Biosynthesis and glycosylation of serine/threonine-rich secreted proteins from Toxocara canis larvae

Parasitology ◽  
1992 ◽  
Vol 105 (2) ◽  
pp. 297-308 ◽  
Author(s):  
A. P. Page ◽  
R. M. Maizels
Keyword(s):  
Molecular Weight ◽  
Apparent Molecular Weight ◽  
Toxocara Canis ◽  
Somatic Tissues ◽  
Minimum Delay ◽  
Glycosylation Inhibition ◽  
Major Surface ◽  
Kinetics Of

SUMMARYToxocara canis infective stage larvae continually produce excretory–secretory (TES) glycoproteins in long-term in vitro culture. The kinetics of synthesis and secretion were studied by metabolic labelling with radioactive [35S]methionine, [14C]serine and [14C]threonine. Maximal incorporation rates required overnight pre-incubation of parasites in medium depleted of the appropriate amino acid. Larvae rapidly incorporated isotope into their somatic tissues, but there was a minimum delay of 10 h before secretion of labelled antigens. Labelling with [14C]serine and [14C]threonine demonstrated a relative abundance of these amino acids in the major surface/secreted glycoproteins of this nematode (TES-32 and 120). Pulse-chase experiments suggested that TES-120 may be derived from a 58 kDa precursor, reflecting extensive post-translational glycosylation. Inhibition of N-glycosylation with tunicamycin and digestion with N-glycanase provided evidence of N-glycosylation in the lower molecular weight ES components (TES-32, 55 and 70). These agents had no effect on the higher molecular weight components (TES-120 and 400) implying that for these molecules glycosylation is predominantly O-linked. The largest ES component (TES-400) was unusual, in incorporating serine and threonine but not methionine, and by exhibiting increased apparent molecular weight following pronase digestion; it is suggested that this molecule is a proteoglycan.

scholarly journals Impact of exocrine pancreatic adaptation on in vitro protein digestibility

1993 ◽  
Vol 69 (2) ◽  
pp. 359-369 ◽  
Author(s):  
P. Valette ◽  
H. Malouin ◽  
T. Corring ◽  
L. Savoie
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Pancreatic Enzyme ◽  
Protein Digestibility ◽  
Low Molecular Weight ◽  
Rapeseed Proteins ◽  
Total Capacity ◽  
Kinetics Of

An in vitro enzymic method was used to study the kinetics of digestion of casein and rapeseed proteins. After a predigestion step with pepsin (EC 3.4.23.1), the protein substrates were submitted to a 24 h hydrolysis either with pancreatin or pancreatic juices of pigs adapted either to casein or rapeseed diets and whose enzyme activities were different. After 3, 6 and 24 h of in vitro digestion, dialysates were collected and analysed for content of nitrogen, amino acids and low-molecular-weight peptides. For a long-term hydrolysis (24 h), overall digestibility of both substrates was not affected by the composition of pancreatic enzyme mixtures. However, at the beginning of hydrolysis a significant effect of pancreatic juices was observed, i.e. individual amino acid digestibility was generally higher when casein pancreatic juice was used for hydrolysis and their relative pattern of release was modified. For both substrates the proportion of amino acids released as low-molecular-weight peptides was not affected by the enzyme mixture used and made up about two-thirds of the total digested material. It is concluded that exocrine pancreatic adaptation to protein sources does not affect the total capacity of protein digestion. However, the changes in initial kinetics of release of amino acids are more dependent on the nature of the protein tested than on the composition of pancreatic enzyme mixtures.

Processing of the precursor to a chloroplast ribosomal protein made in the cytosol occurs in two steps, one of which depends on a protein made in the chloroplast

1985 ◽  
Vol 5 (5) ◽  
pp. 1093-1099
Author(s):  
R J Schmidt ◽  
N W Gillham ◽  
J E Boynton
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Apparent Molecular Weight ◽  
Protein L ◽  
Mature Form ◽  
Chloroplast Protein Synthesis ◽  
Made In

In pulse-chase experiments in which log-phase cells of Chlamydomonas reinhardtii were labeled in vivo for 5 min with H2(35)SO4, fluorographs of immunoprecipitates from whole cell extracts revealed that chloroplast ribosomal proteins L-2, L-6, L-21, and L-29, which are made in the cytosol and imported, appeared in their mature forms. However, in the case of chloroplast ribosomal protein L-18, which is also made in the cytoplasm and imported, a prominent precursor with an apparent molecular weight of 17,000 was found at the end of a 5-min pulse. This precursor was processed to its mature size (apparent molecular weight of 15,500) within the first 5 min of the subsequent chase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the precursor to L-18 formed in vivo was 1.5 kilodaltons smaller than the primary product detected in translations of Chlamydomonas polyadenylated RNA in vitro. Upon a 10-min incubation with a postribosomal supernatant from Chlamydomonas, the 18,500-dalton precursor detected in vitro could be partially converted into a polypeptide that comigrated with the 17,000-dalton precursor detected in extracts of cells labeled in vivo. Under conditions in which the total amounts of chloroplast proteins had been reduced and cells were made to synthesize ribosomes rapidly, the apparent half-life of the 17,000-dalton precursor was extended over that seen in log-phase cells. When chloroplast protein synthesis was inhibited with lincomycin for 3 h before labeling under these conditions, the 17,000-dalton L-18 precursor but not the mature form was found, and the precursor was slowly degraded during a 60-min chase. When cells were placed in the dark for 3 h before labeling, processing of this precursor to the mature form appeared unaffected, but the chloroplast-synthesized ribosomal protein L-26 was detected, indicating that chloroplast protein synthesis was still occurring. We interpret these results to indicate that the maturation of protein L-18 in vivo involves at least two processing steps, one of which depends on a protein made on chloroplast ribosomes.

scholarly journals Processing of the precursor to a chloroplast ribosomal protein made in the cytosol occurs in two steps, one of which depends on a protein made in the chloroplast.

1985 ◽  
Vol 5 (5) ◽  
pp. 1093-1099 ◽  
Author(s):  
R J Schmidt ◽  
N W Gillham ◽  
J E Boynton
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Apparent Molecular Weight ◽  
Protein L ◽  
Mature Form ◽  
Chloroplast Protein Synthesis ◽  
Made In

In pulse-chase experiments in which log-phase cells of Chlamydomonas reinhardtii were labeled in vivo for 5 min with H2(35)SO4, fluorographs of immunoprecipitates from whole cell extracts revealed that chloroplast ribosomal proteins L-2, L-6, L-21, and L-29, which are made in the cytosol and imported, appeared in their mature forms. However, in the case of chloroplast ribosomal protein L-18, which is also made in the cytoplasm and imported, a prominent precursor with an apparent molecular weight of 17,000 was found at the end of a 5-min pulse. This precursor was processed to its mature size (apparent molecular weight of 15,500) within the first 5 min of the subsequent chase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the precursor to L-18 formed in vivo was 1.5 kilodaltons smaller than the primary product detected in translations of Chlamydomonas polyadenylated RNA in vitro. Upon a 10-min incubation with a postribosomal supernatant from Chlamydomonas, the 18,500-dalton precursor detected in vitro could be partially converted into a polypeptide that comigrated with the 17,000-dalton precursor detected in extracts of cells labeled in vivo. Under conditions in which the total amounts of chloroplast proteins had been reduced and cells were made to synthesize ribosomes rapidly, the apparent half-life of the 17,000-dalton precursor was extended over that seen in log-phase cells. When chloroplast protein synthesis was inhibited with lincomycin for 3 h before labeling under these conditions, the 17,000-dalton L-18 precursor but not the mature form was found, and the precursor was slowly degraded during a 60-min chase. When cells were placed in the dark for 3 h before labeling, processing of this precursor to the mature form appeared unaffected, but the chloroplast-synthesized ribosomal protein L-26 was detected, indicating that chloroplast protein synthesis was still occurring. We interpret these results to indicate that the maturation of protein L-18 in vivo involves at least two processing steps, one of which depends on a protein made on chloroplast ribosomes.

scholarly journals Kinetics of sickle cell biorheology and implications for painful vasoocclusive crisis

2015 ◽  
Vol 112 (5) ◽  
pp. 1422-1427 ◽  
Author(s):  
E Du ◽  
Monica Diez-Silva ◽  
Gregory J. Kato ◽  
Ming Dao ◽  
Subra Suresh
Keyword(s):  
Disease Severity ◽  
Cell Density ◽  
Sickle Cell ◽  
Hematological Parameters ◽  
Diagnostic Tools ◽  
Hypoxic Conditions ◽  
Vitro Model ◽  
Kinetics Of

We developed a microfluidics-based model to quantify cell-level processes modulating the pathophysiology of sickle cell disease (SCD). This in vitro model enabled quantitative investigations of the kinetics of cell sickling, unsickling, and cell rheology. We created short-term and long-term hypoxic conditions to simulate normal and retarded transit scenarios in microvasculature. Using blood samples from 25 SCD patients with sickle hemoglobin (HbS) levels varying from 64 to 90.1%, we investigated how cell biophysical alterations during blood flow correlated with hematological parameters, HbS level, and hydroxyurea (HU) therapy. From these measurements, we identified two severe cases of SCD that were also independently validated as severe from a genotype-based disease severity classification. These results point to the potential of this method as a diagnostic indicator of disease severity. In addition, we investigated the role of cell density in the kinetics of cell sickling. We observed an effect of HU therapy mainly in relatively dense cell populations, and that the sickled fraction increased with cell density. These results lend support to the possibility that the microfluidic platform developed here offers a unique and quantitative approach to assess the kinetic, rheological, and hematological factors involved in vasoocclusive events associated with SCD and to develop alternative diagnostic tools for disease severity to supplement other methods. Such insights may also lead to a better understanding of the pathogenic basis and mechanism of drug response in SCD.

Osteoblast Growth Inhibition by Unfractionated Heparin and by Low Molecular Weight Heparins: An in-vitro Investigation

2002 ◽  
Vol 8 (3) ◽  
pp. 251-255 ◽  
Author(s):  
H. J. Kock ◽  
A. E. Handschin
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Cell Cultures ◽  
Term Therapy ◽  
Low Molecular Weight ◽  
High Dose ◽  
Low Molecular Weight Heparins ◽  
In Vitro Investigation

Osteoporosis is a rare but potentially severe complication under high-dose, long-term unfractionated heparin therapy. Low-molecular-weight heparins (LMWHs) have gained increased importance in antithrombotic therapy over the past decade. Whether this heterogeneous group of drugs carries a comparable risk of osteoporosis in long-term application is unknown. In a standardized in vitro model, the effects of 4 different low-molecular-weight heparins (nadroparin, enoxaparin, dalteparin, certoparin) on osteoblast growth were studied at the same dose (50,μg/mL). As control, the effect of unfractionated heparin (Liquemin) was tested on human osteoblasts in vitro at an equal dose. Human osteoblast cell cultures were incubated with equal doses of the heparins, and cell concentrations were measured after 48 and 96 hours. In addition, a fluorescence assay was performed to detect potential cytotoxic effect of heparins on bone cells. In comparison to control groups of non-incubated cell cultures, LMWHs caused a significant inhibition of osteoblast growth (p<0.05). Therefore, the risk of osteoporosis under long-term therapy with high doses of LMWHs cannot be excluded and should be further evaluated in clinical trials.

Changing chemosusceptibility in the second-stage larvae of Toxocara canis by long-term incubation

1993 ◽  
Vol 67 (2) ◽  
pp. 145-150 ◽  
Author(s):  
Nobuaki Akao ◽  
Yoshihisa Goto ◽  
Kaoru Kondo ◽  
Yoshisuke Tsuda
Keyword(s):  
Decanoic Acid ◽  
Toxocara Canis ◽  
Second Stage ◽  
One Year

AbstractSecond-stage larvae of Toxocara canis were maintained in vitro for one year. Susceptibility of the larvae to drugs was evaluated by means of minimal larvicidal concentration (MLC) and larval bursting percentage. MLCs of citral and decanoic acid were almost constant throughout all stages of incubation. However, bursting percentage markedly varied within the first 20 weeks of incubation. Therefore, while larvae are available for use in the MLC assay at any stage of incubation, those beyond the first 20 weeks after incubation should be used for the bursting assay to obtain reproducible results.

scholarly journals Characterization of ornithine decarboxylase of tobacco cells and tomato ovaries.

1982 ◽  
Vol 201 (2) ◽  
pp. 373-376 ◽  
Author(s):  
Y M Heimer ◽  
Y Mizrahi
Keyword(s):  
Molecular Weight ◽  
Ornithine Decarboxylase ◽  
Pyridoxal Phosphate ◽  
Apparent Molecular Weight ◽  
Ph Optimum ◽  
Tobacco Cells ◽  
Thiol Reagent ◽  
Mammalian Origin

Some characteristics of L-ornithine decarboxylase of tomato ovaries and tobacco cells are described. The enzyme has a pH optimum of 8.0. It requires pyridoxal phosphate and thiol reagent (dithiothreitol) for activity. It is specific for L-ornithine and has an apparent Km of 1.4 × 10-4 M. It has an apparent molecular weight of 107000. Putrescine inhibited the activity in vitro. Spermidine and spermine also inhibit the enzyme, but less effectively. It is concluded that the enzyme is similar to that of mammalian origin and likewise fulfils a function related to cell proliferation.

Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells

1987 ◽  
Vol 7 (8) ◽  
pp. 2700-2707
Author(s):  
B Boggs ◽  
F Cabral
Keyword(s):  
Molecular Weight ◽  
Gene Product ◽  
Cho Cells ◽  
Peptide Mapping ◽  
Chinese Hamster ◽  
Apparent Molecular Weight ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Beta Tubulin Gene

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.

Expression of high molecular weight tau in the central and peripheral nervous systems

1993 ◽  
Vol 105 (3) ◽  
pp. 729-737
Author(s):  
I.S. Georgieff ◽  
R.K. Liem ◽  
D. Couchie ◽  
C. Mavilia ◽  
J. Nunez ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Molecular Weight ◽  
Nervous System ◽  
High Molecular Weight ◽  
Tumor Cells ◽  
Apparent Molecular Weight ◽  
In Vitro Transcription ◽  
The Central Nervous System ◽  
Immunohistochemical Studies

Using a novel PCR approach, we have cloned a cDNA encoding the entire high molecular weight tau molecule from rat dorsal root ganglia. The resulting 2080 bp cDNA differs from low molecular weight rat brain tau by the insertion of a novel 762 bp region (exon 4a) between exons 4 and 5. This cDNA clone is identical in sequence with a high molecular weight tau (HMW) cDNA from rat PC12 tumor cells and is closely related to a HMW tau cDNA from mouse N115 tumor cells. In vitro transcription/translation produces a protein that migrates on SDS-PAGE with the same apparent molecular weight as HMW tau purified from rat sciatic nerve. The HMW tau protein is generated from an 8 kb mRNA, which can be detected by northern blots in peripheral ganglia, but not in brain. A more sensitive assay using PCR and Southern blot analysis demonstrates the presence of exon 4a in spinal cord and in retina. In combination with immunohistochemical studies of spinal cord, these data suggest that HMW tau, though primarily in the peripheral nervous system, is also expressed in limited areas of the central nervous system, although its presence cannot be detected in the cerebral cortices.

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