Maintenance treatments with methadone or buprenorphine are more or less efficient procedures for helping heroin addicts to stop or reduce drug abuse. Another approach to treat opiate dependence could be to target the endogenous opioid system by enhancing the effects of enkephalins by protecting them from enzymic degradation by the dual peptidase inhibitor RB101.

No abstract available

Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

Tolerance and dependence induced by chronic delta-9-tetrahydrocannabinol (THC) administration were investigated in mice. The effects on body weight, analgesia and hypothermia were measured during 6 days of treatment (10 or 20 mg kg(-1) THC twice daily). A rapid tolerance to the acute effects was observed from the second THC administration. The selective CB-1 receptor antagonist SR 141716A (10 mg kg(-1)) was administered at the end of the treatment, and somatic and vegetative manifestations of abstinence were evaluated. SR 141716A administration precipitated several somatic signs that included wet dog shakes, frontpaw tremor, ataxia, hunched posture, tremor, ptosis, piloerection, decreased locomotor activity and mastication, which can be interpreted as being part of a withdrawal syndrome. Brains were removed immediately after the behavioural measures and assayed for adenylyl cyclase activity. An increase in basal, forskolin and calcium/calmodulin stimulated adenylyl cyclase activities was specifically observed in the cerebellum of these mice. The motivational effects of THC administration and withdrawal were evaluated by using the place conditioning paradigm. No conditioned change in preference to withdrawal associated environment was observed. In contrast, a conditioned place aversion was produced by the repeated pairing of THC (20 mg kg(-1)), without observing place preference at any of the doses used. This study constitutes a clear behavioural and biochemical model of physical THC withdrawal with no motivational aversive consequences. This model permits an easy quantification of THC abstinence in mice and can be useful for the elucidation of the molecular mechanisms involved in cannabinoid dependence.

Aminopeptidase N (APN) is a zinc metallopeptidase involved in the inactivation of biologically active peptides. The knowledge of its precise distribution is crucial to investigate its physiological role. This requires the use of appropriate probes such as the recently developed highly potent and selective radiolabeled APN inhibitor 2(S)-benzyl-3-[hydroxy(1'(R)-aminoethyl)phosphinyl]propanoyl-L-3-[ (12 5)I]iodotyrosine ([(125)I]RB 129). Its binding properties were investigated using rat brain homogenates (K(d)=3.4 nM) or APN expressed in COS-7 cells (K(d)=0.9 nM). The specific binding was 95% at [K(d)], and preliminary autoradiography in intestine is promising. The decreased affinity of [(125)I]RB 129 (=10(-6) M) for the E(350)D APN mutant, supports the critical role of E(350) in the amino-exopeptidase action of APN.

The selective and potent aminopeptidase N inhibitor [125I]RB 129 has been used for the radioautographic localization of this enzyme in rat brain, spinal cord and intestine. Brain microvessels and intestine brush-border cells were shown to present a high concentration of aminopeptidase N. Moreover, a labeling of various brain structures was observed. A very high level of binding occurred in the meninges, choroid plexus, pineal gland, paraventricular nucleus and pituitary gland. Moderate to high labeling was also observed in the cortex, caudate-putamen, subthalamic nucleus, central periaqueductal gray, thalamus, as well as in the dorsal and ventral horn of the spinal cord, which are known to contain a high concentration of enkephalins, opioid receptors and neutral endopeptidase. This co-localization confirms the physiological implication of aminopeptidase N in the inactivation of enkephalins accounting for the requirement of dual inhibition of neutral endopeptidase and aminopeptidase N to observe highly significant morphine-like effects induced by the protected endogenous opioid peptides. Aminopeptidase N was also visualized in moderate to high levels in other brain structures such as the hippocampus, nucleus accumbens, substantia nigra, hypothalamus (dorsomedial and ventromedial nuclei), raphe nucleus, pontine nucleus, inferior olive, and in high concentration in the granular layer of cerebellum. In summary, aminopeptidase N has been visualized for the first time in numerous brain areas using the selective inhibitor [125I]RB 129. This iodinated probe could allow the ex vivo and in vivo localization of aminopeptidase N in various tissues to be investigated and may also be used to evaluate quantitative changes in aminopeptidase N expression in pathological situations. Aminopeptidase N, which preferably removes NH2-terminal neutral amino acids from peptides, has probably a host of substrates. Nevertheless, a certain in vivo selectivity could be achieved by the presence of the enzyme in structures where the peptide effector and its receptors are also co-localized.

The neutral endopeptidase ("enkephalinase") of the rabbit brush border membrane has been purified to homogeneity by a rapid immunoaffinity method using a monoclonal antibody. In contrast with other methods used so far, a complete extraction of enkephalinase from the brush border membrane can be achieved with octyl glucoside, without loss of activity. The solubilized enzyme can be selectively separated from the other proteins in a single step using an immunoaffinity column consisting of the monoclonal antibody covalently linked to Sepharose CL-4B. It is demonstrated that enkephalinase can then be recovered in an active form by elution at low pH. The purified enzyme obtained by this method is completely inhibited by thiorphan and appears as a single 94,000 dalton protein after polyacrylamide gel electrophoresis under denaturing and reducing conditions.

We report the isolation, cloning and expression, in Bacillus subtilis, of the gene coding for thermolysin, a thermostable metalloprotease which is produced by Bacillus thermoproteolyticus Rokko. The nucleotide sequence has revealed that, like neutral proteases produced by other members of the Bacillus species, thermolysin is probably produced as a preproenzyme carrying a typical N-terminal membrane signal sequence. Further, the thermolysin gene shares a strong homology with two other previously cloned genes from two different strains of Bacillus stearothermophilus. The sequence of the mature secreted protease, inferred from the DNA sequence, is, with two exceptions, identical with the previously published protein sequence of thermolysin [Titani, Hermodson, Ericsson, Walsh and Neurath (1972) Nature (London) 238, 35-37]. The exceptions are Asn37 and Gln119, originally reported to be Asp and Glu respectively. The biochemical characterization of the secreted recombinant protein shows that it is indistinguishable from the wild-type thermolysin.