Clinical and electroencephalographic findings in acutely ill adults with non-convulsive vs convulsive status epilepticus.

BACKGROUND: Non-convulsive status epilepticus (NCSE) indicates a change in the mental state with no motor manifestations, being a clinical expression of prolonged epileptiform activity. In contrast to convulsive status epilepticus (CSE), no unified treatment recommendations have been proposed so far. We were interested to review the clinical and encephalographic characteristics in hospitalized patients with NCSE and CSE and compare their treatment and outcome. PATIENTS AND METHODS: The electroencephalographic recording records of adult patients with electrographic status epilepticus were retrieved. Patients' clinical records were then analyzed. RESULTS: Fifty-three patients with CSE and 25 patients with NCSE were identified. Background diseases, neuroimaging findings and complications were similar in CSE and NCSE. Anoxia was a more frequent etiological factor only for myoclonic SE. Patients with CSE presented more often with coma. The number of drugs used for treatment was similar, but anesthetics drugs were administered more frequently in patients with CSE. The 30-day mortality rate was higher in myoclonic SE and generalized tonic-clonic SE, but the outcome on discharge in terms of survival and recovery was comparable between CSE and NCSE. CONCLUSIONS: The results of the present study show that the clinical parameters of NCSE in acutely ill patients do not substantially differ from those of patients with CSE. Moreover, despite more severe mental changes and the need for more anesthetic drugs for treatment of CSE, the final outcome did not differ between both groups. This might indicate that NCSE in acutely ill patients should be regarded as seriously as CSE.

Substrate and inhibitor specificity of interleukin-1 beta-converting enzyme and related caspases.

Interleukin-1beta-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1beta (pre-IL-1beta) to the mature cytokine and a member of a family of related proteases (the caspases) that includes the Caenorhabditis elegans cell death gene product, CED-3. In addition to their sequence homology, these cysteine proteases display an unusual substrate specificity for peptidyl sequences with a P1 aspartate residue. We have examined the kinetics of processing pre-IL-1beta to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processes pre-IL-1beta, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADP-ribose) polymerase, a DNA repair enzyme that is cleaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 x 10(6) M-1 s-1 for CPP32 to 1.0 x 10(3) M-1 s-1 for TX. In addition, we report kinetic constants for several reversible inhibitors and irreversible inactivators, which have been used to implicate one or more caspases in the apoptotic proteolysis cascade. Ac-Asp-Glu-Val-Asp aldehyde (DEVD-CHO) is a potent inhibitor of CPP-32 with a Ki value of 0.5 nM, but is also potent as inhibitor of CMH-1 (Ki = 35 nM) and ICE (Ki = 15 nM). The x-ray crystal structure of DEVD-CHO complexed to ICE presented here reveals electrostatic interactions not present in the Ac-YVAD-CHO co-complex structure (Wilson, K. P., Black, J.-A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-275), accounting for the surprising potency of this inhibitor against ICE.

Expression and purification of human interleukin-1beta converting enzyme from Trichoplusia ni insect cells using a baculovirus expression system.

Employing a baculovirus expression system, human interleukin-1beta converting enzyme (ICE) has been expressed in Trichoplusia ni High-Five insect cells and purified. ICE was expressed with an N-terminal T7 epitope, thus allowing its purification using immobilized anti-T7 antibodies. The recombinant ICE was purified to >95% homogeneity, the one minor contaminant being the baculovirus anti-apoptotic protein (P35) which was copurified. The purified recombinant ICE was biologically active, cleaving both pIL-1beta and poly(ADP-ribose) polymerase substrates. The kinetic properties of the purified recombinant ICE compare favorably with native ICE purified from a THP-1 monocytic line.

Kinetic characterization of human immunodeficiency virus type-1 protease-resistant variants.

Passage of human immunodeficiency virus type-1 (HIV-1) in T-lymphocyte cell lines in the presence of increasing concentrations of the hydroxylethylamino sulfonamide inhibitor VX-478 or VB-11328 results in sequential accumulation of mutations in HIV-1 protease. We have characterized recombinant HIV-1 proteases that contain these mutations either individually (L10F, M46I, I47V, I50V) or in combination (the double mutant L10F/I50V and the triple mutant M46I/I47V/I50V). The catalytic properties and affinities for sulfonamide inhibitors and other classes of inhibitors were determined. For the I50V mutant, the efficiency (kcat/Km) of processing peptides designed to mimic cleavage junctions in the HIV-1 gag-pol polypeptide was decreased up to 25-fold. The triple mutant had a 2-fold higher processing efficiency than the I50V single mutant for peptide substrates with Phe/Pro and Tyr/Pro cleavage sites, suggesting that the M46I and I47V mutations are compensatory. The effects of mutation on processing efficiency were used in conjunction with the inhibition constant (Ki) to evaluate the advantage of the mutation for viral replication in the presence of drug. These analyses support the virological observation that the addition of M46I and I47V mutations on the I50V mutant background enables increased survival of the HIV-1 virus as it replicates in the presence of VX-478. Crystal structures and molecular models of the active site of the HIV-1 protease mutants suggest that changes in the active site can selectively affect the binding energy of inhibitors with little corresponding change in substrate binding.

Effect of alkaline phosphatase on thromboxane mimetic induced platelet activation.

Recently it has been reported that alkaline phosphatase selectively inhibited thromboxane mimetic induced platelet aggregation and secretion suggesting that the phosphorylation state on the platelet surface may be important for thromboxane induced platelet activation. We report here studies attempting to elucidate the mechanism of action of alkaline phosphatase. Washed human platelet aggregation induced by the thromboxane mimetic IBOP was completely abolished when incubated with alkaline phosphatase (1 unit/ml) for 5 min. The effect was inhibited by co-incubation with 5mM phosphate. Binding studies using [125I]BOP showed that neither the affinity of IBOP for the receptor (control: 9.2 +/- 2.1 nM, alkaline phosphatase: 7.9 +/- 1.8 nM) nor the Bmax (control: 1780 +/- 320 sites/plt. alkaline phosphatase: 1920 +/- 290 sites/plt) were effected by alkaline phosphatase treatment. GTPase activity was measured in platelet membranes treated with and without alkaline phosphatase as measured by IBOP induced hydrolysis of [gamma-32P]GTP. The EC50 values for IBOP induced GTPase were similar whereas the maximum amount of released Pi in the control membranes was more than two fold greater than in alkaline phosphatase treated membranes. These studies suggest that thromboxane induced platelet activation may be dependent upon the phosphorylation state of the thromboxane receptor and/or closely associated protein.

Impeded progression of Friend disease in mice by an inhibitor of retroviral proteases.

The protease of the human immunodeficiency virus type 1 (HIV-1) is essential for the processing of GAG and POL polyproteins and maturation of the virus particles. Using recombinant protease and a truncated GAG polyprotein as substrate, we developed a Western blot assay for the evaluation of inhibitors of the enzyme. Two statine-based inhibitors of the enzyme, KH161 and KH164, were effective in blocking the replication of HIV-1 in acutely infected human T4 lymphoid cells, with potency approaching that of zidovudine (ZDV) when tested in parallel. In chronically infected cells, the production of infectious virus was inhibited by KH161 and KH164, while ZDV was ineffective. Both KH161 and KH164 were also active as antivirals against the replication of murine leukemia virus (MLV) in cultured mouse cells. In an animal model of a murine retroviral disease, KH164 was shown to inhibit in a dose-dependent manner the progression of the disease induced by Friend virus complex (a mixture of Friend MLV and spleen focus-forming virus). The results suggest that the progression of the acquired immune deficiency syndrome (AIDS) may be impeded by inhibitors of HIV-1 protease.

Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors.

A critical step in the replicative cycle of the human immunodeficiency virus HIV-1 involves the proteolytic processing of the polyprotein products Prgag and Prgag-pol that are encoded by the gag and pol genes in the viral genome. Inhibitors of this processing step have the potential to be important therapeutic agents in the management of acquired immunodeficiency syndrome. Current assays for inhibitors of HIV-1 protease are slow, cumbersome, or susceptible to interference by test compounds. An approach to the generation of a rapid, sensitive assay for HIV-1 protease inhibitors that is devoid of interference problems is to use a capture system which allows for isolation of the products from the reaction mixture prior to signal quantitation. In this paper, we describe a novel method for the detection of HIV-1 protease inhibitors utilizing the concept of particle concentration fluorescence. Our approach involves the use of the HIV-1 protease peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val which has been modified to contain a biotin moiety on one side and a fluorescein reporter molecule on the other side of the scissile Tyr-Pro bond. This substrate is efficiently cleaved by the HIV-1 protease and the reaction can be readily quantitated. Known inhibitors of the protease were readily detected using this new assay. In addition, this approach is compatible with existing instrumentation in use for broad screening and is highly sensitive, accurate, and reproducible.

Purification of recombinant HIV-1 protease.

A method is described to purify recombinant HIV-1 protease from soluble extracts of Escherichia coli. The isolation involves QAE-Sepharose anion exchange chromatography, hexyl agarose hydrophobic interaction chromatography, MonoS cation exchange chromatography, and Superose 6 size exclusion chromatography. Approximately 100 micrograms of protease was obtained from 18 g E. coli paste. The protein was judged to be homogeneous due to the presence of a single band on a silver-stained SDS polyacrylamide gel.

Substitutions at the P2' site of gag p17-p24 affect cleavage efficiency by HIV-1 protease.

Heptapeptide substrates containing a single amino acid substitution at the p2' position of the gag p17-p24 junction (S-Q-N-Y-P-X-V where X = G, A, L, I, F, and W) were compared as substrates for HIV-1 protease. Binding of the Ile-, Leu-, and Ala- containing peptides was about equal although hydrolysis was 20-fold lower for the Ala and Leu peptides compared to Ile. Insertion of Gly or Phe at the p2' position resulted in significantly lower cleavage of the peptide while a Trp-containing peptide was not cleaved. These data suggest that a relatively small hydrophobic amino acid is important for hydrolysis and binding at this site. Structure-activity studies such as those described here may be useful in the design of specific inhibitors for HIV-1 protease.