGFR showed nuclear accumulation after EGF treatment. These results suggest that both the wt EGFR and the TM(-) EGFR are required for nuclear accumulation of TM(-) EGFR and may implicate a model of homotypic recognition and translocation of a splice variant of EGFR.

3T3 Cells↗
PubMed2000

Epidermal growth factor is decreased in liver of rats with biliary cirrhosis but does not act as paracrine growth factor immediately after hepatectomy.

BACKGROUND/AIMS: Epidermal growth factor, a potent mitogen for hepatocytes and cholangiocytes, is thought to act as an immediate-early gene after partial hepatectomy. Since regeneration is impaired in cirrhosis, we explored the expression of epidermal growth factor in cirrhotic rat liver immediately after partial hepatectomy. METHODS: Cirrhosis was induced by bile duct ligation (n=21); sham-operated animals served as controls (n=21). Twenty-five days after initial surgery animals were subjected to 70% partial hepatectomy or sham operation; the liver was sampled before surgery and 20, 40 and 90 min thereafter. Epidermal growth factor mRNA levels were assessed by quantitative reverse transcription polymerase chain reaction. Protein expression was estimated by immunohistochemistry using a polyclonal antibody against epidermal growth factor. RESULTS: Before hepatectomy, epidermal growth factor mRNA averaged 70.3+/-39.9 pg/microg of total RNA in controls; this was markedly decreased to 21.9+/-12.7 pg/microg RNA in bile duct ligation (p<0.01). Epidermal growth factor mRNA did not increase after partial hepatectomy in either group, with the exception of sham-operated controls. Immunohistochemistry revealed that partial hepatectomy had no effect on epidermal growth factor expression. Hepatocytes showed uniformly cytosolic epidermal growth factor in controls, while in bile duct ligation immunostaining was faint or absent. Cholangiocytes exhibited a strong cytosolic staining in all experimental groups. CONCLUSIONS: The present study shows that epidermal growth factor is reduced in the cirrhotic liver. This could contribute to the loss of parenchymal liver tissue observed in cirrhosis. The lack of up-regulation after PH sheds doubt on the role of epidermal growth factor as an immediate-early gene in hepatic regeneration. Further, we demonstrate that epidermal growth factor accumulates in cholangiocytes. This observation is strong evidence for involvement of the mitogen epidermal growth factor in the proliferation of bile ducts during cirrhogenesis.

Animals↗
PubMed1999

Regulation of human growth hormone receptor gene transcription by triiodothyronine (T3).

In this study the hypothesis that triiodothyronine (T3) and growth hormone (GH) may have some direct or indirect effect on the regulation of GH-receptor/GH-binding protein (GHR/GHBP) gene transcription was tested. Different concentrations of T3 (0, 0.5, 2, 10 nmol/l) and GH (0, 10, 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally-defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was quantitatively assessed by using PCR amplification. GH at a concentration of 10 ng/ml resulted in a significant increase of GHR/GHBP gene expression whereas a supraphysiological concentration of GH (150 ng/ml) caused a significant decrease of GHR/GHBP mRNA levels. The simultaneous addition of 0.5 nmol/l T3 to the variable concentrations of GH did not modify GHR/GHBP mRNA levels whereas the addition of 2 nmol/l up-regulated GHR/GHBP gene expression already after 1 h, an increase which was even more marked when 10 nmol/l of T3 was added. Interestingly, there was a positive correlation between the increase of GHR/GHBP mRNA levels and the T3 concentration used (r: 0.8). In addition, nuclear run-on experiments and GHBP determinations were performed which confirmed the changes in GHR/GHBP mRNA levels. Cycloheximide (10 microg/ml) did not alter transcription rate following GH addition but blocked GHR/GHBP gene transcription in T3 treated cells indicating that up-regulation of GHR/GHBP gene transcription caused by T3 requires new protein synthesis and is, therefore, dependent on indirect mechanisms. In conclusion, we present data showing that T3 on its own has a stimulatory effect on GHR/GHBP gene transcription which is indirect and additive to the GH-induced changes.

Carrier Proteins↗
PubMed1999

The interaction of Bcl-2 and Bax regulates apoptosis in biliary epithelial cells of rats with obstructive jaundice.

A complex molecular network controls cell homeostasis by inducing apoptosis or proliferation. The balance of Bcl-2 and Bax, members of a protein family, determines whether a cell will become immortal (Bcl-2) or will undergo apoptosis (Bax). To determine the role of Bcl-2 and Bax during proliferation of biliary epithelial cells (BEC) after bile duct ligation (BDL) and their regression after biliary decompression we induced hyperplasia of BEC by BDL in male rats. Regression of hyperplastic BEC by way of apoptosis was induced by biliary decompression through a Roux-en-Y biliodigestive anastomosis. To quantify apoptosis a modified TUNEL assay was used. Expression of Bcl-2 and Bax was visualized by immunohistochemistry and quantified stereologically. BEC increased from <1% to >20% after BDL; this increase was associated with overexpression of Bcl-2 in up to 30% of hyperplastic BEC. After biliodigestive anastomosis, apoptotic BEC increased from <0.1% to a peak of 5.4% after 1 day to reach baseline again 1 week after decompression. This was associated with de novo appearance of Bax. The interaction between Bcl-2 and Bax triggers apoptosis in BEC and acts as a cell rheostat in BEC hyperplasia and its involution after biliary decompression.

Anastomosis, Roux-en-Y↗
PubMed1999

Inhibition of iodine organification and regulation of follicular size in rat thyroid tissue in vitro.

The factors mediating the accumulation of thyroglobulin are of great importance to the understanding of the pathogenesis of human and experimentally induced colloid goiters. To elucidate further the underlying cellular mechanism, thyroid fragments from newborn rats were incorporated into semisolid alginate beads and were cultured as three-dimensional organoids for up to 21 d. In five parallel cultures, the medium contained either no supplements (group A), Nal (group B), thyroid-stimulating hormone (TSH) (group C), Nal plus TSH in the same concentrations as B and C (group D), or Nal and TSH (as in group D) plus methimazole (MMI, group E). The thyroid organoids maintained morphological integrity, functional activity, and ability to proliferate in vitro. Addition of iodine to the cultures significantly increased mean (+/-SEM) follicular diameters from 19.5 +/- 0.7 microm in controls to 33.9 +/- 2.2 microm (p < 0.0001) when NaI was added alone (group B), and 30.4 +/- 1.7 microm (p < 0.0001) when combined with TSH (group D). The effect of NaI on follicular size was abolished by MMI (group E, follicular diameter 23.5 +/- 1.3 microm). The results presented support the recent finding, using a rat colloid goiter model, that not only TSH but also iodine organification or its inhibition are important factors in modulating follicular morphology.

Animals↗
PubMed1999

Follicle-forming cat thyroid cell lines synthesizing extracellular matrix and basal membrane components: a new tool for the study of thyroidal morphogenesis.

Interactions between follicular epithelial cells and extracellular matrix (ECM) are supposed to play an important role in the development and maintenance of thyroid tissue architecture. In the present study we have therefore investigated the synthesis of ECM components by a feline thyroid cell line which is able to form follicle-like structures in vitro, and also in v-ras-transfected and control-transfected sublines. Transfections were performed by lipofection with pZSR (viral Harvey ras gene; neo) and pSV2-neo (control, neo only) plasmids. We have adapted a semisolid culture system composed exclusively of polymerized alginate and therefore devoid of ECM components. Feline cells embedded in alginate gels as single cells and cultured for up to 90 days formed cell clusters within 10 days. Follicle-like structures were formed in the original cell lines and also in the v-ras- and control-transfected cells. Differences in proliferation rates were observed, the v-ras-transfected cells growing up to two to three times faster than the non-transfected cells. Immunostaining was done using rabbit first antibodies directed against mouse collagen IV, human fibronectin, laminin (tumor Engelbreth-Holm-Swarm laminin), perlecan and other ECM components. For comparison, immunostaining was also performed on cryosections of nodular goiters of six hyperthyroid cats. The cell lines and their transfected clones stained strongly positive for collagen IV and fibronectin, and positively but less strongly for laminin and perlecan. The cat goiter tissue stained positively for collagen IV, laminin, perlecan, and fibronectin, and positive staining for S-laminin (containing the beta2-chain) was seen in blood vessel walls in this tissue. In conclusion, cat cell lines grow three-dimensionally in alginate beads over several weeks, they form follicle-like structures and express the same ECM components as the native cat goiter tissue. Transfection with v-ras does increase proliferation rate, but does not fundamentally alter formation of follicle-like structures and ECM expression. Alginate gel culture is a promising new tool for the study of follicular morphogenesis, polarity, the expression pattern of ECM components and of the interaction between thyrocytes and ECM. It avoids interference caused by gels composed of ECM components.

Alginates↗
PubMed1998

Morphological, immunohistochemical and autoradiographic studies of thyroid autonomy.

The values and limits of morphological, immunohistochemical and autoradiographic methods in studies of thyroid autonomy are briefly discussed. For meaningful studies of molecular aspects of thyroid autonomy--such as for example TSH receptor and Gs-alpha gene mutations--it is absolutely crucial that the tissue analysed is well characterized and really is autonomous. This is particularly important in view of the well known heterogeneity of human goiter tissue in respect to many if not all functional and proliferative parameters. To prove functional and proliferative autonomy of thyroid tissue, autoradiography is a very helpful tool, while simple morphology and immunohistochemistry do not contribute substantially to this aim.

Animals↗
PubMed1998

Alginate gel culture allows the retention of extracellular matrix and follicular structure of rat thyroid tissue but does not lead to the formation of follicles by FRTL-5 cells.

Extracellular matrix (ECM) and basement membrane (BM) components were studied by immunohistological methods in native rat thyroid tissue, and in rat thyroid tissue and FRTL-5 cells cultured in a three-dimensional alginate bead system. In all three situations, the presence of collagen IV, laminin, perlecan, and fibronectin was demonstrated. There were marked differences between rat thyroid tissue and FRTL-5 cells in culture. Rat thyroid tissue maintained a follicular structure, whereas FRTL-5 cells did not form follicles. Rat thyroid cells multiplied more slowly than FRTL-5 cells and thyroglobulin (Tg) was visible in the follicular lumen, while in FRTL-5 cells Tg was only seen intracellularly. Tg iodination was much lower in FRTL-5 cells than in rat cells. In rat thyroid cells, positive staining for collagen IV, laminin, and perlecan was seen in thin membranes around individual follicles, and for fibronectin around groups of follicles. In FRTL-5 cells, these ECM/BM components could be identified, but were not organized into equally regular networks around groups of cells. These results demonstrate that of the two types of cells examined, primary cultures of rat thyroid cells in alginate beads maintain structural and functional similarities to native thyroid tissue and would therefore be suitable for future in vitro studies of thyroidal ECM/BM and their interrelationship with growth and function of this organ. FRTL-5 cells cultured in alginate beads show some functional, but not structural similarities to native thyroid tissue and so would be less valuable for use in such studies.

Alginates↗
PubMed1998

False positive staining in the TUNEL assay to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyrocarbonate.

BACKGROUND: The terminal transferase uridyl nick end labelling (TUNEL) assay allows the easy demonstration of cell death as a result of apoptosis. However, when this assay is applied to liver tissue, the number of TUNEL positive cells is dependent on the time of incubation with proteinase K. AIM: To test whether false positive results are the result of the release of endogenous endonucleases by proteinase K and can be abolished by pretreatment with diethyl pyrocarbonate (DEPC). METHODS: Involution of hyperplastic ductules in bile duct ligated rats after biliary decompression by Roux-en-Y anastomosis and acute CCl4 intoxication were studied as models of apoptosis and necrosis, respectively. A standard TUNEL assay was applied to formalin fixed tissue sections mounted with cement. To inhibit putative endogenous endonucleases, tissue slides were pre-incubated with DEPC. RESULTS: In the standard TUNEL assay, the number of positive nuclei was highly dependent upon the length of time that sections were incubated with proteinase K. After pretreatment with DEPC, only cells that also exhibited morphological features of apoptosis stained positive. DEPC pretreatment abolished false positive staining in CCl4 induced hepatocyte necrosis and blocked interference by endogenous alkaline phosphatase in intestine. The method of gluing the tissue section to the glass slide was found to be of utmost importance because the effect of DEPC was abolished on silanised slides. CONCLUSIONS: False positive staining in the TUNEL assay in the liver is caused by the release of endogenous endonucleases as a result of proteinase treatment. This can be abolished by pretreatment of tissue slides with DEPC.

Animals↗
PubMed1998

Phenotypic variability in familial combined pituitary hormone deficiency caused by a PROP1 gene mutation resulting in the substitution of Arg-->Cys at codon 120 (R120C).

As pituitary function depends on the integrity of the hypothalamic-pituitary axis, any defect in the development and organogenesis of this gland may account for a form of combined pituitary hormone deficiency (CPHD). A mutation in a novel, tissue-specific, paired-like homeodomain transcription factor, termed Prophet of Pit-1 (PROP1), has been identified as causing the Ames dwarf (df) mouse phenotype, and thereafter, different PROP1 gene alterations have been found in humans with CPHD. We report on the follow-up of two consanguineous families (n = 12), with five subjects affected with CPHD (three males and two females) caused by the same nucleotide C to T transition, resulting in the substitution of Arg-->Cys in PROP1 at codon 120. Importantly, there is a variability of phenotype, even among patients with the same mutation. The age at diagnosis was dependent on the severity of symptoms, ranging from 9 months to 8 yr. Although in one patient TSH deficiency was the first symptom of the disorder, all patients became symptomatic by exhibiting severe growth retardation and failure to thrive, which was mainly caused by GH deficiency (n = 4). The secretion of the pituitary-derived hormones (GH, PRL, TSH, LH, and FSH) declined gradually with age, following a different pattern in each individual; therefore, the deficiencies developed over a variable period of time. All of the subjects entered puberty spontaneously, and the two females also experienced menarche and periods before a replacement therapy was necessary.

Adrenocorticotropic Hormone↗
PubMed1996

Differential expression of Na+,H(+)-antiporter mRNA in biliary epithelial cells and in hepatocytes.

BACKGROUND/AIMS: Amiloride inhibitable Na+,H(+)-exchange has recently been identified and characterized in rat biliary epithelial cells where its activity at low intracellular pH is significantly higher than in hepatocytes. METHODS: Northern blot and reverse transcription-polymerase chain reaction were used to study the expression of the different Na+,H(+)-antiporter isoforms in isolated biliary epithelial cells and hepatocytes. RESULTS: The present study demonstrates for the first time the expression of Na+,H(+)-antiporter isoform 1 mRNA in rat biliary epithelial cells. Moreover, steady-state levels of this message were several-fold higher in biliary epithelial cells than in hepatocytes. In addition, the expression of Na+,H(+)-antiporter isoform 2 in bile duct epithelial cell but not hepatocytes could be demonstrated by reverse transcription-polymerase chain reaction. CONCLUSIONS: The higher expression of Na+,H(+)-antiporter isoform 1 mRNA may indicate a higher rate of synthesis and therefore a higher Na+,H(+)-exchange activity in biliary epithelial cells than in hepatocytes and is entirely compatible with the results of the previous functional studies.

Animals↗
PubMed1996

Decreased constitutive hepatic nitric oxide synthase expression in secondary biliary fibrosis and its changes after Roux-en-Y choledocho-jejunostomy in the rat.

BACKGROUND/AIMS: Inducible nitric oxide synthase (iNOS) in the liver has been shown to increase after stimulation by different compounds. Its exact role in cirrhosis is unclear, but it is thought to be an important factor in the development of the hyperdynamic state. In contrast, little is known about the constitutive form of nitric oxide synthase (cNOS) in the liver, and in particular in liver disease. We therefore investigated immunohistochemical expression of endothelial NOS (cNOS) and, in addition, of macrophage NOS (iNOS), a constitutive and inducible form of NOS, in secondary biliary fibrosis and after reversing these changes by biliodigestive anastomosis. METHODS: Sprague-Dawley rats were allocated to one of seven groups: sham-operated controls (CTR), bile duct ligation for 3 weeks (BDL) and BDL followed by Roux-en-Y choledochojejunostomy (RY); the latter group was studied after 1, 2, 3, 7 and 45 days (RY1, RY2, RY3, RY7, and RY45). cNOS-positive hepatocytes were stained with a monoclonal antibody specifically recognizing this isoenzyme, and quantitated stereologically. RESULTS: Virtually all hepatocytes were positive for cNOS in CTR (99.9 +/- 0.2%); this was markedly reduced in BDL (73.7 +/- 9.8%; p < 0.05). After RY, cNOS positive hepatocytes increased to reach normal levels (99.7 +/- 0.5%) in RY45. In contrast to cNOS, there was no iNOS positive staining with a monoclonal macrophage NOS antibody for this isoenzyme, which was confirmed by determining total iNOS protein concentration by Western blot. Serum nitrite/nitrate concentrations mirrored these findings and decreased from 7.9 +/- 3.7 mol/l in CTR to 2.1 +/- 0.4 mol/l in BDL. After RY, nitrite/nitrate concentration increased to 22.7 +/- 30.1, 32.4 +/- 21.4 and 44.6 +/- 32.7 mol/l in RY1, RY2 and RY3, respectively; in RY45, serum nitrite/nitrate concentration was normal averaging 6.1 +/- 1.2 mol/l. CONCLUSIONS: The present investigation shows that, in secondary biliary fibrosis, endothelial nitric oxide synthase is decreased in hepatocytes, macrophage nitric oxide synthase is not increased, and that-in contrast to current thinking-nitrate/nitrate production is diminished.

Anastomosis, Roux-en-Y↗