527 research outputs found
Analysis of the Equilibrium and Kinetics of the Ankyrin Repeat Protein Myotrophin
We apply the Wako-Saito-Munoz-Eaton model to the study of Myotrophin, a small
ankyrin repeat protein, whose folding equilibrium and kinetics have been
recently characterized experimentally. The model, which is a native-centric
with binary variables, provides a finer microscopic detail than the Ising
model, that has been recently applied to some different repeat proteins, while
being still amenable for an exact solution. In partial agreement with the
experiments, our results reveal a weakly three-state equilibrium and a
two-state-like kinetics of the wild type protein despite the presence of a
non-trivial free-energy profile. These features appear to be related to a
careful "design" of the free-energy landscape, so that mutations can alter this
picture, stabilizing some intermediates and changing the position of the
rate-limiting step. Also the experimental findings of two alternative pathways,
an N-terminal and a C-terminal one, are qualitatively confirmed, even if the
variations in the rates upon the experimental mutations cannot be
quantitatively reproduced. Interestingly, folding and unfolding pathway appear
to be different, even if closely related: a property that is not generally
considered in the phenomenological interpretation of the experimental data.Comment: 27 pages, 7 figure
Multiplicity of 5' Cap Structures Present on Short RNAs
Most RNA molecules are co- or post-transcriptionally modified to alter their chemical and functional properties to assist in their ultimate biological function. Among these modifications, the addition of 5' cap structure has been found to regulate turnover and localization. Here we report a study of the cap structure of human short (<200 nt) RNAs (sRNAs), using sequencing of cDNA libraries prepared by enzymatic pretreatment of the sRNAs with cap sensitive-specificity, thin layer chromatographic (TLC) analyses of isolated cap structures and mass spectrometric analyses for validation of TLC analyses. Processed versions of snoRNAs and tRNAs sequences of less than 50 nt were observed in capped sRNA libraries, indicating additional processing and recapping of these annotated sRNAs biotypes. We report for the first time 2,7 dimethylguanosine in human sRNAs cap structures and surprisingly we find multiple type 0 cap structures (mGpppC, 7mGpppG, GpppG, GpppA, and 7mGpppA) in RNA length fractions shorter than 50 nt. Finally, we find the presence of additional uncharacterized cap structures that wait determination by the creation of needed reference compounds to be used in TLC analyses. These studies suggest the existence of novel biochemical pathways leading to the processing of primary and sRNAs and the modifications of their RNA 5' ends with a spectrum of chemical modifications
Secondary recrystallization in Fe-3% Si alloy with (110)[001] single-component texture
Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H\u3csub\u3ec\u3c/sub\u3e): Antigen E) by \u3ci\u3ePichia pastoris\u3c/i\u3e
A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(Hc)in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(Hc) gene inserted into pHILD4 Escherichia coli—P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(Hc) sequence. Expression of rBoNTE(Hc) from the Mut+ HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(Hc). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(Hc) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(Hc) per gram wet cell mass as determined by HPLC and Western blot analysis
Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H\u3csub\u3ec\u3c/sub\u3e): Antigen E) by \u3ci\u3ePichia pastoris\u3c/i\u3e
A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(Hc)in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(Hc) gene inserted into pHILD4 Escherichia coli—P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(Hc) sequence. Expression of rBoNTE(Hc) from the Mut+ HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(Hc). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(Hc) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(Hc) per gram wet cell mass as determined by HPLC and Western blot analysis
Assessing the impact of Bacillus strains mixture probiotic on water quality, growth performance, blood profile and intestinal morphology of Nile tilapia, Oreochromis niloticus
The aim of this study was to assess the impact of a commercial probiotic, Sanolife PRO‐F, on water quality, growth performance, blood profiles and intestinal morphometry of monosex Nile tilapia. A field trial was conducted for 10 weeks in which tilapia fingerlings (20 ± 1.26 g) were randomly distributed into three replicate ponds which were subdivided into three treatment groups, receiving Sanolife PRO‐F at 0 (B0), 0.1 (B1) and 0.2 (B2) g/kg diet, respectively. The results showed a significant improvement in growth performance, feed conversion ratio and blood profiles in tilapia fed on treated diets. The whole intestinal lengths, anterior and terminal intestinal villi heights and anterior goblet cells count were greater in tilapia fed on treated diets. There were no noticeable differences in growth and intestinal morphology between tilapia fed on B1 and B2 diets. The ammonia concentration in water was lower with B1 diet while electric conductivity, salinity and total dissolved solids were higher with the B2 diet. The pH level of pond water was enhanced by both diets, B1 and B2. In conclusion, application of Sanolife PRO‐F at 0.1–0.2 g/kg diet might have beneficial effects on growth, immunity, stress responses and gut health and function as well as the water quality of farmed Nile tilapia
Human Polycomb 2 Protein Is a SUMO E3 Ligase and Alleviates Substrate-Induced Inhibition of Cystathionine β-Synthase Sumoylation
Human cystathionine β-synthase (CBS) catalyzes the first irreversible
step in the transsulfuration pathway and commits homocysteine to the synthesis
of cysteine. Mutations in CBS are the most common cause of severe hereditary
hyperhomocysteinemia. A yeast two-hybrid approach to screen for proteins that
interact with CBS had previously identified several components of the
sumoylation pathway and resulted in the demonstration that CBS is a substrate
for sumoylation. In this study, we demonstrate that sumoylation of CBS is
enhanced in the presence of human polycomb group protein 2 (hPc2), an
interacting partner that was identified in the initial yeast two-hybrid screen.
When the substrates for CBS, homocysteine and serine for cystathionine
generation and homocysteine and cysteine for H2S generation, are
added to the sumoylation mixture, they inhibit the sumoylation reaction, but
only in the absence of hPc2. Similarly, the product of the CBS reaction,
cystathionine, inhibits sumoylation in the absence of hPc2. Sumoylation in turn
decreases CBS activity by ∼28% in the absence of hPc2 and by
70% in its presence. Based on these results, we conclude that hPc2
serves as a SUMO E3 ligase for CBS, increasing the efficiency of sumoylation. We
also demonstrate that γ-cystathionase, the second enzyme in the
transsulfuration pathway is a substrate for sumoylation under in vitro
conditions. We speculate that the role of this modification may be for nuclear
localization of the cysteine-generating pathway under conditions where nuclear
glutathione demand is high
Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases
Cystathionine beta-synthase (CBS) is a key regulator of sulfur amino acid metabolism diverting homocysteine, a toxic intermediate of the methionine cycle, via the transsulfuration pathway to the biosynthesis of cysteine. Although the pathway itself is well conserved among eukaryotes, properties of eukaryotic CBS enzymes vary greatly. Here we present a side-by-side biochemical and biophysical comparison of human (hCBS), fruit fly (dCBS) and yeast (yCBS) enzymes. Preparation and characterization of the full-length and truncated enzymes, lacking the regulatory domains, suggested that eukaryotic CBS exists in one of at least two significantly different conformations impacting the enzyme’s catalytic activity, oligomeric status and regulation. Truncation of hCBS and yCBS, but not dCBS, resulted in enzyme activation and formation of dimers compared to native tetramers. The dCBS and yCBS are not regulated by the allosteric activator of hCBS, S-adenosylmethionine (AdoMet); however, they have significantly higher specific activities in the canonical as well as alternative reactions compared to hCBS. Unlike yCBS, the heme-containing dCBS and hCBS showed increased thermal stability and retention of the enzyme’s catalytic activity. The mass-spectrometry analysis and isothermal titration calorimetry showed clear presence and binding of AdoMet to yCBS and hCBS, but not dCBS. However, the role of AdoMet binding to yCBS remains unclear, unlike its role in hCBS. This study provides valuable information for understanding the complexity of the domain organization, catalytic specificity and regulation among eukaryotic CBS enzymes.This work was supported by Postdoctoral Fellowship 0920079G from the American Heart Association (to TM), by National Institutes of Health Grant HL065217, by American Heart Association Grant In-Aid 09GRNT2110159, by a grant from the Jerome Lejeune Foundation (all to JPK) and by a research contract RYC2009-04147 (to ALP). In addition, grant support (P11-CTS-07187, CSD2009-00088 and BIO2012-34937) to Dr. Jose M. Sanchez-Ruiz (University of Granada) and SGIker technical and human support (UPV/EHU, MICINN, GV/EJ, ESF) are gratefully acknowledged
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