scholarly journals SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY

1974 ◽  
Vol 61 (3) ◽  
pp. 789-807 ◽  
Author(s):  
Gert Kreibich ◽  
David D. Sabatini
Keyword(s):  
Serum Proteins ◽  
Specific Activity ◽  
Membrane Vesicles ◽  
Rat Serum ◽  
Coomassie Blue ◽  
Specific Subset ◽  
Low Levels ◽  
Almost All

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [3H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [3H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [3H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.

scholarly journals Cycle initiation and colony formation in culture by murine marrow cells with long-term reconstituting potential in vivo

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4149-4158 ◽  
Author(s):  
M Trevisan ◽  
XQ Yan ◽  
NN Iscove
Keyword(s):  
Colony Formation ◽  
Interleukin 1 ◽  
Methyl Cellulose ◽  
Culture Conditions ◽  
Liquid Suspension ◽  
Almost All ◽  
Marrow Cells

Abstract This investigation was directed at separating long-term reconstituting (LTR) stem cells in normal murine marrow from hematopoietic precursors detectable in short-term assays in vitro and in vivo, and then at determining whether purified LTR cells could themselves form colonies in culture. To do so, it was first necessary to identify culture conditions that would induce their growth while preserving their long- term reconstituting capacity. Marrow was cultured with various cytokines in liquid suspension for 4 days, after which the surviving LTR activity was quantitated in a competitive in vivo assay. Activity was preserved near input levels with combined murine c-kit ligand (KL), interleukin-1 (IL-1), IL-6, and IL-11. When the cultures also included tritiated or unlabeled thymidine, LTR potential was eliminated, indicating that essentially all LTR cells were induced into cell cycle with these cytokines. To purify them, marrow was sorted on the basis of Ly6A expression and Rhodamine 123 retention. The Ly6AhiRh123ls fraction contained 85% of total recovered LTR activity but only 1% of the recovered cells measured by multilineage colony formation in spleens or in vitro. This fraction was cultured in methyl cellulose with KL, IL-1, IL-6, and IL-11 for 4 to 6 days, after which colonies were isolated and injected into mice. High levels of permanent reconstitution were achievable in sublethally irradiated W41/W41 mice after the injection of a single reconstituting unit, and limiting dilution analysis estimated the frequency of multilineage LTR at 1 in 11,200 unpurified adult marrow cells. In either lethally irradiated normal or sublethally irradiated W41/W41 mice, 1-year lymphomyeloid reconstitutions were obtained from 1 in 65 to 84 colonies of 2 to 16 dispersed cells, but not from larger colonies or those with clumped cells. The results establish that resting marrow LTR cells can be separated from almost all of the more advanced clonogenic cells that are still pluripotential, can be induced to cycle in culture by defined cytokines with preservation of their reconstituting potential, and can be manipulated and assayed efficiently at single-cell and colony levels.

Cannabinoid effects on adenylate cyclase and phosphodiesterase activities of mouse brain

1977 ◽  
Vol 55 (4) ◽  
pp. 934-942 ◽  
Author(s):  
Thomas W. Dolby ◽  
Lewis J. Kleinsmith
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Mouse Brain ◽  
Biogenic Amine ◽  
Specific Activity ◽  
Intraperitoneal Administration ◽  
The Brain

The experiments presented in this paper examine the mechanisms underlying the ability of cannabinoids to alter the in vivo levels of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) in mouse brain. It was found that changes in cyclic AMP levels are a composite result of direct actions of cannabinoids on adenylate cyclase (EC 4.6.1.1) activity and indirect actions involving the potentiation or inhibition of biogenic amine induced activity of adenylate cyclase. Furthermore, the long-term intraperitoneal administration of 1-(−)-Δ-tetrahydrocannabinol to mice produced a form of phosphodiesterase (EC 3.1.4.17) in the brain whose activity is not stimulated by Ca2+, although its basal specific activity is similar to that of control animals. In vitro, the presence of the cannabinoids caused no significant changes in activity of brain PDE at the concentrations tested. Some correlations are presented which imply that many of the observed behavioral and physiological actions of the cannabinoids in mammalian organisms may be mediated via cyclic AMP mechanisms.

Structural properties of rat serum proteins which correlate with their degradative rates in vivo

Nature ◽  
1976 ◽  
Vol 262 (5568) ◽  
pp. 514-516 ◽  
Author(s):  
J. FRED DICE ◽  
ALFRED L. GOLDBERG
Keyword(s):  
Structural Properties ◽  
Serum Proteins ◽  
Rat Serum

scholarly journals Biochemical Characterization of Phenylacetaldehyde Dehydrogenases from Styrene-degrading Soil Bacteria

2020 ◽  
Author(s):  
Juliane Zimmerling ◽  
Michel Oelschlägel ◽  
Carolin Großmann ◽  
Matthias Voitel ◽  
Michael Schlömann ◽  
...  
Keyword(s):  
Esterase Activity ◽  
Soil Bacteria ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Aldehyde Dehydrogenases ◽  
Long Term Stability ◽  
Almost All ◽  
Different Characteristics

Abstract Four phenylacetaldehyde dehydrogenases (designated as FeaB or StyD) originating from styrene-degrading soil bacteria were biochemically investigated. In this study, we focused on the Michaelis-Menten kinetics towards the presumed native substrate phenylacetaldehyde and the obviously preferred co-substrate NAD+. Furthermore, the substrate specificity on four substituted phenylacetaldehydes and the co-substrate preference were studied. Moreover, these enzymes were characterized with respect to their temperature as well as long-term stability. Since aldehyde dehydrogenases are known to show often dehydrogenase as well as esterase activity, we tested this capacity, too. Almost all results showed clearly different characteristics between the FeaB and StyD enzymes. Furthermore, FeaB from Sphingopyxis fribergensis Kp5.2 turned out to be the most active enzyme with an apparent specific activity of 17.8 ± 2.1 U mg-1. Compared with that, both StyDs showed only activities less than 0.2 U mg-1 except the overwhelming esterase activity of StyD-CWB2 (1.4 ± 0.1 U mg-1). The clustering of both FeaB and StyD enzymes with respect to their characteristics could also be mirrored in the phylogenetic analysis of twelve dehydrogenases originating from different soil bacteria.

Ontogeny of prolyl endopeptidase, pyroglutamyl peptidase I, TRH, and its metabolites in rat pancreas

1992 ◽  
Vol 262 (6) ◽  
pp. E845-E850
Author(s):  
P. Salers ◽  
L. H. Ouafik ◽  
P. Giraud ◽  
J. Y. Maltese ◽  
A. Dutour ◽  
...  
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Early Stage ◽  
Prolyl Endopeptidase ◽  
Neonatal Rat ◽  
Rat Pancreas ◽  
Low Levels ◽  
Intact Rats

We demonstrate that two enzymes, soluble unspecific pyroglutamyl peptidase I and prolyl endopeptidase, able to degrade thyrotropin-releasing hormone (TRH) in vitro were present in pancreas at the early stage of rat development. Specific particulate pyroglutamyl peptidase II remained undetectable during ontogenesis. Pyroglutamyl peptidase I specific activity increased until day 3 and decreased after day 5. Furthermore, prolyl endopeptidase specific activity rose slightly to a peak on postnatal day 20. A good correlation between immunoreactive TRH and deaminated TRH (TRH-OH) was found in the 1st wk after birth. However, His-Pro diketopiperazine (DKP) levels were stable and low during development. We show that hot acidic extraction conditions could artefactually generate His-Pro DKP. In vivo, active site-directed inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase enzymes do not show any TRH-deamidating and/or pyroglutamyl peptidase I pathways in neonatal rat pancreas. The data suggest that these two enzymes are not involved in intra- or extracellular control of TRH levels in neonatal rat pancreas and that pancreatic TRH content appears to be principally regulated by biosynthetic steps. Nevertheless, low levels of endogenous His-Pro DKP and TRH-OH identified in neonatal rat pancreas suggest that TRH or TRH-like peptides may be metabolized in this tissue in intact rats, albeit at low rates.

scholarly journals THE SECRETORY PATHWAYS OF RAT SERUM GLYCOPROTEINS AND ALBUMIN

1972 ◽  
Vol 52 (2) ◽  
pp. 231-245 ◽  
Author(s):  
Colvin M. Redman ◽  
M. George Cherian
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Rough Endoplasmic Reticulum ◽  
Serum Proteins ◽  
Rat Serum ◽  
Secretory Pathways ◽  
Specific Antisera ◽  
Microsome Fraction

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein. N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membrane-attached polysomes and not into proteins from free polysomes. Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes. Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae. Albumin was mostly recovered (98%) in the cisternae, with negligible amounts in the membranes. To determine whether the radio-active sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the 14C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins. The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae.

THIOCYANATE ION IN SERUM AS AN INTERFERING FACTOR IN THE IN-VITRO BIOASSAY OF THYROTROPHIN

1973 ◽  
Vol 57 (1) ◽  
pp. 1-6 ◽  
Author(s):  
J. R. BOURKE ◽  
S. W. MANLEY ◽  
R. W. HAWKER
Keyword(s):  
Specific Activity ◽  
Cobalt Nitrate ◽  
Ferric Nitrate ◽  
In Vitro Bioassay ◽  
Rat Serum ◽  
Thiocyanate Ion ◽  
Serum Thiocyanate ◽  
In Vitro Bioassays

SUMMARY The presence of thiocyanate ion in human and rat serum has been shown to account entirely for the non-specific activity of sera in an in-vitro bioassay for thyrotrophin. Thiocyanate was identified by its chromatographic behaviour on Sephadex G-10, G-15 and G-25, and by the ferric nitrate and cobalt nitrate tests. Cigarette smoking increased mean serum thiocyanate levels (as NaSCN) from 0·2 to 0·56 mg/100 ml. It is suggested that serum thiocyanate levels are sufficient to inhibit significantly iodide trapping in vivo and that these findings may be relevant to the non-specific responses observed with other in-vitro bioassays based on radioiodine dynamics.

scholarly journals Cycle initiation and colony formation in culture by murine marrow cells with long-term reconstituting potential in vivo

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4149-4158 ◽  
Author(s):  
M Trevisan ◽  
XQ Yan ◽  
NN Iscove
Keyword(s):  
Colony Formation ◽  
Interleukin 1 ◽  
Methyl Cellulose ◽  
Culture Conditions ◽  
Liquid Suspension ◽  
Almost All ◽  
Marrow Cells

This investigation was directed at separating long-term reconstituting (LTR) stem cells in normal murine marrow from hematopoietic precursors detectable in short-term assays in vitro and in vivo, and then at determining whether purified LTR cells could themselves form colonies in culture. To do so, it was first necessary to identify culture conditions that would induce their growth while preserving their long- term reconstituting capacity. Marrow was cultured with various cytokines in liquid suspension for 4 days, after which the surviving LTR activity was quantitated in a competitive in vivo assay. Activity was preserved near input levels with combined murine c-kit ligand (KL), interleukin-1 (IL-1), IL-6, and IL-11. When the cultures also included tritiated or unlabeled thymidine, LTR potential was eliminated, indicating that essentially all LTR cells were induced into cell cycle with these cytokines. To purify them, marrow was sorted on the basis of Ly6A expression and Rhodamine 123 retention. The Ly6AhiRh123ls fraction contained 85% of total recovered LTR activity but only 1% of the recovered cells measured by multilineage colony formation in spleens or in vitro. This fraction was cultured in methyl cellulose with KL, IL-1, IL-6, and IL-11 for 4 to 6 days, after which colonies were isolated and injected into mice. High levels of permanent reconstitution were achievable in sublethally irradiated W41/W41 mice after the injection of a single reconstituting unit, and limiting dilution analysis estimated the frequency of multilineage LTR at 1 in 11,200 unpurified adult marrow cells. In either lethally irradiated normal or sublethally irradiated W41/W41 mice, 1-year lymphomyeloid reconstitutions were obtained from 1 in 65 to 84 colonies of 2 to 16 dispersed cells, but not from larger colonies or those with clumped cells. The results establish that resting marrow LTR cells can be separated from almost all of the more advanced clonogenic cells that are still pluripotential, can be induced to cycle in culture by defined cytokines with preservation of their reconstituting potential, and can be manipulated and assayed efficiently at single-cell and colony levels.

scholarly journals Deep conservation of histone variants in Thermococcales archaea

2021 ◽  
Author(s):  
Kathryn M Stevens ◽  
Antoine Hocher ◽  
Tobias Warnecke
Keyword(s):  
Histone Variants ◽  
Prima Facie ◽  
Transcriptional Responses ◽  
Functional Roles ◽  
Histone Fold ◽  
Dna Accessibility ◽  
Almost All

AbstractHistones are ubiquitous in eukaryotes where they assemble into nucleosomes, binding and wrapping DNA to form chromatin. One process to modify chromatin and regulate DNA accessibility is the replacement of histones in the nucleosome with paralogous variants. Histones are also present in archaea but whether and how histone variants contribute to the generation of different physiologically relevant chromatin states in these organisms remains largely unknown. Conservation of paralogs with distinct properties can provide prima facie evidence for defined functional roles. We recently revealed deep conservation of histone paralogs with different properties in the Methanobacteriales, but little is known experimentally about these histones. In contrast, the two histones of the model archaeon Thermococcus kodakarensis, HTkA and HTkB, have been examined in some depth, both in vitro and in vivo. HTkA and HTkB exhibit distinct DNA-binding behaviours and elicit unique transcriptional responses when deleted. Here, we consider the evolution of HTkA/B and their orthologs across the order Thermococcales. We find histones with signature HTkA- and HTkB-like properties to be present in almost all Thermococcales genomes. Phylogenetic analysis indicates the presence of one HTkA- and one HTkB-like histone in the ancestor of Thermococcales and long-term maintenance of these two paralogs throughout Thermococcales diversification. Our results support the notion that archaea and eukaryotes have convergently evolved histone variants that carry out distinct adaptive functions. Intriguingly, we also detect more highly diverged histone-fold proteins, related to those found in some bacteria, in several Thermococcales genomes. The functions of these bacteria-type histones remain entirely unknown, but structural modelling suggests that they can form heterodimers with HTkA/B-like histones.

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