Protein synthesis in vitro by mantle tissue of the land snail Otala lactea: possible insulin-like peptide function

2000 ◽  
Vol 78 (9) ◽  
pp. 1527-1535 ◽  
Author(s):  
A M Abdraba ◽  
A SM Saleuddin
Keyword(s):  
Protein Synthesis ◽  
Cerebral Ganglion ◽  
Land Snail ◽  
Porcine Insulin ◽  
Land Snails ◽  
Size Exclusion ◽  
Cerebral Ganglia ◽  
Molecular Masses ◽  
Otala Lactea

Mantle-collar tissue from adult land snails Otala lactea continuously incorporated labelled amino acids over a 72-h period of incubation in modified culture medium. Acid-saline extract of cerebral ganglia stimulated protein synthesis by the mantle-collar tissue in vitro. This effect was dose-dependent, with the minimum and maximum doses at 0.5 and 2 cerebral ganglion equivalents, respectively. The protein synthesis-stimulating factor(s) from the cerebral ganglia appeared to be proteinaceous and hydrophobic in nature. The cerebral ganglion extract was fractionated by means of a size-exclusion HPLC column. The biological activity was induced by three fractions with estimated molecular masses of 0.82, 1.88, and 4.33 kilodaltons (kDa). Porcine insulin antiserum abolished the activity of the 4.33- and 1.88-kDa fractions but had no significant effect on the activity of the 0.82-kDa fraction. The results suggest the existence in the cerebral ganglia of more than one factor with protein synthesis-stimulating activity. One of these factors could be related to mammalian insulin. Porcine insulin, however, had no significant effect on protein synthesis by the mantle collar in vitro.

Localization and immunological characterization of insulin-like peptide(s) in the land snail Otala lactea (Mollusca: Pulmonata)

2000 ◽  
Vol 78 (9) ◽  
pp. 1515-1526 ◽  
Author(s):  
A M Abdraba ◽  
A SM Saleuddin
Keyword(s):  
Digestive Tract ◽  
Ethanol Extract ◽  
Land Snail ◽  
Size Exclusion ◽  
Connective Tissue Layer ◽  
Cerebral Ganglia ◽  
Molecular Masses ◽  
Otala Lactea ◽  
Vitro Analysis

Insulin-like peptides were detected by means of immunological techniques in tissues of the land snail Otala lactea. Insulin-positive cells were detected in all the ganglia except the right parietal ganglion and visceral ganglion. In the digestive tract, insulin-positive cells were found in the muscle and connective tissue layer of the intestine. The amount of insulin-like peptide detected in acid-ethanol extract of brains and digestive tracts from active snails did not differ significantly from that in the corresponding tissues from estivating (dormant) ones. More insulin-like peptide was detected in hemolymph from active snails than in hemolymph from estivating ones. Brains from active snails released insulin-like peptide in vitro. Analysis of the cerebral ganglia or digestive tract extracts by size-exclusion chromatography and insulin RIA revealed more than one fraction with insulin immunoreactivity. Some of these fractions contained material with molecular masses close to those of mammalian insulin or its subunits. Further analysis of the extracts by reverse-phase chromatography also revealed more than one fraction with immunoreactivity. The immunoreactive material from the digestive tract was found to be less hydrophobic than insulin. Western blot analysis of the cerebral ganglia extract revealed more than one band with insulin immunoreactivity. Three of these bands had molecular masses very similar to those of vertebrate insulin, its subunits, and its precursor.

Depression of Aerobic Metabolism and Intracellular pH by Hypercapnia in Land Snails, Otala Lactea

1988 ◽  
Vol 138 (1) ◽  
pp. 289-299 ◽  
Author(s):  
M. CHRISTOPHER BARNHART ◽  
BRIAN R. McMAHON
Keyword(s):  
Oxygen Consumption ◽  
Intracellular Ph ◽  
Respiratory Acidosis ◽  
Land Snail ◽  
Land Snails ◽  
Whole Body ◽  
Extracellular Acidosis ◽  
Critical Po2 ◽  
Otala Lactea ◽  
La Jolla

The pulmonate land snail Otala lactea undergoes simultaneous hypercapnia, hypoxia, extracellular acidosis and metabolic depression during dormancy. We tested the effects of ambient hypercapnia and hypoxia on oxygen consumption (VO2) and on extracellular and intracellular pH of active (i.e. non-dormant) individuals. Active snails reduced VO2, by 50% within l h when exposed to 65mmHg (1 mmHg = 133.3Pa) ambient PCO2, and by 63% in 98mmHg. These levels of CO2 are within the range that occurs naturally in the lung and blood during dormancy. VO2 of hypercapnic snails remained below that of controls for the duration of exposure (up to 9 h) and returned to control levels within 1 h when CO2 was removed. Both pHe and whole-body pHi (measured using [14C]DMO) fell with increasing haemolymph PCO2 by approximately 0.7logPCO2 Critical (VO2- limiting) ambient PO2 of active snails was 90mmHg in the absence of CO2 and dropped to 50 mmHg when VO2 was reduced 45% by exposure to CO2. Estimated critical PO2 at the lower VO2 typical of dormancy is well below the typical lung PO2 of dormant Otala, suggesting that PO2 in the lung does not normally limit oxygen consumption during dormancy. These results support the hypothesis that hypercapnia or resulting respiratory acidosis depresses metabolic rate during dormancy, and argue against a limitation of VO2 by hypoxia. Note: Present address: Physiological Research Laboratory, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093, USA.

Heat Dissipation, Gas Exchange and Acid-Base Status in the Land Snail Oreohelix During Short-Term Estivation

1990 ◽  
Vol 152 (1) ◽  
pp. 77-92 ◽  
Author(s):  
BERNARD B. REES ◽  
STEVEN C. HAND
Keyword(s):  
Heat Dissipation ◽  
Respiratory Acidosis ◽  
Land Snail ◽  
Land Snails ◽  
O2 Consumption ◽  
Short Term ◽  
R Ratio ◽  
Acid Base Status ◽  
Quantity Of Heat

Within 4 days following entry into estivation, heat dissipation and oxygen consumption by the land snail Oreohelix spp. decreased by 83% compared to standard non-estivating rates. During both non-estivating and estivating conditions, the quantity of heat dissipated per mole of O2 consumed was indicative of a completely aerobic metabolism. This calorimetric-respirometric (C/R) ratio was −461±12 kJ mol−1O2 (S.E.M., N=5) under standard non-estivating conditions and −464±26 kJ mol−1O2 (N=4) during estivation. Respiratory exchange ratios reflected a primary dependence upon carbohydrate as a metabolic substrate during both states. Carbon dioxide retention occurred during the first 36h of estivation, resulting in an increase in hemolymph PCOCO2 and a decrease in pH. The respiratory acidosis during short-term estivation was not compensated by elevation of hemolymph [HCO3−] above levels predicted from the in vitro nonbicarbonate buffer value of hemolymph. A brief period of rapid CO2 release, which caused hemolymph PCOCO2 and pH to return to pre-estivation values, preceded the increase in O2 consumption during arousal. Exposure of nonestivating snails to 4.67 kPa PCOCO2 (1 kPa=7.5 mmHg) caused a rapid and fully reversible 50% suppression of respiration rate. The temporal nature of CO2 retention and release during entry into and arousal from estivation, and the suppression of O2 consumption by artificial hypercapnia, support the hypothesis that elevated PCOCO2. or the resultant acidosis may contribute to metabolic suppression during estivation by land snails.

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity

2019 ◽  
Vol 476 (16) ◽  
pp. 2297-2319 ◽  
Author(s):  
Marta Grzechowiak ◽  
Milosz Ruszkowski ◽  
Joanna Sliwiak ◽  
Kamil Szpotkowski ◽  
Michal Sikorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Inorganic Pyrophosphatase ◽  
Prolonged Storage ◽  
Mitochondrial Targeting ◽  
Cleavage Rate ◽  
Inorganic Pyrophosphate ◽  
Putative Signal Peptide ◽  
Targeting Peptide

Abstract Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31–Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro

1976 ◽  
Vol 35 (02) ◽  
pp. 350-357 ◽  
Author(s):  
Hana Bessler ◽  
Galila Agam ◽  
Meir Djaldetti
Keyword(s):  
Protein Synthesis ◽  
Fold Increase ◽  
Human Platelets ◽  
Polystyrene Latex ◽  
Latex Particles ◽  
The Third ◽  
Platelet Protein ◽  
Dose Dependent ◽  
Phagocytotic Activity

SummaryA three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased.Vincristine (20 μg/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.

EFFECT OF ACTINOMYCIN D ON TSH-INDUCED STIMULATION OF THYROIDAL PROTEIN SYNTHESIS IN THE RAT

1974 ◽  
Vol 77 (1) ◽  
pp. 64-70 ◽  
Author(s):  
Gustav Wägar
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
The Other ◽  
Leucine Incorporation ◽  
Short Term ◽  
Other Hand ◽  
Thyroid Glands ◽  
Translational Level ◽  
Stimulation Of

ABSTRACT Whether the short-term regulation of thyroidal protein synthesis by TSH occurs at the transcriptional or the translational level was tested by measuring the effect of actinomycin D (act D) on the TSH-induced stimulation of L-14C-leucine incorporation into the thyroidal proteins of rats. TSH was injected 6 h before the rats were killed. The thyroid glands were then removed and incubated in vitro in the presence of L-14C-leucine for 2 h. The pronounced stimulation of leucine incorporation in the TSH-treated animals was depressed as compared with controls but still significant even when the animals had been pre-treated with 100 μg act D 24 and 7 h before sacrifice. On the other hand, act D strongly decreased incorporation of 3H-uridine into RNA. Short-term regulation of thyroidal protein synthesis by TSH appears to be partly but not wholly dependent on neosynthesis of RNA. Hence regulation may partly occur at the translation level of protein synthesis.

INHIBITION OF THYROGLOBULIN BIOSYNTHESIS AND DEGRADATION BY EXCESS IODIDE. SYNERGISM WITH LITHIUM

1976 ◽  
Vol 81 (2) ◽  
pp. 495-506 ◽  
Author(s):  
A. Radvila ◽  
R. Roost ◽  
H. Bürgi ◽  
H. Kohler ◽  
H. Studer
Keyword(s):  
Protein Synthesis ◽  
Thyroid Gland ◽  
Inhibitory Action ◽  
Inhibit Protein Synthesis ◽  
Thyrotrophic Hormone ◽  
Thyroid Glands ◽  
Pre Treatment ◽  
Excess Iodide ◽  
Kidney Liver

ABSTRACT Lithium and excess iodide inhibit the release of thyroid hormone from preformed stores. We thus tested the hypothesis that this was due to an inhibition of thyroglobulin breakdown. Rats were pre-treated with propylthiouracil (PTU) for 3 weeks in order to deplete their thyroids of thyroglobulin. While the PTU was continued, lithium chloride (0.25 mEq./100 g weight) or potassium iodide (3 mg per rat) were injected every 12 h for 3 days. Thereafter the thyroglobulin content in thyroid gland homogenates was measured. PTU pre-treatment lowered the thyroglobulin content from 4.21 to 0.22 mg/100 mg gland. Lithium caused a marked re-accumulation of thyroglobulin to 0.60 mg/100 mg within 3 days. While iodide alone had only a borderline effect, it markedly potentiated the action of lithium and a combination of the two drugs increased the thyroglobulin content to 1.04 mg/100 mg. Thyroxine was injected into similarly pre-treated animals to suppress secretion of thyrotrophic hormone. This markedly inhibited the proteolysis of thyroglobulin and 1.3 mg/100 mg gland accumulated after 3 days. Excess iodide, given in addition to thyroxine, decreased the amount of thyroglobulin accumulated to 0.75 mg/100 mg gland. To study whether this could be explained by an inhibitory action of iodide on thyroglobulin biosynthesis, thyroid glands from animals treated with excess iodide were incubated in vitro in the presence of 0.2 mm iodide for 3 h. Iodide decreased the incorporation of radioactive leucine into total thyroidal protein and into thyroglobulin by 25 and 35 % respectively. Iodide did not inhibit protein synthesis in the kidney, liver or muscle tissue. Thus, large doses of iodide selectively inhibit thyroglobulin biosynthesis.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

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