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Biomedical subjects

R Landgraf

Publications and source records attributed to R Landgraf.

At least 145 records · Page 8Linked to original sources
PubMed1996

Kinetics of spontaneous displacement of RNA from heteroduplexes by DNA.

We have used R-loop formation and direct hybridization techniques to analyze the kinetics by which RNA is displaced from a heteroduplex by DNA of identical sequence. Using random walk simulations we were able to calculate the step times for a single displacement reaction. For RNA with a GC content of 57-60% the data indicate an RNA exchange probability of 50.06%, which is indicative of a modest destabilization of the heteroduplex compared with a DNA duplex in the presence of magnesium. The average step time for the reversible exchange of a single nucleotide is 345.0 (+/- 1.3) ms/step. An acceleration of the displacement reaction was observed in the absence of magnesium. A comparison with step times for elongation shows that RNA displacement would not be rate limiting to transcription elongation under two conditions: (i) if magnesium is eliminated from the newly synthesized heteroduplex; (ii) if displacement is kept in a forward only exchange mode through binding of the emerging RNA. Distamycin, a minor groove binding drug, is very effective as a 'catalyst' of RNA displacement. This effect is likely to be due to preferential binding of distamycin to the minor groove of the DNA duplex as opposed to the heteroduplex. This kinetic assay could therefore serve as a convenient assay for the determination of binding preferences of nucleic acid ligands.

Base Sequence↗
PubMed1996

Release of vasopressin within the rat suprachiasmatic nucleus: no effect of a V1/V2 antagonist.

The release of arginine vasopressin (AVP) within the suprachiasmatic nucleus (SCN) of anaesthetized male rats was monitored by microdialysis. Dialysis of the nucleus with 56 mM K+ solution (to 400%) as well as with 1 M NaCl-hypertonic medium (to 530%) triggered a significant increase in intra-SCN release of AVP (p < 0.01). To investigate whether endogenous AVP influences its own release, we administered in a second experiment the combined V1/V2 receptor antagonist d(CH2)5[D-Tyr(Et)2,Val4]AVP directly into the dialysed area. Compared with vehicle-treated controls, the antagonist had no effects on intranuclear release of AVP during basal or stimulated conditions. Thus, in contrast to recent observations in the supraoptic nucleus, we found no indications for autoregulatory mechanisms of AVP release within the SCN.

Animals↗
PubMed1996

Release of vasopressin from supraoptic neurons within the median eminence in vivo. A combined microdialysis and push-pull perfusion study in the rat.

The present study was designed to investigate whether or not arginine vasopressin (AVP) is released from magnocellular neurons within the median eminence (ME) in vivo. Urethane-anesthetized adult male Wistar rats were equipped with a microdialysis probe aimed at the supraoptic (SON) or paraventricular nucleus (PVN), a push-pull perfusion probe resting in the ME, and a blood microdialysis probe within the jugular vein. Dialysis of the SON (but not the PVN) with Ringer's solution containing 56 mmol l-1 K+ resulted in an increase in AVP release within the ME (to 492 +/- 192% of release during basal conditions, P < 0.05) and into blood (to 138 +/- 9%, P < 0.01) whereby the release probably occurred from axonal swellings and nerve terminals of supraoptic neurons which project through the internal zone of the ME to the posterior pituitary. The calculated amount of AVP released into the extracellular fluid of the ME was high enough (approximately 1 pg/microliter) to hypothesize that the neuropeptide could enter the portal blood capillaries in physiologically relevant concentrations. Taken together, the present study indicates that activation of magnocellular neurons is accompanied by release of AVP within the median eminence. We assume that AVP released in this way might mediate a communication between the hypothalamic-neuro-hypophysial system and the hypothalamic-pituitary-adrenal axis in response to selected stressful stimuli.

Animals↗
PubMed1996

Acamprosate and alcohol: I. Effects on alcohol intake following alcohol deprivation in the rat.

Acamprosate (calcium-acetyl homotaurinate) is a new compound in the treatment of alcoholism. Its efficacy has been proven in several clinical trials and registration is now pending in most European countries. The basic mechanisms by which acamprosate elicits its anti-craving action, thereby leading to reduced relapse rates, is not known at the moment. In the present study we describe a rat model of long-term alcohol-drinking which mimics relapse behavior in human alcoholics. The effect of acamprosate was studied in this model. Wistar rats had a free choice between water and alcohol solutions of different concentrations (5, 10, 20% v/v). After two months of continuous alcohol access, rats were deprived of alcohol for three days. Following this deprivation phase, all alcohol solutions were presented again. This procedure was repeated monthly for the following six months. The rats consumed 3.5 +/- 0.3 g/kg alcohol a day. After alcohol deprivation, alcohol intake rose to 5.2 +/- 0.3 g/kg per day resulting in blood alcohol levels of 30 +/- 6 mg/dl. Interestingly, the addition of quinine to the alcohol solutions or the additional presentation of a 5% sucrose solution did not affect the alcohol-deprivation effect after eight months of this intermittent alcohol exposure. However, when acamprosate (50-200 mg/kg i.p.) was administered twice daily, alcohol-drinking following an alcohol-deprivation phase was decreased dose dependently. Given at the highest dose alcohol intake even dropped significantly below baseline drinking. Together, these results show that acamprosate effectively diminishes the alcohol-deprivation effect. Furthermore, the described model seems to be a suitable animal model to screen compounds for their anti-relapse properties and subsequently for their anti-craving action.

Acamprosate↗
PubMed1996

Behavioral consequences of intracerebral vasopressin and oxytocin: focus on learning and memory.

Since the pioneering work of David de Wied and his colleagues, the neuropeptides arginine vasopressin and oxytocin have been thought to play a pivotal role in behavioral regulation in general, and in learning and memory in particular. The present review focuses on the behavioral effects of intracerebral arginine vasopressin and oxytocin, with particular emphasis on the role of these neuropeptides as signals in interneuronal communication. We also discuss several methodological approaches that have been used to reveal the importance of these intracerebral neuropeptides as signals within signaling cascades. The literature suggests that arginine vasopressin improves, and oxytocin impairs, learning and memory. However, a critical analysis of the subject indicates the necessity for a revision of this generalized concept. We suggest that, depending on the behavioral test and the brain area under study, these endogenous neuropeptides are differentially involved in behavioral regulation; thus, generalizations derived from a single behavioral task should be avoided. In particular, recent studies on rodents indicate that socially relevant behaviors triggered by olfactory stimuli and paradigms in which the animals have to cope with an intense stressor (e.g., foot-shock motivated active or passive avoidance) are controlled by both arginine vasopressin and oxytocin released intracerebrally.

Animals↗
PubMed1996

Influence of glycemic control on viscosity and density of plasma and whole blood in type-1 diabetic patients.

The hemorheological properties of blood play an important role in determining blood flow. Blood inertia, as characterized by blood density, controls blood flow in the large arteries, whereas blood viscosity becomes increasingly important with decreasing vessel diameter. In order to evaluate the impact of glycemic control on the rheological properties of blood, we examined viscosity (shear range: 600-0.2 s-1) and density of plasma and whole blood in 26 Type-1 diabetic patients and in 24 healthy controls, matched for age and sex. The diabetic subjects were subdivided into two groups according to their degree of glycemic control: 14 patients with good (HbA1c = 7.1 +/- 0.6%), and 12 patients with poor control (HbA1c = 8.7 +/- 0.7%). Diabetic patients as a whole did not differ from healthy controls in any of the rheological parameters. Subdivision of the patients due to their degree of glycemic control led to a marked rheological separation of Type-1 diabetic subjects with significantly lower plasma (P < 0.008) and whole blood viscosity (P < 0.03 at 10 and 25 s-1), and plasma density (P < 0.05) in well controlled patients. Compared with healthy controls, well controlled diabetic patients had significantly lower values of viscosity (P < 0.005) and density (P < 0.05) of plasma. Poorly controlled patients, on the other hand, did not differ from healthy controls in the examined rheological parameters. There seems to be a positive influence of good glycemic control on hemorheology in Type-1 diabetic patients.

Adult↗
PubMed1996

Endocrine and behavioral effects of airpuff-startle in rats.

Adult male rats chronically implanted with cannulae in the jugular vein were used to characterize the endocrine and behavioral consequences of airpuff-startle. In the first series of experiments, resting animals subjected to three blocks of airpuff (blocks of three airpuffs each with each block separated by 1 min) showed a 10-fold increase in plasma adrenocorticotropin (ACTH) and corticosterone levels, indicating a significant but moderate activation of the hypothalamo-pituitary-adrenal (HPA) axis when compared with the untreated controls (n = 5 each). In the second series of experiments, monitoring of anxiety-related behavior in the defensive withdrawal paradigm revealed a significant increase in anxiety induced by airpuff-startle application compared with the untreated controls (n = 10 each). This behavioral effect, however, was not correlated with plasma hormone levels, as blood samples taken immediately before and 12 min after exposure to airpuff-startle and exposure to the defensive withdrawal paradigm showed a significant rise in ACTH and corticosterone in both groups. In summary, the findings of the present study indicate the airpuff-startle (1) is a potent stimulus in the activation of the HPA axis, and (2) increases anxiety-related behavior as measured in the defensive withdrawal paradigm. Furthermore, the results of our study support the hypothesis that peripheral endocrine parameters that are used to measure activation of the HPA axis do not necessarily correlate with behavioral data obtained in tests with are thought to measure anxiety.

Adrenocorticotropic Hormone↗
PubMed1996

Oxytocin released within the supraoptic nucleus of the rat brain by positive feedback action is involved in parturition-related events.

Oxytocin is released within the supraoptic nucleus during parturition and suckling. During suckling, such release is important in positive feedback stimulation of oxytocin neurons. We have investigated whether oxytocin released within this hypothalamic nucleus during parturition (1) acts on local receptors to further amplify its own release in a positive feedback manner and (2) is critically involved in the regulation of the delivery process. To examine the effect of the oxytocin antagonist on oxytocin release within the supraoptic nucleus, microdialysates were sampled before and during parturition and either vehicle or the antagonist was infused adjacent to the microdialysis probe directly into the supraoptic nucleus after delivery of the second pup. Intranuclear infusion of an oxytocin receptor antagonist (des-Gly-NH2d(CH2)5[Tyr(Me)2Thr4]OVT; 50 ng/0.5 microliters) significantly (P < 0.01) diminished the parturition-related rise in oxytocin release within the supraoptic nucleus and reduced the number of pups delivered during the first and second 30-min dialysis period compared to vehicle-treated controls. Bilateral infusion of the oxytocin receptor antagonist into the supraoptic nucleus after delivery of the second pup significantly slowed parturition (P < 0.05), although the parturition-related rise in plasma oxytocin concentration was unchanged. In addition, the onset of suckling was significantly affected by the antagonist as indicated by fewer liver pups and fewer surviving pups with milk in their stomachs 24 hours after parturition (P < 0.05). To seek other, periventricular sites of oxytocin action during parturition, oxytocin or the oxytocin antagonist was infused into the lateral cerebral ventricle from the birth of pup 2. Via this route, oxytocin speeded up parturition, but the antagonist was ineffective; thus it appears that periventricular oxytocin-sensitive sites are not normally active in promoting parturition, and can do so. The findings indicate a receptor-mediated positive feedback action of oxytocin on its own release within the supraoptic nucleus during parturition, which seems to be involved in the progress of parturition without significantly affecting circulation oxytocin levels. Oxytocin released within the supraoptic nucleus might be important for the coordinated activation of oxytocin neurons and for the synergistic central and peripheral oxytocin effects involved in the regulation of parturition-related events necessary for the survival of the newborn, including the onset of lactation.

Animals↗
PubMed1996

Engineering of DNA binding proteins into site-specific cutters: reactivity of Trp repressor-1,10-phenanthroline chimeras.

Trp repressor (TrpR) can be converted into a site-specific nuclease by chemical modification of the cysteine mutants TrpR D46C or TrpR E49C with 5-iodoacetamido-1,10-phenanthroline (OP). In the presence of cupric ion and 3-mercaptopropionic acid, TrpR-regulated operators are cleaved. The properties of these semisynthetic scission reagents have been compared. The E49C construct cleaves efficiently at two sites within the operator and the D46C cleaves at multiple sites. Molecular modeling indicates that the reason for the focused reactivity of E49C is that the OP is rigidly oriented in the protein-DNA complexes whereas the OP can adopt several orientations in TrpR D46C. Mutations and reaction conditions that increase the affinity of the repressor enhance the scission efficiency which approaches 100% within the acrylamide matrix. TrpR E49C-OP smoothly cleaves the trpEDCBA operator in a plasmid in a reaction dependent on the corepressor L-tryptophan. In the absence of tryptophan, non-specific cleavage of the plasmid is observed under the same conditions. Therefore, tryptophan not only directs cleavage to a specific site but also blocks it at non-specific sites. The analysis of the cleavage pattern of the trpEDCBA operator provides strong evidence for the tandem binding model in which protein-protein interactions stabilize binding on the DNA. TrpR E49C-OP should serve as the basis for the engineering of a family of highly specific semisynthetic scission reagents.

Bacterial Proteins↗
PubMed1996

Human intestinal trefoil factor is expressed in human hypothalamus and pituitary: evidence for a novel neuropeptide.

Human intestinal trefoil factor, hITF, a secretory polypeptide found mainly in the human gastrointestinal tract, is a member of the newly characterized trefoil factor or P-domain peptide family representing putative growth factors. Here we describe the identification of this gut peptide in the human brain and pituitary. With reverse transcriptase polymerase chain reaction, we were able to isolate and clone the transcript from human hypothalamus. An antibody generated against a synthetic peptide derived from the carboxyl terminus of hITF was used for immunohistochemical studies of appropriate tissue sections. Neurons expressing hITF were identified in two magnocellular hypothalamic nuclei, the paraventricular and periventricular nuclei. hITF polypeptide was also observed in Herring bodies of the neurohypophysis and in secretory cells of the adenohypophysis. Double immunostaining with antigrowth hormone antibody showed partial coexistence in a selected subpopulation of adenohypophysial cells. Localization of hITF in the hypothalamo-neurohypophysial system may suggest a modulatory action on the classical magnocellular nonapeptides vasopressin and oxytocin, and further indicates an adenohypophysial importance of this peptide. It is likely that hITF represents a novel neuropeptide of yet unknown function.

Adult↗
PubMed1996

Neural input modulates osmotically stimulated release of vasopressin into the supraoptic nucleus.

The effects of lesioning of the anteroventral third ventricle (AV3V) region on vasopressin (VP) release into the supraoptic nucleus (SON) and blood in response to central and systemic osmotic stimulation were determined. Microdialysis probes were implanted bilaterally within the SON of male urethan-anesthetized rats with sham or AV3V lesions. Osmotic stimuli were administered intraperitoneally (3.5 M NaCl, 600 microliters/100 g body wt) and then via the microdialysis probes (1 M NaCl-artificial cerebrospinal fluid). AV3V lesions attenuated the response to systemic osmotic stimulation. The lesioned rats showed no increase in intranuclear VP release and reduced plasma VP (increase of 42.6 +/- 8.4 vs. 78.0 +/- 16.4 pg/ml) and blood pressure responses (7.1 +/- 2.3 vs. 19.6 +/- 3.2 mmHg) to intraperitoneal NaCl. In contrast, the endocrine and cardiovascular responses to direct osmotic stimulation of the nucleus were as seen in previous studies and seemed to be unaffected by the lesion. These results show that lesion of the AV3V region interrupts neuronal inputs which trigger VP secretion from the posterior pituitary as well as release into the extracellular compartment of the SON.

Analysis of Variance↗
PubMed1996

Combined dexamethasone/CRH test in rats: hypothalamo-pituitary-adrenocortical system alterations in aging.

Alterations of the hypothalamo-pituitary-adrenocortical (HPA) system are well-known phenomena in human aging as well as under stressful conditions and in psychiatric disorders. Among the various neuroendocrine function tests developed so far, the combined dexamethasone (DEX)/corticotropin-releasing hormone (CRH) test, in which DEX-pretreated subjects receive a single dose of CRH, has proved to be the most sensitive measure of subtle changes in HPA system regulation. To further explore the mechanisms underlying these neuroendocrine abnormalities in an animal model, a combined DEX/CRH test was established in young male Wistar rats. Five days before the experiment, the jugular vein was catheterized under halothane anesthesia for subsequent drug infusion and blood sampling. DEX (30 micrograms/kg) administered at 12.00 h, during the diurnal trough, suppressed the diurnal increase in circulating corticotropin (ACTH) and corticosterone between 18.00 and 20.00 h, during the acrophase. Subsequent CRH (50 ng/kg) infused at 20.00 h provoked a minimal escape from DEX suppression, indicated by a slight increase in ACTH and corticosterone secretion. Therefore, the combination of 30 micrograms/kg DEX given at 12.00 h followed by pituitary-adrenal system stimulation with 50 ng/kg CRH at 20.00 h was defined as the standard DEX/CRH test procedure and was then used in young (3-6 months) and aged male Wistar rats (20-24 months). After DEX treatment, basal ACTH levels between 18.00 and 20.00 h were significantly higher in aged than in young rats (77.6 +/- 23.2 vs. 19.9 +/- 0.9 pg/ml; p < 0.01), indicating resistance of the HPA system to the suppressive effect of DEX. In addition, the ACTH response to subsequent CRH was significantly higher in aged than in young animals (area under the concentration time curve: 3,670 +/- 2,230 vs. 294 +/- 112; p < 0.05). Thus, the HPA system appeared to be profoundly dysregulated in aged male Wistar rats. The elevated basal ACTH levels reflect glucocorticoid nonsuppression, suggesting negative feedback impairment. This is further supported by the elevated ACTH response to a subsequent CRH challenge, which, in addition, may indicate changes in the endogenous synergistic mechanisms of CRH with other corticotropic factors, for instance vasopressin. In summary, the DEX/ CRH test revealed HPA system alterations in aging and can be applied in future studies to further explore the mechanisms underlying the neuroendocrine disturbances during (psycho) pathological states.

Adrenal Cortex↗
PubMed1996

Evaluation of the quality of life of patients with insulin-dependent diabetes mellitus before and after organ transplantation with the SF 36 health survey.

OBJECTIVE: To find out if patients with insulin-dependent diabetes mellitus who had undergone successful pancreas and kidney transplantation thought that their quality of life was better than that of patients before transplantation or patients who had rejected the grafts. DESIGN: Cross-sectional study. SETTING: Teaching hospital, Germany. SUBJECTS: 110 of 143 patients to whom questionnaires had been sent. Patients were divided into those awaiting transplantation who did not require dialysis (n = 9), those awaiting transplantation who were receiving dialysis (n = 27), those with functioning grafts after transplantation (n = 34), those with functioning kidneys taking insulin (n = 34); and those who had rejected both grafts and were being treated with both dialysis and insulin (n = 6). INTERVENTIONS: Short Form (SF) 36 health survey and two visual analogue scales. MAIN OUTCOME MEASURES: Comparison of quality of life scores. RESULTS: The duration of dialysis before transplantation was the only feature that differed significantly between the responders (median 24 (range 0-133) months) compared with the non-responders (16 (0-70) months), p < 0.01. The psychometric quality of the SF 36 yielded good variance and reliability in the subscales, and excellent "scale fit" values (between 90% and 100%). Patients who had undergone successful transplantation scored significantly higher than those who had rejected their pancreatic graft in the subscales "vitality" (mean (SD) transformed score 64.4 (15.2) compared with 55.5 (18.9), p < 0.05), and "general health perception" (60.8(18.2) compared with 50.2 (22.5), p < 0.05). The worst ratings of quality of life were given by patients awaiting transplantation (whether or not they were being dialysed) and those who had rejected both grafts. CONCLUSIONS: The SF 36 health survey is a valid instrument for testing patients' perceptions of outcome after transplantation. Those patients who had undergone successful pancreas and kidney transplantation gave the highest scores.

Adult↗
PubMed1995

Chronic infusion of a CRH1 receptor antisense oligodeoxynucleotide into the central nucleus of the amygdala reduced anxiety-related behavior in socially defeated rats.

We studied the role of central amygdala CRH receptors in behavioral responses to an anxiogenic stimulus. An antisense oligodeoxynucleotide corresponding to the rat CRH1 receptor mRNA was infused chronically into the central amygdaloid nucleus of male rats via osmotic minipumps (0.25 micrograms/0.5 microliters/h). Control groups received infusions of either a scrambled sequence oligodeoxynucleotide or vehicle. On the 4th day of treatment, rats were subjected to 10 min of social defeat and immediately afterwards tested on the elevated plus-maze. Antisense oligodeoxynucleotide-treated rats spent significantly more time exploring the open arms of the plus-maze than scrambled sequence- and vehicle-treated animals, both of which did not differ from each other. The social discrimination test, on the other hand, revealed no difference in juvenile recognition abilities among the treatment groups. Using in situ hybridization and receptor autoradiography, we were not able to detect clear signals of CRH1 receptor mRNA and CRH binding sites in the central amygdaloid nucleus of either group, confirming the reportedly low expression and density of CRH receptors in this brain area. The present data support the view that CRH receptors in the central nucleus of the amygdala are involved in the mediation and expression of anxiety-related behavior, but simultaneously raise questions as to the mechanisms of antisense oligodeoxynucleotide action.

Amygdala↗
PubMed1995

R-loop stability as a function of RNA structure and size.

The sequence-specific formation of R-loops can be assayed using RNAs which overlap a HindIII cleavage site in a 3.5 kb plasmid. Chemical modification of the displaced DNA strand has permitted stabilization of these R-loops and allowed a systematic investigation of the dependence of these triple-stranded structures on the chain length and structure of the input RNA. RNAs as short as 50 nt form stable R-loops if 5-allylamine uridines (Uaa-RNA) are used in place of normal uridines; normal RNAs must be 100 nt long to form R-loops quantitatively. Since acetic anhydride decreases the hybridization efficiency of Uaa-RNAs, the positive charge of the RNAs must diminish the electrostatic repulsion of the three negatively charged phosphodiester backbones. The dependence of R-loop stability on the length of RNA can be stimulated with a random walk model, which also applies to strand migration within Holiday junctions. R-loop hybridization provides a versatile method to generate single-stranded DNA in a sequence-selective manner.

DNA↗