scholarly journals Use of glycyl-l-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles

1994 ◽  
Vol 300 (1) ◽  
pp. 229-236 ◽  
Author(s):  
T O Berg ◽  
P E Strømhaug ◽  
T Løvdal ◽  
P O Seglen ◽  
T Berg
Keyword(s):  
Acid Phosphatase ◽  
Degradation Products ◽  
Rat Hepatocytes ◽  
Endocytic Pathway ◽  
Marker Enzyme ◽  
Major Peak ◽  
Isolated Rat Hepatocytes ◽  
Cathepsin C ◽  
Minor Peak ◽  
A Minor

Lysosome-disrupting enzyme substrates have been used to distinguish between lysosomal and prelysosomal compartments along the endocytic pathway in isolated rat hepatocytes. The cells were incubated for various periods of time with 125I-labelled tyramine cellobiose (125I-TC) covalently coupled to asialoorosomucoid (AOM) (125I-TC-AOM); this molecule is internalized by receptor-mediated endocytosis and degraded in lysosomes, where the degradation products (acid-soluble, radio-labelled short peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN) and methionine O-methyl ester (MOM), which are hydrolysed by lysosomal cathepsin C and a lysosomal esterase respectively, both diffused into hepatocytic lysosomes after electrodisruption of the cells. Intralysosomal accumulation of the hydrolysis products (amino acids) of these substrates caused osmotic lysis of more than 90% of the lysosomes, as measured by the release of acid-soluble radioactivity derived from 125I-TC-AOM degradation. The acid-soluble radioactivity coincided in sucrose-density gradients with a major peak of the lysosomal marker enzyme acid phosphatase at 1.18 g/ml; in addition a minor, presumably endosomal, acid phosphatase peak was observed around 1.14 g/ml. The major peak of acid phosphatase was almost completely released by GPN (and by MOM), while the minor peak seemed unaffected by GPN. Acid-insoluble radioactivity, presumably in endosomes, banded (after 1 h of 125I-TC-AOM uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in sucrose gradients, and was not significantly released by GPN. GPN thus appears to be an excellent tool by which to distinguish between endosomes and lysosomes. MOM, on the other hand, released some radioactivity and acid phosphatase from endosomes as well as from lysosomes.

Some aspects of the ecology of bagrid catfishes in a southern Nigerian river

1991 ◽  
Vol 7 (2) ◽  
pp. 221-232 ◽  
Author(s):  
George Idodo-Umeh ◽  
Reginald Victor
Keyword(s):  
Spatial Distribution ◽  
Species Composition ◽  
Rainy Season ◽  
Major Peak ◽  
Fish Yield ◽  
Southern Nigeria ◽  
Minor Peak ◽  
A Minor ◽  
Principal Species ◽  
Dry And Rainy Seasons

ABSTRACTSome aspects of the ecology of bagrid catfishes in River Ase, southern Nigeria were studied for a period of two years. Nine species of Bagridae were recorded and these accounted for 15.0% of the number and 24.4% of the weight of all fish captured. Chrysichthys nigrodigitatus and Chrysichthys auratus longifilis were the principal species. C. nigrodigitatus was a rainy season species, while C. auratus longifilis was abundant in both dry and rainy seasons. Both species showed a major peak in catches between 0600 and 0900 h. C. nigrodigitatus exhibited a minor peak in catches between 1500 and 2100h, while C. auratus longifilis showed a minor peak between 1500 and 1800h. The spatial distribution of C. nigrodigitatus and C. auratus longifilis populations was heterogeneous. Bagrid fishes were an important component in the fish yield of the study river and its species composition has been compared with those of other Nigerian waters. The distribution and abundance of C. nigrodigitatus and C. auratus longifilis are discussed in detail.

A survey of microorganisms for thermonuclease production

1980 ◽  
Vol 26 (4) ◽  
pp. 532-535 ◽  
Author(s):  
C. E. Park ◽  
A. de Melo Serrano ◽  
M. Landgraf ◽  
J. C. Huang ◽  
Z. Stankiewicz ◽  
...  
Keyword(s):  
The Other ◽  
Major Peak ◽  
Coagulase Negative Staphylococci ◽  
Ph Profile ◽  
Streptococcus Faecalis ◽  
Heat Stable ◽  
Minor Peak ◽  
A Minor ◽  
Group D ◽  
Coagulase Positive Staphylococci

A total of 1204 cultures comprising 16 genera were surveyed for production of thermonuclease (TNase) in milk. Cultures other than Staphylococcus capable of TNase production were restricted to two genera, Streptococcus and Bacillus. Nineteen percent of 338 group D streptococci comprising four species (85% of which were Streptococcus faecalis) and 17% of 60 streptococci belonging to other groups produced TNase. Nine percent of 130 Bacillus cultures comprising six species produced the enzyme. On the other hand, 99% of coagulase-positive staphylococci produced TNase and only 18% of the coagulase-negative staphylococci produced the enzyme. The amount of TNase produced by streptococci and bacilli was significantly lower than that produced by coagulase-positive staphylococci. The pH profile of the streptococci and Bacillus TNases was similar to that of the staphylococcal TNase; each enzyme exhibited a minor peak at pH 7.0 and a broad major peak ranging from pH 8.5 to 10. The nuclease produced by coagulase-positive Staphylococcus was more heat stable than the nucleases produced by Streptococcus and Bacillus; there was little loss in activity of the staphylococcal enzyme after 60 min at 100 °C, whereas 50% of the activity of the streptococcal and Bacillus nucleases was destroyed in 40–60 min and 60–80 min, respectively.

Seasonal variation in human births

1990 ◽  
Vol 22 (1) ◽  
pp. 113-119 ◽  
Author(s):  
William H. James
Keyword(s):  
Seasonal Variation ◽  
Seasonal Pattern ◽  
The Other ◽  
European Countries ◽  
Major Peak ◽  
The Us ◽  
Minor Peak ◽  
Human Births ◽  
The Difference ◽  
A Minor

SummaryDuring the first half of this century, the seasonal pattern of births in European countries showed a major peak in the spring and a minor peak in the autumn. In contrast, the pattern in the US was of a minor peak in spring and a major peak in autumn. Over the last 20 years, the pattern in England and Wales has changed to resemble the US pattern, and the same seems to be true of several other European countries. A hypothesis is offered to account for the difference between the European and the US patterns and for the change from one to the other in some countries.The magnitude of seasonality correlates positively with latitude: it is suggested that this is partially consequent on variation in luminosity.

Characterization of immunoreactive dynorphin in human phaeochromocytomas

1988 ◽  
Vol 117 (1) ◽  
pp. 123-132 ◽  
Author(s):  
T. A. Howlett ◽  
G. M. Besser ◽  
L. H. Rees
Keyword(s):  
Acetic Acid ◽  
Gel Filtration ◽  
Molecular Size ◽  
Major Peak ◽  
Gel Filtration Chromatography ◽  
Post Translational Modifications ◽  
Minor Peak ◽  
Wet Weight ◽  
A Minor

ABSTRACT The prodynorphin-derived opioids, dynorphin (DYN) and α-neoendorphin (αNE) were studied in 24 human phaeochromocytomas and related tumours. Nineteen tumours, extracted in HCl (0·1 mol/l), contained concentrations of immunoreactive DYN (ir-DYN) ranging from < 0·5 to 794 pmol/g wet weight. None of the extracts in HCl contained ir-αNE (all < 2·4 pmol/g). Sephadex G-50 gel filtration chromatography of ir-DYN in HCl (0·1 mol/l) extracts of six tumours revealed three small peaks of ir-DYN of higher molecular size (approximately 12 000, 6000 and 3000 daltons), a minor peak of ir-DYN eluting just after DYN(1–17), and a broad major peak, consisting of at least three components, which was significantly retarded and eluted after the salt volume of the column. High-pressure liquid chromatography (HPLC) of these extracts revealed multiple peaks of ir-DYN, most of which did not coelute with any synthetic DYN peptides. On both gel filtration chromatography and HPLC, one of the minor peaks coeluted with DYN(1–32). None of the peaks of ir-DYN coeluted with DYN(1–17) which had been acetylated using acetic anhydride. Extracts of the same tumours in acetic acid (0·1 mol/l) yielded similar values for ir-DYN content, but parallelism in the assay was improved. Sephadex G-50 chromatography revealed a different pattern of ir-DYN with a major peak coeluting with DYN(1–17) and, in two tumours, a minor peak coeluting with DYN(1–8). Studies with HPLC revealed, however, that substantial degradation of synthetic DYN occurred during extraction in acetic acid (0·1 mol/l) in spite of the precautions taken. Phaeochromocytomas frequently contain ir-DYN in concentrations which may approach that of the mammalian pituitary. These tumours did not, however, contain ir-αNE and, with the possible exception of a small amount of DYN(1–32), the ir-DYN present did not correspond with any known sequences. Thus, whilst prodynorphin is expressed in phaeochromocytomas, it does not seem to be processed to the usual end-products, and post-translational modifications therefore seem likely. Enzymatic degradation of DYN may occur during extraction in acetic acid (0·1 mol/l), and this medium should, therefore, be avoided in studies of such labile peptides. J. Endocr. (1988) 117, 123–132

Breeding and Moult in Honeyeaters (Aves : Meliphagidae) near Adelaide, South Australia

1980 ◽  
Vol 7 (3) ◽  
pp. 453 ◽  
Author(s):  
HA Ford
Keyword(s):  
South Australia ◽  
Major Peak ◽  
Limited Data ◽  
Nectar Production ◽  
Similar Time ◽  
Minor Peak ◽  
A Minor ◽  
Increasing Temperature ◽  
Breeding Seasons ◽  
Extensive Breeding

'New Holland honeyeaters Phylidonyris novaehollandiae moult their primary feathers between mid- October and late April. Most adults probably have completed their moult by February, some immatures moult later. Individual birds may take over 100 days to complete moulting their primaries. The secondary and tail feathers are moulted towards the end of the primary moult and slightly after it. Limited data for 15 other species suggest that they moult at a similar time and for a similar duration. Breeding, in the Adelaide area, occurred in all months of the year in at least some species of honeyeaters, with a major peak from July to November, and a minor peak in April and May. Insectivorous species breed somewhat later than nectarivorous species and breed less often in autumn. Breeding of the former group may be influenced chiefly by increasing temperature in spring, whereas autumn rainfall probably influences the timing of peak nectar production. Although there is some overlap between the extensive breeding seasons and moulting periods of honeyeaters I have no evidence that individual honeyeaters breed while they are moulting.

The so-called of Escherichia coli is a stable denatured conformer of the major isoacceptor

1986 ◽  
Vol 64 (4) ◽  
pp. 315-322 ◽  
Author(s):  
Thomas-Louis Tremblay ◽  
Jacques Lapointe
Keyword(s):  
Escherichia Coli ◽  
Salt Concentration ◽  
Growth Conditions ◽  
Major Peak ◽  
E Coli ◽  
Minor Peak ◽  
Trna Species ◽  
Resolution Analysis ◽  
A Minor ◽  
Two Peaks

A single peak of tRNAGlu is obtained upon chromatography of unfractionated tRNA from Escherichia coli on DEAE-Sephadex A-50 if this tRNA was previously renatured, whereas two peaks of tRNAGlu are resolved if the sample chromatographed is a mixture of native (renatured) and denatured tRNA. Higher resolution analysis of native E. coli tRNA by RPC-5 chromatography showed that most of the tRNAGlu is present in one peak, eluted shortly after a minor peak containing about or less than 5% of the total amount of tRNAGlu; these two peaks were also observed with commercially available tRNAGlu purified from E. coli. When denatured, the tRNAGlu present in each of these two peaks was eluted from the RPC-5 column at a much lower salt concentration. The properties of the denatured conformers obtained from native tRNAGlu present in the major and minor peaks, and the variation, with growth conditions of E. coli, in the relative amount of tRNAGlu in the minor peak suggest that the tRNAGlu present in the minor peak is an undermodified form of the tRNAGlu present in the major peak. This [Formula: see text] (or [Formula: see text] when modified in the anticodon) would then be the only tRNA species acceptor of glutamate in E. coli.

Free-flow electrophoresis of the low-density structures that contain asialoglycoproteins in the rat liver

1984 ◽  
Vol 62 (11) ◽  
pp. 1051-1058 ◽  
Author(s):  
Maria T. Debanne ◽  
Maria Bolyos ◽  
Erwin Regoeczi
Keyword(s):  
Acid Phosphatase ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Complete Separation ◽  
Free Flow ◽  
Major Protein ◽  
Differential Centrifugation ◽  
Marker Enzymes ◽  
Minor Peak ◽  
A Minor

Rats were given an intravenous dose (1–2 μg/100 g) of iodine-labelled asialotransferrin, asialofetuin, or asialoorosomucoid either alone or in combinations, and the distribution of the radioactivity in the liver, removed 10–20 min after the injection, was analyzed by free-flow electrophoresis in an Elphor VaP 11 apparatus. Liver homogenates were prepared for electrophoresis according to an elaborate ultracentrifugation scheme that is outlined in detail with respect to conditions and yields. The scheme involved differential centrifugation, followed by density gradient centrifugation in a linear sucrose gradient and gel filtration using Sepharose 2B. Two ligand-containing fractions were obtained during differential centrifugation, each associated with a different complement of subcellular marker enzymes. On free-flow electrophoresis, the ligand present in either fraction exhibited a major and a minor peak. They were incompletely separated, the minor peak shouldering on the major one. The major peak had a higher electrophoretic mobility than the peaks of the acid phosphatase and phosphodiesterase I activities, but it had the same mobility as the sialyltransferase activity. The minor, less electronegative peak comigrated with the peaks of acid phosphatase and phosphodiesterase I activities and also with the major protein component of the subcellular fraction. It is concluded that the asialoglycoprotein-transporting subcellular vesicles are heterogeneous in regard to charge and that their complete separation from subcellular marker enzymes cannot be accomplished by free-flow electrophoresis.

scholarly journals Phosphoinositide 3-kinase regulates maturation of lysosomes in rat hepatocytes

2003 ◽  
Vol 372 (3) ◽  
pp. 861-869 ◽  
Author(s):  
Seyed Ali MOUSAVI ◽  
Andreas BRECH ◽  
Trond BERG ◽  
Rune KJEKEN
Keyword(s):  
Intracellular Transport ◽  
Degradation Products ◽  
Buoyant Density ◽  
Rat Hepatocytes ◽  
Amino Acid Mixture ◽  
Endocytic Pathway ◽  
Acid Mixture ◽  
Density Distributions ◽  
Late Endosomes ◽  
Phosphoinositide 3 Kinase

To obtain information about the role of phosphoinositide 3-kinase (PI3K) in the endocytic pathway in hepatocytes, the uptake and intracellular transport of asialo-orosomucoid (ASOR) was followed in cells treated with wortmannin or LY294002. The two inhibitors, at concentrations known to inhibit the enzyme, did not affect internalization or the number of surface asialoglycoprotein receptors, but they caused a paradoxical increase (approx. 50% above control values) in the degradation of ASOR labelled with [125I]tyramine cellobiose ([125I]TC). Wortmannin or LY204002 inhibited the autophagic sequestration of lactate dehydrogenase very effectively, and the enhanced degradation of [125I]TC-ASOR could be an indirect effect of reduced autophagy, as an amino acid mixture known to inhibit autophagy also caused increased degradation of [125I]TC-ASOR, and its effect was not additive to that of wortmannin or LY294002. Wortmannin or LY294002 had pronounced effects on the late parts of the endocytic pathway in the hepatocytes: first, dense lysosomes disappeared and were replaced by swollen vesicles; secondly, degradation of [125I]TC-ASOR took place in an organelle of lower buoyant density (in a sucrose gradient) than the bulk of lysosomes (identified in the gradient by lysosomal marker enzymes). With increasing length of incubation with wortmannin or LY294002, the density distributions of the lysosomal markers also shifted to lower density and gradually approached that of the labelled degradation products. The labelled degradation products formed from [125I]TC-labelled proteins were trapped at the site of formation, because they did not penetrate the vesicle membranes. The results obtained indicate that internalization and intracellular transport of ASOR to lysomes may take place in the absence of PI3K activity in rat hepatocytes. On the other hand, fusion of late endosomes with lysosomes seems to produce ‘hybrid organelles’ (active lysosomes) that are unable to mature into dense lysosomes.

scholarly journals The Autophagic and Endocytic Pathways Converge at the Nascent Autophagic Vacuoles

1997 ◽  
Vol 136 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Willisa Liou ◽  
Hans J. Geuze ◽  
Math. J.H. Geelen ◽  
Jan W. Slot
Keyword(s):  
Rat Hepatocytes ◽  
Endocytic Pathway ◽  
Fusion Partner ◽  
Isolated Rat Hepatocytes ◽  
Autophagic Vacuoles ◽  
Exponential Increase ◽  
Morphological Criteria ◽  
Endosomal Vesicles ◽  
Multivesicular Endosomes ◽  
Endocytic Pathways

We used an improved cryosectioning technique in combination with immunogold cytochemistry and morphometric analysis to study the convergence of the autophagic and endocytic pathways in isolated rat hepatocytes. The endocytic pathway was traced by continuous uptake of gold tracer for various time periods, up to 45 min, while the cells were incubated in serum-free medium to induce autophagy. Endocytic structures involved in fusion with autophagic vacuoles (AV) were categorized into multivesicular endosomes (MVE) and vesicular endosomes (VE). Three types of AV—initial (AVi), intermediate (AVi/d), and degradative (AVd)—were defined by morphological criteria and immunogold labeling characteristics of marker enzymes. The entry of tracer into AV, manifested as either tracer-containing AV profiles (AV+) or fusion profiles (FP+) between AV and tracer-positive endosomal vesicles/vacuoles, was detected as early as 10 min after endocytosis. The number of AV+ exhibited an exponential increase with time. FP+ between MVE or VE and all three types of AV were observed. Among the 112 FP+ scored, 36% involved VE. Of the AV types, AVi and AVi/d were found five to six times more likely to be involved in fusions than AVd. These fusion patterns did not significantly change during the period of endocytosis (15–45 min). We conclude that the autophagic and endocytic pathways converge in a multistage fashion starting within 10 min of endocytosis. The nascent AV is the most upstream and preferred fusion partner for endosomes.

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