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Biomedical subjects

A Ehret

Publications and source records attributed to A Ehret.

13 recordsLinked to original sources
PubMed2003

High potassium-induced activation of choline-acetyltransferase in human neocortex: implications and species differences.

The role of electrical and potassium (K(+))-induced depolarisation on choline-acetyltransferase (ChAT) activity in human and mouse neocortical slices was studied. When [3H]-ACh release was evoked by two K(+) stimulations in human neocortex, the mean S(2)/S(1) ratio was significantly below unity. ChAT inhibitors, like bromo-acetylcholine and ocadaic acid, raised this ratio by 79 and 63%, respectively, suggesting that the diminished S(2)/S(1) value in the absence of ChAT inhibitors reflected an increased ChAT activity at S(2) following K(+) depolarisation at S(1). When stimulated electrically, however, the S(2)/S(1) ratio in human neocortex was near unity and ocadaic acid remained without effect. In parallel experiments on mouse neocortical slices, the S(2)/S(1) ratio was near unity in both electrically or K(+)-evoked [3H]-ACh release and was not altered by ChAT inhibition. ChAT activity following K(+) depolarisation was also determined directly. ChAT activation in human neocortical slices was highest at 10 and 20mM K(+). ChAT activity in mouse neocortical tissue was not altered by K(+) depolarisation. These results suggest that in human, but not in mouse, neocortex ChAT activity may be increased due to ongoing K(+) depolarisation. This increase of ChAT activity supports a cholinergic degeneration hypothesis which has been entitled "autocannibalism" by Wurtman [TINS 15 (1992) 177].

Acetylcholine↗
PubMed2003

Neurotransmitter release and its presynaptic modulation in the rat hippocampus after selective damage to cholinergic or/and serotonergic afferents.

UNLABELLED: Male Long-Evans rats sustained injections of 5,7-dihydroxytryptamine (5,7-DHT) into the fimbria-fornix and the cingular bundle or/and intraseptal injections of 192 IgG-saporin to induce serotonergic or/and cholinergic hippocampal denervations; Sham-operated rats served as controls. Four to ten weeks after lesioning, we measured (i). the electrically evoked release of acetylcholine ([3H]ACh), noradrenaline ([3H]NA) and serotonin ([3H]5-HT) in hippocampal slices in the presence of drugs acting on auto- or heteroreceptors, (ii). the nicotine-evoked release of NA and (iii). the choline acetyltransferase (ChAT) activity and the concentration of monoamines in homogenates. Saporin lesions reduced the accumulation of [3H]choline, the release of [3H]ACh and the ChAT activity, but increased the concentration of NA and facilitated the release of [3H]NA evoked by nicotine. 5,7-DHT lesions reduced the accumulation and the release of [3H]5-HT, the concentration of 5-HT, and also facilitated the release of [3H]NA evoked by nicotine. Accumulation and electrically evoked release of [3H]NA were not altered by either lesion. The combination of both toxins resulted in an addition of their particular effects. The 5-HT(1B) receptor agonist, CP 93129, and the muscarinic agonist, oxotremorine, reduced the release of [3H]ACh in control and 5,7-DHT-lesioned rats; in rats injected with saporin, their effects could not be measured reliably. CP 93129 and the alpha(2)-adrenoceptor agonist, UK 14304, reduced the release of [3H]5-HT in all groups by about 65%. IN CONCLUSION: (i). selective neurotoxins can be combined to enable controlled and selective damage of hippocampal transmitter systems; (ii). 5-HT exerts an inhibitory influence on the nicotine-evoked release of NA, but partial serotonergic lesions do not influence the release of ACh at a presynaptic level and (iii). presynaptic modulatory mechanisms involving auto- and heteroreceptors may be conserved on fibres spared by the lesions.

5,7-Dihydroxytryptamine↗
PubMed2002

Phase I and pharmacokinetic study of BIBX 1382 BS, an epidermal growth factor receptor (EGFR) inhibitor, given in a continuous daily oral administration.

The pyrimido-pyrimidine BIBX 1382 BS inhibits the intracellular tyrosine kinase domain of the epidermal growth factor receptor (EGFR), thus specifically reverting the aberrant enzymatic activity from overexpressed and constitutively activated EGFR. A phase I and pharmacokinetic study of this new specific molecule was carried out. After initially performing an accelerated titration design from the first toxicities onwards, a modified Fibonacci scheme was used to escalate the daily oral dose. The following dosages and cycles (defined as treatment during 28 days) were applied: 25 mg: 6; 50 mg: 3; 100 mg: 6; 200 mg: 7; 150 mg: 3. Over a 10 months accrual phase, 11 patients (pts) (7 females, 4 males) with a median age of 63 years (range 50-73 years), World Health Organization Performance Status (WHO PS) 0:5 pts, 1:6 pts and miscellaneous solid tumours were entered. The median number of cycles applied per pt was 2 (range 1-7). Reversible, dose-dependent increase of liver enzymes (maximal Common Toxicity Criteria (CTC) grades: gamma-glutamyl transferase (GGT): 4, aspartate aminotransferase (GOT): 3, alanine aminotransferase (GPT): 3, alkaline phosphatase (AP): 3, bilirubin: 3) occurred. Oral medication yielded plasma levels far below those expected to be efficacious. In conclusion, target plasma levels could not be reached via the oral route at a reasonable dosage. Meanwhile, a preclinically unknown metabolite was identified from the urine of one patient. Subsequently, this metabolite was found to be abundant in patient plasma. The metabolite was demonstrated to be pharmacologically inactive. Due to a dose-limiting increase of liver enzymes, low bioavailability of BIBX 1382 BS and the detection of a pharmacologically inactive metabolite, this trial was discontinued.

Administration, Oral↗
PubMed2001

Methylated analogues of methyl (R)-4-(3,4-dichlorophenylacetyl)- 3-(pyrrolidin-1-ylmethyl)piperazine-1-carboxylate (GR-89,696) as highly potent kappa-receptor agonists: stereoselective synthesis, opioid-receptor affinity, receptor selectivity, and functional studies.

Analogues of the kappa-receptor agonist methyl (R)-4-(3,4-dichlorophenylacetyl)-3-(pyrrolidin-1-ylmethyl)piperazine-1-carboxylate (GR-89,696, 6) bearing an additional methyl substituent in the side chain are synthesized and evaluated for their kappa-receptor affinity and selectivity. A key step in the synthesis is the stereoselective reductive amination of the ketones 9, 18, and 19 with pyrrolidine and NaBH(3)CN, which succeeds only in the presence of the Lewis acid Ti(OiPr)(4). Whereas the BOC-substituted ketone 9 affords the unlike and like diastereomers of 10 in a ratio of 70:30, the diastereoselectivity during the reductive amination of the butyl and phenyl substituted ketones 18 and 19 is enhanced to 85:15 (butyl derivative) and >95:<5 (phenyl derivative) in favor of the unlike diastereomers. In receptor binding studies using the radioligand [(3)H]U-69,593 the (S,S)-configured methyl carbamate (S,S)-14 reveals the highest kappa-receptor affinity (K(i) = 0.31 nM) within this series, even exceeding the lead kappa-agonist 6 (GR-89,696). A slightly reduced kappa-receptor affinity is observed with the propionamide (S,S)-13 (K(i) = 0.67 nM). The kappa-receptor affinity of piperazines with acyl or alkoxycarbonyl residues at both nitrogen atoms (11, 13, 14) decreases in the order (S,S) > (R,R) > (S,R) > (R,S). The methyl carbamate (S,S)-14 discloses a unique activity profile also binding at mu-receptors in the subnanomolar range (K(i) = 0.36 nM). In a functional assay, i.e., by measuring acetylcholine release in rabbit hippocampus slices, the agonistic effects of the methyl carbamate (S,S)-14 and the propionamide (S,S)-13 are demonstrated. Only weak kappa- and mu-receptor affinities are found with the butyl- and phenyl-substituted piperazines 22 and 23. However, considerable sigma(1)-receptor affinity is determined for the enantiomeric, unlike-configured butyl derivatives (R,S)-22 and (S,R)-22 with K(i)-values of 40.2 nM and 81.0 nM, respectively.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed2001

The effect of HIV-1 regulatory proteins on cellular genes: derepression of the IL-2 promoter by Tat.

In HIV-infected individuals dysregulation of the immune system is characterized by severe disorders of the cytokine network. Increase secretion of IL-2, the major T cell growth and differentiation cytokine, may play a decisive role in sensitization of T cells for activation induced apoptosis and indirect death of activated T cells through augmented virus replication. We investigated the cause of enhanced IL-2 secretion and found that the HIV Tat induces this effect. We demonstrate that increased IL-2 secretion is due to Tat-enhanced IL-2 promoter activation. Tat derepresses and activates the distal AP-1 site (position -185 to -177) in the IL-2 promoter. In nonstimulated T cells a repressor complex containing NF-IL6, JunB, c-Fos and Fra-1 is formed on the AP-1(IL-2/d) site and represses IL-2 promoter activity. After T cell activation, a heterodimeric activator containing p65 and c-Jun binds to the AP-1(IL-2/d) site. HIV Tat enhances activation of NF-kappaB and consequently, activates the AP-1(IL-2/d) site. Our data provide evidence for a novel mechanism by which HIV Tat dysregulates IL-2 production and therefore may contribute to the HIV-1 infection in a way yet to be clarified.

Binding Sites↗
PubMed2001

Modulation of electrically evoked acetylcholine release in cultured rat septal neurones.

The electrically evoked release of acetylcholine and its modulation via auto- and heteroreceptors were studied in primary cell cultures prepared from embryonic rat septum (ED 17). Cultures were grown for 1, 2 or 3 weeks on circular, poly D-lysine-coated glass coverslips. They developed a dense network of non-neuronal and neuronal cells, only some of which were immunopositive for choline acetyltransferase. To measure acetylcholine release, the cells on the coverslips were pre-incubated with [3H]choline (0.1 micromol/L), superfused with modified Krebs-Henseleit buffer at 25 degrees C and electrically stimulated twice for 2 min (S1, S2; 3 Hz, 0.5 ms, 90-100 mA). The electrically evoked overflow of [3H] from the cells consisted of approximately 80% of authentic [3H]Ach, was largely Ca2+-dependent and tetrodotoxin sensitive, and hence represents an action potential-evoked, exocytotic release of acetylcholine. Using pairs of selective agonists and antagonist added before S2, muscarinic autoreceptors, as well as inhibitory adenosine A1- and opioid mu-receptors, could be detected, whereas delta-opioid receptors were not found. Evoked [3H] overflow from cultures grown for 1 week, although Ca2+ dependent and tetrodotoxin sensitive, was insensitive to the muscarinic agonist oxotremorine, whereas the effect of oxotremorine on cells grown for 3 weeks was even more pronounced than that in 2-week-old cultures. In conclusion, similar to observations on rat septal tissue in vivo, acetylcholine release from septal cholinergic neurones grown in vitro is inhibited via muscarinic, adenosine A1 and mu-opioid receptors. This in vitro model may prove useful in the exploration of regulatory mechanisms underlying the expression of release modulating receptors on septal cholinergic neurones.

Acetylcholine↗
PubMed1998

DEDD, a novel death effector domain-containing protein, targeted to the nucleolus.

The CD95 signaling pathway comprises proteins that contain one or two death effector domains (DED), such as FADD/Mort1 or caspase-8. Here we describe a novel 37 kDa protein, DEDD, that contains an N-terminal DED. DEDD is highly conserved between human and mouse (98. 7% identity) and is ubiquitously expressed. Overexpression of DEDD in 293T cells induced weak apoptosis, mainly through its DED by which it interacts with FADD and caspase-8. Endogenous DEDD was found in the cytoplasm and translocated into the nucleus upon stimulation of CD95. Immunocytological studies revealed that overexpressed DEDD directly translocated into the nucleus, where it co-localizes in the nucleolus with UBF, a basal factor required for RNA polymerase I transcription. Consistent with its nuclear localization, DEDD contains two nuclear localization signals and the C-terminal part shares sequence homology with histones. Recombinant DEDD binds to both DNA and reconstituted mononucleosomes and inhibits transcription in a reconstituted in vitro system. The results suggest that DEDD is a final target of a chain of events by which the CD95-induced apoptotic signal is transferred into the nucleolus to shut off cellular biosynthetic activities.

Amino Acid Sequence↗
PubMed1997

AIDS and the death receptors.

Activation-induced cell death (AICD) of T cells involves the CD95 receptor/ligand system. T cell activation through the T cell receptor results in expression of the CD95 ligand (CD95L) that acts on CD95+ cells by direct binding and in a paracrine or autocrine fashion. In AIDS, upregulation of CD95L in T cells is accelerated by two viral gene products, HIV-1 Tat and gp120. The CD95 signaling pathway is, therefore, likely to represent an important road to cell death of the CD4+ T cells in AIDS. Recently, the early events in the CD95 signaling pathway have been identified. A key role hereby plays a receptor-interacting member of the interleukin 1 beta-converting enzymes (ICE), FLICE, that could be a target for therapeutic intervention. In addition to CD95, the role of other members of the TNF receptor superfamily in AIDS is discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Resistance of chimpanzee T cells to human immunodeficiency virus type 1 Tat-enhanced oxidative stress and apoptosis.

CD4+ T-cell depletion in AIDS patients involves induction of apoptosis in human immunodeficiency virus (HIV)-infected and noninfected T cells. The HIV type 1 (HIV-1)-transactivating protein Tat enhances apoptosis and activation-induced cell death (AICD) of human T cells. This effect is mediated by the CD95 (APO-1/Fas) receptor-CD95 ligand (CD95L) system and may be linked to the induction of oxidative stress by Tat. Here we show that HIV-1 Tat-induced oxidative stress is necessary for sensitized AICD in T cells caused by CD95L expression. Tat-enhanced apoptosis and CD95L expression in T cells are inhibited by neutralizing anti-Tat antibodies, antioxidants, and the Tat inhibitor Ro24-7429. Chimpanzees infected with HIV-1 show viral replication resembling early infection in humans but do not show T-cell depletion or progression towards AIDS. The cause for this discrepancy is unknown. Here we show that unlike Tat-treated T cells in humans, Tat-treated chimpanzee T cells do not show downregulation of manganese superoxide dismutase or signs of oxidative stress. Chimpanzee T cells are also resistant to Tat-enhanced apoptosis, AICD, and CD95L upregulation.

Acquired Immunodeficiency Syndrome↗
PubMed1995

APO-1 (CD95)-dependent and -independent antigen receptor-induced apoptosis in human T and B cell lines.

Certain B and T cell lines respond to activation signals, e.g. through the antigen receptor, by undergoing apoptotic cell death. In T cells it has been recently shown that TCR-mediated apoptosis involves APO-1/Fas(CD95) receptor-ligand interaction. To investigate whether the TCR-CD3 complex can trigger alternative apoptosis pathways we generated subclones of the T cell line Jurkat which were completely resistant towards APO-1-mediated apoptosis. These JurkatR cells differed phenotypically from sensitive parental JurkatS cells only by the lack of APO-1 protein expression. Although JurkatR cells responded normally to anti-CD3 stimulation by expression of APO-1 ligand they failed to undergo anti-CD3-induced apoptosis. Thus, in Jurkat cells APO-1-mediated apoptosis was the main, and might be the only, mechanism for anti-CD3-induced cell death. However, BL-60 B cells, highly sensitive to anti-IgM-induced apoptosis, did not use the APO-1 receptor-ligand system because they failed to express APO-1 ligand mRNA. Taken together, our results suggest that malignant T and B cell lines may use APO-1 receptor-ligand-dependent and -independent antigen receptor-induced apoptosis pathways respectively. Similarly, differential pathways may be used by T and B cell subsets.

Apoptosis↗
PubMed1976

Carboxamidomethyl esters as reactive substrates for alpha-chymotrypsin. Orientational effects of hydrogen-bonding interactions.

Effects of hydrogen-bonding interactions of amide groups on reactivity of esters to alpha-chymotrypsin were studied. Of the methyl esters studied, only that from acetyl-L-phenylalanine has k3 rate-limiting. In methyl beta-phenylpropionates an alpha-acetamido substituent increased k2 greater than 550 times, k3 approximately 5 times; an alpha-acetylclycyloxy substituent increased k2 approximately 2 times, k3 approximately 6 times, both in comparison with the alpha-acetoxy esters. Essentially all carboxamidomethyl esters studied have k3 rate-limiting; reactivity to hydroxide is only 4 times that of methyl esters. In alpha-substituted beta-phenylpropionates, carboxamido-methyl esters show k2 values greater than 110 times greater than 280 times, greater than 26 times, and 7 times the k2 values of the methyl esters for the alpha substituents, acetoxy, acetylglycyloxy, hydroxy, and hydrogen, respectively. In esters of alpha-acetamido acids, carboxamidomethyl esters show k2 values 44 times, greater than 110 times, greater than 12 times, and approximately 33 times the k2 values of the methyl esters of glycine, alanine, leucine, and phenylalanine, respectively. Cyanomethyl esters also had k3 rate-limiting. Hydrogen-bonding to the enzyme of either an alpha-acetamido group or a carboxamidomethyl group combined with bonding of the beta-aryl group, orients the hydrolyzing groups properly, increasing k2. Hydrogen-bonding of both alpha-acetamido and carboxamido-methyl groups is effective to a lesser degree. The amide group appears to have small effects on Ks as hydrogen bonding is balanced by desolvation. It is proposed that desolvation during bonding increases k2 and Ks.

Amides↗