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Biomedical subjects

C Wagner

Publications and source records attributed to C Wagner.

At least 127 records · Page 7Linked to original sources
PubMed1998

Overexpression of functional hydrolase domain of rat liver 10-formyltetrahydrofolate dehydrogenase in Escherichia coli.

Rat liver 10-formyltetrahydrofolate dehydrogenase (FDH) is a tetrameric enzyme composed of four identical 902-amino-acid-residue (99 kDa) monomers. We expressed the enzyme and its 310-amino-acid-residue amino-terminal domain, which is 10-formyltetrahydrofolate hydrolase, in Escherichia coli BL21 (DE3) cells using the pRSET expression vector. We removed the entire translated region of the vector including the polyhistidyl tag and the recombinant proteins were expressed, not as a fusion constructs, but as unmodified sequences. The expressed full-length enzyme was found to be an insoluble protein and was not purified and characterized, while the amino-terminal domain was expressed as a soluble protein possessing hydrolase activity. The recombinant amino-terminal domain was purified in one step on a DEAE MemSep 1000 HP Ion-Exchange Membrane Chromatography Cartridge (Millipore) using a ConSep LC100 chromatographic system (Millipore). The chromatography gave a homogenous and active preparation of the recombinant protein with a yield of about 2 mg per 100 ml of bacterial culture. Kinetic parameters of the hydrolase reaction displayed by the amino-terminal domain expressed in E. coli were similar to those of the recombinant full-length enzyme and its amino-terminal domain previously expressed in insect cells. The purified recombinant enzyme remained active for at least 4 weeks at 4 degreesC. These results show that the hydrolase amino-terminal domain of FDH can be overexpressed as a functional enzyme in E. coli cells and purified in one step by a simple chromatographic procedure.

Animals↗
PubMed1998

Positive feedback regulation of angiotensin II-AT1B receptor gene expression in rat adrenal glands.

This study aimed to investigate the role of endogenous angiotensin II (ANGII) in the upregulation of ANG-II AT1 receptors in adrenal glands during a low-salt intake. To this end male Sprague-Dawley rats were fed a low-salt diet (0.2 mg/g) for 10 days and were treated with the ANGII-AT1 receptor antagonist losartan (40 mg/kg per day) for 2 days, and adrenal mRNA levels for ANGII AT1A and AT1B receptors were determined by RNase protection. The low-salt diet increased AT1A and AT1B receptor mRNA levels by 90% and 220%, respectively. Losartan treatment did not change the basal AT1A mRNA level, but decreased AT1B mRNA by 50%. Treatment of rats on a low-salt diet with losartan did not change the increase of AT1A mRNA but significantly attenuated the increase of AT1B mRNA to 90% of the control value. Stimulation of endogenous ANGII levels by unilateral renal artery clipping for 2 days lowered AT1A mRNA by 25% and increased AT1B mRNA by 30%. Additional treatment with losartan did not affect the decreased AT1A mRNA levels in rats with a unilateral renal artery clip, but significantly attenuated the increase of AT1B mRNA. These findings suggest that sodium deficiency stimulates adrenal AT1A and AT1B receptor mRNA levels primarily via an ANGII-AT1-independent mechanism. The preferential increase of adrenal AT1B mRNA during a low-salt intake could be explained by the elevation of endogenous ANGII levels during sodium deficiency, suggesting that endogenous ANGII acts as an enhancer for adrenal AT1B but not for AT1A receptor gene expression via ANGII-AT1 receptors.

Adrenal Glands↗
PubMed1998

Interleukin-1 inhibits renin gene expression in As4.1 cells but not in native juxtaglomerular cells.

Cardiovascular effects of inflammatory interleukins (IL) have been suggested to be mediated by the renin-angiotensin system in vivo. To address the direct cellular effect of IL, we examined the influence of IL-1beta on renin secretion and renin mRNA in cultures of mouse juxtaglomerular granular (JG) cells and in the mouse tumor cell line As4.1, which expresses renin mRNA. Renin mRNA levels and secretion of active renin were not significantly changed by IL-1beta in native JG cells. Activation of adenylyl cyclase by forskolin increased renin secretion and renin mRNA levels three- and fivefold, respectively. These stimulatory responses to forskolin were not altered by IL-1beta. In contrast to native JG cells, renin mRNA abundance was markedly suppressed by IL-1beta in As4.1 cells, whereas secretion of active renin and the stability of renin mRNA were not changed. In As4.1 cells forskolin did not change renin secretion or renin mRNA abundance in the absence or in the presence of IL-1beta. These findings suggest that IL-1beta has no direct influence on renin secretion and renin mRNA abundance at the level of native JG cells.

Animals↗
PubMed1998

Effects of static forehead and forearm muscle tension on total respiratory resistance in healthy and asthmatic participants.

The impact of static muscle tension on total respiratory resistance (TRR) was examined. Participants (24 healthy, 24 asthmatic) performed biofeedback-assisted sequences of tensing (15 s) and relaxing (20 s) forehead and forearm muscles. Muscle tension levels were 40% or 80% of the maximum individual force. Oscillatory TRR, electromyograms, ventilation, heart period, and respiratory sinus arrhythmia were recorded. Baseline TRR did not change over the session as a whole. Decreases in TRR during forehead tension in both groups were accompanied by increases in end-expiratory volume, which could have mediated TRR changes. During forearm tension, decreases in TRR with minimal ventilation changes were only observed in healthy participants, whereas asthmatic patients revealed marked increases in respiratory volume and flow. These results indicate that static muscle activity and TRR are negatively related. Ventilatory changes can exaggerate or diminish evidence for this relationship.

Adult↗
PubMed1998

Rubrobramide, a cytotoxic and phytotoxic metabolite from Cladobotyrum rubrobrunnescens.

Cladobotrin (1) and rubrobramide (2) were isolated from the culture filtrate of Cladobotryum rubrobrunnescens, a mycophilic deuteriomycete. Rubrobramide (2), possessing a unique oxidized tricyclic system, is a new compound structurally related to flavipucine (3), which also is produced by the fungus. The structure of 2 was determined by spectroscopic methods. Cladobotrin (1) exhibited weak nematicidal activity toward Meldogyne incognita, while 2 showed weak cytotoxic and phytotoxic activities.

Anti-Infective Agents↗
PubMed1998

T-type and L-type calcium channel blockers exert opposite effects on renin secretion and renin gene expression in conscious rats.

1. This study aimed to investigate and to compare the effects of pharmacological T-type calcium channel and of L-type calcium channel blockade on the renin system. To this end, male healthy Sprague-Dawley rats were treated with the T-channel blocker mibefradil or with the L-channel blocker amlodipine at doses of 5 mg kg(-1), 15 mg kg(-1) and 45 mg kg(-1) per day for four days and their effects on plasma renin activity (PRA) and kidney renin mRNA levels were determined. 2. Whilst amlodipine lowered basal systolic blood pressure at 5 mg kg(-1), mibefradil had no effect on basal blood pressure in the whole dose range examined. Amlodipine dose-dependently induced up to 7 fold elevation of PRA and renin mRNA levels. Mibefradil significantly lowered PRA and renin mRNA levels at 5 mg kg(-1) and moderately increased both parameters at a dose of 45 mg kg(-1), when PRA and renin mRNA levels were increased by 100% and 30%, respectively. In primary cultures of renal juxtaglomerular cells neither amlodipine nor mibefradil (0.1-10 microM) changed renin secretion. 3. In rats unilateral renal artery clips (2K-1C) mibefradil and amlodipine at doses of 15 mg kg(-1) day(-1) were equally effective in lowering blood pressure. In contrast mibefradil (5 mg kg(-1) and 15 mg kg(-1) day(-1)) significantly attenuated the rise of PRA and renin mRNA levels, whilst amlodipine (15 mg kg(-1)) additionally elevated the rise of PRA and renin mRNA levels in response to renal artery clipping. 4. These findings suggest that T-type calcium channel blockers can inhibit renin secretion and renin gene expression in vivo, whilst L-type calcium channel blockers act as stimulators of the renin system. Since the inhibitory effect of T-type antagonists is apparent in vivo but not in vitro, one may infer that the effect on the renin system is indirect rather than directly mediated at the level of renal juxtaglomerular cells.

Amlodipine↗
PubMed1998

Homozygous deletion of the p16INK4a and the p15INK4b tumour suppressor genes in a subset of human sporadic cutaneous malignant melanoma.

Chromosome 9p21 is frequently deleted in malignant melanoma, and one familial malignant melanoma gene has been linked to 9p21-22. Recently, the cyclin D-dependent kinase inhibitors (CDKIs) p16INK4a and p15INK4b have been localized within chromosome 9p21, and the presence of p16INK4a point mutations has been demonstrated in familial melanoma and melanoma cell lines in vitro. To analyse the role of these CDKIs in sporadic human cutaneous non-metastatic malignant melanoma, we examined 36 primary tumour specimens representing different stages of melanoma progression and their corresponding normal skin samples for the three mechanisms of CDKI inactivation described so far. Homozygous codeletion of the p16INK4a and the p15INK4b gene was detected by Southern blot analysis in two tumour samples. By direct sequencing of polymerase chain reaction (PCR)-amplified microdissected genomic DNA; no somatic or germline p16INK4a point mutations or small deletions were detected in the remaining 34 tumour samples; one individual exhibited the previously described germline codon 148 (Ala-->Thr) polymorphism. In these tumour specimens, comparative semiquantitative reverse PCR analysis of p16INK4a transcript levels revealed no evidence for repression of p16INK4a gene transcription and thus for p16INK4a promoter inactivation by DNA methylation. These results indicate homozygous p16INK4a and p15INK4b loss to occur in a subset of cutaneous melanomas and suggest, in view of the frequent loss of heterozygosity on chromosome 9p21, the presence of another tumour suppressor gene within this chromosomal region.

Blotting, Southern↗
PubMed1998

Predominant expression of CD44 splice variant v10 in malignant and reactive human skin lymphocytes.

The remarkable functional diversity of the cell surface receptor CD44 may be due to expression of multiple variant isoforms generated by alternative splicing of variant exons. Functional and correlative data implicate a role of CD44 variant isoforms in adhesion dependent processes such as lymphocyte recirculation and tumor progression and metastasis. We have analyzed 25 primary cutaneous lymphomas and 35 reactive lymphoid cell skin infiltrates or T cell-mediated skin diseases for the expression of CD44 variant isoforms. Irrespective of histologic typing, staging, and grading, cutaneous lymphomas as well as nonmalignant skin-infiltrating CD3+ CD4+ and CD8+ T and CD19+ B lymphocytes exhibited a strong expression of CD44v10 and a moderate expression of CD44v3 as determined by immunohistochemistry, immunofluorescence microscopy, and mRNA analysis. Expression of v5, v6, v7, and v9-containing CD44 variant isoforms was not detected. Furthermore, flow cytometry revealed expression of CD44v10 on a significant proportion of peripheral blood lymphocytes from Sézary's syndrome patients and a remarkable co-expression with cutaneous lymphocyte antigen. These results indicate a distinct CD44 variant isoform expression pattern associated with skin-homing lymphocytes different to lymphatic cells at noncutaneous sites. This differential expression pattern of CD44 variant isoforms may contribute to the development of lymphocyte skin infiltrates and/or the unique biologic behavior of cutaneous lymphomas.

Alternative Splicing↗
PubMed1998

Fibronectin synthesis by human tubular epithelial cells in culture: effects of PDGF and TGF-beta on synthesis and splicing.

BACKGROUND: Enhanced synthesis of extracellular matrix proteins including fibronectin (FN) is associated with the development of sclerosis. In this context we studied FN synthesis by tubular epithelial cells in response to transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF). METHODS: FN protein synthesis by human tubular epithelial cells in culture (TEC) was measured by biosynthetic labeling and ELISA. Splicing of FN was assessed by RT-PCR and by Northern blotting. RESULTS: Cultivated TEC synthesized and released FN, the majority of which was deposited as an unsoluble protein and a minor portion (10 to 15%) was released into the supernatant. TGF-beta and, to a lesser degree, PDGF, up-regulated FN synthesis. All three FN splice variants (EDA, EDB, and IIICS) were produced. PDGF did not influence the splicing. TGF-beta preferentially up-regulated the EDA splice variant, but had no effect on the splicing of the other domains. CONCLUSIONS: PDGF and TGF-beta both up-regulate FN synthesis of TEC. TGF-beta, but not PDGF, also changed the quality of the de novo synthesized FN, and thus has a different role in the development of sclerosis.

Cells, Cultured↗
PubMed1998

Control of the renal renin system by local factors.

Local factors, such as prostaglandins (PGs), nitric oxide (NO), and endothelins (ETs), produced in the immediate vicinity of juxtaglomerular (JG) cells can exert significant effects on renin secretion and renin gene expression. PGE2, as the main renotubular PG, and PGI2, as the main endothelial prostanoid, both stimulate renin secretion and renin gene expression by activating cAMP formation in JG cells. Although the direct effect of NO on JG cells is less clear, its overall effect in vivo seems to be to stimulate the renin system. Evidence is emerging that stimulation by NO is related to the cAMP pathway, and cGMP-induced inhibition of cAMP-phosphodiesterase III (PDE-III) may mediate this effect. ETs, on the other hand, appear to inhibit the renin system, in particular in those pathways activated by cAMP, acting via Ca2+- and protein kinase C-related mechanisms. There is increasing evidence that both NO and PGs could be involved in the physiological regulatory mechanisms by which salt intake affects the renin system.

Animals↗
PubMed1998

Relevance of markers of hemostasis activation in obstetrics/gynecology and pediatrics.

Pregnancy and puerperium are considered to be hypercoagulable states with increased incidence of thromboembolic events. During normal pregnancy, changes in the hemostatic mechanism involve increased stasis and increased coagulation factors and/or decreased levels of anticoagulant proteins such as protein C and protein S as well as enhanced thrombin generation and decreased fibrinolytic activity. The physiological or pathophysiological activation of hemostasis during pregnancy results in the generation of the so-called activation markers which increase, reflecting hypercoagulability and therefore representing an imbalance in the hemostatic system. The most interesting markers of hemostasis activation and, thus, of thrombin generation are: thrombin-antithrombin III complex (TAT), antithrombin III itself, prothrombin fragment 1+2 (F 1+2), fibrin monomer (soluble fibrin) and D-Dimer (which indicates also an increased fibrinolytic activity). Together with fibrinogen levels and platelet counts, the activation markers are useful tools in different pathological situations in pregnancy to predict and monitor the severity of the condition. Recently, a higher incidence of factor V Leiden mutation has been demonstrated in selected populations in whom thrombotic events developed during pregnancy and puerperium. Therefore, the combination of APC resistance/FV Leiden mutation and pregnancy may predict a high risk for thromboembolic phenomena. In newborns, the activation markers are elevated immediately after birth and decline to near adult levels during the first 24 h of life. During infections the activation markers are increased showing the same behavior as in the mature adult system. In neonates and children, the same etiologies can be responsible for acquired and inherited pathological hypercoagulable states as in the adult.

Adult↗
PubMed1998

Regulation of renal renin release.

Renal renin release is affected by several systemic and intrarenal factors. Systemic factors include sympathetic nerves, circulating angiotensin II, blood pressure and salt balance of the organism. Intrarenal factors involved are nitric oxide and the prostaglandins, which stimulate renin secretion.

Angiotensin II↗
PubMed1998

Genesis of the ultradian rhythm of GH secretion: a new model unifying experimental observations in rats.

Growth hormone (GH) induces growth in animals and humans and also has important metabolic functions. The GH neuroendocrine axis consists of a signaling cascade from the hypothalamus to the pituitary, the liver, and peripheral tissues, including two major feedback mechanisms. GH is secreted from the pituitary into the circulating blood according to the effect on the somatotrophs of two hypothalamic peptides, GH-releasing hormone (GHRH) and its antagonist, somatostatin (SRIF). The typical GH profile in the male rat shows secretory episodes every 3.3 h, which are subdivided into two peaks. Focusing on the mechanisms for generation of this ultradian GH rhythm, we simulated the time course of GH secretion under a variety of conditions. The model that we propose is based on feedback of GH on its own release mediated both by GH receptors on SRIF neurons in the brain and by a delayed SRIF release into both the brain and portal blood. SRIF, with a resultant periodicity of 3.3 h, affects both the somatotroph cells in the pituitary and the GHRH neurons in the hypothalamus. The secretion of GHRH is postulated to occur in an approximately 1-h rhythm modulated by the level of SRIF in the hypothalamus. The model predicts a possible mechanism for the feminization of the male GH rhythm by sex steroids and vice versa, and suggests experiments that might reveal the proposed intrinsic 1-h GHRH rhythm.

Activity Cycles↗
PubMed1998

Stimulation of renin secretion by NO donors is related to the cAMP pathway.

This study aimed to characterize the cellular pathways along which nitric oxide (NO) influences the secretion of renin from the kidney. Using the isolated perfused rat kidney model, we found that the NO donor sodium nitroprusside (SNP) (1-30 mumol/l) induced a prompt, concentration-dependent fourfold increase of basal renin secretion. The membrane-permeable cGMP analogs 8-bromo-cGMP and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP; each 5-50 mumol/l) inhibited basal renin secretion and attenuated the stimulation of renin secretion by SNP. Conversely, the renin stimulatory effect of SNP was enhanced in the presence of the G kinase inhibitor Rp-8-CPT-cGMPS (10 mumol/l). The renin stimulatory effect of SNP was amplified in nominally calcium-free perfusate and was abolished in the presence of angiotensin II (1 nmol/l). Renin secretion stimulated by SNP was clearly attenuated by the A kinase inhibitor Rp-8-CPT-cAMPS (25 mumol/l). These findings indicate that the renin stimulatory effect of NO donors in renal juxtaglomerular cells cannot be explained by activation of G kinase and is also less likely to be causally related to the regulation of renin secretion by calcium. Because A kinase activity is required for the stimulation of renin secretion by SNP, it appears as if the renin stimulatory effect is causally related to the cAMP pathway controlling renin secretion.

Animals↗
PubMed1998

Role of nitric oxide in the control of renin secretion.

Because of the significant constitutive expression of NO synthases in the juxtaglomerular apparatus, nitric oxide (NO) is considered as a likely modulator of renin secretion. In most instances, NO appears as a tonic enhancer of renin secretion, acting via inhibition of cAMP degradation through the action of cGMP. Depending on as yet unknown factors, the stimulatory effect of NO on renin secretion may also switch to an inhibitory one that is compatible with the inhibition of renin secretion by cGMP-dependent protein kinase activity. Whether NO plays a direct regulatory role or a more permissive role in the control of renin secretion remains to be answered.

Animals↗
PubMed1998

Angiotensin AT1B receptor mediates calcium signaling in vascular smooth muscle cells of AT1A receptor-deficient mice.

Our studies on angiotensin II receptor subtype 1A (AT1A) knockout mice define how endogenous receptors other than AT1A receptors stimulate changes in cytosolic calcium concentration ([Ca2+]i) in cultured aortic vascular smooth muscle cells (VSMCs). Wild-type cells have a 1.7 ratio of AT1A/AT1B receptor mRNA as determined by semiquantitative reverse transcriptase-polymerase chain reaction. Mutant cells express AT1B receptor mRNA but not that for the AT1A receptor. In wild-type cells with AT1A present, Ang II (10(-7) mol/L) produces a characteristic rapid peak increase in [Ca2+]i of 150 to 180 nmol/L, followed by a plateau phase characterized by a sustained 70 to 80 nmol/L increase in [Ca2+]i. An unexpected finding was that the magnitude and time-dependent pattern of [Ca2+]i changes produced by Ang II were similar in cells that lacked AT1A receptors but possessed AT1B receptors. The response in mutant cells indicates effective coupling of an Ang II receptor to one or more second messenger systems. The similarity of response patterns between cells with and without AT1A receptors suggests that non-AT1A receptors are functionally linked to similar signal transduction pathways in mutant cells. The fact that mutant and wild-type cells exhibit similar patterns of calcium mobilization and entry supports the notion that AT1A and non-AT1A receptors share common signal transduction pathways. The AT2 receptor ligands PD-123319 and CGP-42112 do not alter Ang II effects in either VSMC type, suggesting a paucity of AT2 receptors and/or an absence of their linkage to [Ca2+]i pathways. The nonpeptide AT1 receptor blocker losartan antagonizes Ang II-induced [Ca2+]i increases in both cell groups, supporting mediation by native AT1B receptors and effective coupling of this subtype to second messenger systems leading to calcium entry and mobilization. Our results demonstrate that Ang II causes calcium signaling in AT1A-deficient VSMCs that is mediated by an endogenous losartan-sensitive AT1B receptor.

Angiotensin II↗
PubMed1998

Impact of peritoneal dialysis modality and acidosis on nutritional status in peritoneal dialysis patients.

Continuous ambulatory (CAPD) and continuous cyclic peritoneal dialysis (CCPD) differ in solute transport, and variances in mass balance could impact nutritional parameters. Chronic acidosis may decrease albumin synthesis and increase catabolism. We prospectively studied 50 peritoneal dialysis patients (age: 55 +/- 16 yrs; CAPD = 33; CCPD = 17) over 24 months. Acidosis was defined by an anion gap > 18 mEq/L. Bioimpedance analysis was used to estimate body cell mass and fat-free mass. Patients on CAPD had a lower body mass index than CCPD patients (27 +/- 5 kg/m2 vs. 29 +/- 9 kg/m2 respectively; P = 0.039). However no differences were observed in body cell mass (25 +/- 8 kg vs. 26 +/- 9 kg respectively; P = 0.787) or fat-free mass (53 +/- 14 kg vs. 50 +/- 18 kg respectively; P = 0.404). Urea kinetic modeling showed no differences in Kpt/V or nPCR (0.26 +/- 0.06 vs. 0.24 +/- 0.07; P = 0.709 and 0.67 +/- 0.29 g/kg per day vs. 0.65 +/- 0.23 g/kg per day; P = 0.709 for CAPD and CCPD respectively). When categorized by the presence of acidosis no differences were observed in body cell mass (24 +/- 8 kg vs. 27 +/- 9 kg respectively; P = 0.131) or fat-free mass (54 +/- 15 kg vs. 50 +/- 16 kg respectively; P = 0.348), while body mass index was greater in acidotic than nonacidotic individuals (30 +/- 8 kg/m2 vs. 26 +/- 6 kg/m2 respectively; P = 0.005). Dietary protein intake assessed as nPCR was similar in both groups (0.64 +/- 0.26 vs. 0.71 +/- 0.28 g/kg per day; P = 0.319, for CAPD and CCPD respectively). We conclude that over a 24-month period neither peritoneal dialysis modality nor the presence of acidosis has a detrimental influence on nutritional parameters in well dialyzed patients.

Acidosis↗