scholarly journals In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques.

1978 ◽  
Vol 79 (1) ◽  
pp. 217-234 ◽  
Author(s):  
P J Oates ◽  
O Touster
Keyword(s):  
Electron Microscope ◽  
Light Microscope ◽  
Fusion Reaction ◽  
Biochemical Properties ◽  
New Techniques ◽  
Vacuole Fusion ◽  
Low Concentrations ◽  
Reproducible Manner

To investigate the properties of phagolysosome (PL) fusion in Acanthamoeba homogenates, it was necessary to develop reliable methods for measuring in vitro PL fusion. The need to distinguish PL fusion from PL adhesion was met by the development of a quantitative electron microscope assay. Initial characterization of the fusion reaction by this method was followed by the development of a more rapid light microscope assay. Results obtained by the two methods were found to be in close agreement. By use of these new techniques, the in vitro PL fusion reaction was demonstrated to occur in a quantitatively reproducible manner. Under the present conditions employed, PL breakdown was not detected at any time during the in vitro incubation, while PL fusion was observed to proceed linearly for approximately 10 min, at which time the reaction ceased. Incubation of mixtures of two distinct PL types resulted in increases in hybrid PL types that were paralleled by decreases in nonhybrid PL types. The relative changes in PL concentrations observed were quantitatively consistent with PL fusion occurring randomly with respect to PL type. PL fusion was strongly inhibited by low concentrations of KF (50% inhibition at 2.7 mM), and by approximately tenfold higher concentrations of KCl, while KCN and 2,4-dinitrophenol (2,4-DNP) had little effect. In addition to further defining the nature of the PL fusion reaction in this system, these results demonstrate that, by use of the techniques described, quantitative study of the biochemical properties of this reaction is now possible.

scholarly journals Plasticity in Neuroblastoma Cell Identity Defines a Noradrenergic-to-Mesenchymal Transition (NMT)

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2904
Author(s):  
Margot Gautier ◽  
Cécile Thirant ◽  
Olivier Delattre ◽  
Isabelle Janoueix-Lerosey
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Biochemical Properties ◽  
Poor Overall Survival ◽  
Cell Identity ◽  
Mesenchymal Transition ◽  
Neuroblastoma Cell Lines

Neuroblastoma, a pediatric cancer of the peripheral sympathetic nervous system, is characterized by an important clinical heterogeneity, and high-risk tumors are associated with a poor overall survival. Neuroblastoma cells may present with diverse morphological and biochemical properties in vitro, and seminal observations suggested that interconversion between two phenotypes called N-type and S-type may occur. In 2017, two main studies provided novel insights into these subtypes through the characterization of the transcriptomic and epigenetic landscapes of a panel of neuroblastoma cell lines. In this review, we focus on the available data that define neuroblastoma cell identity and propose to use the term noradrenergic (NOR) and mesenchymal (MES) to refer to these identities. We also address the question of transdifferentiation between both states and suggest that the plasticity between the NOR identity and the MES identity defines a noradrenergic-to-mesenchymal transition, reminiscent of but different from the well-established epithelial-to-mesenchymal transition.

Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

1985 ◽  
Vol 22 (4) ◽  
pp. 375-386 ◽  
Author(s):  
H. C. Wimberly ◽  
D. O. Slauson ◽  
N. R. Neilsen
Keyword(s):  
Functional Activity ◽  
Biochemical Characterization ◽  
Platelet Activating Factor ◽  
Biochemical Properties ◽  
Naturally Occurring ◽  
Rabbit Platelets ◽  
Rapid Reaction ◽  
Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

scholarly journals Characterization of human pre-elafin mutants: full antipeptidase activity is essential to preserve lung tissue integrity in experimental emphysema

2007 ◽  
Vol 405 (3) ◽  
pp. 455-463 ◽  
Author(s):  
Alain Doucet ◽  
Dominique Bouchard ◽  
Marie France Janelle ◽  
Audrey Bellemare ◽  
Stéphane Gagné ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Neutrophil Elastase ◽  
Tight Binding ◽  
Biochemical Properties ◽  
Van Der Waals Interactions ◽  
Tissue Destruction ◽  
Methionine Residue ◽  
Experimental Emphysema

Pre-elafin is a tight-binding inhibitor of neutrophil elastase and myeloblastin; two enzymes thought to contribute to tissue damage in lung emphysema. Previous studies have established that pre-elafin is also an effective anti-inflammatory molecule. However, it is not clear whether both functions are linked to the antipeptidase activity of pre-elafin. As a first step toward elucidating the structure/function relationship of this protein, we describe here the construction and characterization of pre-elafin variants with attenuated antipeptidase potential. In these mutants, the P1′ methionine residue of the inhibitory loop is replaced by either a lysine (pre-elafinM25K) or a glycine (pre-elafinM25G) residue. Both mutated variants are stable and display biochemical properties undistinguishable from WT (wild-type) pre-elafin. However, compared with WT pre-elafin, their inhibitory constants are increased by one to four orders of magnitude toward neutrophil elastase, myeloblastin and pancreatic elastase, depending on the variants and enzymes tested. As suggested by molecular modelling, this attenuated inhibitory potential correlates with decreased van der Waals interactions between the variants and the enzymes S1′ subsite. In elastase-induced experimental emphysema in mice, only WT pre-elafin protected against tissue destruction, as assessed by the relative airspace enlargement measured using lung histopathological sections. Pre-elafin and both mutants prevented transient neutrophil alveolitis. However, even the modestly affected pre-elafinM25K mutant, as assayed in vitro with small synthetic substrates, was a poor inhibitor of the neutrophil elastase and myeloblastin elastolytic activity measured with insoluble elastin. We therefore conclude that full antipeptidase activity of pre-elafin is essential to protect against lung tissue lesions in this experimental model.

scholarly journals Synthesis and characterization of silver nanoparticle-decorated cobalt nanocomposites (Co@AgNPs) and their density-dependent antibacterial activity

2019 ◽  
Vol 6 (5) ◽  
pp. 182135 ◽  
Author(s):  
Zakia Kanwal ◽  
Muhammad Akram Raza ◽  
Saira Riaz ◽  
Saher Manzoor ◽  
Asima Tayyeb ◽  
...  
Keyword(s):  
Metallic Nanoparticles ◽  
Cobalt Acetate ◽  
X Ray Diffraction ◽  
Characteristic Analysis ◽  
Density Dependent ◽  
Antibacterial Performance ◽  
Magnetic Cores ◽  
Low Concentrations

Magnetic cores loaded with metallic nanoparticles can be promising nano-carriers for successful drug delivery at infectious sites. We report fabrication, characteristic analysis and in vitro antibacterial performance of nanocomposites comprising cobalt cores (Co-cores) functionalized with a varied concentration of silver nanoparticles (AgNPs). A two-step polyol process synchronized with the transmetalation reduction method was used. Co-cores were synthesized with cobalt acetate, and decoration of AgNPs was carried out with silver acetate. The density of AgNPs was varied by changing the amount of silver content as 0.01, 0.1 and 0.2 g in the synthesis solution. Both AgNPs and Co-cores were spherical having a size range of 30–80 nm and 200 nm to more than 1 µm, respectively, as determined by scanning electron microscopy. The metallic nature and face-centred cubic crystalline phase of prepared nanocomposites were confirmed by X-ray diffraction. Biocompatibility analysis confirmed high cell viability of MCF7 at low concentrations of tested particles. The antibacterial performance of nanocomposites (Co@AgNPs) against Escherichia coli and Bacillus subtilis was found to be AgNPs density-dependent, and nanocomposites with the highest AgNPs density exhibited the maximum bactericidal efficacy. We therefore propose that Co@AgNPs as effective drug containers for various biomedical applications.

Differential Regulation of Articular Cartilage Biomechanical and Biochemical Properties by IGF-1 and TGF-β1 During In Vitro Growth

2009 ◽  
Author(s):  
Michael E. Stender ◽  
Christian R. Flores ◽  
Kristin J. Dills ◽  
Gregory M. Williams ◽  
Kevin M. Stewart ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Growth Models ◽  
Biochemical Properties ◽  
Low Friction ◽  
Cartilage Growth ◽  
Detailed Characterization ◽  
Naturally Occurring ◽  
Tgf Β1

Articular cartilage (AC) is a load bearing material that provides a low friction wear resistant interface in synovial joints. Naturally-occurring and stimulated intrinsic repair of damaged AC is ineffective. Thus, there is a desire to engineer effective replacement tissue that could be used for AC repair. Previous studies [1] have shown that culture of immature cartilage with medium including TGF-β1 will result in a more mature tissue than culture with IGF-1. Detailed characterization of tissue mechanical properties would be helpful for development of cartilage growth models [2].

scholarly journals RADIOAUTOGRAPHIC VISUALIZATION OF THE INCORPORATION OF GALACTOSE-3H AND MANNOSE-3H BY RAT THYROIDS IN VITRO IN RELATION TO THE STAGES OF THYROGLOBULIN SYNTHESIS

1969 ◽  
Vol 43 (2) ◽  
pp. 289-311 ◽  
Author(s):  
P. Whur ◽  
Annette Herscovics ◽  
C. P. Leblond
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Golgi Apparatus ◽  
Rough Endoplasmic Reticulum ◽  
Light Microscope ◽  
Detectable Effect ◽  
Apical Vesicles ◽  
Rat Thyroid

Rat thyroid lobes incubated with mannose-3H, galactose-3H, or leucine-3H, were studied by radioautography. With leucine-3H and mannose-3H, the grain reaction observed in the light microscope is distributed diffusely over the cells at 5 min, with no reaction over the colloid. Later, the grains are concentrated towards the apex, and colloid reactions begin to appear by 2 hr. With galactose-3H, the reaction at 5 min is again restricted to the cells but it consists of clumped grains next to the nucleus. Soon after, grains are concentrated at the cell apex and colloid reactions appear in some follicles as early as 30 min. Puromycin almost totally inhibits incorporation of leucine-3H and mannose-3H, but has no detectable effect on galactose-3H incorporation during the 1st hr. Quantitation of electron microscope radioautographs shows that mannose-3H label localizes initially in the rough endoplasmic reticulum, and by 1–2 hr much of this reaction is transferred to the Golgi apparatus. At 3 hr and subsequently, significant reactions are present over apical vesicles and colloid, while the Golgi reaction declines. Label associated with galactose-3H localizes initially in the Golgi apparatus and rapidly transfers to the apical vesicles, and then to the colloid. These findings indicate that mannose incorporation into thyroglobulin precursors occurs within the rough endoplasmic reticulum; these precursors then migrate to the Golgi apparatus, where galactose incorporation takes place. The glycoprotein thus formed migrates via the apical vesicles to the colloid.

scholarly journals Characterization of a Novel Lytic Protein Encoded by the Bacillus cereus E33L GeneampDas a Bacillus anthracis Antimicrobial Protein

2012 ◽  
Vol 78 (8) ◽  
pp. 3025-3027 ◽  
Author(s):  
Feliza A. Bourguet ◽  
Brian E. Souza ◽  
Angela K. Hinz ◽  
Matthew A. Coleman ◽  
Paul J. Jackson
Keyword(s):  
Bacillus Cereus ◽  
Bacillus Anthracis ◽  
Antimicrobial Protein ◽  
Cell Wall Biosynthesis ◽  
Bacterial Genomes ◽  
Content Type ◽  
Highly Active ◽  
Low Concentrations

ABSTRACTLytic proteins encoded by bacterial genomes have been implicated in cell wall biosynthesis and recycling. TheBacillus cereusE33LampDgene encodes a putativeN-acetylmuramoyl-l-alanine amidase. This gene, expressedin vitro, produced a very stable, highly active lytic protein. Very low concentrations rapidly and efficiently lyse vegetativeBacillus anthraciscells.

scholarly journals Characterization of Streptococcus gordonii SecA2 as a Paralogue of SecA

2009 ◽  
Vol 191 (11) ◽  
pp. 3482-3491 ◽  
Author(s):  
Barbara A. Bensing ◽  
Paul M. Sullam
Keyword(s):  
Protein Transport ◽  
Maximal Activity ◽  
Biochemical Properties ◽  
Streptococcus Gordonii ◽  
Lower Solubility ◽  
Highly Sensitive ◽  
Sec System ◽  
High Degree

ABSTRACT The accessory Sec system of Streptococcus gordonii is essential for transport of the glycoprotein GspB to the bacterial cell surface. A key component of this dedicated transport system is SecA2. The SecA2 proteins of streptococci and staphylococci are paralogues of SecA and are presumed to have an analogous role in protein transport, but they may be specifically adapted for the transport of large, serine-rich glycoproteins. We used a combination of genetic and biochemical methods to assess whether the S. gordonii SecA2 functions similarly to SecA. Although mutational analyses demonstrated that conserved amino acids are essential for the function of SecA2, replacing such residues in one of two nucleotide binding folds had only minor effects on SecA2 function. SecA2-mediated transport is highly sensitive to azide, as is SecA-mediated transport. Comparison of the S. gordonii SecA and SecA2 proteins in vitro revealed that SecA2 can hydrolyze ATP at a rate similar to that of SecA and is comparably sensitive to azide but that the biochemical properties of these enzymes are subtly different. That is, SecA2 has a lower solubility in aqueous solutions and requires higher Mg2+ concentrations for maximal activity. In spite of the high degree of similarity between the S. gordonii paralogues, analysis of SecA-SecA2 chimeras indicates that the domains are not readily interchangeable. This suggests that specific, unique contacts between SecA2 and other components of the accessory Sec system may preclude cross-functioning with the canonical Sec system.

Biochemical characterization of human DNA polymerase i provides clues to its biological function

2001 ◽  
Vol 29 (2) ◽  
pp. 183-187 ◽  
Author(s):  
A. Tissier ◽  
E. G. Frank ◽  
J. P. McDonald ◽  
A. Vaisman ◽  
A. R. Fernàndez deHenestrosa Henestrosa ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Biochemical Characterization ◽  
Short Review ◽  
Biochemical Properties ◽  
Dna Polymerase I ◽  
Polymerase I

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase i (poli). The role of poli within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poli and to speculate as to how these biochemical properties might relate to its in vivo function.

The effects of oestradiol and relaxin on extensibility and collagen organisation of the pregnant rat cervix

1995 ◽  
Vol 146 (2) ◽  
pp. 331-337 ◽  
Author(s):  
S H Cheah ◽  
K H Ng ◽  
V T Johgalingam ◽  
M Ragavan
Keyword(s):  
Electron Microscope ◽  
Light Microscope ◽  
Late Pregnancy ◽  
Collagen Fibres ◽  
Thin Sections ◽  
Pregnant Rat ◽  
And Control ◽  
Collagen Organisation

Abstract The effects of exogenously introduced oestradiol-17β (E) and relaxin (RLX) on cervical extensibility and collagen organisation were tested in rats ovariectomised in late pregnancy. When the cervices were stretched in vitro by 1 mm increments, it was found that those from rats given E alone generated significantly higher tensions than those from control rats, while cervices from rats given both E and RLX had tensions similar to controls. Examination of cervical sections under the light microscope and ultra-thin sections under the electron microscope showed that the collagen fibres in the cervices from E-treated rats were highly organised, whereas those from animals given E+RLX and control animals were disorganised and dispersed. It was concluded that E decreased cervical extensibility, while RLX counteracted the effect of E to maintain a soft and easily extensible cervix. Journal of Endocrinology (1995) 146, 331–337

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