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Biomedical subjects

R Landgraf

Publications and source records attributed to R Landgraf.

At least 163 records · Page 9Linked to original sources
PubMed1995

Double stranded scission of DNA directed through sequence-specific R-loop formation.

R-loop formation with short (100 nt) RNAs provides a highly flexible and stringent method to achieve sequence-specific separation of target DNA at any given sequence. After stabilization of R-loops with glyoxal and removal of the RNA through RNase treatment the remaining single-stranded DNA bubble provides a highly favorable substrate for attenuated micrococcal nuclease. We investigated this method for sequence-specific scission of double-stranded DNA and achieved quantitative scission of 3-5 kb plasmids. The applicability to larger size DNA is demonstrated through specific excision of the intervening segment between two R-loops from a P1 plasmid of approximately 120 kb.

Base Sequence↗
PubMed1995

In vivo measurement of a diurnal variation in vasopressin release in the rat suprachiasmatic nucleus.

Diurnal changes in the intranuclear release of vasopressin (VP) and oxytocin (OT) in the suprachiasmatic (SCN), paraventricular (PVN) and supraoptic nuclei (SON) of the rat were studied by means of brain microdialysis. A significant diurnal variation in VP release in the SCN was detected, with the highest levels occurring during midday and a trough around midnight. OT release from the SCN was below detection limit. The release of neither of these neurohypophysial peptides showed diurnal variations within the PVN or SON.

Animals↗
PubMed1995

Interleukin-1 beta stimulates both central and peripheral release of vasopressin and oxytocin in the rat.

Simultaneous microdialysis in the brain and blood was used to monitor the release of vasopressin and oxytocin within the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei and into the systemic circulation of urethane-anaesthetized male rats before and after central administration of interleukin-1 beta (IL-1 beta). Following intracerebroventricular infusion of the cytokine (200 ng/5 microliters), the content of vasopressin (up to 278% compared to vehicle-treated control, P < 0.01 compared to vehicle-treated control and preinfusion baseline) but not oxytocin (up to 148%, not significant) in 30-min blood microdialysates was found to be increased. This peripheral release was accompanied by a transient rise in vasopressin (up to 163%, P < 0.05) and oxytocin (up to 182%, P < 0.05) release within the SON, the peak typically occurring during the first and second 30-min collection intervals after IL-1 beta respectively. In contrast, in the simultaneously microdialysed PVN, both vasopressin and oxytocin failed to respond to intracerebroventricular IL-1 beta. In another series of experiments, IL-1 beta was directly infused (20 ng/0.5 microliters) into either the SON or PVN during microdialysis of the corresponding nucleus. The cytokine caused a significant and immediate rise in intra-SON release of both vasopressin (up to 225%, P < 0.01) and oxytocin (up to 178%, P < 0.05). Again, in the PVN, nonapeptide release, although tending to be stimulated in response to intranuclear IL-1 beta, failed to reach statistical significance. The cytokine-induced central and peripheral release pattern appeared to be independent of the rise in body temperature observed after IL-1 beta administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Drosophila engrailed-1,10-phenanthroline chimeras as probes of homeodomain-DNA complexes.

We have converted the Drosophila engrailed homeodomain into a sequence-specific nuclease by linking the protein to the chemical nuclease 1,10-phenanthroline-copper (OP-Cu). Unique cysteines were introduced at six positions into the homeodomain by site-directed mutagenesis for the covalent attachment of OP-Cu. The varied DNA-binding affinity and specificity of these mutants and the DNA cleavage pattern of their OP-Cu derivatives allowed us to assess the crystal structure of the engrailed homeodomain-DNA complex. We have also achieved site-specific double-stranded DNA scission with one of the homeodomain mutants, E28C, which has the potential of being used to identify engrailed binding sites in the genome. Because the homeodomain is so well conserved among members of the homeodomain-containing protein family, other homeodomain proteins can be converted into nucleases by attaching OP-Cu at position 28 of their homeodomains.

Animals↗
PubMed1995

Anxiety: a potential predictor of vulnerability to the initiation of ethanol self-administration in rats.

Anxiolytic effects of ethanol have been proposed to be important factors in the initiation of ethanol consumption. To examine this hypothesis, drug-naive Wistar rats were tested in the elevated plus-maze to determine their initial level of anxiety. Based on their response, we separated the animals into anxious and non-anxious groups. After that, animals went through an oral ethanol self-administration procedure. Rats that were initially classified as anxious showed a significantly (P < 0.01) higher intake and preference for ethanol during the initiation phase of the voluntary drinking procedure than non-anxious animals. In another experiment, intraperitoneal (IP) injections of ethanol (0.5-1.5 g/kg) produced dose-dependent anxiolytic effects in rats when tested in the elevated plus-maze procedure. Blood ethanol levels following IP injections during the plus-maze test were similar to those reached during the oral ethanol self-administration procedure, which shows that the rats indeed drank sufficient amounts of ethanol to experience its anxiolytic effects. These findings indicate that the basal level of anxiety plays an important role in vulnerability to alcohol drinking.

Alcohol Drinking↗
PubMed1995

Social discrimination procedure: an alternative method to investigate juvenile recognition abilities in rats.

Experiments were performed to establish the social discrimination procedure as an alternative method to the widely used social recognition test for investigating short-term olfactory memory processes in rats. The time that 4-mo old male animals spent investigating conspecific juveniles was taken as an index of their juvenile recognition/discrimination abilities. When the same juvenile was reexposed to the adult 30 min after its initial exposure, it was investigated at a significantly lower intensity compared to a simultaneously presented novel juvenile. If the second exposure to the previously exposed juvenile occurred 2 h later, however, both juveniles were investigated equally, indicating an extinction of olfactory memory. The simultaneous presentation of the previously exposed juvenile and novel juvenile provides not only an internal control under identical experimental conditions (thus reducing the number of sessions for a given experimental series), but also the opportunity to separate specific (i.e., memory-related) from nonspecific (i.e., investigatory behavior-suppression) effects in pharmacological studies. Furthermore, the social discrimination procedure enables even in sexually naive adult male rats the detection of juvenile recognition abilities which seem to be masked in the social recognition test by sexual/aggressive behavior-motivated investigation. The method described here might be an attractive alternative to the conventional social recognition procedure.

Animals↗
PubMed1995

Microdialysis with high NaCl causes central release of amino acids and dopamine.

Recent studies have shown that the neuropeptides arginine-8-vasopressin (AVP) and oxytocin (OXT) are released within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to microdialysis of these nuclei with high-NaCl perfusion media. These results suggest an inherent osmosensitivity of SON and PVN neurons. To investigate whether the observed release of AVP/OXT is a unique phenomenon to these neuropeptides, several brain regions were examined for the release of amino acids or dopamine in response to high- or low-NaCl stimulation. Urethane-anesthetized male Sprague-Dawley rats were perfused with five-ion solution using U-shaped microdialysis probes. Samples were collected at 30-min intervals and analyzed for amino acids and dopamine by HPLC. In the dialysates of all perfusion areas, including the SON, PVN, hippocampus, and striatum, concentrations of Asp, Glu, Ser, Gln, Gly, taurine (Tau), and gamma-aminobutyric acid (GABA) were significantly increased during perfusion with high-NaCl medium. This release was found to be dose dependent when tested in the hippocampus and striatum with perfusion medium containing 0.5 or 1.0 M NaCl. However, only the release of Glu and Ser was found to be Ca2+ dependent. In contrast, the use of mannitol, a nonionic osmolyte, for perfusions in the striatum in concentrations of 0.5 and 1 M resulted in reduced levels of amino acids in the dialysates (Glu, Ser, Gln, and Tau). Low-NaCl perfusion medium (0.01 M) resulted in significantly increased Glu, Tau, Gly, and GABA levels in the striatum. In addition, dopamine levels in striatal dialysates were significantly increased during stimulation with 1 M NaCl. These results indicate that stimulation with high NaCl concentrations affects the release of several neurotransmitters and is not specific for AVP and OXT. The described phenomenon of the release of amino acids in response to this stimulation seems to be a response to the changed ionic concentration rather than to the osmolality. In light of these findings shown for amino acids and dopamine as well as those previously reported for AVP, OXT, and angiotensin, it would appear that sensitivity to tonicity changes brought about by microdialysis may be a feature of many transmitter systems.

Amino Acids↗
PubMed1995

Long-term antidepressant treatment reduces behavioural deficits in transgenic mice with impaired glucocorticoid receptor function.

Impaired cognitive function and enhanced activity of the hypothalamic-pituitary-adrenocortical system are among the cardinal symptoms of major depression in humans that resolve after successful antidepressant treatment. We used a transgenic mouse model expressing antisense RNA complementary to that of glucocorticoid receptor (GR) mRNA to test the hypothesis that reduced GR function can cause these clinical disturbances. The transgenic mice show profound behavioural changes in a number of animal tests that are indicative of cognitive impairment. These mice also have elevated plasma corticotropin concentrations in response to stress. After long-term treatment with moclobemide, a reversible inhibitor of monoamine oxidase type A that acts clinically as an antidepressant, both the behavioural deficits and the hormonal alterations disappeared. These observations suggest that a transgenic mouse with GR dysfunction may be a useful model for investigation of drug effects on the cognitive and neuroendocrine aspects of depression.

Animals↗
PubMed1995

Osmotic responsiveness and cross talk involving oxytocin, but not vasopressin or amino acids, between the supraoptic nuclei in virgin and lactating rats.

Lactation is associated with complex changes of the hypothalamo-neurohypophysial system, and oxytocin released within the hypothalamic supraoptic (SON) and paraventricular nuclei may serve as a signal of communication between the magnocellular nuclei in lactating rats. In the first study, the intranuclear and peripheral release patterns of oxytocin and vasopressin in response to intraperitoneal hypertonic saline were studied in virgin and lactating rats to determine if the reduced osmoresponsiveness of the oxytocinergic and vasopressinergic systems during lactation is reflected by reduced release not only into blood, but also within the SON. Simultaneous microdialysis was performed within the SON and the jugular vein before and up to 6 hr after peripheral osmotic stimulation (3.0 M NaCl, 0.6 ml/100 gm body weight, i.p.). There was an immediate increase in secretion of both oxytocin and vasopressin into blood, whereas peptide release within the SON was delayed and peaked after 4-5 hr. Peripheral release of both peptides was significantly reduced in lactating animals, whereas within the SON release of oxytocin, but not vasopressin, was significantly reduced during lactation. In the second study, cross talk between the SONs--another phenomenon which seems to be characteristic for lactation--was studied. Microdialysis of one SON with hypertonic perfusion medium (with 1 M NaCl) significantly increased the release of oxytocin, vasopressin, and various amino acids (aspartate, glutamate, serine, glutamine, gamma amino butyric acid, and arginine) within the ipsilateral SON. In contrast to virgin female and male animals, this unilateral stimulation of the SON resulted in a transiently increased release of oxytocin in the contralateral SON of lactating rats. The release of vasopressin and amino acids within the contralateral SON of lactating rats remained unchanged, indicating specific activation of contralateral oxytocinergic neurons.

Amino Acids↗
PubMed1995

V1 vasopressin receptor antisense oligodeoxynucleotide into septum reduces vasopressin binding, social discrimination abilities, and anxiety-related behavior in rats.

To develop and validate a vasopressin (AVP) receptor knockdown strategy, we infused an antisense oligodeoxynucleotide to the V1 subtype mRNA into the septum of male rats with osmotic minipumps and measured behavioral, cellular and molecular parameters. Compared to vehicle and scrambled-sequence oligo controls, chronic antisense administration for up to 4 d diminished the ability of the animals to distinguish a previously exposed juvenile from a novel one and to respond to exogenous AVP (1 ng/5 microliters, intracerebroventricular) with an improved social memory. Furthermore, anxiety-related behavior was reduced. As measured in the behaviorally tested rats, antisense treatment resulted in a reduced binding of radiolabeled AVP in the septum, but not in other limbic brain areas (receptor autoradiography), and an increased amount of V1 receptor mRNA (reverse transcriptase PCR), indicating translational arrest and ongoing transcriptional activity. In sense oligo-treated rats, on the other hand, both the social and the anxiety-related behavior scores lay between levels obtained in control and antisense-treated animals. These sense-treated rats showed a slightly reduced V1 receptor density in the septum and reduced receptor mRNA levels, indicating hybridization of the sense oligo to the DNA. The data show the potential of antisense targeting to further reveal relationships between local gene expression, neuropeptide-receptor interactions in distinct brain areas, and behavioral performance.

Analysis of Variance↗
PubMed1995

Measurement of transit disorders in different gastrointestinal segments of patients with diabetes mellitus in relation to duration and severity of the disease by use of the metal-detector test.

The existence of gastrointestinal transit disorders in other intestinal segments beside the stomach in Type-1 diabetes mellitus (DM) and occurrence in Type-2 DM and in uremia has yet been confirmed only in few studies. Eleven healthy volunteers, 34 patients with Type-1, 32 patients with Type-2 DM in different stages of their disease and 34 non-diabetic patients with endstage-renal disease were investigated by use of the metal detector test. Patients were divided in three subgroups, depending on the duration of their disease: < 1 year: "Short", 1 - 10 years: "Middle", > 10 years: "Long". For comparison with the metal detector test scintigraphic studies of esophageal and gastric transit were performed in 17 patients and small intestinal transit was studied by use of the H2-lactulose breath test in 20 patients with long-standing DM Type-1. In Type-1 DM there is an increase of gastric (135 +/- 18, p < 0.01; 218 +/- 26, p < 0.0001 vs. 73 +/- 7 min.) and large intestinal transit times (79 +/- 18, P < 0.02; 76 +/- 11, p < 0.04 vs. 40 +/- 5 h) in patients with middle or long standing DM. In Type-2 DM similar transit disturbances occur (gastric emptying, long group: 120 +/- 15 min., p < 0.02; colonic transit, long group: 80 +/- 13 h, p < 0.01). In uremia transit disturbances were only found in patients with chronic ambulatory peritoneal dialysis (colonic transit: 71 +/- 9 h, p < 0.05). In 65% gastric scintigraphy and in 55% of cases the H2-lactulose breath test showed a prolongation of gastric emptying or a prolonged mouth-to-cecum transit. Transit disorders can occur in every stage of DM with preferential involvement of the stomach and the colon. These findings are of clinical relevance, since transit disturbances can result in instable metabolic condition.

Adult↗
PubMed1995

Release of oxytocin within the supraoptic nucleus. Mechanisms, physiological significance and antisense targeting.

In addition to its secretion from neurohypophysial terminals, oxytocin (OT) is released within the hypothalamic magnocellular nuclei--the supraoptic (SON) and paraventricular nuclei--in response to various stimuli as mainly shown by microdialysis studies. During parturition and suckling, OT is released within the SON in a positive feedback role to further amplify its own local and/or neurohypophysial release depending upon the reproductive conditions. Whereas, for instance, intra-SON OT is important for appropriate OT secretion and milk ejection during suckling, it seems to be involved in the timing of the parturition process without directly affecting OT secretion during birth. As a consequence of various morphological and physiological adaptations of the OT system during lactation, the release pattern of OT into blood and within the SON is altered in response to other stimuli, like swim stress and peripheral osmotic stimulation. In addition, in lactating, but not virgin female or male rats, unilateral stimulation of the SON results in the release of OT, but not vasopressin or various amino acids, within the contralateral SON, which implicates a functional role of intranuclear OT in the plastic coupling of the paired SON during lactation. To manipulate OT synthesis and release, antisense targeting was used. Novel, acute effects (within 5 hours) of an OT antisense oligodeoxynucleotide on suckling-related parameters and, specifically, on the electrophysiological excitability of OT neurons as well as their ability to express the Fos-protein after CCK-8 are described. Since the OT content in the hypothalamo-neurohypophysial system was still unaffected at this time, antisense oligonucleotides may induce feedforward alterations in neuronal responsiveness which precede or parallel effects on neuropeptide synthesis. Taken together, the functional significance of OT released within the brain and into blood during reproduction provides an example of how a neuropeptide might regulate complex physiological and behavioral performances in a synergistic manner.

Adaptation, Physiological↗
PubMed1994

Assessment of soluble adhesion molecules (sICAM-1, sVCAM-1, sELAM-1) and complement cleavage products (sC4d, sC5b-9) in urine. Clinical monitoring of renal allograft recipients.

Increasing evidence exists that inducible adhesion molecules are involved in cell-mediated allograft rejection. In addition, complement activation during rejection has been described. This study investigated, whether specific molecules derived from either pathway are excreted into urine during rejection and whether they can provide useful diagnostic tools for the monitoring of renal transplant recipients. Urinary concentrations of soluble adhesion molecules (sICAM-1, sVCAM-1, sE-selectin) and of complement cleavage products (sC4d and sC5b-9), were determined by standardized ELISA in 30 normal controls and 80 samples from 49 recipients of renal allografts. In contrast to the low amounts of adhesion molecules and complement components uniformly excreted by healthy persons (group 0), marked differences were observed among allograft recipients. To prove the clinical relevance of these differences in excretion, patient samples were assigned to 5 categories according to clinical and histopathological criteria: group I--acute steroid-resistant rejection (n = 10); group II--acute steroid-sensitive rejection (n = 10); group III--chronic rejection (n = 23); group IV--stable graft function (n = 27); and group V--miscellaneous disorders (n = 10), including infections, CsA overdoses, and glomerulonephritis. Urinary levels of sICAM-1, sVCAM-1, and sC4d were significantly higher in group I compared with all other groups (P < 0.01). The difference in sICAM-1 excretion between groups III and IV also reached statistical significance (P < 0.05). Urinary concentrations of sICAM-1, sVCAM-1, and sC4d were reflective of their histological distribution in corresponding graft biopsies. None of the patients excreted E-selectin in detectable amounts. Excretion of the terminal membrane attack complex C5b-9 was not significantly associated with any diagnosis. It is concluded that for clinical purposes the combined evaluation of sICAM-1, sVCAM-1, and sC4d is most useful and can provide valuable information with regard to the severity and the type of allograft rejection.

Cell Adhesion Molecules↗