Biomedical subjects

Michael Bader

Publications and source records attributed to Michael Bader.

121 records · Page 7Linked to original sources

PubMed2002

Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning.

Hematopoietic stem cells have been successfully employed for tolerance induction in a variety of rodent and large animal studies. However, clinical transplantation of fully allogeneic bone marrow or blood-borne stem cells is still associated with major obstacles, such as graft-versus-host disease or cytoreductive conditioning-related toxicity. Here we show that when rat embryonic stem cell-like cells of WKY origin are injected intraportally into fully MHC-mismatched DA rats, they engraft permanently (>150 days) without supplementary host conditioning. This deviation of a potentially alloreactive immune response sets the basis for long-term graft acceptance of second-set transplanted WKY cardiac allografts. Graft survival was strictly correlated with a state of mixed chimerism, which required functional thymic host competence. Our results provide a rationale for using preimplantation-stage stem cells as vehicles in gene therapy and for the induction of long-term graft acceptance.

Animals↗

PubMed2002

Locally synthesized angiotensin modulates pineal melatonin generation.

We aimed to study the mechanisms and the significance of the influence exerted by the renin-angiotensin system (RAS) on the pineal melatonin production. Pineal melatonin and other indoles were determined by HPLC with electrochemical detection after angiotensin AT1-receptor blockade with Losartan in vivo or in cultured glands. N-acetyltransferase (NAT) activity was radiometricaly measured. To test the in vivo relevance of the local RAS, pineal melatonin and its indole precursors were determined in transgenic rats with inhibited production of angiotensinogen exclusively in astrocytes, TGR(ASrAOGEN). Tryptophan hydroxylase (TPH) and NAT mRNA levels were determined by real-time RT-PCR. Pineal melatonin content was significantly decreased by AT1-receptor blockade in vivo, in cultured glands and in TGR(ASrAOGEN) (35%, 32.4% and 17.5% from control, respectively). Losartan produced a significant decrease of pineal 5-hydroxytryptophan, serotonin, 5-hydroxyindole acetic acid and N-acetylserotonin in pineal cultures. Also, the pineal content of the precursor indoles in TGR(ASrAOGEN) rats was significantly lowered. The reduction of 5-hydroxytryptophan levels by 33-75% in both in vivo and in vitro studies suggests a decreased activity of TPH. Moreover, the TPH mRNA levels in TGR(ASrAOGEN) rats were significantly lower than control rats. On the other hand, NAT activity was unaffected by Losartan in pineal culture and its expression was not significantly different from control in TGR(ASrAOGEN) rats. Our results demonstrate that a local pineal RAS exerts a tonic modulation of indole synthesis by influencing the activity of TPH via AT1-receptors.

Angiotensin Receptor Antagonists↗

PubMed2002

Determination of mercury in blood, urine and saliva for the biological monitoring of an exposure from amalgam fillings in a group with self-reported adverse health effects.

It has been argued that the release of mercury from amalgam fillings is of toxicological relevance. The aim of the study was to determine the internal mercury exposure of two groups differing in their attitude towards possible health hazards by mercury from amalgam fillings. It was to be examined if the two groups differ with regard to the mercury concentration in different biological matrices and to compare the results with current reference values. Blood, urine and saliva samples were analyzed from 40 female subjects who claimed to suffer from serious health damage due to amalgam fillings ("amalgam sensitive subjects"). 43 female control subjects did not claim any association ("amalgam non-sensitive controls"). Mercury was determined by means of cold vapour atomic absorption spectrometry. Number and surfaces of amalgam fillings were determined by dentists for each subject. Median (range) mercury levels in blood were 2.35 (0.25-13.40) micrograms/l for "amalgam sensitive subjects" and 2.40 (0.25-10.50) micrograms/l for "amalgam non-sensitive controls". In urine, the median mercury concentrations were 1.55 (0.06-14.70) micrograms/l and 1.88 (0.20-8.43) micrograms/g creatinine respectively. No significant differences could be found between the two groups. Mercury levels in blood and urine of the examined subjects were within the range of background levels in the general population including persons with amalgam fillings. Stimulated saliva contained 76.4 (6.7-406.0) micrograms mercury/l in "amalgam sensitive subjects" and 57.0 (2.8-559.0) micrograms mercury/l in controls (not significant). Mercury levels in saliva did not correlate with the concentrations in blood and urine, but merely with the number of amalgam fillings or of the filling surfaces. Mercury in saliva is therefore not recommended for a biological monitoring.

Adult↗

PubMed2002

Ischemic injury in experimental stroke depends on angiotensin II.

Since pharmacological interactions of the renin-angiotensin system appear to alter the neurological outcome of stroke patients significantly, we examined the effect of elevated levels of angiotensin II and the role of its receptor subtype AT1 in brain infarction in transgenic mice after focal cerebral ischemia. Angiotensinogen-overexpressing and angiotensin receptor AT1 knockout mice underwent 1 h or 24 h permanent middle cerebral artery occlusion (MCAO). The current study revealed a much smaller penumbra size, i.e., brain tissue at risk, in angiotensinogen-overexpressing animals compared with their wild-type subgroup after 1 h MCAO, but an enlarged infarct size after 24 h. In contrast, a smaller lesion area of energy failure and a much larger penumbral area were found in AT1 knockout mice compared with wild-type littermates. Lower perfusion thresholds for ATP depletion and protein synthesis inhibition after MCAO in AT1-deficient mice and reduced cell damage in an in vitro model using embryonic neurons of AT1 knockout mice suggest injury mechanisms independent of arterial blood pressure. Our data, therefore, demonstrate a direct correlation between brain angiotensin II and the severity of ischemic injury in experimental stroke.

Angiotensin II↗

PubMed2002

Differential effects of angiotensin II and angiotensin-(1-7) at the nucleus tractus solitarii of transgenic rats with low brain angiotensinogen.

OBJECTIVES: In this study, we investigated the effects of angiotensin II and angiotensin-(1-7) at the nucleus tractus solitarii (nTS) in transgenic rats with a severe deficit in brain angiotensinogen production, TGR(ASrAOGEN) (TGR). METHODS: Angiotensin II (10 pmol), angiotensin-(1-7) (10 pmol) or NaCl (0.9%/50 nl) were microinjected into the nTS of urethane-anaesthetized TGR (n = 36) and Sprague Dawley (SD) (n = 34) rats. Mean arterial pressure (MAP) and heart rate were measured via a femoral artery catheter and the baroreflex control of heart rate was evaluated after increases in MAP induced by phenylephrine (baroreflex bradycardia). RESULTS: Angiotensin II microinjections into the nTS of the TGR induced a higher decrease in MAP and heart rate (-37 +/- 5 mmHg and -69 +/- 12 b.p.m., respectively) in comparison to SD rats (-18 +/- 1 mmHg and -43 +/- 5 b.p.m., respectively). In contrast, changes after angiotensin-(1-7) microinjections into the nTS of TGR (-6 +/- 1 mmHg and -13 +/- 4 b.p.m.) were significantly smaller than that induced in SD (-11 +/- 2 mmHg and -24 +/- 6 b.p.m.). The baseline baroreflex sensitivity to phenylephrine of TGR was accentuated in comparison to SD rats (0.70 +/- 0.06 versus 0.44 +/- 0.03 ms/mmHg). Angiotensin II microinjection into the nTS produced similar attenuation in the baroreflex bradycardia in both SD (0.28 +/- 0.07 versus 0.5 +/- 0.07 ms/mmHg, before injection) and TGR (0.44 +/- 0.1 versus 0.82 +/- 0.1 ms/mmHg, before injection). In contrast, angiotensin-(1-7) microinjection elicited a facilitation of the baroreflex bradycardia in SD (0.68 +/- 0.12 versus 0.41 +/- 0.03 ms/mmHg, before injection), while in TGR, angiotensin-(1-7) induced an attenuation of baroreflex bradycardia (0.34 +/- 0.07 ms/mmHg versus 0.55 +/- 0.05 ms/mmHg, before injection). CONCLUSIONS: These results indicate that a permanent inhibition of angiotensinogen synthesis in the brain can lead to an increase in the sensitivity of the baroreflex control of heart rate (baroreflex bradycardia) and an increase in angiotensin II responsiveness at the nTS. However, the nTS effect of angiotensin-(1-7) was significantly attenuated in these transgenic rats. These data further indicate that the decrease in brain angiotensins in the transgenic rats may be functionally relevant and support the concept of differential regulatory mechanisms for the effects of the two angiotensin peptides.

Angiotensin I↗

PubMed2002

Abolition of hypertension-induced end-organ damage by androgen receptor blockade in transgenic rats harboring the mouse ren-2 gene.

A sexual dimorphism in hypertension has been observed in both human and laboratory animal studies. The mechanisms by which male sex hormones regulate cardiovascular homeostasis are still not yet fully understood and represent the subject of this study. The possible involvement of androgen receptors in the development of hypertension and end-organ damage in transgenic rats harboring the mouse Ren-2 renin gene [TGR(mREN2)27] was studied. Male TGR(mREN2)27 rats were treated with the androgen receptor antagonist Flutamide starting at 4 wk of age. Also, an androgen receptor mutation (testicular feminization mutation [tfm]) was introduced in these rats by crossbreeding male TGR(mREN2)27 rats with tfm rats. The resulting offspring male rats that contain the tfm mutation are insensitive to androgens. Flutamide treatment or tfm mutation produced a significant attenuation of the development of hypertension. Besides a reduction in cardiac hypertrophy, urinary albumin excretion was blunted and no histologic characteristics of end-organ damage were observed in the kidney after Flutamide treatment. Testosterone levels increased 15-fold after Flutamide treatment and 2.7-fold by the tfm mutation. Also, plasma estrogens and luteinizing and follicle-stimulating hormones were significantly increased. Plasma renin concentrations and activity but not plasma angiotensinogen were reduced. Our results indicate that androgens contribute not only to the development of hypertension, but even more importantly to end-organ damage in TGR(mREN2)27 rats.

Androgen Antagonists↗

PubMed2002

Transcriptional regulation of the rat bradykinin B2 receptor gene: identification of a silencer element.

Kinins are involved in a variety of physiological and pathophysiological processes related to cardiovascular homeostasis, inflammation, blood flow, and nociception. Under physiological conditions, the bradykinin B2 (BKB2) receptor is constitutively expressed and mediates most of kinins' actions. However, the mechanisms regulating BKB2 receptor gene expression are still poorly understood. In this study, 4.6 kilobases of the 5'-flanking region from the rat BKB2 receptor gene were sequenced, and computer analysis revealed several sites for transcriptional factors. Nine promoter mutants were cloned in luciferase reporter gene vectors and transfected in NG108-15 cells and rat aorta vascular smooth muscle cells (VSMCs), showing several positive and negative regulatory elements. A classical silencer with 56 base pairs (bp) caused a decrease in reporter gene activity in NG108-15 cells and VSMCs and was able to inhibit the thymidine kinase promoter. Using electrophoretic mobility shift assay and surface plasmon resonance assay, protein-DNA interactions in the silencer region were determined and specific sets of protein-silencer complexes were detected in both cell types. More intense complexes were observed in the central 21 bp of the silencer and mutation in a putative SRE-1 site strongly impaired the protein-DNA binding. Down-regulation of the BKB2 receptor population in NG108-15 cells promoted by N(6), 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate was paralleled by an increase in the amount of nuclear proteins bound to the silencer sequence showing an inverse relationship between protein-silencer complexes and the transcription of the BKB2 receptor gene. In summary, these data highlight the cell-specific regulation of the BKB2 receptor and the importance of a silencer element present in the regulatory region of the gene.

5' Flanking Region↗

PubMed2002

Blood pressure response to chronic episodic hypoxia: the renin-angiotensin system.

By using an inspired oxygen fraction that produces oxyhemoglobin desaturation equivalent to that seen in human sleep apnea, we have demonstrated that 35 days of recurrent episodic hypoxia (every 30 s for 7 h/day) results in an 8-13 mmHg persistent increase in diurnal systemic mean arterial blood pressure (MAP) in rats. Blockade of angiotensin II receptors (AT(1a)) eliminates this response. Separate groups of male Sprague-Dawley rats were fed high-salt (8%), ad libitum-salt, or low-salt (0.1%) diets for 7 wk: 2 wk of wash-in for baseline blood pressure measurement and 5 wk of experimental conditions. Rats in each salt group were subjected to episodic hypoxia whereas controls remained unhandled under normoxic conditions. MAP remained at basal levels in all nonepisodic hypoxia controls as well as high-salt-diet episodic hypoxia-exposed rats. Ad lib and low-salt episodic hypoxia rats showed an increase in MAP from 106 and 104 mmHg at baseline to 112 and 113 mmHg, respectively (P < 0.05). Whole kidney renin mRNA was suppressed in high-salt controls and episodic hypoxia rats, whereas kidney AT(1a) mRNA showed opposite changes. Suppression of the renin-angiotensin system with a high-salt diet blocks the increase in MAP in episodic hypoxia-challenged rats, in part by suppressing local tissue renin levels. Upregulation of the tissue angiotensin II system appears to be necessary for the chronic blood pressure changes that occur from episodic hypoxia.

Animals↗

PubMed2002

Tonin and kallikrein in the brain of transgenic rat line expressing human tissue kallikrein.

A transgenic rat line harboring the human tissue kallikrein gene was investigated for expression and activity of tonin and kallikrein in different regions of the brain. The introduction of the transgene into the rat genome produced a significant augmentation of the expression levels and activity of rat tissue kallikrein. The possibility that human kallikrein does not hydrolyze the rat substrate is probably responsible for the augmented expression of the rat enzyme. On the other hand, although expression of tonin was significantly reduced, tonin activity was not altered in most brain structures, except for cerebellum and neurohypophysis.

Animals↗

PubMed2002

In vivo bradykinin B2 receptor activation reduces renal fibrosis.

Angiotensin-converting enzyme (ACE) inhibitors reduce the progression of various fibrotic renal diseases both in humans and in animal models. Unilateral ureteral obstruction (UUO) is an animal model of accelerated renal tubulointerstitial fibrosis that is attenuated by ACE inhibition. Although ACE inhibitors increase bradykinin concentrations in addition to their effect on angiotensin II formation, the role of bradykinin in renal fibrosis has not been studied. We show here that genetic ablation (B2(-/-) mice) or pharmacological blockade of the bradykinin B2 receptor increases UUO-induced interstitial fibrosis in mice, whereas transgenic rats expressing increased endogenous bradykinin show reduced UUO-induced interstitial fibrosis. The increased interstitial fibrosis in B2(-/-) mice was accompanied by a decreased activity of plasminogen activators (PAs) and metalloproteinase-2 (MMP-2), enzymes involved in ECM degradation, suggesting that the protective effects of bradykinin involve activation of a B2 receptor/PA/MMP-2 cascade. This ability of bradykinin to increase PA activity was confirmed in primary culture proximal tubular cells. Thus, in both mice and rats, bradykinin B2 receptor activation reduces renal tubulointerstitial fibrosis in vivo, most likely by increasing ECM degradation.

Animals↗

PubMed2002

Cell type-specific expression of the Mas proto-oncogene in testis.

The Mas proto-oncogene encodes a G-protein-coupled receptor with the common seven transmembrane domains and may be involved in the actions of angiotensins. Because Mas is highly expressed in testis, we investigated the cell type-specificity and the onset of expression of the gene in this organ. Using an RNase protection assay, it could be shown that neither whole testes nor cultured Sertoli and Leydig cells of 12-day-old mice express Mas mRNA. Mas expression is first detected in 18-day-old mice and thereafter increases continuously until 6 months of age. By in situ hybridization, the expression could be localized to Leydig cells and Sertoli cells, the signals being much more pronounced in the former. A weak signal was detected in primary spermatocytes. The strong ontogenetically controlled and cell type-specific expression of this membrane-bound receptor in testis implicates a role for the Mas proto-oncogene in testis maturation and function.

Animals↗

PubMed2002

Molecular structure and alternative splicing of the human carboxypeptidase M gene.

Using RACE technology the 5' and 3' ends of human carboxypeptidase M (CPM) mRNA were determined and found to be divergent from the published sequence. With these results the complete structure of the human CPM gene was established based on the human genome sequence in the GenBank database. The gene was shown to contain 9 exons comprising at least 75 kb of genomic sequence. A novel first exon of 30 bp was identified and an upstream promoter sequence containing several transcription factor binding sites was found by computer analysis. Furthermore, the ATG starting codon was detected defining an open reading frame of 1329 bp that codes for a protein of 443 residues. Additionally, the polyadenylation site was discovered, determining a 3' noncoding region of 2000 nucleotides. The exon-intron boundaries diverged substantially compared to those of the other basic carboxypeptidases, CPD, CPE, CPN, and AEBP1. Cloning and sequencing of RT-PCR products from different tissues revealed alternative splicing of exons 3 and 5, which results in the generation of four different mRNA isoforms. RNA extracted from tumor tissues contained more CPM mRNA than control tissue, suggesting an upregulation of CPM expression in tumors and raising the question of the role of this enzyme in cancer.

Alternative Splicing↗

PubMedDate not supplied

Structure and expression of two kininogen genes in mice.

Kininogens serve dual functions by forming a scaffold for the assembly of the protein complex initiating the surface-activated blood coagulation cascade and as precursors for the kinin hormones. While rats have three kininogen genes, for mice, cattle, and humans only one gene has been described. Here, we present sequence and expression data of a second mouse kininogen gene. The two genes, kininogen-I and kininogen-II, are located in close proximity on chromosome 16 in a head-to-head arrangement. In liver and kidney, both genes are expressed and for each gene three alternative splice variants are synthesized. Two of them are the expected high and low molecular weight isoforms known from all mammalian kininogens. However, for both genes also a third, hitherto unknown splice variant was detected which lacks part of the high molecular weight mRNA due to splicing from the low molecular weight donor site to alternative splice acceptor sites in exon 10. The physiological functions of the six kininogen isoforms predicted by these findings need to be determined.

Alternative Splicing↗