The homologous tryptophan critical for cytochrome c peroxidase function is not essential for ascorbate peroxidase activity

The crystal structures of ascorbate peroxidase (APX) and cytochrome c peroxidase (CCP) show that the active site structures are nearly identical. Both enzymes contain a His-Asp-Trp catalytic triad in the proximal pocket. The proximal Asp residue hydrogen bonds with both the His proximal heme ligand and the indole ring nitrogen of the proximal Trp. The Trp is stacked parallel to and in contact with the proximal His ligand. This Trp is known to be the site of free radical formation in CCP compound I and also is essential for activity. However, APX forms a porphyrin radical and not a Trp-centered radical, even though the His-Asp-Trp triad structure is the same in both peroxidases. We found that conversion of the proximal Trp to Phe has no effect on APX enzyme activity and that the mutant crystal structure shows that changes in the structure are confined to the site of mutation. This indicates that the paths of electron transfer in CCP and APX are distinctly different. The Trp-to-Phe mutant does alter the stability of the APX compound I porphyrin radical, by a factor of two. Electrostatic calculations and modeling studies show that a potassium cation located about 8?Å from the proximal Trp in APX, but absent in CCP, makes a significant contribution to the stability of a cation Trp radical. This underscores the importance of long-range electrostatic effects in enzyme catalyzed reactions.     

Preparation of coenzymic activity of soluble polyethyleneimine-bound NADP+ derivatives.

Alkylation at N-1 of the NADP+ adenine ring with 3,4-epoxybutanoic acid gave 1-(2-hydroxy-3-carboxypropyl)-NADP+. Enzymic reduction of the latter, followed by alkaline Dimroth rearrangement and enzymic reoxidation, gave N6-(2-hydroxy-3-carboxypropyl)-NADP+. On the other hand, bromination at C-8 of the NADP+ adenine ring, followed by reaction with the disodium salt of 3-mercaptroproionic acid, gave 8-(2-carboxyethylthio)-NADP+. Carbodimide coupling of the three carboxylic NADP+ derivatives to polyethyleneimine afforded the corresponding macromolecular NADP+ analogues. The carboxylic and the polyethyleneimine derivatives synthesized have been shown to be co-enzymically active with yeast glucose-6-phosphate dehydrogenase, liver glutamate dehydrogenase and yeast aldehyde dehydrogenase. The degree of efficiency relative to NADP+ with the three enzymes ranged from 17% to 100% for the carboxylic derivatives and from 1% to 36% for the polyethyleneimine analogues. On comparing the efficiences with the three enzymes of the N-1 derivatives to the one of the corresponding N6 anc C-8 analogues, the order of activity was N-1 greater than N6 greater C-8, except in the case of the carboxylic compounds with glutamate dehydrogenase, where this order was inverted. None of these modified cofactors were active with pig heart isocitrate dehydrogenase.     

The effect of the elongation of the proximal aorta on the estimation of the aortic wall distensibility

The compliance of the proximal aortic wall is a major determinant of cardiac afterload. Aortic compliance is often estimated based on cross-sectional area changes over the pulse pressure, under the assumption of a negligible longitudinal stretch during the pulse. However, the proximal aorta is subjected to significant axial stretch during cardiac contraction. In the present study, we sought to evaluate the importance of axial stretch on compliance estimation by undertaking both an in silico and an in vivo approach. In the computational analysis, we developed a 3-D finite element model of the proximal aorta and investigated the discrepancy between the actual wall compliance to the value estimated after neglecting the longitudinal stretch of the aorta. A parameter sensitivity analysis was further conducted to show how increased material stiffness and increased aortic root motion might amplify the estimation errors (discrepancies between actual and estimated distensibility ranging from − 20 to − 62%). Axial and circumferential aortic deformation during ventricular contraction was also evaluated in vivo based on MR images of the aorta of 3 healthy young volunteers. The in vivo results were in good qualitative agreement with the computational analysis (underestimation errors ranging from − 26 to − 44%, with increased errors reflecting higher aortic root displacement). Both the in silico and in vivo findings suggest that neglecting the longitudinal strain during contraction might lead to severe underestimation of local aortic compliance, particularly in the case of women who tend to have higher aortic root motion or in subjects with stiff aortas.

     

Evolution of a Complex Locus for Terpene Biosynthesis in Solanum

Functional gene clusters, containing two or more genes encoding different enzymes for the same pathway, are sometimes observed in plant genomes, most often when the genes specify the synthesis of specialized defensive metabolites. Here, we show that a cluster of genes in tomato (Solanum lycopersicum; Solanaceae) contains genes for terpene synthases (TPSs) that specify the synthesis of monoterpenes and diterpenes from cis-prenyl diphosphates, substrates that are synthesized by enzymes encoded by cis-prenyl transferase (CPT) genes also located within the same cluster. The monoterpene synthase genes in the cluster likely evolved from a diterpene synthase gene in the cluster by duplication and divergence. In the orthologous cluster in Solanum habrochaites, a new sesquiterpene synthase gene was created by a duplication event of a monoterpene synthase followed by a localized gene conversion event directed by a diterpene synthase gene. The TPS genes in the Solanum cluster encoding cis-prenyl diphosphate–utilizing enzymes are closely related to a tobacco (Nicotiana tabacum; Solanaceae) diterpene synthase encoding Z-abienol synthase (Nt-ABS). Nt-ABS uses the substrate copal-8-ol diphosphate, which is made from the all-trans geranylgeranyl diphosphate by copal-8-ol diphosphate synthase (Nt-CPS2). The Solanum gene cluster also contains an ortholog of Nt-CPS2, but it appears to encode a nonfunctional protein. Thus, the Solanum functional gene cluster evolved by duplication and divergence of TPS genes, together with alterations in substrate specificity to utilize cis-prenyl diphosphates and through the acquisition of CPT genes.     

A highly sensitive molecular structural probe applied to in situ biosensing of metabolites using PEDOT:PSS

A large amount of research within organic biosensors is dominated by organic electrochemical transistors (OECTs) that use conducting polymers such as poly(3,4-ethylene dioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS). Despite the recent advances in OECT-based biosensors, the sensing is solely reliant on the amperometric detection of the bioanalytes. This is typically accompanied by large undesirable parasitic electrical signals from the electroactive components in the electrolyte. Herein, we present the use of in situ resonance Raman spectroscopy to probe subtle molecular structural changes of PEDOT:PSS associated with its doping level. We demonstrate how such doping level changes of PEDOT:PSS can be used, for the first time, on operational OECTs for sensitive and selective metabolite sensing while simultaneously performing amperometric detection of the analyte. We test the sensitivity by molecularly sensing a lowest glucose concentration of 0.02 mM in phosphate-buffered saline solution. By changing the electrolyte to cell culture media, the selectivity of in situ resonance Raman spectroscopy is emphasized as it remains unaffected by other electroactive components in the electrolyte. The application of this molecular structural probe highlights the importance of developing biosensing probes that benefit from high sensitivity of the material's structural and electrical properties while being complimentary with the electronic methods of detection.     

Human TTRV30M localization within podocytes in a transgenic mouse model of transthyretin related amyloidosis: does the environment play a role?

Transthyretin related amyloidosis is a nosological entity that leads to disability, diminished quality of life, all stages of chronic kidney disease and eventually death. Podocytes are polarized, highly differentiated epithelial cells important for proper nephron function. In the present study we investigated whether deposited TTRVal30Met (TTRV30M) molecules could be localized within podocytes in situ under the effect of different housing conditions (i.e. specific pathogen free [SPF] vs. non-SPF). Murine renal glomeruli from human TTRV30M (hTTRV30M) transgenic mice were examined via direct and indirect immunofluorescence techniques for the presence of hTTRV30M, murine serum amyloid P, activated caspase-3 and NPHS1. Association strength and amount of colocalization for NPHS1?ChTTRV30M, NPHS1-activated caspase-3, hTTRV30M-murine serum amyloid P were estimated. Localization of hTTRV30M in podocytes was demonstrated by immuno-electron microscopy. Renal hTTRV30M gene and NPHS1 gene expression levels were estimated. Non-SPF transgenic mice showed increased glomerular hTTRV30M deposition compared to their SPF counterparts. Furthermore increased podocytic localization of hTTRV30M was noticed in non-SPF mice. Glomerular caspase-3 activation was increased only in the non SPF housing conditions. Podocytic caspase-3 activation was increased in SPF and in non-SPF transgenic mice when compared to non transgenic controls. Environmental conditions influence glomerular deposition and podocytic localization of hTTRV30M. In this context increased caspase-3 activation occurred.     

Endogenous Purification Reveals GREB1 as a Key Estrogen Receptor Regulatory Factor

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Highlights? A proteomic method identifies protein-protein interaction in primary tumors ? GREB1 is the top estrogen-induced ER-interacting protein ? GREB1 is an essential ER cofactor recruited to chromatin ? GREB1 is an independent prognostic marker     

Drug Resistance in Natural Isolates of Leishmania donovani s.l. Promastigotes Is Dependent of Pgp170 Expression

Resistance of pathogens to drugs is a growing concern regarding many diseases. Parasites like Leishmania, Plasmodium and Entamoeba histolytica; and neoplastic cells, present the multidrug-resistant phenotype rendering chemotherapy ineffective. The acquired resistance of Leishmania to antimony has generated intense research on the mechanisms involved but the question has not yet been resolved. To test the hypothesis that drug efflux in Leishmania, as measured by flow cytometry using the fluorescent dye Rhodamine-123, is largely dependent on the number of efflux pumps an isolate can express, the amount of Pgp 170 molecules was assessed in ten field isolates (5 “resistant” and 5 “susceptible”) using: Western Blotting, Confocal and Transmission Electron Microscopy, and proteomics. Their survival after exposure to three antileishmanial drugs, in vitro, was evaluated and clinical data were compared to the in vitro results. All isolates were resistant to Glucantime but susceptible to Miltefosine, whilst Amphotericin B was more effective on the “susceptible” isolates. The MDR gene, expressing the transmembrane efflux pump Pgp 170, appears to play a key role in the phenomenon of drug resistance. When “susceptible” versus “resistant” parasites were compared, it was shown that the higher the number of Pgp 170 molecules the higher the Rhodamine-123 efflux from the parasite body and, when exposed to the drug, the number of efflux pumps increased. However, the rate of this increase was not linear and it is possible that there is a maximum number of Pgp 170 molecules an isolate can express. Nevertheless, the phenomenon is a complex one and other factors and proteins are involved in which the HSP-70 group proteins, detected in the “resistant” isolates, may play a significant role.     

Sleep Promotes the Extraction of Grammatical Rules

Grammar acquisition is a high level cognitive function that requires the extraction of complex rules. While it has been proposed that offline time might benefit this type of rule extraction, this remains to be tested. Here, we addressed this question using an artificial grammar learning paradigm. During a short-term memory cover task, eighty-one human participants were exposed to letter sequences generated according to an unknown artificial grammar. Following a time delay of 15 min, 12 h (wake or sleep) or 24 h, participants classified novel test sequences as Grammatical or Non-Grammatical. Previous behavioral and functional neuroimaging work has shown that classification can be guided by two distinct underlying processes: (1) the holistic abstraction of the underlying grammar rules and (2) the detection of sequence chunks that appear at varying frequencies during exposure. Here, we show that classification performance improved after sleep. Moreover, this improvement was due to an enhancement of rule abstraction, while the effect of chunk frequency was unaltered by sleep. These findings suggest that sleep plays a critical role in extracting complex structure from separate but related items during integrative memory processing. Our findings stress the importance of alternating periods of learning with sleep in settings in which complex information must be acquired.