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The association and dissociation of the homodimeric p66/p66 form of HIV-1 reverse transcriptase were investigated. The effects on the dimerization process of different salt concentrations, pH and the presence of a template/primer and nucleotide substrates were monitored by measuring polymerase activity and analytical size-exclusion HPLC. At submicromolar concentrations of enzyme and physiological salt concentrations, most of the enzyme exists in the inactive monomeric form. Increasing NaCl concentration from 0.05 to 1 m decreased the equilibrium dissociation constant from 2.0 to 0.34 µm. Analysis of the kinetics of the dimerization process indicated it followed a two-step mechanism, with rapid initial association of the two subunits to form an inactive homodimer followed by a slow isomerization step rendering the active enzyme form. The presence of poly(rA)/dT20 decreased the equilibrium dissociation constant of the homodimer about 30-fold, while the addition of 5 µm dTTP had no effect. The kinetics of the process showed that the template/primer favored dimerization by binding to the inactive homodimer and promoting its isomerization to the active form. These results were confirmed by analyzing the reverse reaction, i.e. the dissociation of the enzyme, by dilution in a low-ionic-strength buffer. The results suggest that binding of immature HIV-1 reverse transcriptase to its natural template/primer may be relevant in both the dimerization process and the selection of its natural primer.

A set of new pyrimido[5,4-b]indole derivatives that are structurally related to some non-nucleside HIV-1 reverse transcriptase inhibitors were synthesized and biologically evaluated for their activity as inhibitors of wild and mutant HIV-1 RT types in an 'in vitro' recombinant HIV-1 RT screening assay, as well as anti-infectives in HLT4lacZ-1IIIB cells. Preliminary structure-activity relationships suggest that activity is promoted by simultaneous substitution in positions 2 and 4, especially when chains of alkyldiamine type are present, and by electron-releasing substituents (methoxy) in positions 7 and 8. The inactivity or the very low activity of title derivatives does not suggest interest in AIDS therapy.

Glucocorticoids and cAMP regulate, either in a synergistic or additive fashion, the transcription of multiple genes, although some antagonistic effects of dexamethasone on cAMP-activated transcription have been described. The increased glucocorticoid receptor (GR) mediated response of some cell types, as a result of augmented cAMP, has been considered to be mainly due to an increased stability of GR mRNA, although other plausible explanations should not be ruled out. We studied the possibility that GR transcription itself could be affected by cAMP levels. HeLa cells were transfected with human GR (hGR) promoter constructs and their transcriptional activity determined after inducing a cAMP increase with forskolin. We found that forskolin almost doubled the transcriptional activity of the promoter construct spanning −2995 to +38 of the hGR, whereas no significant variations were observed with shorter chimeras containing sequences downstream −979. Shift mobility showed binding of CREB in vitro to a putative cAMP responsive element located at −1000, suggesting that hGR may be upregulated by cAMP at the transcriptional level, thus adding a new mechanism ascribable to this second messenger, which in conjunction with the cAMP-induced GR mRNA increased stability, would lead to a more precise control of the amount of GR protein within the cell.

Activated and differentiated human monocytes with a CD14+CD16+ phenotype were found to contain a DNase activity associated with secretion granules. Activated cells were obtained from patients with autoimmune diseases. Activation and differentiation of monocytes were also achieved after incubation of PBMC from healthy subjects with protein A (SpA) or immunopotentiating peptides. DNase activity corresponded to a 66-kDa protein, similar to that described in granules from T lymphocytes, active preferentially on double-strand DNA. DNA fragmentation activity increased when NO donors were present; the activity was higher in the presence of Ca2+, and at low pH values. The Ca2+-dependent activity was inhibited by Zn2+. NO-dependent activity was additive with that of Ca2+-dependent and it was not inhibited by Zn2+. Dithiothreitol did not modify the effect of NO on DNase activity. Incubation of PBMC in the presence of NMLA, an inhibitor of NO synthases, decreased this DNase activity. Data reported clearly suggest a regulatory role of NO in granule-associated DNase activity.

We have found a common structural motif in human autoantigens, heat shock proteins and viral proteins. Peptides modelled after sequences present in those molecules were synthesized and immunomodulating properties tested. They share a core of 15 amino acid residues and a common pattern ('2-6-11' motif) characterized by requirements at fixed positions with respect to a Pro (position 6); an apolar residue or a Lys at position 2; and a Glu, Asp or Lys at position 11. Any of these peptides, when added to cultures of lymphomononuclear cells, caused the activation of monocytes manifested by a release of IL-1 alpha, IL-1 beta and TNF alpha. A release of INF gamma and IL-2 took also place; this release was abolished by anti-DR antibodies. Neither IL-4 nor IL-5 could be detected. This suggests a presentation by APCs and the appearance of cells with a Th1 phenotype. Monocytes and Th1 cells freshly obtained from 12 patients of Graves' disease, 8 of Hashimoto's disease and 8 of primary biliary cirrhosis exhibited activation features similar to those found in cells from healthy subjects incubated in the presence of peptides with a "2-6-11' motif and representing fragments of autoantigens. Their immunopotentiating properties suggest their involvement in the initiation or progression of the autoimmune response mediated by activated monocytes and Th1 cells.

The presence of inducible nitric oxide synthase (iNOS) in fresh monocytes from patients with Graves' disease was demonstrated for the first time. Immunophenotypic analysis showed a profile reflecting a state of activation and differentiation of monocytes. Incubation of lymphomononuclear cells from healthy volunteers in the presence of synthetic peptides with sequences related to thyroid autoantigens (TSH receptor, thyroid peroxidase, or thyroglobulin) led to a stimulation of monocytes manifested by a change in phenotype and expression of iNOS. This expression did not take place on isolated monocytes, unless products associated with Th1 activity were present in the medium. Active peptides contained a characteristic "2-6-11" motif already described [López-Moratalla et al. (1995) Biochim. Biophys. Acta 1265, 181-188]. These results are suggestive of a new role for autoantigens in the pathogenesis of Graves' disease: that of inducing the expression of iNOS and activating the monocyte possibly underlying the autoimmune response.

Summary The synthesis and the study of the activity of new indol-2-carboxamides and pyridazino[4,5- b ]indoles as inhibitors of HIV-1 reverse transcriptase (RT) are presented. The activity of the compounds synthesized as inhibitors of different types of HIV-1 RT (wild type enzyme and mutant forms P236L, Y 181C and P236L/Y181C) was evaluated. The activity of the most active compounds was investigated in the syncytia reduction in vitro assay, in HIV-1 IIIB -infected HT4lacZ-1 cells. Their potential cytotoxicity was determined in parallel. Two lead compounds, N -[1-[2-(3-isopropylamino)pyridyl]piperazin]-5,6-methylenedioxy indol-2-carboxamide 7q and N -[1-[2-(3-ethylamino)pyridyl]piperazin]-5,6-methylenedioxyindol-2-carboxamide 7s have been identified.

It has been reported previously that a short synthetic immunomodulating peptide (Pa) and the neuropeptide beta-endorphin modulate the immune system. We have found now that NF-kappaB participates in the stimulation of monocytes by both peptides and we investigated the molecular mechanism by which these stimuli activate NF-kappaB. Pa and beta-endorphin induce accumulation of cyclic 3('),5(')-adenosine monophosphate (cAMP) in a calcium/calmodulin-dependent fashion since it was completely inhibited by the calmodulin antagonist W-7. The effect of these complexes seems to be mediated, at least in part, by nitric oxide (NO) synthesized by constitutive NO synthase since the NO synthase inhibitor N-methyl-L-arginine (NMLA) reduced the elevation of cAMP. Furthermore, the NO donor SIN-1 provoked nitration of G(S)alpha, leading to the cAMP elevation that was suppressed by the G(S)alpha-selective antagonist NF-449. Interestingly, the rapid degradation of NF-kappaB inhibitor IkappaBalpha induced by Pa- and beta-endorphin was reversed by a pretreatment with H-89 and cyclosporin A, inhibitors of protein kinase A (PKA) and protein phosphatase 2B (PP2B), respectively. These observations are consistent with the inhibition caused by W-7, NMLA, H-89, and cyclosporin A on NF-kappaB induction by these agonists, indicating the involvement of PKA and PP2B in the regulation of NF-kappaB in human monocytes.

A short immunomodulating peptide (Pa) containing a defined structural motif present in a number of extracellular matrix proteins and autoantigens was found to stimulate human monocytes. Pa-induced apoptosis of isolated monocytes, as indicated by internucleosomal DNA cleavage, increased annexin V binding capacity and cleavage of caspase substrates, such as poly(ADP)ribosylpolymerase. In addition, Bcl-2 protein levels were downregulated during Pa-induced cell death. Nuclear extracts of monocytes incubated with Pa showed higher neutral, Ca(2+)-dependent DNase activity than those obtained from nontreated monocytes. Caspase inhibitors prevented Pa-induced apoptosis, Bcl-2 depletion, and DNase activation. Treatment of monocytes with Pa activated c-Jun N-terminal kinases and p38 kinase, in an acidic sphingomyelinase- and caspase-dependent fashion. Pa-induced apoptosis was blocked by selective inhibitors of p38 kinase (SB203580) and acidic sphingomyelinase (SR33557). These results indicate that JNK and p38 kinase stimulation as well as monocyte apoptosis induced by Pa could depend, at least in part, on early activation of acidic sphingomyelinase.

Human monocyte subsets, isolated from cultures of mononuclear cells, or freshly obtained from patients with multiple sclerosis, Graves' disease or pemphigus vulgaris, differed in phenotype, apoptotic features, mRNA levels of arginase II (A-II) and the inducible form of nitric oxide synthase (iNOS). Liver-type arginase I mRNA was present in all subsets. Apoptosis was followed by the expression of T cell intracellular antigen (TIA) and the simultaneous detection of DNA stainability by propidium iodine and annexin V binding. Apoptosis was practically absent both in activated CD14(++)CD33(++)DR(++)CD25(++)CD69(++)CD71(++/+) CD16(-) cells, expressing A-II mRNA and having arginase activity, but not iNOS mRNA, and in not fully mature large CD14(++)CD16(+)CD23(+)DR(++) monocytes, expressing simultaneously both mRNAs and having both enzyme activities. However, differentiated small CD14(+/++)CD16(+)CD69(+)CD25(+/-)CD71(++)CD23(+) DR(++) monocytes, expressing high levels of iNOS mRNA, exhibited apoptotic signs. Amounts of NO synthesised by monocytes co-expressing iNOS and arginase changed with the addition of arginine or an iNOS inhibitor; in that case a correlation of NO production and apoptotic features was observed. Data suggest a regulatory role for endogenous NO in apoptosis of stimulated and differentiated monocytes, and also that iNOS and A-II, when simultaneously present, could control the production of NO as a consequence of their competition for arginine.

Lipophilic and hydrophilic extracts of four Argentine plants (Gamochaeta simplicaulis Cabr. 1, Achyrocline flaccida Wein. D. C. 2, Eupatorium buniifolium H. et A. 3, and Phyllanthus sellowianus Muell. Arg. 4) were examined in vitro for their ability to inhibit the polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (wild and Y181C mutant types). The active extracts were also examined as inhibitors of viral replication in HLT4LacZ-1 IIIB cell cultures, evaluating their cytotoxicity in parallel. Infusions 21 and 41, among the crude extracts, showed the highest activity. These extracts were refractioned into four fractions; 2I 4 and 4I 4 were active as inhibitors of DNA-polymerase (wild and Y181C types) and RNase H activities. These fractions were potent as inhibitors of viral replication and were not cytotoxic. Refractionation of 214 yielded five new fractions, two of which, 2I 4 -4 and 2I 4 -5, showed notable activity. Refractionation of 4I 4 yielded four new fractions; of these, 414-3 and 414-4 were active. The marked biological activity found in the infusion of A. flaccida and P. sellowianus makes them sufficiently attractive to be considered in the combined chemotherapy of the disease. Copyright © 1999 John Wiley & Sons, Ltd.

Evidence suggesting a relationship between neuroendocrine and immune systems is steadily growing. We demonstrate now that inducible nitric oxide synthase (iNOS) is expressed in human peripheral blood monocytes after incubation of lymphomononuclear cells in the presence of beta-endorphin, a neuropeptide released by the pituitary in response to mental or physical stress or by activated lymphocytes. beta-endorphin raised cAMP level in monocytes. The possible relationship between cAMP and iNOS expression on monocytes was investigated. Immunostaining for iNOS decreased, when besides beta-endorphin an inhibitor of protein kinase A (H-89) was added to the medium at the beginning of the incubation. The cAMP level raised by beta-endorphin was lowered by naloxone, which also reduced slightly iNOS expression. These results clearly point to the monocyte as a link between neuroendocrine and immune systems, an observation of potential relevance in our understanding of how stress and autoimmunity could be interconnected.

Immunophenotypic analysis of peripheral blood leukocytes from patients with multiple sclerosis (MS) showed a profile reflecting a state of activation and differentiation of monocytes. A subset of CD16+ monocytes with high HLA-DR expression was more prominent in patients with MS than in healthy subjects. The presence of the inducible form of nitric oxide synthase (iNOS) in these differentiated and activated monocytes freshly obtained from patients with MS was demonstrated by immunocytochemistry and flow cytometry analysis with two different antibodies. Incubation of lymphomononuclear cells from healthy volunteers in the presence of an immunomodulating peptide (NVLGAPKKLNESQAV) led to stimulation and maturation of monocytes manifested by changes in phenotype and an increase in both iNOS mRNA and protein, as well as HLA-DR expression. In this case also iNOS was expressed mainly on subsets of CD16+ monocytes with high HLA-DR expression. NO produced by human monocytes seems to have a function in the upregulation of membrane HLA-DR. These results are suggestive of a role for monocytic iNOS in the autoimmune response underlying the pathogenesis of multiple sclerosis.

This paper presents the synthesis of new indole, pyridazino[4,5-b]-indole, and pyridazino[4,5-a]indole analogs as well as a study of their "in vitro" activity as inhibitors of different phosphodiesterases isolated from dog cardiac tissue, dog aorta, and bovine platelets; the study of their activity as inhibitors of platelet aggregation in guinea pig whole blood, with ADP and arachidonic acid (AA) as pro-aggregants, is also included. The selected compounds 8-benzyloxy-3,4-dihydro-1-(3,4,5-trimethoxy)benzylideneaminopyridazin o[4,5- b]indole 14g, and 8-benzyloxy-4-[(3,4-dimethyl)pyrazolyl]pyridazino[4,5-b]indo le 20 present an interesting profile as potential inodilators, with a complementary, beneficial activity as inhibitors of the aggregation, activities which could possibly be related to the inhibition of the PDE's. Among the other compounds studied, 8-benzyloxy-3,4-dihydro-1-[4-(methyl)piperazino]acetamidopyrida zino[4,5- b]indol-4-one 16c and 8-benzyloxy-3,4-dihydro-1-[4-(2- methoxyphenyl)piperazino]acetamidopyridazino[4,5-b]indol-4-o ne 16f stood out as inhibitors of platelet aggregation, with a mechanism that could possibly be related to the AA cascade.

Synthetic peptides with sequences present in extracellular matrix proteins are capable of causing the expression of the inducible form of nitric oxide synthase (iNOS), detected by immunocytochemistry, and the release of NO by human lymphomononuclear cells incubated in their presence. Active peptides are 15-mers containing a characteristic 2-6-11 motif in which the amino acid residue at position 2 is Leu, Ile, Val, Gly, Ala or Lys; the residue at position 6 is always Pro; and residue 11 is Glu or Asp. The induction of iNOS in human monocytes and macrophages could be involved in the cytotoxicity against tumor cell lines also elicited by these peptides.

Some fused pyridazino[4,5- b ]indoles (7) were synthesized. These new compounds present a planar topography and some resemblance to carbazeram, imadozan, and other pyridazlno agents with cardiotonic activity. These compounds also possess a complementary effect as inhibitors of platelet aggregation. 6-(3,5-Dimethylpyrazolyl)- 1,2,4-triazolo[4,3- b ]pyridazino[4,5- b ]indole (7a) has a good profile as an inodilatador with antiaggregate activity due to the inhibition of phosphodiesterase.

1. 1. Pyridoxal-5′-phosphate (PLP), a marker of primary amines, bound covalently to S-1 in an approximate ratio of 1:1. PLP was localized within the tryptic 25,000 mol. wt fragment. 2. 2. Fluorescein isothiocyanate (FITC), which is known to bind covalently to primary amino groups located at nucleotide binding sites, readily bound to S-1 in a 2:1 ratio. FITC was localized within the 20,000 and 50,000 mol. wt tryptic peptides, in a ratio of 1:1 in each one. 3. 3. These results are consistent with the existence of nucleotide binding sites on myosin different from those of the catalytic sites.

Purified F1-ATPase from Micrococcus lysodeikticus contains zinc in the amount of 1 mol/mol of enzyme. This zinc content correlates with standard values of ATPase activity (assayed with Ca2+-ATP as substrate) of the protein, i.e. 5--6 mumol substrate hydrolysed . min-1 . mg-1. Prolonged dialysis against EDTA results in a zinc-free protein which concomitantly loses its ATPase activity. Chelators such as Zincon, EDTA and L-cysteine inhibit the ATPase activity in concentration and/or time dependence related to their affinity for the metal ion involved. Reconstitution of the metallo (Zn2+) protein is demonstrated by the incorporation to the zinc-free protein of 65Zn2+ in amount near the 1 mol/mol of enzyme. This incorporation was concomitant with the regain of ATPase activity. The inhibition by EDTA and Zincon is reversed specifically by Zn2+ while the inhibition by EDTA is prevented by Zn2+ and Mn2+ and to, a minor extent, by Cd2+. Zn2+ and Ca2+ ions are involved and are probably mandatory in the ATPase activity of M. lysodeikticus F1 but their roles appear to be different and not exchangeable. Other divalent metal ions inhibit the Ca2+-ATPase activity of the Zn2+ protein by the following decreasing order; Hg2+, Fe2+, Co2+, Cd2+, Mn2+, Mg2+. M. lysodeikticus F1-ATPase is thus identified as a metallo (zinc) protein, which requires additional divalent metal ions for ATP hydrolysis.

Redox titration of F1-ATPase from rat liver mitochondria referred to the modification of the hydrolytic activity on Mg-ATP has resulted in a three-step pattern, with three distinct jumps of activity separated by clear plateaus. The measured potentials ranged from −400 mV to +400 mV and were obtained by the addition of dithionite and ferricyanide. Electron exchange was facilitated with a mixture of different redox mediators. At pH 7.4 the midpoint potentials were +210 mV, +40 mV and −230 mV. These three midpoint potentials were displaced towards more negative values by 2,4-dinitrophenol or by an increase of the pH of the medium. The titration curves were described by n=2 Nernst equations.

The hydrolytic activity of rat liver F 1 -ATPase was stimulated up to 60% by FAD concentrations as low as 10-10 M. Stimulations as high as 100% were obtained with similar concentrations of CoQ.

1. 1. The inhibition of F 1 -ATPase by its natural peptide inhibitor is mixed non-competitive with two pH optimum values (5.5 and 8.2). 2. 2. A two-step model for the interaction is suggested in which two enzyme conformations would exhibit different affinities for the peptide. 3. 3. At low pH, interaction would be favoured. At high pH, a conformation (not susceptible to inhibition) changes into another (susceptible to inhibition) through the hydrolytic reaction stimulation, due to hiah pH.

1. 1. ATPase natural inhibitor interacted in a mixed non-competitive manner with compounds affecting hydrolytic activity. 2. 2. k a 's for DNP, HCO 3 − and free ATP, and K 1 's for SCN − and ADP became smaller as inhibitor peptide concentration increased, reflecting an increase in affinity of f 1 -ATPase for these compounds induced by the peptide. 3. 3. Activators increased the peptide inhibitory effect, whereas inhibitors decreased it. 4. 4. A two-step model for the peptide-enzyme interaction is suggested in which ATP hydrolysis is a key factor.

The effects of citrate, free ATP, and bicarbonate on the activity of isolated rat liver mitochondrial ATPase have been studied. Citrate behaved as a weak noncompetitive inhibitor; free ATP at low concentrations was found to be an activator, whereas at high concentrations behaved as an inhibitor. Citrate competed with free ATP. Neither citrate nor free ATP competed with bicarbonate. The results obtained suggest the existence of at least two catalytic sites with ATP hydrolyzing activity and at least three regulatory sites: one for bicarbonate, and two for free ATP, one with low and another with high affinity. Citrate would compete with free ATP for the same sites. The interaction of free ATP with the high affinity site activated the enzyme, whereas its interaction with the low affinity site would lead to an inhibition.

A series of peptides of 15 amino acids with sequences contained in human extracellular matrix (ECM) proteins (fibronectin, laminin A, laminin B1, tenascin, undulin, alpha 1-chain of type IV and VIII collagen and alpha 2-chain of type VIII collagen) have been synthesized. The selected structures conformed to the following pattern: (i) Pro at position 6, (ii) Leu, Lys, Ile, Val, Ala or Gly at position 2, (iii) Glu or Asp at position 11. Fibronectin and the indicated peptides, when present in cultures of lymphomononuclear cells from healthy donors, promoted stimulation of monocytes manifested by a release of IL-1 alpha, IL-beta, IL-6 and TNF alpha; an increase in the percentage of cells expressing CD14, CD16, CD11b and CD14/CD16; an increase in cytotoxicity against HT-29. Cytotoxicity against K562 and Daudi cells (targets of NK and LAK cells) was also observed together with an increase in the percentage of cells expressing CD56, CD56/CD16 (corresponding to NK cells), and CD56/CD8 (corresponding to NK-like lymphocytes), indicating a stimulation of lymphocytes. Activated monocytes and lymphocytes contained a large number of granules with DNAse activity. These results suggest that at least some of the immunological properties of ECM proteins could be accounted for by motifs fulfilling a characteristic sequence pattern shared by all of them.

Activation of human T helper 1 cells took place when lymphomononuclear cells from healthy donors were incubated in the presence of short synthetic peptides encompassing sequences present in extracellular matrix proteins. Active peptides conformed to a common structural pattern ("2-6-11 motif") [N.Lopez-Moratalla et al., Biochem. Biophys. Acta (1994) 1221, 153-158] conferring immnunomodulating properties. The release of IL-2 and IFNγ, as well as LAK and NK-dependent cytotoxicity induced by these peptides, could be blocked by anti-HLA-DR antibody. Activated CD4 + cells isolated from the mixed incubated population contained secretion granules with DNAase activity. These results suggest that these immunomodulating peptides presented by HLA-II play a key role in the differentiation of CD4 + T cells towards a Th1 functional phenotype.