Caspase inhibitors induce a switch from apoptotic to proinflammatory signaling in CD95-stimulated T lymphocytes

CD95 is a major apoptosis receptor that induces caspase activation and programmed cell death in susceptible cells. CD95-induced apoptosis can be blocked by peptidic caspase inhibitors such as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or Ile-Glu-Thr-Asp-fluoromethyl ketone. Here we show that stimulation of CD95 in the presence of these inhibitors induces necrosis and expression of various proinflammatory cytokines in primary T lymphocytes, such as TNF-alpha, IFN-gamma and granulocyte/macrophage colony-stimulating factor. In the absence of caspase inhibition CD95 stimulation did not result in cytokine expression, indicating that this proinflammatory signaling pathway is suppressed by active caspases. Further analysis with A3.01 T cells revealed that the proinflammatory signaling activity of CD95 was mediated by MEK/ERK, p38 and NF-kappaB signaling pathways. These findings point to a pivotal role of caspases not only as mediators of apoptosis but also as enzymes that prevent proinflammatory signaling during CD95-induced apoptosis. Moreover, our findings may be useful for the development of novel pharmacological strategies.     

A rapid and simple method for efficient coating of microtiter plates using low amounts of antigen in the presence of detergent

Bio-Beads SM-2 have previously been used for the removal of non-ionic detergents from protein solutions. Addition of Bio-Beads SM-2 to detergent solubilized antigen significantly enhanced the immobilization of antigen to microtiter wells. Depending on the incubation time used 35-45% of the applied antigen could be immobilized to the microtiter wells. Using this method and a subsequent ELISA procedure it was possible to detect monoclonal antibodies in hybridoma supernatants after coating microtiter wells with 100 microliters of a solution containing 16 ng antigen/ml in the presence of 0.01% Triton X-100.     

Benzodiazepine receptors: Multiple receptors or multiple conformations?

Summary Several lines of evidence from reversible binding studies seem to indicate there are at least two central benzodiazepine receptor subtypes, the BZ₁ and BZ₂ receptors. Irreversible binding studies, using³H-flunitrazepam as a photoaffinity label for benzodiazepine receptors, not only are in perfect agreement with the data from reversible binding studies but extend these studies by identifying P₅₁, a protein with apparent molecular weight 51,000, as a protein associated with the BZ₁ receptor and by suggesting that the BZ₂ receptor might actually consist of several different benzodiazepine receptors associated with different and distinct proteins irreversibly labeled by³H-flunitrazepam.Other reversible binding studies have accumulated indicating the existence of several different conformations of benzodiazepine receptors. Irreversible binding studies support this conclusion and in addition suggest the existence of four different benzodiazepine binding sites within the GABA-benzodiazepine receptor complex. It is therefore hypothesized that there are several different GABA-benzodiazepine receptor subtypes all of which have four distinct benzodiazepine binding sites which can exist in at least three different but freely interconvertible conformations. This hypothesis can account for all experimental observations obtained so far and might partially explain the distinct clinical effects of structurally similar benzodiazepines.     

The role of the envelope glycoprotein in the depletion of T helper cells in human immunodeficiency virus infection

Infection with the human immunodeficiency virus (HIV) causes gradual depletion of CD4+ T helper lymphocytes and destruction of the lymphoid tissue, which ultimately leads to a fatal defect of the cellular immune system. Paramount to the understanding of the pathogenesis of HIV infection is to elucidate the mechanism which underlies the loss of T helper cells. Various ideas have been proposed in order to explain this issue. Several hypotheses have focused on the role of the envelope glycoprotein in this process. This review summarizes the data obtained and concepts proposed regarding the involvement of the HIV glycoprotein in the pathology of CD4+ T cell depletion. This work was supported by the Deutsche Forschungsgemeinschaft, the Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie and the Stifterverband für die Deutsche Wissenschaft     

Clusters of GABAA receptors on cultured hippocampal cells correlate only partially with functional synapses

We describe a method to label gamma-aminobutyric acid (GABA)A receptors on the surface of living hippocampal neurons in primary culture, and we compare the distribution of receptors with that of active synapses. To visualize GABAA receptors, the affinity-purified antibody beta3(1-13), recognizing the extracellular N-termini of the GABAA receptor beta2- and beta3-subunits, was used in combination with fluorescent secondary antibodies. The beta2- and beta3-subunits belong to the predominant GABAA receptor subunits in the hippocampus. As expected for aggregates of GABAA receptors in the somato-dendritic plasma membrane, a patchy staining pattern similar to that seen by labelling neurons after fixation was obtained. An antiserum recognizing an intracellular epitope of GABAA receptor beta3-subunits did not label the receptors in living neurons. Whole-cell recordings of GABA-evoked Cl - currents were not affected after decorating GABAA receptors with antibody beta3(1-13). Combining the staining of GABAA receptors with the labelling of active presynaptic terminals with the fluorescent dyes FM1-43 or FM4-64, consistently resulted in the detection of GABAA receptor clusters that were not located at active synapses. These amounted to approximately 50% of all labelled GABAA receptor clusters. GABAA receptor clusters that were not associated with active presynaptic terminals partially colocalized with the synaptic vesicle marker protein sv2, while another fraction had no presynaptic counterpart at all. These findings suggest the presence of presynaptically silent GABAergic synapses in cultured hippocampal neurons. They also indicate that for the maintenance of GABAA receptor aggregates, the release of GABA from an opposing active terminal is not essential.     

Special relationship of gamma-aminobutyric acid to the ventromedial nucleus of the hypothalamus during embryonic development

The ventromedial nucleus of the hypothalamus (VMH) is a key nucleus for regulating homeostatic, neuroendocrine, and behavioral functions. We conducted immunocytochemical analyses by using antisera directed against gamma-aminobutyric acid (GABA), its synthetic enzyme glutamic acid decarboxylase (GAD67), GABA-A receptor subunits (alpha2, beta3, epsilon), estrogen receptor-alpha, and Neuropeptide Y (NPY) in the region of the VMH in embryonic mice to identify potential patterning elements for VMH formation. Cells and fibers containing GABA and GAD67 encircled the primordial VMH as early as embryonic day 13 (E13) when the cytoarchitecture of the VMH was not recognizable by Nissl stain. At E16-17 the cytoarchitecture of the VMH became recognizable by Nissl stain as GABAergic fibers invaded the nucleus, continued postnatally, and by adulthood the density of GABAergic fibers was greater inside than outside the VMH. GABA-A receptor subunit expression (beta3 by E13 and alpha2 by E15) within the primordial VMH suggested potential sensitivity to the surrounding GABA signal. Brain slices were used to test whether fibers from distal or proximal sites influenced VMH development. Coronal Vibratome slices were prepared and maintained in vitro for 0-3 days. Nissl stain analyses showed a uniform distribution of cells in the region of the VMH on the day of plating (E15). After 3 days in vitro, cellular aggregation suggesting VMH formation was seen. Nuclear formation in vitro suggests that key factors resided locally within the coronal plane of the slices. It is suggested that either GABA intrinsic to the region nearby the VMH directly influences the development and organization of the VMH, or along with other markers provides an early indicator of pattern determination that precedes the cellular organization of the VMH.     

The citalopram challenge test in patients with major depression and in healthy controls

Neuroendocrine challenge tests in depressed patients have revealed a blunted hormonal reaction to serotonergic stimuli. In the present study, citalopram was chosen as the serotonergic agent for neuroendocrine stimulation. Compared to earlier challenge agents, citalopram has the advantage of serotonergic selectivity, its application is well tolerated and the possibility of intravenous application reduces pharmacokinetic interference. Sixteen patients suffering from an acute episode of major depression and 16 healthy controls underwent the stimulation procedure with 20 mg of citalopram and placebo. Whereas significant differences in the secretion of prolactin and cortisol between citalopram and placebo challenge were observed in the control group, no differences were found in the group of depressed patients. Comparison of depressed patients and controls showed a significantly blunted prolactin secretion in patients. Differences in cortisol secretion following serotonergic stimulation with citalopram did not become significant. The stimulation procedure was well tolerated in all subjects, although a higher number of side effects was observed in the control group. The amount of side effects did not correlate with the hormone responses. These results are in line with the hypothesis of serotonergic hypofunction in depressed patients. In conclusion, the 20-mg citalopram challenge test is thought to be a promising tool for further investigation of serotonergic function in psychiatric illness.     

Dual mode of stimulation by the beta-carboline ZK 91085 of recombinant GABA(A) receptor currents: molecular determinants affecting its action.

In electrophysiological measurements the beta-carboline ethyl 6-benzyloxy-beta-carboline-3-carboxylate (ZK 91085) acts as a positive allosteric modulator on rat recombinant alpha1beta2gamma2 GABA(A) receptors and binds with high affinity (IC50-1.5 nM) to the [3H]-flunitrazepam site. Flumazenil was able to partially counteract the current modulation. These observations indicate an action of ZK 91085 at the benzodiazepine binding site. At the dual subunit combination alpha1beta2, which lacks the gamma subunit required for benzodiazepine modulation, we still observed a potentiation of GABA currents. Thus ZK 91085 acts via an additional site on the channel. At the subunit combination alpha1beta1, ZK 91085 potentiation is strongly reduced as compared to alpha1beta2. In binding studies, ZK 91085 was able to decrease [35S]-TBPS binding in alpha1beta2gamma2 and alpha1beta2 but not in alpha1beta1. This selectivity of ZK 91085 for receptors containing the beta2 isoform over those containing the beta1 isoform is reminiscent of the action of loreclezole. To identify amino acid residues important for the second type of modulation, we functionally compared wild type alpha1beta2 and mutant receptors for stimulation by ZK 91085. The mutation beta2N265S, that abolishes loreclezole effects, also abolishes ZK 91085 stimulation. The mutation beta2Y62L increased stimulation by ZK 91085 3-4 fold, locating an influencing entity of the second type of action of ZK 91085 at an alpha/beta subunit interface. Structural intermediates of ZK 91085 and the beta-carboline abecarnil, the latter of which only slightly potentiated GABA currents in alpha1/beta2, were analysed to determine structural requirements for modulation. ZK 91085 thus allosterically stimulates the GABA(A) receptor through two sites of action: the benzodiazepine site and the loreclezole site in contrast to classical beta-carbolines, that confer negative allosteric modulation through the benzodiazepine site.     

Homozygosity for a conserved Mhc class II DQ-DRB haplotype is associated with rapid disease progression in simian immunodeficiency virus-infected macaques: results from a prospective study

In human immunodeficiency virus type 1 (HIV-1)-infected individuals, disease progression varies considerably. This is also observed after experimental infection of macaques with simian immunodeficiency virus (SIV). Major histocompatibility complex (MHC) genes may influence disease progression in both species. Homozygosity for Mhc-Mamu (Macaca mulatta)-DQB1*0601 was previously identified to be associated with rapid disease progression in SIV-infected macaques. To validate the association of this genotype with disease progression, a prospective study was carried out. Six unrelated monkeys homozygous for Mamu-DQB1*0601 and DRB1*0309-DRB*W201 and 6 heterozygous monkeys were infected with SIVmac. Five of the homozygous and only 1 of the heterozygous monkeys died rapidly after infection, with manifestations of AIDS. These results were validated by a retrospective survival analysis of 71 SIV-infected monkeys. The identified DQ-DRB genotype is frequent among monkeys of different breeding colonies and allows a fairly reliable selection before infection of monkeys predisposed for rapid disease progression.