Parkinson's disease in the limelight.

The 2014 Lasker-DeBakey Clinical Medical Research Award -one of three prestigious awards granted by the Lasker Foundation in recognition of scientists, clinicians and public servants who have made major advances in the understanding, diagnosis, treatment, cure or prevention of human disease- has been granted to two pioneers in the field of Parkinson's disease therapy. In spite of the availability of more than two dozen drugs and fixed-dose combination products to treat the symptoms of Parkinson's disease -most notably the gold standard levodopa, a dopamine precursor- as well as nonpharmacological treatments like deep brain stimulation, many patients do not respond to available drugs or experience breakthrough symptoms, and the disease is ultimately uncurable. This article reviews currently available therapies as well as biomarkers and novel diagnostics.

Efficacy of anti-scorpion venom serum over prazosin in the management of severe scorpion envenomation.

BACKGROUND: Scorpion venoms cause a massive release of neurotransmitters. Either anti-scorpion venom serum (AScVS) or prazosin has been used in the management of severe scorpion envenomation. AIMS: To compare the time taken for clinical recovery by patients with severe scorpion envenomation after AScVS therapy with that following prazosin therapy. SETTINGS AND DESIGN: A prospective, open-labeled clinical trial was undertaken to compare the effects of the AScVS and/or prazosin on clinical recovery in scorpion-stung patients. MATERIALS AND METHODS: Eighty-one patients from rural districts of Maharashtra presenting with severe scorpion envenomation were assigned to three treatment groups (AScVS: n = 28; prazosin: n = 25; AScVS + prazosin: n = 28). Severity of scorpion envenomation was graded using a proposed composite clinical scoring system to assess the therapeutic efficacy. AScVS was administered as an intravenous slow bolus, ranging from 40 to 100 ml, depending on the severity of envenomation. Prazosin was given as 1 mg every 3 h. STATISTICAL ANALYSIS USED: The non-parametric "Kruskal-Wallis" test was used in the statistical analysis and a P-value of 0.05 was considered significant. RESULTS: Mean composite scores of patients from the three groups at the time of admission were comparable. Complete clinical recovery was noted in 4.14 ± 1.6 h and 19.28 ± 5.03 h in the subjects who were administered AScVS and prazosin, respectively (P < 0.001). There was no incidence of anaphylactic reaction to AScVS. CONCLUSIONS: Intravenous slow bolus of AScVS given based on the clinical severity of envenomation leads to early recovery than prazosin alone and is well tolerated.

Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer.

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.

Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli.

Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.