A Unique Modification of the Eukaryotic Initiation Factor 5A Shows the Presence of the Complete Hypusine Pathway in Leishmania donovani

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of an unusual amino acid hypusine (N€-(4-amino-2-hydroxybutyl) lysine), which is present on only one cellular protein, eukaryotic initiation factor 5A (eIF5A). We present here the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of α-helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A ∼42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp), in vitro. L. donovani DOHH (LdDOHH) showed ∼40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues) and 174-183 (ten amino acid residues) which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. This report, for the first time, confirms the presence of a complete hypusine pathway in a kinetoplastid unlike eubacteria and archaea. The structural differences between the L. donovani DOHH and the human homolog may be exploited for structure based design of selective inhibitors against the parasite

Age and sex differences in ischemic stroke treatment in a nationwide analysis of 1.11 million hospitalized cases

Background and Purpose-To date, there is still uncertainty about age and sex differences in access to stroke unit treatmentand use of intravenous thrombolysis (IVT), while age and sex differences have not been investigated for the new treatmentoption of mechanical thrombectomy (MT). We, therefore, undertook a complete nationwide analysis of all hospitalizedischemic stroke patients in Germany from 2013 to 2017.Methods-We used the nationwide administrative database of the German Federal Statistical Office and investigated accessto stroke unit treatment, IVT, MT, and in-hospital mortality. Patients were subdivided into 6 predefined age groups(20-44, 45-59, 60-69, 70-79, 80-89, and >90 years). Pooled overall and age group estimates were calculated using therandom-effects model. To evaluate potential sex disparities, we estimated odds ratios (ORs) with 95% CIs.Results-A total of 1 112570 patients were hospitalized for first or recurrent ischemic stroke from 2013 to 2017. Overall,stroke unit treatment increased significantly from 66.8% in 2013 to 73.5% in 2017, as did IVT (from 12.4% to 15.9%)and MT (from 2.4% to 5.8%; all P<0.001). Although the difference became smaller over time, patients =80 years of agestill received significantly less often treatments. Men of all age groups had a significantly higher probability receivingstroke unit treatment (OR, 1.11; 95% CI, 1.09-1.12) and lower in-hospital mortality (OR, 0.91; 95% CI, 0.89-0.93). Nodisparity was observed in the use of IVT (OR, 1.00; 95% CI, 0.98-1.01), while women of all ages were treated more oftenwith MT (OR, 1.26; 95% CI, 1.22-1.30).Conclusions-Access to stroke unit treatment has to be increased in both older patients and women of all ages. While therewas no sex difference in IVT use, it is important to further investigate the significantly higher frequency of MT in womenwith ischemic stroke irrespective of age. © 2019 American Heart Association, Inc