Mindfulness-based relapse prevention for cannabis regular users: Preliminary outcomes of a randomized clinical trial.

Mindfulness-based approaches have shown their effectiveness in caring for patients with substance use disorders (SUD). Mindfulness-based relapse prevention (MBRP) integrates practices from mindfulness-based interventions and cognitive-behavioral relapse prevention (RP) approaches. This article presents the preliminary results of a study that measures the effectiveness of an MBRP protocol for volunteer cannabis users willing to reduce or stop their consumptions. Twenty cannabis users were randomly assigned to either receive an eight-week outpatient MBRP program or treatment as usual (TAU). Cannabis use was assessed weekly through the timeline follow back (TLFB). Eighty percent of individuals received MBRP treatment and 60% of individuals received TAU completed treatment. Preliminary results did not find significant difference at the end of treatment (week 8) regarding the number of joints smoked. Despite the absence of any significant difference between the two groups, the contribution of mindfulness in the caring of SUD seems encouraging and promising. Many MBRP group participants reported qualitative changes in the way they consumed. This study will be continued in order to evaluate the effectiveness of MBRP on a larger number of subjects.

Maturation of HIV envelope glycoprotein precursors by cellular endoproteases.

The entry of enveloped viruses into its host cells is a crucial step for the propagation of viral infection. The envelope glycoprotein complex controls viral tropism and promotes the membrane fusion process. The surface glycoproteins of enveloped viruses are synthesized as inactive precursors and sorted through the constitutive secretory pathway of the infected cells. To be infectious, most of the viruses require viral envelope glycoprotein maturation by host cell endoproteases. In spite of the strong variability of primary sequences observed within different viral envelope glycoproteins, the endoproteolytical cleavage occurs mainly in a highly conserved domain at the carboxy terminus of the basic consensus sequence (Arg-X-Lys/Arg-Arg downward arrow). The same consensus sequence is recognized by the kexin/subtilisin-like serine proteinases (so called convertases) in many cellular substrates such as prohormones, proprotein of receptors, plasma proteins, growth factors and bacterial toxins. Therefore, several groups of investigators have evaluated the implication of convertases in viral envelope glycoprotein cleavage. Using the vaccinia virus overexpression system, furin was first shown to mediate the proteolytic maturation of both human immunodeficiency virus (HIV-1) and influenza virus envelope glycoproteins. In vitro studies demonstrated that purified convertases directly and specifically cleave viral envelope glycoproteins. Although these studies suggested the participation of several enzymes belonging to the convertases family, recent data suggest that other protease families may also participate in the HIV envelope glycoprotein processing. Their role in the physiological maturation process is still hypothetical and the molecular mechanism of the cleavage is not well documented. Crystallization of the hemagglutinin precursor (HA0) of influenza virus allowed further understanding of the molecular interaction between viral precursors and the cellular endoproteases. Furthermore, relationships between differential pathogenicity of influenza strains and their susceptibility to cleavage are molecularly funded. Here we review the most recent data and recent insights demonstrating the crucial role played by this activation step in virus infectivity. We discuss the cellular endoproteases that are implicated in HIV gp160 endoproteolytical maturation into gp120 and gp41.

Purification and characterization of the nuclease NucM of Erwinia chrysanthemi.

The major periplasmic nuclease of Erwinia chrysanthemi strain 3937, NucM, has been purified near to homogeneity by a one step purification procedure, using chromatography on a sulfopropyl column. NucM cleaves randomly single and double-stranded DNA and RNA. It does not need divalent cations for its action, and is more active in low salt buffers. A serine and a histidine residue could be present in the catalytic site. Formation of disulfide bonds is necessary for NucM activity. NucM is probably synthesized as a reduced inactive polypeptide and becomes active in the periplasm once disulfide bonds are formed.

Evaluation of structure-antigenicity relationship of peptides from human immunodeficiency virus type 1 (HIV-1) p18 protein by circular dichroism.

The antigenicity of Human Immunodeficiency Virus type 1 (HIV-1) matrix p18 protein was evaluated by analyzing the specificity of anti-p18 antibodies elicited either in HIV-1 infected humans, or in HIV-1 infected or immunized chimpanzees, against a panel of long and short overlapping synthetic peptides [from 12 to 46 amino acid (aa) residues] covering the entire sequence of p18. The relationship between peptide structure and antigenicity was further investigated by probing the secondary structures of the peptides by circular dichroism. The results obtained clearly showed the immunodominance of the N-terminal region mimicked by peptide P1 (aa 2-45), which reacted with 52 and 100% of human and chimpanzee anti-p18 sera, respectively. In contrast smaller 15 aa long peptides C1, C2, C3, C4 and P3 which cover the entire sequence of immunodominant peptide P1, showed only weak or no reactivity. In contrast to widely accepted hypotheses, circular dichroism analysis of both small and large peptides secondary structures did not show any obvious correlation between antigenicity and the ability of peptides to adopt an ordered conformation.

Effects of calcium ions on proteolytic processing of HIV-1 gp160 precursor and on cell fusion.

Complete activation of human immunodeficiency virus type 1 (HIV-1) requires the endoproteolytic cleavage by cellular protease of the envelope glycoprotein precursor (gp160) into the external glycoprotein gp120, and the transmembrane glycoprotein gp41. We report here the effect of depletion of cellular calcium ions on maturation of precursor gp160 and its concomitant effect on syncytium formation. We show that the cellular endoprotease activity responsible for gp160 maturation and the capacity for HIV-1 to induce syncytium formation are calcium-dependent. In addition, we show that endoproteolytic maturation is a key step in syncytium formation induced by HIV-1.

Identification of a procarboxypeptidase A-truncated protease E binary complex in human pancreatic juice.

The characterization, in human pancreatic juice, of a binary complex associating procarboxypeptidase A with a 32 kDa inactive glycoprotein (G32) is reported in this paper. Free G32 was isolated after dissociation of the binary complex. N-terminal sequence analysis revealed a complete homology between this protein and human protease E (HPE 1), except for the two strongly hydrophobic N-terminal residues (Val-Val) which are missing in G32. This protein might be a truncated protease E highly analogous to the subunit III of the ruminant procarboxypeptidase A-S6 ternary complex. The analogy with bovine subunit III is further supported by interspecies reassociation experiments showing that bovine procarboxypeptidase A can specifically bind human G32.

Further studies on the human pancreatic binary complexes involving procarboxypeptidase A.

In contrast to procarboxypeptidase B which has always been reported to be secreted by the pancreas as a monomer, procarboxypeptidase A occurs as a monomer and/or associated to one or two functionally different proteins, depending on the species. Recent studies showed that, in the human pancreatic secretion, procarboxypeptidase A is mainly secreted as a 44 kDa protein involved in at least three different binary complexes. As previously reported, two of these complexes associated procarboxypeptidase A to either a glycosylated truncated protease E or zymogen E. In this paper, we identified proelastase 2 as the partner of procarboxypeptidase A in the third complex, thus reporting for the first time the occurrence of a proelastase 2/procarboxypeptidase A binary complex in vertebrates. Moreover, from N-terminal sequence analyses, the 44 kDa procarboxypeptidase A involved in these complexes was identified as being of the A1 type. Only one type of procarboxypeptidase B, the B1 type, has been detected in the analyzed pancreatic juices, thus emphasizing the previously observed genetic differences between individuals.

Crosslinking of Dam methyltransferase with S-adenosyl-methionine.

Highly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 microM S-adenosyl-methionine; it was inhibited in the presence of substances which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins, which do not bind S-adenosyl-methionine, as well as heat inactivated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106.

Cytotoxic effect on lymphocytes of Tat from human immunodeficiency virus (HIV-1).

The human immunodeficiency virus type 1 (HIV-1) genome codes for trans-activator Tat, an 86-residue protein whose expression is critical for viral replication. Full-length Tat and Tat peptides from HIV-1 were chemically synthesized using optimized solid phase technique. Synthetic Tat2-86 was found not only to inhibit antigen-induced human peripheral blood lymphocyte (PBL) proliferation in vitro, as described by Viscidi et al. [1989, Science 246, 1606-1608], but also mitogen-induced PBL proliferation, with 50% inhibition obtained at 0.9 and 8 microM, respectively. To assess the mechanism by which Tat exert its inhibitory effect, we analysed its interaction and effect on CD4(+)-cells. Direct fluorescence and indirect immunofluorescence assays analysed by flow cytometry showed that fluorescein isothiocyanate-labeled and -unlabeled Tat interact (> 0.2 microM) with CD4-expressing lymphoid cells (CEM cell line). Experiments of chromium-51 release and Trypan blue exclusion on these tumor cells in vitro have demonstrated the capacity of Tat to modify cellular membrane permeability and cell viability, in a dose-dependent manner. The use of Tat peptides revealed that those containing the Tat basic region from 49 to 57 were able to bind to the cell membrane and to exhibit a cytotoxic activity on lymphocytes. Together, the data suggest that the potential cytotoxicity of Tat on lymphocytes could be directly implicated in virus-induced immune dysfunction observed in HIV-1 infected patients.

T4-lymphocyte endoprotease responsible for the proteolytic processing of HIV-1 gp160, like Kex2p endoprotease, is a calcium-dependent enzyme.

In the present study we show that precursor gp160 is cleaved in the HIV-1 infected CEM (CD4+) cell line preferentially in the presence of calcium ions demonstrating that the responsible cellular endoprotease is a calcium-dependent enzyme. Taking into account this similarity, a synthetic peptide modelling the cleavage site of HIV-1 envelope glycoprotein precursor was used as substrate for Kex2p. Results obtained clearly showed that the processing enzyme Kex2p (EC 3.4.21.61), a subtilisin-like serine protease that is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae is able to cleave correctly this peptide at the potential cleavage site.

Processing and routage of HIV glycoproteins by furin to the cell surface.

Proteolytic activation of HIV-1 and HIV-2 envelope glycoprotein precursors (gp160 and gp140, respectively) occurs at the carboxyl side of a consensus motif consisting of the highly basic amino acid sequence. We have shown previously (Hallenberger et al., 1997) and confirmed in this report, that furin and PC7 can be considered as the putative physiological enzymes involved in the proteolytic activation of the HIV-1 and HIV-2 envelope precursors. In this study, we show by cell surface biotinylation and immunoprecipitation of the cell surface associated viral glycoproteins with antibodies that the mature viral envelope glycoproteins are correctly transported to the cell. membrane. Furthermore, we show that the uncleaved forms of the glycoproteins (gp160HIV-1 and gp140HIV-2) are also highly represented at the cell surface. First, transient expression of gp160 and gp140 into CV1, a cell line known to be inefficient in the proteolytic processing of the env gene, results in the expression of gp160 and gp140 at the cell surface. Moreover, HIV-1 infection of T cells also showed that gp160 is directed to the cell surface. In addition, we show that the precursor is not incorporated in the virus particle following the budding from the cell surface. Furthermore, a gp160 mutant (deficient for three carbohydrate sites on the gp41), shown to be poorly processed with the coexpressed endoproteases, is found to be transported as an uncleaved precursor to the cell surface. In contrast to HIV envelope glycoproteins, the influenza hemagglutinin precursor (HA0), that is thought to be matured by the furin-like enzymes as well, is found to be retained within the cell and is not able to reach the cell surface. Taken together, these results show that the proteolytic maturation of the viral envelope precursors of human immunodeficiency viruses type 1 and type 2 is not a prerequisite for cell surface targeting of the HIV glycoproteins. Implications of these results for antiviral immune response are discussed.

Antibody neutralization of HIV-1 and the potential for vaccine design.

Neutralisation by antibody is, for a number of viruses, an in vitro correlate for protection in vivo. For HIV-1 this is controversial. However, the induction of a potent anti-HIV neutralising antibody response remains one of the principal goals in vaccine development. A greater knowledge of the fundamental mechanisms underlying the neutralisation process would help direct research towards suitable vaccine immunogens. The primary determinant of HIV neutralisation appears to be antibody affinity for the trimeric envelope glycoprotein spike on the virion, suggesting that epitope-specific effects are secondary and implying a single, dominant mechanism of neutralisation. Antibody interference with virion attachment to the target cell appears to be a major mechanism of neutralisation by gp120-specific antibodies. This is probably achieved both by antibody-induced dissociation of gp120 from gp41 and by direct inhibition of virus binding to receptor-coreceptor complexes. A gp41-specific antibody neutralises by interfering with post-attachment steps leading to virus membrane fusion. Recent advances in structural analyses of the HIV envelope glycoproteins coupled with data obtained from antibody mapping and neutralisation studies allow a greater understanding of Env function and its inhibition. This in turn should lead to a more rational basis for vaccine design aimed at stimulating highly effective neutralising antibodies.

Selective HIV-1-induced downmodulation of CD4 and coreceptors.

CD4 and members of the chemokine receptor family are required for infection of host cells, in vitro and in vivo, by the human immunodeficiency virus type-1. Although it is established that HIV-1 gp 120 interacts with CD4 and the coreceptors CCR5 or CXCR4 at the plasma membrane during HIV entry, longer-term interactions taking place between these molecules and HIV Env are less well understood. We have measured the cell surface expression of CD4, CCR5 and CXCR4 on a CD4+/CXCR4+CCR5+ T cell line following infection by cell line-adapted X4 and primary X4, X4R5 and R5 viruses. We report a selective downmodulation of CD4 by X4 and R5X4 viruses, but not by R5 viruses. None of the viruses tested significantly reduced CXCR4 expression at any time after infection. CCR5 protein and mRNA expression was eliminated by chronic infection with R5 viruses. These results indicate that chronic HIV-1 infection has distinct effects on CD4 and coreceptor membrane expression that depends on viral origin and coreceptor usage.

Characterization of the nucM gene coding for a nuclease of the phytopathogenic bacteria Erwinia chrysanthemi.

The gene nucM encoding a nuclease was cloned from a genomic library of Erwinia chrysanthemi. The nucM gene was subcloned, and mutagenized by insertion of a uidA-KanR cartridge. This mutation was introduced by recombination into the Erwinia chrysanthemi chromosome. The nucM mutant lost NucM activity when tested on a DNA plate after 24 hours, but still possessed secondary weak nuclease activity. The nucleotide sequence of nucM was determined. It presents a 798 bp open reading frame, coding for a 266-amino-acid protein, with a predicted molecular mass of 29,910 Da. The deduced NucM protein shows 59% sequence identity with the DNase I precursor from Vibrio cholerae. It contains a typical leader sequence. Experiments of cell fractionation showed that NucM is periplasmic in E. chrysanthemi. The transcription start has been determined by S1 mapping. The -10 and -35 regions do not show homology with consensus sequence of the promoters recognized by sigma 70. In fact, the promoter seems to be dependent on the sigma 70, but the first transcription nucleotide is unusually far from the -10 region. nucM seems to be expressed constitutively.

Structure of the phenylalanine hydroxylase gene in Drosophila melanogaster and evidence of alternative promoter usage.

The complete Drosophila melanogaster phenylalanine hydroxylase gene isolated from a genomic library was sequenced. Gene structure consisted of five exons covering a region of around 3 kb. Position of introns in the C-terminal domain was conserved with mammalian aromatic amino acid hydroxylase genes. Putative promoter sequences in the 5'UTR and intron 1 were identified. A novel transcript was detected differing from that previously reported by the inclusion of a part of the intron 1 sequence. It could be produced using an alternative promoter. The deduced open reading frame would code a protein with a small difference at the N-terminus. Expression of the alternative transcripts was examined throughout development.

Systematic use of the incomplete factorial approach in the design of protein crystallization experiments.

We have used the Incomplete Factorial Approach (Carter, C. W., and Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219-12223) in conjunction with the program Cristal (Roussel, A., Serre, L., Frey M., and Fontecilla-Camps, J. (1990) J. Crystal Growth 106, 405-409) to crystallize six different proteins. We were able to obtain crystals and to identify the critical factors for crystallization for each of these six proteins. In some of the cases, we succeeded on the first try while using only minute amounts of protein. This study proves that the Incomplete Factorial Approach is a powerful tool in identifying the factors that need to be varied to achieve crystallization. Single crystals of adequate size were obtained for all the proteins reported here, although some did not diffract well enough to be studied by x-ray diffraction methods; the asymmetric units of these latter crystals contain a large metric units of these latter crystals contain a large number of molecules, which is most likely due to the presence of significant amounts of carbohydrate in the proteins.

Kex2p: a model for cellular endoprotease processing human immunodeficiency virus type 1 envelope glycoprotein precursor.

The endoproteolytic cleavage of the envelope glycoprotein precursor (gp160) of the human immunodeficiency virus type 1 (HIV-1) by a cellular protease is required for full activation of the virus. In this study, processing of gp160 was analyzed in vitro using the Kex2p endoprotease from the yeast Saccharomyces cerevisiae as a processing enzyme model. Endoproteolytic processing was examined using a synthetic peptide that mimics the cleavage site of HIV-1 glycoprotein, and a recombinant gp160 bearing the entire sequence of the env gene product, including the conserved cleavage site Arg508-Glu-Lys-Arg511. Coexpression in BHK-21 of Kex2p and gp160 by recombinant vaccinia viruses demonstrates that Kex2p can correctly process the HIV-1 glycoprotein to gp120 and gp41. Furthermore, recombinant gp160 and peptide were used as substrates and subjected to proteolysis with purified membranes from an S. cerevisiae strain overproducing the Kex2p endoprotease. Treatment of recombinant gp160, which has an apparent molecular mass of 127 kDa, with Kex2p and Western blot analysis showed that the precursor was cleaved into two products of about 101 and 34 kDa apparent molecular mass. Amino acid sequencing of the NH2-terminus of the 34-kDa product showed that the cleavage site of recombinant gp160 was between Arg511 and Ala512. Recombinant gp160 mutated at the sequence coding for the potential cleavage site, and mature recombinant gp120, however, were not cleaved when treated with Kex2p. In summary, our results show that Kex2p cleaves both the HIV-1 envelope glycoprotein precursor and a synthetic peptide mimicking the cleavage site of HIV-1 gp160 at the dibasic site, suggesting functional analogy between yeast Kex2p and the cellular protease responsible for the maturation of HIV-1 envelope glycoproteins in infected human cells.

The role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells.

Proteolytic activation of the precursor envelope glycoproteins gp160 of human immunodeficiency virus type 1 (HIV-1) and gp140 of HIV-2, a prerequisite for viral infection, results in the formation of gp120/gp41 and gp125/gp36, respectively. Cleavage is mediated by cellular proteases. Furin, a member of the eukaryotic subtilisin family, has been shown to be an activating protease for HIV. Here, we compared the presence of furin and other mammalian subtilisins in lymphatic cells and tissues. Northern blot analyses revealed that furin and the recently discovered protease LPC/PC7 were the only subtilisin-like enzymes transcribed in such cells. Furin was identified as an enzymatically active endoprotease present in different lymphocytic, as well as monocytic, cell lines. When expressed from vaccinia virus vectors, the proprotein convertases were correctly processed, transported, and secreted into the media and enzymatically active. Coexpression of different subtilisins with the HIV envelope precursors revealed that furin and LPC/PC7 are able to cleave HIV-1 gp160. Moreover, both enzymes proteolytically processed the envelope precursor of HIV-2. gp140 was also cleaved to some extent by PC1, which is not, however, present in lymphatic cells. Furin- and LPC/PC7-catalyzed cleavage of HIV-1 gp160 resulted in biologically active envelope protein. In conclusion, among the known members of the subtilisin family, only furin and LPC/PC7 fulfill the requirements of a protease responsible for in vivo activation of HIV envelope glycoproteins.