Inhibition of HIV-1 replication by a nonnucleoside reverse transcriptase inhibitor.

A series of dipyridodiazepinones have been shown to be potent inhibitors of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT). One compound, BI-RG-587, had a Ki of 200 nanomolar for inhibition of HIV-1 RT that was noncompetitive with respect to deoxyguanosine triphosphate. BI-RG-587 was specific for HIV-1 RT, having no effect on feline and simian RT or any mammalian DNA polymerases. BI-RG-587 inhibited HIV-1 replication in vitro as demonstrated by in situ hybridization, inhibition of protein p24 production, and the lack of syncytia formation in cultured human T cell lines and freshly isolated human peripheral blood lymphocytes. Cytotoxicity studies of BI-RG-587 on human cells showed a high therapeutic index (greater than 8000) in culture.

Harmonisation of ethics committees' practice in 10 European countries.

BACKGROUND: The Directive 2001/20/EC was an important first step towards consistency in the requirements and processes for clinical trials across Europe. However, by applying the same rules to all types of drug trials and transposing the Directive's principles into pre-existing national legislations, the Directive somewhat failed to meet its facilitation and harmonization targets. In the field of ethics, the Directive 2001/20/EC conditioned the way of understanding and transposing the "single opinion" process in each country. This led to a situation in which two models of research ethics committees organisation systems exist, being the model in which the "single opinion" is considered to be the decision made by a single ethics committee more effective and simpler in terms of administrative and logistic workload. METHOD: A survey was conducted in 10 European countries. Members of the European Clinical Research Infrastructures Network working party number 1, with expertise in the field of ethics, responded. RESULTS: There is a major heterogeneity in the composition of ethics committees among the surveyed countries based on the number of members, proportion of experts versus lay members and expertise of the scientific members. A harmonized education of the ethics committees' membership based in common curricula is recommended by the majority of countries. CONCLUSIONS: Despite the efforts for harmonization of the European Clinical Trial Directive, from an ethical point of view, there remains a plurality of ethics committees' systems in Europe. It is important to comprehend the individual national systems to understand the problems they are facing.

The specificity of two proteinases that cleave adjacent to arginine, C1 esterase and acrosin, for peptide p-nitroanilide substrates.

Relative values of Vmax/Km for hydrolysis of 40 peptide p-nitroanilides catalyzed by human Cl-s and human acrosin are reported. For Cl-s, Ac-Lys(gamma Cbz)-Gly-Arg is the optimum sequence, but 25% of the substrates have (Vmax/Km)rel greater than 0.25 compared to this sequence. The best acrosin substrate tested has the sequence Tos-Gly-Pro-Arg, although (Vmax/Km)rel greater than 0.15 for more than half of the substrates. Proline at P2 is preferred by acrosin. Both enzymes prefer arginine at P1 greater than or equal to 3-fold over lysine and will not accept citrulline. In addition, occupancy of site S3 may yield an increase in Vmax/Km of greater than or equal to 10-fold with either enzyme, but many residues are accepted at S2, S3 and S4. Thus, an acrosin assay using Tos-Gly-Pro-Arg p-nitroanilide as a substrate is more than 20-times as sensitive as existing assays with blocked arginine derivatives.

Inhibition of human leukocyte elastase by N-substituted peptides containing alpha,alpha-difluorostatone residues at P1.

A series of tripeptides which contain alpha,alpha-difluorostatone residues at P1-P1' and span the S3-S1' subsites have been shown to be potent inhibitors of human leukocyte elastase (HLE). The tripeptides described contain the nonproteinogenic achiral residue N-(2,3-dihydro-1H-inden-2-yl)glycine at the P2-position. This redidue has previously been shown in the case of HLE to be a good bioisosteric replacement for L-proline. Of the peptides prepared, those which contain the alpha,alpha-difluoromethylene keton derivative of L-valine (difluorostatone) are the preferred residue at the P1-primary specificity position. Substitution at P1 by the corresponding alpha,alpha-difluoromethylene ketones of L-leucine and L-phenylalanine gives inactive compounds. Of the tripeptides described the most potent in vitro compound is ethyl N-[N-CBZ-L-valyl-N-(2,3-dihydro-1H-inden-2-yl)glycyl]- 4(S)-amino-2,2-difluoro-3-oxo-5-methylhexanoate (17B) (IC50 = 0.635 microM). It is presumed that the inhibitor 17b interacts with the S3-S1' binding regions of HLE. Additionally extended binding inhibitors were prepared which interact with the S3-S3' binding subsites of HLE. In order to effect interaction with the S1'-S3' subsites of HLE, the leaving group side of cleaved peptides, spacers based upon Gly-Gly, and those linked via the N epsilon of L-lysine were utilized. One of the most potent extended compounds (P3-P3') in vitro is methyl N6-[4(S)-[[N-[N-CBZ-L-valyl-N- (2,3-dihydro-1H-inden-2-yl)glycyl]amino]-2,2-difluoro-3-oxo-5- methylhexanoyl]-2(S)-(acetylamino)-6-aminohexanoate (24b) (IC50 = 0.057 microM). The described in vitro active inhibitors were also evaluated in hamsters in an elastase-induced pulmonary hemorrhage (EPH) model. In this model, intratracheal (it.) administration of 22c, 5 min prior to HLE challenge (10 micrograms, it.) effectively inhibited hemorrhage (94.6%) in a dose-dependent manner. The described alpha,alpha-difluoromethylene ketone inhibitors are assumed to act as transition-state analogs. The inhibition process presumably acts via hemiketal formation with the active site Ser195 of HLE, and is facilitated by the strongly electron withdrawing effect of the alpha,alpha-difluoromethylene functionality.

Substituted naphthalenones as a new structural class of HIV-1 reverse transcriptase inhibitors.

A novel substituted naphthalenone (TGG-II-23A) has been found that inhibits HIV-1 infection of CEM-SS cells at concentrations that are not cytotoxic. Time of addition experiments indicate that TGG-II-23A functions at a stage of the HIV-1 life cycle at or near reverse transcription. Cell free assays confirmed that TGG-II-23A inhibits HIV-1 reverse transcriptase. Similar to other non-nucleoside inhibitors, TGG-II-23A was specific for HIV-1 and failed to inhibit the replication of HIV-2. The binding site of TGG-II-23A appears to be in close proximity to that of the TIBO-like inhibitors, since a TIBO-resistant HIV-1 was also resistant to TGG-II-23A treatment. TGG-II-23A is a mixed non-competitive inhibitor that exhibits the same template:primer selectivity as other non-nucleoside inhibitors. TGG-II-23A therefore represents a new structural entry into the TIBO/Nevirapine class of inhibitors of HIV-1 reverse transcriptase.

Internal supply of coenzyme to an amperometric glucose biosensor based on a chemically modified electrode.

A biosensor for glucose using glucose dehydrogenase immobilized on a chemically modified graphite electrode was supplied with coenzyme, nicotinamide adenine dinucleotide (NAD+), through pores in the material. A graphite rod was hollowed out, leaving 0.3 mm at the end contacting the solution, filled with 10 mM NAD+ and pressurized. The response factor was 40% of that obtained when 2 mM NAD+ was mixed with the sample solution in a flow system. The coenzyme consumption was 11 microliters h-1 representing a 500-fold saving compared to supply through the bulk solution. The biosensor had a linear calibration curve from the detection limit, 1 microM, to 2 mM glucose and a repeatability of 0.3%. The graphite electrode was modified by adsorption of a bis-(benzophenoxazinyl)-terephthaloyl derivative in order to be able to oxidize NADH at 0 mV versus Ag/AgCl, 0.1 M KCl.

Inhibition of 5-lipoxygenase by substituted 3,4-dihydro-2H-1,4-thiazines.

A series of substituted 3,4-dihydro-2H-1,4-thiazines inhibit 5-lipoxygenase from rat leukocytes and exhibit submicromolar IC50 values. A novel synthesis of these compounds was developed based on the formation of hydroxymethyleneamine 13 and its cyclization to the title compounds. The dihydrothiazines have low oxidation potentials, typically E1/2 is near 0.3 V, and a representative compound reduces Fe(III)(phen)3, with k = 10(5) M-1s-1. We propose that these lipophilic compounds bind to 5-lipoxygenase and reduce the iron in the active site, thus inactivating the enzyme.

Rapid, sensitive and efficient HPLC assays for HIV-1 proteinase.

The proteinase encoded by human immunodeficiency virus type 1 (HIV-1) cleaves peptide substrates of sequences derived from processing sites in HIV-1 gag-pol polypeptide. Based on this cleavage, assays that utilize HPLC to measure activity of HIV-1 proteinase are reported herein. In the assay first described, a baseline separation of unlabeled substrate and products is achieved with a run time of 10 min and UV detection. Enzyme concentrations as low as 1 nM, which is the lowest reported for an assay employing underivatized peptide substrate, are attained. Even more powerful, versatile and sensitive, a second method that takes advantage of a peptide substrate labeled at its N-terminus with the fluorescein derivative is described as well. Because of the fluorescein label, this method offers several superior features, including very fast analysis of substrate and product in less than 3 min and fluorescence detection which provides essentially total freedom from interference. Synthesis of fluorescein-labeled peptide substrate is accomplished by solid-phase peptide synthesis.

Rhinovirus 3C protease catalyzes efficient cleavage of a fluorescein-labeled peptide affording a rapid and robust assay.

The 3C protease encoded by human rhinovirus type 2 catalyzes with equal efficiency cleavage of a peptide substrate with or without a fluorescein label attached to the amino acid at the P7' position. Substrates Ac-MEALFQGPLQYKDL-NH2 and MEALFQGPLQYKE(fluorescein)L are hydrolyzed with values of Vmax/KM of 970 M-1 s-1 and 1100 M-1 s-1, respectively. With the labeled substrate, HPLC achieves separation of substrate and product in 2.5 min. Separation in as little as 12 s is feasible. Fluorescein was derivatized so that it could be incorporated into peptides using automated solid-phase peptide synthesis.

Mechanism and activation for allosteric adenosine 5'-monophosphate nucleosidase. Kinetic alpha-deuterium isotope effects for the enzyme-catalyzed hydrolysis of adenosine 5'-monophosphate and nicotinamide mononucleotide.

The kinetic alpha-deuterium isotope effect on Vmax/Km for hydrolysis of NMN catalyzed by AMP nucleosidase at saturating concentrations of the allosteric activator MgATP2- is kH/kD = 1.155 +/- 0.012. This value is close to that reported previously for the nonenzymatic hydrolysis of nucleosides of related structure, suggesting that the full intrinsic isotope effect for enzymatic NMN hydrolysis is expressed under these conditions; that is, bond-changing reactions are largely or completely rate-determining and the transition state has marked oxocarbonium ion character. The kinetic alpha-deuterium isotope effect for this reaction is unchanged when deuterium oxide replaces water as solvent, corroborating this conclusion. Furthermore, this isotope effect is independent of pH over the range 6.95-9.25, for which values of Vmax/Km change by a factor of 90, suggesting that the isotope-sensitive and pH-sensitive steps for AMP-nucleosidase-catalyzed NMN hydrolysis are the same. Values of kH/kD for AMP nucleosidase-catalyzed hydrolysis of NMN decrease with decreasing saturation of enzyme with MgATP2- and reach unity when the enzyme is less than half-saturated with this activator. This requires that the rate-determining step changes from cleavage of the covalent C-N bond to one which is isotope-independent. In contrast to the case for NMN hydrolysis, AMP nucleosidase-catalyzed hydrolysis of AMP at saturating concentrations of MgATP2- shows a kinetic alpha-deuterium isotope effect of unity. Thus, covalent bond-changing reactions are largely or completely rate-determining for hydrolysis of a poor substrate, NMN, but make little or no contribution to rate-determining step for hydrolysis of a good substrate, AMP, by maximally activated enzyme. This behavior has several precedents.

Measurement and differentiation of the cellular infiltrate in experimental toxic contact dermatitis.

Toxic (irritant) contact dermatitis was elicited by epicutaneous application of dinitrochlorobenzene (DNCB) and croton oil in unsensitized guinea pigs. The course of the skin reaction was studied with the naked eye, low-power microscopy, and a method based on counting of infiltrating cells in the upper corium. A weakly toxic dose of DNCB (100 microgram/cm2) gave marked erythema and moderate swelling, more pronounced 6 h after application of the DNCB than at 24 h or 48 h. A significant increase in mononuclear cells in particular but also in neutrophil and eosinophil granulocytes was noted in the upper corium. A more strongly toxic dose (500 microgram/cm2) gave a similar visible reaction, this too most marked at 6 h, but here there was an increase in neutrophil granulocytes in particular. Over and above predominant mononuclear-cell infiltration, croton oil (10 microgram/cm2) caused a slight increase in basophil cells. The reaction thus resembled a contact allergic reaction. In the light of earlier findings in allergic contact reactions the results suggest that weak stimuli, toxic or allergenic, elicit a non-specific skin response. With stronger stimulation the histological picture is modified and the cellular response acquires a pattern characteristic of toxic or allergic contact dermatitis.

Ambulation in patients with myelomeningocele: a multivariate statistical analysis.

Factors determining ambulation in 163 patients with myelomeningocele were studied by a multivariate statistical method. Neurological dysfunction unrelated to the plaque was analyzed by magnetic resonance imaging. There were no ambulators at the thoracic or L1-L2 level. At the L3 level, 54% ambulated, and at the L4 level, 67% ambulated. Eighty percent were ambulators at L5 and all at the sacral level. Below L1-L2, one-half of the nonambulators had neurological deficiencies caused by syringohydromyelia or Chiari malformations preventing ambulation. Severe scoliosis was closely, age moderately, and hip flexion contracture slightly related to the inability of the other nonambulators to walk, while pelvic obliquity, hip dislocation, or knee flexion contracture was not.

The cellular infiltrate in experimental allergic and toxic contact dermatitis. Effect of systemically administered corticosteroids.

The effect of systemically administered corticosteroids on allergic and toxic contact dermatitis in the guinea pig was studied. Dermatitis was provoked with dinitrochlorobenzene (DNCB). The influence of corticosteroids on the sensitization phase was also investigated. The epicutaneous test reactions were assessed with the naked eye, low-power microscopy, and a method based on the counting of infiltrating cells in the upper corium. Prednisolone injected in a dose of 5 mg/kg body weight daily for 4 days starting on the 1st day of sensitization or the 1st day of testing tended to shorten the duration of the contract allergic skin reaction as seen with the naked eye. Microscopically, a decrease in the number of infiltrating basophil granulocytes was found. Prednisolone had no convincing influence on the toxic contract eczematous reaction macroscopically or microscopically. Responses to weakly toxic doses of DNCB tended to fade earlier and to show small displacements of granulocytes in the differential count of infiltrating cells compared to controls. The results suggest that prednisolone given systemically influences only slightly the inflammatory cell infiltration in contact dermatitis in the guinea pig.

Measurement and differentiation of the cellular infiltrate in expermiental allergic contact dermatitis.

We describe a quantitative method for the grading of contact allergic reactions in guinea pigs. These reactions are characterized by marked cellular infiltration, and the method is based on total and differential counting of cells in the upper corium. A varying and objectively gradable increase in mononuclear and basophil polymorphonuclear cells was found. In naked-eye-positive reactions this increase was highly significant 24, 48, and 72 hours after epicutaneous application of dinitrochlorobenzene (DNCB). The degree of cellular infiltration reflects aspects of a cell-mediated immune response other than the visible reaction ordinarily made use of. The method can be used to study how systemically or topically administered drugs affect cellular features in contact allergy.

Effect of cyclophosphamide on the cellular infiltrate in experimental allergic contact dermatitis.

The influence of cyclophosphamide on contact allergy was studied by a method based on the count of cells infiltrating epicutaneous test sites in sensitized guinea pigs. Cyclophosphamide was injected 72 h before sensitization with dinitrochlorobenzene (DNCB). On testing 2 weeks later, marked mononuclear cell infiltration and dramatic increase in the count of basophil polymorphonuclear cells in the upper corium were found, compared to the control group. Cell infiltration in tests performed 1 and 3 weeks after sensitization differed from the reaction after 2 weeks. When cyclophosphamide was given 72 h before testing done 2 weeks after sensitization, both mononuclear and polymorphonuclear counts were found to be greatly reduced. This in vivo method of assessing the degree and composition of cellular infiltration in a contact allergic reaction after different periods of time throws new light on the influence of drugs on a cell-mediated immune response and also the variation in response with time.

5-lipoxygenase from rat PMN lysate.

The products of arachidonic acid metabolism in the 15,000xg supernatant of sonicated rat PMN are described. Only products derived from 5-lipoxygenase are observed. These products are 5-HETE and products derived from hydrolysis of LTA4, particularly LTB4. Some minor products derived from decomposition of 5-HPETE are also observed. The dependence of the activity of 5-lipoxygenase on enzyme and on substrate concentrations is presented and discussed in terms of a kinetic model that includes enzyme inactivation during turnover and substrate inhibition. The 5-lipoxygenase activity is stimulated by Ca++ and ATP.

Activity of soybean lipoxygenase in the absence of lipid hydroperoxide.

Soybean lipoxygenase was assayed under conditions such that the concentration of the enzyme was in excess of the concentration of the substrate, arachidonic acid. Under these conditions, the concentration of lipid hydroperoxides present as contaminants in the substrate was negligible relative to the enzyme concentration, and the concentration of lipid hydroperoxide product could be determined accurately. The ferric form of the enzyme was observed to be fully active and to catalyze the oxidation of arachidonic acid at a near-diffusion-controlled rate, 1.4 X 10(7) M-1 s-1 at 0 degree C, at concentrations of lipid hydroperoxides as low as 5% of the enzyme concentration. From this, it can be concluded that the higher oxidation states that would be accessible by oxidation of Fe(III) by hydroperoxide are not required for catalysis by soybean lipoxygenase. Surprisingly, the activation of the ferrous form of the enzyme was also observed at insignificantly low lipid hydroperoxide concentrations. This activation presumably involves oxidation of the ferrous to the ferric form of the enzyme and must be more facile than has hitherto been reported. This result may rationalize previous reports that the ferrous and the ferric forms of the enzyme are both active.

Microfluorometry using fluorescein diacetate reflects the integrity of the plasma membrane in UVA-irradiated cultured skin fibroblasts.

The damaging effect of long-wave ultraviolet radiation (UVA) on the plasma membranes of cultured human skin fibroblasts was evaluated by cytofluorometry performed after vital staining with fluorescein diacetate. The damage was associated with lipid peroxidation, as shown by accumulation of malondialdehyde and 4-hydroxyalkenals; such accumulation was induced by a UVA dose of only 8 J/cm2. Pretreatment with the effective membrane peroxidation inhibitor alpha-tocopherol (added in the form of alpha-tocopherol succinate) or the singlet oxygen quencher beta-carotene protected the cells from membrane damage. Further, depletion of intracellular glutathione by exposure of the cells to buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase aggravated the membrane-damaging effects. The results confirm the photodynamic effects of UVA, which presupposes the excitation of endogenous photosensitizer(s) and the production of reactive oxygen species. The present results indicate that this method of detection of alterations in plasma membrane stability may be highly suitable for studying various photobiological phenomena and for use as a model for testing substances that could protect skin from UVA damage. The trypan blue exclusion test proved to be too insensitive to detect these changes.

Ambulation in patients with myelomeningocele: a 12-year follow-up.

Factors determining change in ambulatory status were studied over a 12-year observation time in 60 ambulating patients with myelomeningocele. There were 26 female and 34 male subjects with a median age of 22 years (range, 12-54). We used the method of Lindseth to define the neurologic level of the lesion and classified walking ability according to the criteria of Hoffer. The prevalence of spasticity and spine and lower-limb deformities was assessed. Orthopedic and neurosurgical interventions and other medical events were registered, as well as occurrence of pressure sores, musculoskeletal pain, and use of orthoses. There were 19 patients with downward transitions in ambulatory level during the follow-up time. Factors explaining deterioration in these 19 patients included deterioration of the neurologic level of lesion, spasticity, knee and hip flexion contractures, low-back pain, lack of motivation, as well as those of major medical events like stroke, recurrent septicemia, lower limb edema, and invasive surgical interventions.