Influence of metal ions on structure and catalytic activity of papain

Papain is an endoprotease belonging to cysteine protease family. The catalytic activity of papain in presence of two different metal ions namely zinc and cadmium has been investigated. Both the metal ions are potent inhibitors of the enzyme activity in a concentration dependent manner. The enzyme loses 50% of its activity at 2 x 10(-4) M of CdCl2 and 4 x 10(-4) M of ZnCl2. It is completely inactivated above 1 x 10(-3) M concentration of either ZnCl2 or CdCl2. Of the two metal ions zinc with a ki value of 5 x 10(-5) M is a more potent inhibitor than cadmium which has a ki value of 8 x 10(-5) M. Both the metal ions have higher affinity for active site than the substrate. At concentrations above 1 x 10(-2) M of metal ions the inhibition is not reversible. Calorimetric studies showed decreased thermal stability of papain upon binding of these metal ions. Far UV circular dichroic spectral data showed only small changes in the beta-structure content upon binding of these metal ions. These data are also supported by decrease in the apparent thermal transition temperature of papain by 5 degrees C upon binding of metal ions indicating destabilization of the papain molecule. The mechanism of both partial and complete inactivation of papain in presence of these two metal ions both at lower and higher concentration has been explained.     

Transcriptional inactivation of p53 by deletions and single amino acid changes in mouse mot-1 protein

Mouse mortalin proteins, mot-1 and mot-2, differ by only two amino acid residues in their C-terminus. In previous studies we showed that they differ in their subcellular distributions and interactions with the tumor suppressor protein, p53. By using mot-1 deletion mutants and amino acid substitution constructs, we report here that inability of mot-1 to affect p53 activity in vivo is dependent on the presence of both of the unique mot-1 amino acids and all three of the predicted hsp70, EF hand, and leucine zipper motif regions. The two proteins and their single amino acid mutants showed different mobilities on SDS-polyacrylamide gel presenting an evidence for their different secondary structures. Taken together, the data suggest that each of the two differing amino acids between mot-1 and mot-2 is an important determinant of their secondary structures and in vivo activities.     

Impact of Rural Residence on Warfarin Use and Clinical Events in Patients with Non-Valvular Atrial Fibrillation: A Canadian Population Based Study

Background and Purpose

We studied whether anticoagulant use and outcomes differed between rural versus urban Canadian non-valvular atrial fibrillation (NVAF) patients prior to the introduction of direct oral anticoagulant drugs.

Methods

Retrospective cohort study of 25,284 adult Albertans with NVAF between April 1, 1999 and December 31, 2008.

Results

Compared to urban patients, rural patients were older (p = 0.0009) and had more comorbidities but lower bleeding risk at baseline. In the first year after NVAF diagnosis, urban patients were less likely to be hospitalized (aOR 0.82, 95%CI 0.77–0.89) or have an emergency department visit for any reason (aOR 0.61, 95%CI 0.56–0.66) but warfarin dispensation rates (72.2% vs 71.8% at 365 days, p = 0.98) and clinical outcomes were similar: 7.8% died in both groups, 3.2% rural vs. 2.8% urban had a stroke or systemic embolism (SSE) (aOR 0.92, 95%CI 0.77–1.11), and 6.6% vs. 5.7% (aOR 0.93, 95%CI 0.81–1.06) had a bleed. Baseline SSE risk did not impact warfarin dispensation (73.0% in those with high vs. 72.8% in those with low CHADS₂ score, p = 0.85) but patients at higher baseline bleeding risk were less likely to be using warfarin (69.2% high vs. 73.6% low HASBLED score, p<0.0001) in the first 365 days after diagnosis. In warfarin users, bleeding was more frequent (7.5% vs 6.2%, aHR 1.51 [95%CI 1.33–1.72]) but death or SSE was less frequent (7.0% vs 18.1%, aHR 0.60 [0.54–0.66]).

Conclusion

Warfarin use and clinical event rates did not differ between rural and urban NVAF patients in a universal access publically-funded healthcare system.     

TLRR (lrrc67) interacts with PP1 and is associated with a cytoskeletal complex in the testis

Background information. Spermatozoa are formed via a complex series of cellular transformations, including acrosome and flagellum formation, nuclear condensation and elongation and removal of residual cytoplasm. Nuclear elongation is accompanied by the formation of a unique cytoskeletal structure, the manchette. We have previously identified a leucine‐rich repeat protein that we have named TLRR (testis leucine‐rich repeat), associated with the manchette that contains a PP1 (protein phosphatase‐1)‐binding site. Leucine‐rich repeat proteins often mediate protein–protein interactions; therefore, we hypothesize that TLRR acts as a scaffold to link signalling molecules, including PP1, to the manchette near potential substrate proteins important for spermatogenesis. Results. TLRR and PP1 interact with one another as demonstrated by co‐immunoprecipitation and the yeast two‐hybrid assay. TLRR binds more strongly to PP1γ2 than it does to PP1α. Anti‐phosphoserine antibodies immunoprecipitate TLRR from testis lysate, indicating that TLRR is a phosphoprotein. TLRR is part of a complex in testis that includes cytoskeletal proteins and constituents of the ubiquitin–proteasome pathway. The TLRR complex purified from 3T3 cells contains similar proteins, co‐localizes with microtubules and is enriched at the microtubule‐organizing centre. TLRR is also detected near the centrosome of elongated, but not mid‐stage, spermatids. Conclusion. We demonstrate here that TLRR interacts with PP1, particularly the testis‐specific isoform, PP1γ2. Immunoaffinity purification confirms that TLRR is associated with the spermatid cytoskeleton. In addition, proteins involved in protein stability are part of the TLRR complex. These results support our hypothesis that TLRR links signalling molecules to the spermatid cytoskeleton in order to regulate important substrates involved in spermatid transformation. The translocation of TLRR from the manchette to the centrosome region suggests a possible role for this protein in tail formation. Our finding that TLRR is associated with microtubules in cultured cells suggests that TLRR may play a common role in modulating the cytoskeleton in other cell types besides male germ cells.     

Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis

The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.     

Small-molecule cyclic alpha V beta 3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion

Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial alpha V beta 3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block alpha V beta 3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA (alpha-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind alpha V beta 3. An inactive peptide, EMD 135981 (cR beta-ADFV) was used as control. Cyclization and the introduction of D-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind alpha V beta 3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either alpha V beta 3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic alpha V beta 3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.     

Dau c 1.01 and Dau c 1.02-silenced transgenic carrot plants show reduced allergenicity to patients with carrot allergy

Pathogenesis-related protein-10 (PR10) is a ubiquitous small plant protein induced by microbial pathogens and abiotic stress that adversely contributes to the allergenic potency of many fruits and vegetables, including carrot. In this plant, two highly similar genes encoding PR10 isoforms have been isolated and designated as allergen Dau c 1.01 and Dau c 1.02. The aim of the study was to generate PR10-reduced hypoallergenic carrots by silencing either one of these genes in transgenic carrots by means of RNA interference (RNAi). The efficiency of gene silencing by stably expressed hairpin RNA (hnRNA) was documented by means of quantitative RT-PCR (qPCR) and immunoblotting. Quantification of the residual protein revealed that PR10 accumulation was strongly decreased compared with untransformed controls. Treatment of carrot plants with the PR protein-inducing chemical salicylic acid resulted in an increase of PR10 isoforms only in wild-type but not in Dau c 1-silenced mutants. The decrease of the allergenic potential in Dau c 1-silenced plants was sufficient to cause a reduced allergenic reactivity in patients with carrot allergy, as determined with skin prick tests (SPT). However, simultaneous silencing of multiple allergens will be required to design hypoallergenic carrots for the market. Our findings demonstrate the feasibility of creating low-allergenic food by using RNAi. This constitutes a reasonable approach to allergen avoidance.