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R Landgraf

Publications and source records attributed to R Landgraf.

At least 19 recordsLinked to original sources
PubMed2002

The tumour necrosis factor-alpha induced vascular permeability is associated with a reduction of VE-cadherin expression.

BACKGROUND: Vascular permeability is controlled by endothelial cell-to-cell junctions. Vascular endothelial (VE)-cadherin, the major component of adherence junctions (AJ) in the endothelium, is the target of the permeability-increasing agent tumour necrosis factor-a (TNF-alpha). We investigated whether TNF-alpha regulates the synthesis of VE-cadherin on the transcriptional level. MATERIALS AND METHODS: Human endothelial cells, isolated from aorta (aEC) and umbilical cord (HUVEC), were exposed to TNF-alpha (200 U/ml) for 6 h or 12 h, with and without subsequent incubation with TNF-alpha for 24 h. VE-cadherin mRNA was evaluated by semi-quantitative RT-PCR. The VE-Cadherin protein expression was analyzed by flow cytometry (FACS). RESULTS: The VE-cadherin amplification curve of TNF-alpha treated cells was shifted to the right compared to controls, indicating a lower mRNA amount. The number of cycles at which half-maximal amplification (N50) was achieved, was lower for control aEC (30.7) than for TNF-alpha treated aEC (33.0). The N50 of HUVEC treated with TNF-alpha for 12 h and 12 h + 24 h medium (N50 = 32.1), was higher compared to controls (N50 = 29.7) and cells treated with TNF-alpha for 6 h (N50 = 30.8). As determined by FACS analysis, incubation with TNF-alpha caused a small decrease of VE-cadherin protein expression from 7.44 to 6.05 mean channel intensity. CONCLUSION: Our results indicate that TNF-alpha affects VE-cadherin gene expression on the transcriptional level, inducing a downregulation of the VE-cadherin expression.

Antigens, CD↗
PubMed2002

Weight in adolescents with type 1 diabetes mellitus during continuous subcutaneous insulin infusion (CSII) therapy.

Continuous subcutaneous insulin infusion (CSII) has become increasingly popular as a form of intensified insulin therapy in adolescents with type 1 diabetes mellitus (DM). One reported drawback was increased weight gain in adolescents after initiation of insulin pump therapy. In a prospective, longitudinal, non-randomized and case controlled study, we followed 12 adolescents (mean age 13.6 yr, 8 males, 4 females) from 6 months before the start of CSII to 12 months on CSII. These 12 adolescents with DM on CSII were matched for age, gender, HbA1c, duration of DM, and body mass index (BMI) with 12 adolescents who continued on multiple injection therapy (MIT). In addition, six of the 12 adolescents on CSII intended to control their weight by means of the insulin pump. These six vs six adolescents within the CSII group were further analyzed for weight development and eating habits. Clinical indications for CSII were dawn phenomenon, night-time hypoglycemia and patient request for more flexibility in DM management. All patients had been in satisfactory metabolic control on MIT. After 12 months of CSII, the daily insulin requirement remained significantly lower than 18 months before (0.79 +/- 0.11 vs 1.02 +/- 27 U/kg/d, p = 0.034) and number of daily meals was lower (4.1 +/- 0.9 vs 6.5 +/- 0.7, p = 0.006). Mean initial HbA1c was 7.4% in the MIT and CSII patients, and remained comparable between these two groups. BMI was not different between the CSII and MIT group over the entire study period. However, those adolescents on CSII who intended to control their weight by means of the insulin pump were able to achieve relative weight loss during the,first 6 months on CSII. Two patients of the CSII group had one severe hypoglycemic episode with loss of consciousness. In conclusion, CSII does not lead to weight gain by itself, but allows sufficient weight control without a negative effect on metabolic control. The general threat of weight gain in patients who switch to insulin pump therapy must be pointed out, and the role of eating habits and caloric content of food should play a central role in insulin pump educational programs.

Adolescent↗
PubMed2001

Artificial nucleases.

The oxidation of DNA and RNA provides a facile approach for investigating the interaction of nucleic acids with proteins and oligonucleotides. In this article, we have outlined our understanding of the mechanism of DNA scission by 1,10-phenanthroline-copper(I) in the presence of hydrogen peroxide. We also discuss results obtained by using 1,10-phenanthroline-oligonucleotide conjugates in probing the size of the transcriptionally active open complex. Finally, we outline an effective method for converting DNA-binding proteins into site-specific modification agents by using 1,10-phenanthroline-copper(I).

Base Sequence↗
PubMed2001

Identification of a heregulin binding site in HER3 extracellular domain.

HER3 (also known as c-Erb-b3) is a type I receptor tyrosine kinase similar in sequence to the epidermal growth factor (EGF) receptor. The extracellular segment of this transmembrane receptor contains four domains. Domains I and II are similar in sequence to domains III and IV, respectively, and domains II and IV are cysteine-rich. We show that the EGF-like domain of heregulin (hrg) binds to domains I and II of HER3, in contrast to the EGF receptor, for which prior studies have shown that a construct consisting of domains III and portions of domain IV binds EGF. Next, we identified a putative hrg binding site by limited proteolysis of the recombinant extracellular domains of HER3 (HER3-ECD(I-IV)) in both the presence and absence of hrg. In the absence of hrg, HER3-ECD(I-IV) is cleaved after position Tyr(50), near the beginning of domain I. Binding of hrg to HER3-ECD(I-IV) fully protects position Tyr(50) from proteolysis. To confirm that domain I contains a hrg binding site, we expressed domains I and II (HER3-ECD(I-II)) and find that it binds hrg with 68 nm affinity. These data suggest that domains I and II of HER3-ECD(I-IV) act as a functional unit in folding and binding of hrg. Thus, our biochemical findings reinforce the structural hypothesis of others that HER3-ECD(I-IV) is similar to the insulin-like growth factor-1 receptor (IGF-1R), as follows: 1) The protected cleavage site in HER3-ECD(I-IV) corresponds to a binding footprint in domain I of IGF-1R; 2) HER3-ECD(I-II) binds hrg with a 68 nm dissociation constant, supporting the hypothesis that domain I is involved in ligand binding; and 3) the large accessible surface area (1749 A) of domain L1 of IGF-1R that is buried by domain S1, as well as the presence of conserved contacts in this interface of type 1 RTKs, suggests that domains L1 and S1 of IGF-1R function as a unit as observed for HER3-ECD(I-II). Our results are consistent with the proposal that HER3 has a structure similar to IGF-1R and binds ligand at a site in corresponding domains.

Amino Acid Sequence↗
PubMed2001

Anxiolytic and anti-stress effects of brain prolactin: improved efficacy of antisense targeting of the prolactin receptor by molecular modeling.

We provide the first evidence that prolactin is a neuromodulator of behavioral and neuroendocrine stress coping in the rat. In virgin female and male rats, intracerebral infusion of ovine prolactin (oPRL) into the lateral cerebral ventricle (intracerebroventricular) exerted an anxiolytic effect on the elevated plus-maze in a dose-dependent manner (0.1 and 1.0 microg/5 microl; p < 0.01). In contrast, downregulation of the expression of the long form of brain prolactin receptors by chronic intracerebroventricular infusion of an antisense oligodeoxynucleotide (ODN) (osmotic minipump, 0.5 microg. 0.5 microl(-1). hr(-1); 5 d) increased anxiety-related behavior on the plus-maze compared with mixed bases-treated and vehicle-treated rats (p < 0.01), again demonstrating an anxiolytic effect of PRL acting at brain level. Furthermore, in jugular vein-catheterized female rats, the stress-induced increase of corticotropin secretion was decreased after chronic intracerebroventricular infusion of oPRL (osmotic minipump, 1.0 microg. 0.5 microl(-1). hr(-1); p < 0.05) and, in contrast, was further elevated by antisense targeting of the brain prolactin receptors (p < 0.01). This provides evidence for a receptor-mediated attenuation of the responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis by prolactin. The antisense ODN sequence was selected on the basis of secondary structure molecular modeling of the target mRNA to improve antisense ODN-mRNA hybridization. Receptor autoradiography confirmed the expected improvement in the efficacy of downregulation of prolactin receptor expression [empirically designed antisense, 30%; p > 0.05, not significant; adjustment of target position after mRNA modeling, 72%; p < 0.05). Taken together, prolactin acting at brain level has to be considered as a novel regulator of both emotionality and HPA axis reactivity.

Adrenocorticotropic Hormone↗
PubMed2001

Three-dimensional cluster analysis identifies interfaces and functional residue clusters in proteins.

Three-dimensional cluster analysis offers a method for the prediction of functional residue clusters in proteins. This method requires a representative structure and a multiple sequence alignment as input data. Individual residues are represented in terms of regional alignments that reflect both their structural environment and their evolutionary variation, as defined by the alignment of homologous sequences. From the overall (global) and the residue-specific (regional) alignments, we calculate the global and regional similarity matrices, containing scores for all pairwise sequence comparisons in the respective alignments. Comparing the matrices yields two scores for each residue. The regional conservation score (C(R)(x)) defines the conservation of each residue x and its neighbors in 3D space relative to the protein as a whole. The similarity deviation score (S(x)) detects residue clusters with sequence similarities that deviate from the similarities suggested by the full-length sequences. We evaluated 3D cluster analysis on a set of 35 families of proteins with available cocrystal structures, showing small ligand interfaces, nucleic acid interfaces and two types of protein-protein interfaces (transient and stable). We present two examples in detail: fructose-1,6-bisphosphate aldolase and the mitogen-activated protein kinase ERK2. We found that the regional conservation score (C(R)(x)) identifies functional residue clusters better than a scoring scheme that does not take 3D information into account. C(R)(x) is particularly useful for the prediction of poorly conserved, transient protein-protein interfaces. Many of the proteins studied contained residue clusters with elevated similarity deviation scores. These residue clusters correlate with specificity-conferring regions: 3D cluster analysis therefore represents an easily applied method for the prediction of functionally relevant spatial clusters of residues in proteins.

Adenosine Triphosphate↗
PubMed2001

Post-prandial decrease of circulating human ghrelin levels.

Ghrelin, an endogenous ligand of the GH secretagogue-receptor, has recently been shown to stimulate GH secretion and to have orexigenic and adipogenic effects in rodents, but little is known about its regulation and biological function in humans. Gastric motor function is under control of the central nervous system; however, the afferent and efferent loops of this feedback control mechanism remain to be elucidated. In the study presented here we investigated the effect of nutrient intake on circulating human ghrelin levels, and a possible association between ghrelin levels and gastric emptying. Ten healthy volunteers received a standard meal after an overnight fast. Food intake significantly decreased plasma ghrelin levels from 248.5 +/- 15.0 to 179.5 +/- 17.9 fmol/ml (120 min after meal, p=0.047). Gastric emptying half-time (non-invasive 13C-octanoic acid breath test) was correlated with fasting plasma ghrelin levels (r=0.74, p=0.0013). Ghrelin appears to be one possible candidate to provide feedback signaling between nutrient intake, gastric motor function and the central nervous system.

Adult↗
PubMed2001

[Intraoperative monitoring of intact parathyroid hormone (iPTH) in surgery of primary hyperparathyroidism with a new rapid test].

OBJECTIVES: Intraoperative differentiation between adenoma and hyperplasia during surgery for primary hyperparathyroidism (pHPT) is sometimes difficult, but essential for good results. The aim of our study was to evaluate a new highly sensitive electrochemiluminescence immunoassay (ECLIA) for intraoperative monitoring of intact parathyroid hormone (iPTH) following parathyroidectomy as an adjunct for identification of solitary adenoma in patients with pHPT. METHODS: Thirty consecutive patients with pHPT (2 with recurrent pHPT) were examined following a standardized protocol: Immediately before and 5, 10 and 15 min following parathyroidectomy of the enlarged gland, iPTH was measured with a new ECLIA (Roche-Diagnostics, Mannheim, Germany). The results were available within 15-20 min. Besides 20 conventional bilateral neck explorations, parathyroidectomy was carried out in a minimally invasive video-assisted technique (MI-VAP) in 10 patients. RESULTS: Among the 30 patients we found 24 with solitary adenoma (80%), 5 with hyperplasia (17%) and one with a double adenoma (3%). Five minutes after removal of a solitary adenoma the level of iPTH had decreased by 65 (12)% [mean (+/- SD)], after 10 min by 76 (8)% and after 15 min by 81 (8)%. All patients with multiple gland disease could be clearly identified, as iPTH after 15 min did not fall below 50% of basal value. Only after removal of all hyperplastic glands did iPTH decrease to the normal range. Sensitivity and specificity for prediction of a solitary adenoma were 92% and 100% (decline of iPTH more than 50% from baseline value 5 min after parathyroidectomy). In one patient with recurrent pHPT intraoperative sampling from different sites in both internal jugular veins could predict the quadrant of the enlarged gland. Correlation (r) between the results of the quick and the conventional assay, which requires 24 h of incubation, was 0.955. All patients had normal or low calcium levels postoperatively. CONCLUSIONS: (1) Intraoperative monitoring of iPTH with this new quick assay allows safe identification of patients with solitary adenoma during surgery for pHPT. (2) It represents a valuable adjunct to surgical skill not only in primary operations for pHPT but especially in cases of recurrent surgery for pHPT. (3) With this test available minimally invasive techniques for parathyroidectomy may be employed in cases of preoperatively localized adenoma (ultrasound, sesta-mibi scan), avoiding bilateral neck exploration with its higher potential for complications.

Adenoma↗
PubMed2001

Neuroendocrine and behavioral effects of repetitive transcranial magnetic stimulation in a psychopathological animal model are suggestive of antidepressant-like effects.

The neuroendocrine and behavioral effects of repetitive transcranial magnetic stimulation (rTMS) were investigated in two rat lines selectively bred for high and low anxiety-related behavior. The stimulation parameters were adjusted according to the results of accurate computer-assisted and magnetic resonance imaging-based reconstructions of the current density distributions induced by rTMS in the rat and human brain, ensuring comparable stimulation patterns in both cases. Adult male rats were treated in two 3-day series under halothane anesthesia. In the forced swim test, rTMS-treatment induced a more active coping strategy in the high anxiety-related behavior rats only (time spent struggling; 332% vs. controls), allowing these animals to reach the performance of low anxiety-related behavior rats. In contrast, rTMS-treated low anxiety-related behavior rats did not change their swimming behavior. The development of active coping strategies in high anxiety-related behavior rats was accompanied by a significantly attenuated stress-induced elevation of plasma corticotropin and corticosterone concentrations. In summary, the behavioral and neuroendocrine effects of rTMS of frontal brain regions in high anxiety-related behavior rats are comparable to the effects of antidepressant drug treatment. Interestingly, in the psychopathological animal model repetitive transcranial magnetic stimulation induced changes in stress coping abilities in the high-anxiety line only.

Adaptation, Psychological↗
PubMed2001

Cross-fostering and cross-breeding of HAB and LAB rats: a genetic rat model of anxiety.

Recently, two Wistar rat lines, bred and selected for either high (HAB) or low (LAB) anxiety-related behavior on the elevated plus-maze, were described as a novel psychopathologic animal model. The behavioral and neuroendocrine responses to exposure to an emotional stressor were markedly enhanced in HAB rats compared with LAB rats, thus resembling patients suffering from psychiatric diseases. The present study focused on the developmental and genetic basis of the line-specific differences by using cross-fostering and cross-breeding approaches. For the cross-fostering paradigm, neonate HAB offspring were nursed by a LAB foster mother, and vice versa, until weaning. In the cross-breeding approach, HAB females were mated with LAB males, and vice versa, to create an intermediate F1 generation. Thereafter, the F1 animals were strictly sibling-mated to produce a segregating F2 generation. At 10 weeks of age, anxiety-related behavior of all animals was tested on the elevated plus-maze. The robustness of emotionality was assessed in rats of both lines throughout their entire lifetime. Serving this purpose, the frequency of ultrasound isolation calls, indicative of the emotionality of newborn rats, was monitored in regularly-fostered HAB and LAB pups on postnatal day 11. In addition, the timecourse of anxiety-related behavior was studied by repeated testing on the elevated plus-maze at the ages of 10 weeks, 6 months, 16 months, and 19 months. The cross-fostering approach failed to reveal behavioral differences between regularly and cross-fostered HAB and LAB rats, indicating no line-specific differences in maternal care or maternally-influenced development, at least after postnatal day 1. In contrast, cross-breeding resulted in F1 and F2 offspring displaying intermediate behavioral patterns on the elevated plus-maze which were exactly in between those shown in HAB and LAB control rats, thus confirming a genetic basis of the differences in anxiety. Cross-breeding revealed no differences related to the gender of the offspring or to the line-derivation of sire or dam, indicating an autosomal, rather than heterosomal, heredity of the divergent emotionality in HABs and LABs. Further, we were able to show stable and robust emotional differences in rats of both lines during their entire lifetime. HAB rats showed an enhanced frequency of ultrasound isolation calls on postnatal day 11 (p < 0.05) and a lower open arm exploration of the elevated plus-maze throughout adulthood (p < 0.01) compared with the same-aged LABs. In conclusion, the extremely divergent anxiety levels of HAB and LAB rats are maintained during their whole lives and are determined genetically, rather than being learned. These findings may be important for further studies on the genetic basis of emotionality.

Aging↗
PubMed2001

The anxiolytic effect of the CRH(1) receptor antagonist R121919 depends on innate emotionality in rats.

Hyperactivity of central corticotropin-releasing hormone (CRH) circuits appears to contribute to the symptomatology of affective and anxiety disorders and therefore CRH receptor antagonists have attracted attention as potential therapeutic agents. R121919, a novel high-affinity nonpeptide CRH(1) receptor antagonist, displaced (125)I-oCRH in rat pituitary, cortex and amygdala, but not in choroid plexus or cerebral blood vessels in vitro and in vivo, which is consistent with CRH(1) receptor antagonism. In vivo, R121919 significantly inhibited stress-induced corticotropin release in rats selectively bred for high- and low-anxiety-related behaviour but displayed anxiolytic effects in high-anxiety rats only. These data, corroborated by ex vivo receptor occupancy studies, suggest that this animal model is appropriate for the evaluation of CRH(1) receptor antagonists and that compounds such as R121919 will be beneficial whenever the central stress hormone system is hyperactive.

Adrenocorticotropic Hormone↗
PubMed2001

Social isolation after a single defeat reduces striatal dopamine transporter binding in rats.

A single social defeat in male rats has long lasting physiological and behavioural consequences, which are similar to those seen in depressive patients. In addition, the housing conditions after social defeat appear to be crucial for the development of depression-like symptoms. Because the dopaminergic system is thought to be altered in depressive illness, we investigated the impact of individual and group housing on the temporal development of changes of dopamine transporter (DAT) binding in male rats after a single social defeat. The number of striatal DAT binding sites was reduced in animals that remained isolated after being defeated. The isolation length after social defeat amplified this effect, indicating a temporal development of the changes on the striatal DAT. In animals which returned to the familiar group after social defeat the density of striatal DAT binding sites was not affected. We conclude that social isolation after a single defeat reduces the number of DAT binding sites. In contrast, a familiar environment after a single social defeat appears to prevent the stress-induced alterations on the dopaminergic system. This finding suggests that housing conditions are critical when investigating the central nervous effects of social defeat in male rats.

Animals↗
PubMed2001

Forced swimming stimulates the expression of vasopressin and oxytocin in magnocellular neurons of the rat hypothalamic paraventricular nucleus.

Previous studies have shown that a 10-min forced swimming session triggers the release of both vasopressin and oxytocin into the extracellular fluid of the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) in rats. At the same time oxytocin, but not vasopressin, was released from the axon terminals into the blood. Here we combined forced swimming with in situ hybridization to investigate whether (i) the stressor-induced release of vasopressin and oxytocin within the PVN originates from parvo- or magnocellular neurons of the nucleus, and (ii) central release with or without concomitant peripheral secretion is followed by changes in the synthesis of vasopressin and/or oxytocin. Adult male Wistar rats were killed 2, 4 or 8 h after a 10-min forced swimming session and their brains processed for in situ hybridization using 35S-labelled oligonucleotide probes. As measured on photo-emulsion-coated slides, cellular vasopressin mRNA concentration increased in magnocellular PVN neurons 2 and 4 h after swimming (P < 0.05). Similarly, oxytocin mRNA concentration was significantly increased in magnocellular neurons of the PVN at 2 and 8 h (P < 0.05). We failed to observe significant effects on vasopressin and oxytocin mRNA levels in the parvocellular PVN and in the SON. Taken together with results from previous studies, our data suggest that magnocellular neurons are the predominant source of vasopressin and oxytocin released within PVN in response to forced swimming. Furthermore, in the case of vasopressin, central release in the absence of peripheral secretion is followed by increased mRNA levels, implying a refill of depleted somato-dendritic vasopressin stores. Within the SON, however, mRNA levels are poor indicators of the secretory activity of magnocellular neurons during stress.

Animals↗
PubMed2001

Taurine selectively modulates the secretory activity of vasopressin neurons in conscious rats.

Previous experiments have shown that a 10-min forced swimming session triggers the release of vasopressin from somata and dendrites, but not axon terminals, of neurons of the hypothalamic-neurohypophysial system. To further investigate regulatory mechanisms underlying this dissociated release, we forced male Wistar rats to swim in warm (20 degrees C) water and monitored release of the potentially inhibitory amino acids gamma amino butyric acid (GABA) and taurine into the hypothalamic supraoptic nucleus using microdialysis. Forced swimming caused a significant increase in the release of taurine (up to 350%; P < 0.05 vs. prestress release), but not GABA. To reveal the physiological significance of centrally released taurine, the specific taurine antagonist 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide was administered into the supraoptic nucleus via retrodialysis. Administration of this antagonist caused a significant increase in the release of vasopressin within the supraoptic nucleus and into the blood both under basal conditions and during stress (up to 800%; P < 0.05 vs. basal values), without affecting hypothalamic or plasma oxytocin. Local administration of the GABA(A) receptor antagonist bicuculline, in contrast, failed to influence vasopressin secretion at either time point. In a separate series of in vivo electrophysiological experiments, administration of the same dosage of the taurine antagonist into the supraoptic nucleus via microdialysis resulted in an increased electrical activity of identified vasopressinergic, but not oxytocinergic, neurons. Taken together our data demonstrate that taurine is released within the supraoptic nucleus during physical/emotional stress. Furthermore, at the level of the supraoptic nucleus, taurine inhibits not only the electrical activity of vasopressin neurons but also acts as an inhibitor of both central and peripheral vasopressin secretion during different physiological states.

Action Potentials↗
PubMed2001

Insulin antibodies are associated with lipoatrophy but also with lipohypertrophy in children and adolescents with type 1 diabetes.

AIM: To evaluate clinical and immunological factors that are associated with lipodystrophy, i.e. lipoatrophy and lipohypertrophy, in diabetic children and adolescents. METHODS: We investigated in a cross-sectional study 112 children and adolescents (age 1.1-19.1 yrs.) with type 1 diabetes. To grade lipodystrophy, we developed a clinical score ranging from normal (grade 0), moderate hypertrophy of subcutaneous tissue (grade 1), severe hypertrophy with increased density of tissue (grade 2) to lipoatrophy (grade 3). In all children, grade of lipodystrophy, antibodies against insulin (IA) or beta cell antigens (IA-2 and GAD) and clinical parameters were documented. RESULTS: The antibodies against insulin (IA) increased significantly after diabetes manifestation and initiation of insulin treatment, while beta cell specific antibodies (IA-2, GAD) did not. Lipoatrophy (grade 3) was seen in 4 children, severe lipohypertrophy (grade 2) in 18 and moderate lipohypertrophy (grade 1) in 27 children. No alteration of injection sites was found in 63 children. Amongst clinical and immunological parameters, IA levels were significantly associated with hypertrophy or atrophy of injection sites. CONCLUSION: The strong association of lipoatrophy and lipohypertrophy with insulin antibodies might suggest that autoimmune phenomena with insulin play a role in the development of both. Despite an association of IA and lipodystrophy in type 1 diabetic children, the causal link between the two remains unproven and requires further longitudinal exploration.

Adolescent↗