scholarly journals The cruciated microtubule-associated fibers of the green alga Dunaliella bioculata consist of a 31 kDa SF-assemblin

1996 ◽  
Vol 109 (4) ◽  
pp. 827-835
Author(s):  
K.F. Lechtreck ◽  
S. Frins ◽  
J. Bilski ◽  
A. Teltenkotter ◽  
K. Weber ◽  
...  
Keyword(s):  
Green Alga ◽  
Gel Filtration ◽  
Coiled Coil ◽  
Immunoblot Analysis ◽  
Complete Sequence ◽  
Major Protein ◽  
Helical Proteins ◽  
Related Proteins

Cytoskeletons of Dunaliella bioculata, the biflagellate wallless green alga, were isolated and analyzed using a monoclonal and a polyclonal antibody raised against SF-assemblin, the major protein of the two striated microtubule-associated fibers of the alga Spermatozopsis similis. Indirect immunofluorescence showed antigenic structures associated with the four microtubular flagellar roots. SDS-PAGE followed by immunoblot analysis revealed a cross-reacting polypeptide of 31 kDa. This protein of D. bioculata was isolated using gel filtration chromatography in 8 M urea and in vitro reassembly of striated fibers. Microsequencing of the purified protein yielded various peptides, which could be aligned along the sequence of SF-assemblin from S. similis. A complete sequence of the Dunaliella protein was obtained by cDNA cloning. It documents the non helical head domain followed by a helical rod domain with a 29 residue repeat pattern based on four heptads followed by a skip residue. Compared to SF-assemblin of S. similis the SF-assemblin of Dunaliella has a shorter head and a slightly longer rod domain. The two algal SF-assemblins share only 57% sequence identity. We conclude that SF-assemblin and related proteins in various protists are representatives of a new class of alpha-helical proteins characterized by the ability to form a special segmented coiled coil and to assemble into striated fibers of 2 nm protofilaments in vivo and in vitro.

scholarly journals Coiled-Coil Trigger Motifs in the 1B and 2B Rod Domain Segments Are Required for the Stability of Keratin Intermediate Filaments

2000 ◽  
Vol 11 (10) ◽  
pp. 3539-3558 ◽  
Author(s):  
Kenneth C. Wu ◽  
Janine T. Bryan ◽  
Maria I. Morasso ◽  
Shyh-Ing Jang ◽  
Jeung-Hoon Lee ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Coiled Coils ◽  
Detectable Effect ◽  
Keratin 5 ◽  
Molecular Stability ◽  
Potential Trigger ◽  
Helical Proteins ◽  
The Stability

Many α-helical proteins that form two-chain coiled coils possess a 13-residue trigger motif that seems to be required for the stability of the coiled coil. However, as currently defined, the motif is absent from intermediate filament (IF) protein chains, which nevertheless form segmented two-chain coiled coils. In the present work, we have searched for and identified two regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger motifs. We made a series of point substitutions with the keratin 5/keratin 14 IF system. Combinations of the wild-type and mutant chains were assembled in vitro and in vivo, and the stabilities of two-chain (one-molecule) and two-molecule assemblies were examined with use of a urea disassembly assay. Our new data document that there is a region located between residues 100 and 113 of the 2B rod domain segment that is absolutely required for molecular stability and IF assembly. This potential trigger motif differs slightly from the consensus in having an Asp residue at position 4 (instead of a Glu) and a Thr residue at position 9 (instead of a charged residue), but there is an absolute requirement for a Glu residue at position 6. Because these 13 residues are highly conserved, it seems possible that this motif functions in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residues 79 and 91 of the 1B rod domain segment, which may also be conserved in all IF chains. However, we were unable to find a trigger motif in the 1A rod domain segment. In addition, many other point substitutions had little detectable effect on IF assembly, except for the conserved Lys-23 residue of the 2B rod domain segment. Cross-linking and modeling studies revealed that Lys-23 may lie very close to Glu-106 when two molecules are aligned in the A22 mode. Thus, the Glu-106 residue may have a dual role in IF structure: it may participate in trigger formation to afford special stability to the two-chain coiled-coil molecule, and it may participate in stabilization of the two-molecule hierarchical stage of IF structure.

Identification of the Herbicide Binding Region of the Qв-Protein by Photoaffinity Labeling with Azidoatrazine

1984 ◽  
Vol 39 (5) ◽  
pp. 425-429 ◽  
Author(s):  
Paul K. Wolber ◽  
Katherine E. Steinback
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Single Amino Acid ◽  
Rhodopseudomonas Capsulata ◽  
Major Protein ◽  
Peptide Pattern ◽  
Single Amino Acid Change ◽  
Spinach Thylakoids

Spinach thylakoids have been photoaffinity labeled with [14C]azidoatrazine in vitro and with specific [3H]amino acids in vivo. The radiolabeled protein has been isolated, cleaved with trypsin, and the resulting fragments have been identified by their characteristic [3H]amino acid incorporation. The peptide pattern has been interpreted using the known gene sequence for the QB-protein of spinach thylakoids, which is the major protein labeled by both the in vivo and in vitro procedures. When analyzed by gel filtration, over 80% of the 14C-activity migrated as two peaks: one as undigested protein at the void volume and the other at an apparent MW of 8.35 ± 0.76 kilodaltons. The remaining activity migrated with the included volume as free azidoatrazine. Fragments were analysed for covalent [14C]azidoatrazine binding; the tryptic peptide covalently labeled by [14C]azidoatrazine was found to be the fragment Pro-141 to Arg-225. This peptide is well removed from the site of a single amino acid change at Ser-264 implicated in triazine resistance, but includes a region suggested to be involved in Qв-binding by homology with sequences of two reaction center proteins from Rhodopseudomonas capsulata.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

scholarly journals Dipterocarpus tuberculatus Roxb. Ethanol Extract Has Anti-Inflammatory and Hepatoprotective Effects In Vitro and In Vivo by Targeting the IRAK1/AP-1 Pathway

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2529
Author(s):  
Haeyeop Kim ◽  
Woo Seok Yang ◽  
Khin Myo Htwe ◽  
Mi-Nam Lee ◽  
Young-Dong Kim ◽  
...  
Keyword(s):  
Ethanol Extract ◽  
Serum Levels ◽  
Hepatoprotective Activity ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Related Proteins ◽  
Pro Inflammatory Cytokines ◽  
Inflammatory Effect

Dipterocarpus tuberculatus Roxb. has been used traditionally as a remedy for many diseases, especially inflammation. Therefore, we analyzed and explored the mechanism of the anti-inflammatory effect of a Dipterocarpus tuberculatus Roxb. ethanol extract (Dt-EE). Dt-EE clearly and dose-dependently inhibited the expression of pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β in lipopolysaccharide (LPS)-treated RAW264.7 cells. Also, Dt-EE suppressed the activation of the MyD88/TRIF-mediated AP-1 pathway and the AP-1 pathway related proteins JNK2, MKK4/7, and TAK1, which occurred as a result of inhibiting the kinase activity of IRAK1 and IRAK4, the most upstream factors of the AP-1 pathway. Finally, Dt-EE displayed hepatoprotective activity in a mouse model of hepatitis induced with LPS/D-galactosamine (D-GalN) through decreasing the serum levels of alanine aminotransferase and suppressing the activation of JNK and IRAK1. Therefore, our results strongly suggest that Dt-EE could be a candidate anti-inflammatory herbal medicine with IRAK1/AP-1 inhibitory and hepatoprotective properties.

scholarly journals miR-146a-5p Promotes Chondrocyte Apoptosis and Inhibits Autophagy of Osteoarthritis by Targeting NUMB

Cartilage ◽  
2021 ◽  
pp. 194760352110235
Author(s):  
Hongjun Zhang ◽  
Wendi Zheng ◽  
Du Li ◽  
Jia Zheng
Keyword(s):  
Caspase 3 ◽  
Cartilage Tissue ◽  
Luciferase Reporter ◽  
Chondrocyte Apoptosis ◽  
Knee Cartilage ◽  
Before And After ◽  
Related Proteins ◽  
Human And Mouse

Objective miR-146a-5p was found to be significantly upregulated in cartilage tissue of patients with osteoarthritis (OA). NUMB was shown to be involved in the autophagy regulation process of cells. We aimed to learn whether NUMB was involved in the apoptosis or autophagy process of chondrocytes in OA and related with miR-146a-5p. Methods QRT-PCR was used to detect miR-146a-5p level in 22 OA cartilage tissues and 22 controls. The targets of miR-146a-5p were analyzed using software and the luciferase reporter experiment. The apoptosis and autophagy, and related proteins were detected in chondrocytes treated with miR-146a-5p mimic/inhibitor or pcDNA3.1-NUMB/si-NUMB and IL-1β, respectively. In vivo experiment, intra-articular injection of miR-146a-5p antagomir/NC was administered at the knee of OA male mice before and after model construction. Chondrocyte apoptosis and the expression of apoptosis and autophagy-related proteins were also detected. Results miR-146a-5p was highly expressed in knee cartilage tissue of patients with OA, while NUMB was lowly expressed and negatively regulated by miR-146a-5p. Upregulation of miR-146a-5p can promote cell apoptosis and reduce autophagy of human and mouse chondrocytes by modulating the levels of cleaved caspase-3, cleaved PARP, Bax, Beclin 1, ATG5, p62, LC3-I, and LC3-II. Increasing the low level of NUMB reversed the effects of miR-146a-5p on chondrocyte apoptosis and autophagy. Intra-articular injection of miR-146a-5p antagomir can also reverse the effects of miR-146a-5p on the apoptosis and autophagy of knee joint chondrocytes in OA mice. Conclusion Downregulation of miR-146a-5p suppresses the apoptosis and promotes autophagy of chondrocytes by targeting NUMB in vivo and in vitro.

scholarly journals 1455. Potential Mechanisms of Cefiderocol MIC Increase in Enterobacterales in In Vitro Resistance Acquisition Studies

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S730-S730
Author(s):  
Yoshinori Yamano ◽  
Rio Nakamura ◽  
Miki Takemura ◽  
Roger Echols
Keyword(s):  
Iron Transport ◽  
Resistance Mechanisms ◽  
Altered Expression ◽  
Carbapenem Resistant ◽  
Mueller Hinton ◽  
Two Component ◽  
Mueller Hinton Agar ◽  
Related Proteins

Abstract Background Cefiderocol (CFDC) is a novel siderophore, iron-chelating cephalosporin, which is transported into bacteria via iron transporters. CFDC has potent in vitro and in vivo activity against all aerobic Gram-negative bacteria, including carbapenem-resistant strains. To date, clinical isolates with cefiderocol MIC >4 µg/mL have been found infrequently, in which the presence of a few β-lactamases or altered iron transport was found. We investigated potential new mechanisms causing CFDC MIC increases in non-clinical studies. Methods The mutation positions were determined by whole genome sequencing using four K. pneumoniae mutants including two KPC producers and one NDM producer that had shown CFDC MIC increases in previous in vitro resistance-acquisition studies. The mutant strains were obtained at the frequency of 10-7 to < 10-8 by spreading bacteria on standard Mueller‒Hinton agar medium containing CFDC at concentrations of 10× MIC, with or without apo-transferrin (20 μg/mL). CFDC MIC was determined by broth microdilution using iron-depleted cation-adjusted Mueller-Hinton broth based on Clinical and Laboratory Standards Institute guidelines. The emergence of MIC increase mutants was also assessed by in vitro chemostat models under humanized plasma pharmacokinetic exposures of CFDC. Results The possible resistance mechanisms were investigated. Mutation of baeS or envZ, sensors of two-component regulation systems, were found in three or two mutants among the tested four isolates, respectively, and caused the MIC to increase by 4–32-fold. The altered expression level of specific genes by the baeS or envZ mutation could affect CFDC susceptibility, but the specific genes have not been identified. In addition, the mutation of exbD, an accessory protein related to iron transport, was identified in one case and caused the MIC to increase by >8-fold. In vitro chemostat studies using two isolates (one NDM producer and one KPC producer) showed no resistance acquisition during 24-hour exposure. Table. Overview of mutation emergence in five isolates of K. pneumoniae Conclusion The mutation of two-component regulation systems (BaeSR and OmpR/EnvZ) and iron transport-related proteins were shown to be possible mechanisms causing CFDC MIC increases, but these mutants did not appear under human exposures. Disclosures Yoshinori Yamano, PhD, Shionogi & Co., Ltd. (Employee) Rio Nakamura, BSc, Shionogi & Co., Ltd. (Employee) Miki Takemura, MSc, Shionogi & Co., Ltd. (Employee) Roger Echols, MD, Shionogi Inc. (Consultant)

scholarly journals The inhibitory effect of silencing CDCA3 on migration and proliferation in bladder urothelial carcinoma

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dexin Shen ◽  
Yayun Fang ◽  
Fenfang Zhou ◽  
Zhao Deng ◽  
Kaiyu Qian ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Urothelial Carcinoma ◽  
Carcinoma Cells ◽  
Expression Level ◽  
Tumor Cell Growth ◽  
Migration Ability ◽  
Bladder Urothelial Carcinoma ◽  
Related Proteins

Abstract Background CDCA3 is an important component of the E3 ligase complex with SKP1 and CUL1, which could regulate the progress of cell mitosis. CDCA3 has been widely identified as a proto-oncogene in multiple human cancers, however, its role in promoting human bladder urothelial carcinoma has not been fully elucidated. Methods Bioinformatic methods were used to analyze the expression level of CDCA3 in human bladder urothelial carcinoma tissues and the relationship between its expression level and key clinical characteristics. In vitro studies were performed to validate the specific functions of CDCA3 in regulating cell proliferation, cell migration and cell cycle process. Alterations of related proteins was investigated by western blot assays. In vivo studies were constructed to validate whether silencing CDCA3 could inhibit the proliferation rate in mice model. Results Bioinformatic analysis revealed that CDCA3 was significantly up-regulated in bladder urothelial carcinoma samples and was related to key clinical characteristics, such as tumor grade and metastasis. Moreover, patients who had higher expression level of CDCA3 tend to show a shorter life span. In vitro studies revealed that silencing CDCA3 could impair the migration ability of tumor cells via down-regulating EMT-related proteins such as MMP9 and Vimentin and inhibit tumor cell growth via arresting cells in the G1 cell cycle phase through regulating cell cycle related proteins like p21. In vivo study confirmed that silencing CDCA3 could inhibit the proliferation of bladder urothelial carcinoma cells. Conclusions CDCA3 is an important oncogene that could strengthen the migration ability of bladder urothelial carcinoma cells and accelerate tumor cell growth via regulating cell cycle progress and is a potential biomarker of bladder urothelial carcinoma.

scholarly journals Expression of laminin γ 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter

1996 ◽  
Vol 313 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Françoise LEVAVASSEUR ◽  
Jocelyne LIÉTARD ◽  
Kohei OGAWA ◽  
Nathalie THÉRET ◽  
Peter D. BURBELO ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Basement Membranes ◽  
Major Protein ◽  
Cultured Hepatocytes

Laminin γ1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin γ1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin γ1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5´ flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin γ1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a Mr 60000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

Biochemical and pharmacokinetic characterisation of two PEGylated variants of dipetarudin

2009 ◽  
Vol 102 (09) ◽  
pp. 454-459 ◽  
Author(s):  
Anne Koehler ◽  
Goetz Nowak ◽  
Mercedes López
Keyword(s):  
Gel Filtration ◽  
Pharmacokinetic Profile ◽  
Peripheral Compartment ◽  
Therapeutic Application ◽  
Similar Activity ◽  
Elimination Half ◽  
K 12 ◽  
Extravascular Compartment

SummaryDipetarudin was coupled to polyethylene glycol (PEG)-5000 residues in order to improve its pharmacokinetic profile and to enhance its anticoagulant efficacy. The resulting compounds, mono-and di-PEGylated dipetarudin were purified by gel filtration. Mono-PEGylated dipetarudin exhibited similar activity like its non-conjugated equivalent both in vitro and in vivo. However, di-PEGylated dipetarudin showed longer distribution and elimination half-lives and higher area under the time-concentration curve in comparison with the unmodified inhibitor which may be attributed to decreased renal clearance. Futhermore, ratio k 12/k 21 decreased when the number of PEG chains coupled to dipetarudin increased. It means that the intercompartment transfer of dipetarudin, characterised by a fast distribution and a high retention in the peripheral compartment, is reverted by coupling to PEG. Thus, the transfer of mono-PEGylated dipetarudin between these compartments is similar in both senses and the transfer of di-PEGylated dipetarudin is slower from vascular to extravascular compartment than vice versa. Our results show that di-PEGylated dipetarudin produces a better and longer anticoagulant effect than unmodified dipetarudin which is a desirable attribute for future therapeutic application.

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