Asparaginyl endopeptidases (AEPs) are proteases that have crucial roles in plant defense and seed storage protein maturation. Select plant AEPs, however, do not function as proteases but as ...transpeptidases (ligases) catalyzing the intra-molecular ligation of peptide termini, which leads to peptide cyclization. These ligase-type AEPs have potential biotechnological applications ranging from in vitro peptide engineering to plant molecular farming, but the structural features enabling these enzymes to catalyze peptide ligation/cyclization rather than proteolysis are currently unknown. Here, we compare the sequences, structures, and functions of diverse plant AEPs by combining molecular modeling, sequence space analysis, and functional testing in planta. We find that changes within the substrate-binding pocket and an adjacent loop, here named the "marker of ligase activity", together play a key role for AEP ligase efficiency. Identification of these structural determinants may facilitate the discovery of more ligase-type AEPs and the engineering of AEPs with tailored catalytic properties.
Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and ...lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para >meta >ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs.
Background: Delivering molecules to selected cellular compartments is important for analytical and practical purposes.
Results: Varying the length and positions of alkyl substituents results in preferential uptake of zinc porphyrins by particular cellular structures.
Conclusion: Uptake, distribution, and phototoxicity of porphyrins depend on charge, lipophilicity, and molecular shape.
Significance: Systematic chemical modification provides the basis for rational design of molecules targeting specific cellular compartments.
After early work by Newton, the eighteenth and early nineteenth century French mathematicians Laplace, Lagrange, Poisson, and Cauchy made real theoretical advances in the linear theory of water ...waves; in Germany, Gerstner considered nonlinear waves, and the brothers Weber performed fine experiments. Later in Britain during 1837-1847, Russell, Green, Kelland, Airy, and Earnshaw all made substantial contributions, setting the scene for subsequent work by Stokes and others.
Mitochondria play a key role in aerobic ATP production and redox control. They harness crucial metabolic pathways and control cell death mechanisms, properties that make these organelles essential ...for survival of most eukaryotic cells. Cancer cells have altered cell death pathways and typically show a shift towards anaerobic glycolysis for energy production, factors which point to mitochondria as potential culprits in cancer development. Targeting mitochondria is an attractive approach to tumor control, but design of pharmaceutical agents based on rational approaches is still not well established. The aim of this study was to investigate which structural features of specially designed Zn(II)N-alkylpyridylporphyrins would direct them to mitochondria and to particular mitochondrial targets. Since Zn(II)N-alkylpyridylporphyrins can act as highly efficient photosensitizers, their localization can be confirmed by photodamage to particular mitochondrial components. Using cultured LS174T adenocarcinoma cells, we found that subcellular distribution of Zn-porphyrins is directed by the nature of the substituents attached to the meso pyridyl nitrogens at the porphyrin ring. Increasing the length of the aliphatic chain from one carbon (methyl) to six carbons (hexyl) increased mitochondrial uptake of the compounds. Such modifications also affected sub-mitochondrial distribution of the Zn-porphyrins. The amphiphilic hexyl derivative (ZnTnHex-2-PyP) localized in the vicinity of cytochrome c oxidase complex, causing its inactivation during illumination. Photoinactivation of critical cellular targets explains the superior efficiency of the hexyl derivative in causing mitochondrial photodamage, and suppressing cellular respiration and survival. Design of potent photosensitizers and redox-active scavengers of free radicals should take into consideration not only selective organelle uptake and localization, but also selective targeting of critical macromolecular structures.
Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at ...the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF; polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.
We describe a fully GPU-based implementation of the first level trigger for the upgrade of the LHCb detector, due to start data taking in 2021. We demonstrate that our implementation, named Allen, ...can process the 40 Tbit/s data rate of the upgraded LHCb detector and perform a wide variety of pattern recognition tasks. These include finding the trajectories of charged particles, finding proton–proton collision points, identifying particles as hadrons or muons, and finding the displaced decay vertices of long-lived particles. We further demonstrate that Allen can be implemented in around 500 scientific or consumer GPU cards, that it is not I/O bound, and can be operated at the full LHC collision rate of 30 MHz. Allen is the first complete high-throughput GPU trigger proposed for a HEP experiment.
Several members of the Rubiaceae and Violaceae families produce a series of cyclotides or macrocyclic peptides of 29-31 amino acids with an embedded cystine knot. We aim to understand the mechanism ...of synthesis of cyclic peptides in plants and have isolated a cDNA clone that encodes the cyclotide kalata B1 as well as three other clones for related cyclotides from the African plant Oldenlandia affinis. The cDNA clones encode prepropeptides with a 20-aa signal sequence, an N-terminal prosequence of 46-68 amino acids and one, two, or three cyclotide domains separated by regions of about 25 aa. The corresponding cyclotides have been isolated from plant material, indicating that the cyclotide domains are excised and cyclized from all four predicted precursor proteins. The exact processing site is likely to lie on the N-terminal side of the strongly conserved GlyLeuPro or SerLeuPro sequence that flanks both sides of the cyclotide domain. Cyclotides have previously been assigned an antimicrobial function; here we describe a potent inhibitory effect on the growth and development of larvae from the Lepidopteran species Helicoverpa punctigera.
High patient weight is a risk factor for mechanical implant failure and some manufacturers list obesity as a contraindication for implant use. We reviewed data from the 2012-2013 UK National Joint ...Registry to determine whether surgical practice reflects these manufacturer recommendations.
The product literature for the most commonly used hip and knee implants was reviewed for recommendations against use in obese patients (body mass index BMI ≥ 30kg/m(2)). The total number of obese patients undergoing hip and knee arthroplasty was calculated, as was the proportion receiving implants against manufacturer recommendations.
Out of 200,054 patient records, 147,691 (74%) had a recorded BMI. The mean BMI for patients undergoing primary total hip arthroplasty was 29kg/m(2), compared with 31kg/m(2) for total knee arthroplasty. Of the 25 components reviewed, 5 listed obesity as a contraindication or recommended against implant use in obese patients. A total of 10,745 patients (16% of all obese patients) received implants against manufacturer recommendations.
A high proportion of patients are receiving implants against manufacturer recommendations. However, there are limitations to using BMI for stratifying risk of implant fatigue failure and manufacturers should therefore provide more detailed guidelines on size specific implant load limits to facilitate surgical decisions.
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