scholarly journals Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

1988 ◽  
Vol 8 (5) ◽  
pp. 1957-1969 ◽  
Author(s):  
R A Shapiro ◽  
D Herrick ◽  
R E Manrow ◽  
D Blinder ◽  
A Jacobson
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Mrna Decay ◽  
Decay Rates ◽  
Time Dependent ◽  
Translational Efficiency ◽  
Cdna Clones ◽  
Dependent Manner ◽  
Significant Difference ◽  
Kinetics Of

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.

scholarly journals Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates

1988 ◽  
Vol 8 (5) ◽  
pp. 1957-1969
Author(s):  
R A Shapiro ◽  
D Herrick ◽  
R E Manrow ◽  
D Blinder ◽  
A Jacobson
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Mrna Decay ◽  
Decay Rates ◽  
Time Dependent ◽  
Translational Efficiency ◽  
Cdna Clones ◽  
Dependent Manner ◽  
Significant Difference ◽  
Kinetics Of

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.

Pseudomonas aeruginosa lectins I and II and their interaction with human airway cilia

2005 ◽  
Vol 119 (8) ◽  
pp. 595-599 ◽  
Author(s):  
Marco Mewe ◽  
Denis Tielker ◽  
Robert Schönberg ◽  
Melitta Schachner ◽  
Karl-Erich Jaeger ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Joint Action ◽  
Mammalian Cells ◽  
Time Dependent ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Similar Time ◽  
Airway Cilia ◽  
Kinetics Of ◽  
Ciliary Beat

The bacterium Pseudomonas aeruginosa (PA) produces two carbohydrate binding lectins, designated PA lectin-I and lectin-II (PA-IL, PA-IIL). Both lectins are used by the bacterium to adhere to the glycocalyx of mammalian cells. In addition, the lectins immobilize ciliary beat. The kinetics of ciliary beat inhibition by each individual lectin have been analysed; however, their joint action on cilia has not been reported. Here we demonstrate that PA-IL and PA-IIL inhibit ciliary beat in a similar time-dependent manner. If applied simultaneously, ciliary beat inhibition after five hours of incubation was weaker than if each lectin was applied separately. Thus it can be hypothesized that the lectins compete for the same binding site(s) of the glycocalyx. Sugar inhibition experiments demonstrate that D-galactose and L-fucose inhibit both lectins, although clear preferences of D-galactose for PA-IL and of L-fucose for PA-IIL exist. These interactions have to be kept in mind when designing sugar-based therapies.

scholarly journals Disposition kinetics of ceftriaxone and determination of its therapeutic dose in dogs

2020 ◽  
Vol 19 (8) ◽  
pp. 1735-1758
Author(s):  
Ifeanyi G. Eke ◽  
Ukamaka U. Eze ◽  
Aruh O. Anaga ◽  
Kennedy F. Chah ◽  
Boniface M. Anene ◽  
...  
Keyword(s):  
Steady State ◽  
Serum Concentration ◽  
Half Life ◽  
Therapeutic Dose ◽  
Maximum Serum ◽  
Elimination Half Life ◽  
Disposition Kinetics ◽  
Elimination Half ◽  
Significant Difference ◽  
Kinetics Of

Purpose: To evaluate the disposition kinetics of ceftriaxone (CFZ) in dogs with a view to determining its therapeutic dose and dosing frequency.Methods: Twelve (12) Basenji dogs (n = 4), divided into 3 groups (A, B and C), were used for the study. Ceftriaxone was administered intramuscularly at doses of 12.5, 25, and 50 mg/kg once to groups A, B and C respectively. Plasma CFZ concentration was determined by agar well diffusion assay at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h post-treatment, and the pharmacokinetic parameters were determined.Results: Intramuscular injection of CFZ to dogs resulted in rapid absorption, distribution and elimination (p < 0.05). The elimination half-life was short and did not change significantly with increase in dose. Serum concentration of CFZ changed significantly (p < 0.05) with increase in dose of CFZ. The maximum serum concentration (Cmax, 15.00 ± 1.18, 141.37 ± 15.87 and 259 ± 5.21 μg/mL) for groups A, B and C respectively were significantly (p < 0.05) different. The steady state CFZ concentrations; 0.94, 8.81 and 16.19 μg/mL for groups A, B and C, respectively, were significantly (p < 0.05) different. However, there was no significant difference in the time to reach steady state concentrations (Tmax, 00±0.021, 4.00±0.10 and 4.30±0.12 for groups A, B and C respectively). The therapeutic dose of CFZ was therefore determined to be 25 – 50 mg/kg every 4 h.Conclusion: Ceftriaxone undergoes rapid elimination in dogs with a short elimination half-life, thus making it an inconvenient prescription for out-patients in veterinary clinics. Keywords: Ceftriaxone, Pharmacokinetic profile, Dogs, Therapeutic dose, Veterinary clinic

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum

1987 ◽  
Vol 7 (2) ◽  
pp. 799-805
Author(s):  
R Kelly ◽  
D R Shaw ◽  
H L Ennis
Keyword(s):  
Protein Synthesis ◽  
Dictyostelium Discoideum ◽  
Spore Germination ◽  
Mrna Decay ◽  
Protein Synthesis Inhibitor ◽  
Cdna Clones ◽  
Cellular Slime Mold ◽  
Suitable Model ◽  
Mrna Synthesis ◽  
Developmentally Regulated

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.

ATP hydrolysis during contraction of permeabilized airway smooth muscle

1999 ◽  
Vol 277 (2) ◽  
pp. L334-L342 ◽  
Author(s):  
Keith A. Jones ◽  
Robert R. Lorenz ◽  
Y. S. Prakash ◽  
Gary C. Sieck ◽  
David O. Warner
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Atp Hydrolysis ◽  
Isometric Force ◽  
Time Dependent ◽  
Tracheal Smooth Muscle ◽  
Hydrolysis Rate ◽  
Shortening Velocity ◽  
Actomyosin Atpase ◽  
Significant Difference

This study determined whether the time-dependent decline in the rate of ATP hydrolysis by actomyosin ATPase during sustained isometric force can occur in the absence of a time-dependent decline in regulatory myosin light chain (rMLC) phosphorylation in Triton X-100-permeabilized canine tracheal smooth muscle. Maximal activation with 10 μM Ca2+ induced sustained increases in isometric force, stiffness, and rMLC phosphorylation; however, the increase in the ATP hydrolysis rate was initially high but then declined to a steady-state level above that of the unstimulated muscle (basal 31.8 ± 5.8 nmol ⋅ cm−3 ⋅ s−1; peak 81.4 ± 11.3 nmol ⋅ cm−3 ⋅ s−1; steady-state 62.2 ± 9.1 nmol ⋅ cm−3 ⋅ s−1). Activation of strips in which the rMLC was irreversibly and maximally thiophosphorylated with adenosine 5′- O-(3-thiotriphosphate) also induced sustained increases in isometric force and stiffness but a nonsustained increase in ATP hydrolysis rate. There was no significant difference in the peak or steady-state isometric force, stiffness, or ATP hydrolysis rate or in the steady-state maximum unloaded shortening velocity between strips activated by 10 μM Ca2+ or rMLC thiophosphorylation (0.058 ± 0.016 and 0.047 ± 0.011 muscle lengths/s, respectively). Mechanisms other than changes in rMLC phosphorylation contribute to the time-dependent decline in actomyosin ATPase activity during sustained activation of canine tracheal smooth muscle.

Ventilatory responses to intravenous and airway CO2 administration in anesthetized dogs

1980 ◽  
Vol 48 (2) ◽  
pp. 337-346 ◽  
Author(s):  
W. E. Fordyce ◽  
F. S. Grodins
Keyword(s):  
Blood Flow ◽  
Steady State ◽  
Loading Rate ◽  
Blood Flow Rate ◽  
Time Dependent ◽  
Base Line ◽  
Ventilatory Responses ◽  
Significant Difference ◽  
Anesthetized Dogs ◽  
Randomized Protocol

The ventilatory responses to steady-state venous CO2 loading (iv CO2) and CO2 inhalation have been observed in chloralose-urethan-anesthetized dogs. Intravenous CO2 was administered by increasing the CO2 fraction of gas ventilating a membrane gas exchanger in an arteriovenous bypass; blood flow rate was fixed at 30 ml/min. During the study, we identified a time-dependent hyperventilation in all 14 experimentally treated dogs and in 4 additional sham-treated dogs. When we tested 8 of these animals with a protocol having small progressive increments in iv CO2 loading rate, we observed a response approaching isocapnia during iv CO2 and a large hypocapnia when we returned to control conditions. The use of a randomized protocol in 6 animals demonstrated the necessity of accounting for this systematic base-line shift, because before doing so the response depended more on the passage of time than on the nature of the CO2 load. After this analytical adjustment was made, there was no significant difference between the respiratory controller gains (delta nu E/delta Paco2) for inhaled and iv CO2.

scholarly journals Effect ofEnterococcus faecalisPyelonephritis on the Intracortical Accumulation Kinetics of Gentamicin in Rats

1990 ◽  
Vol 1 (4) ◽  
pp. 117-120 ◽  
Author(s):  
Martial Jullien ◽  
Guy Thériault ◽  
Michel G Bergeron ◽  
Denis Beauchamp
Keyword(s):  
Steady State ◽  
Left Kidney ◽  
Severe Infection ◽  
Serum Levels ◽  
Linear Increase ◽  
Least Squares Regression ◽  
Sprague Dawley ◽  
Significant Difference ◽  
Accumulation Kinetics ◽  
Kinetics Of

The role of serum levels on the intracortical accumulation kinetics of gentamicin was evaluated in normal andEnterococcus faecalis-infected kidneys using a short term infusion model in conscious rats. Female Sprague-Dawley rats were infused over a period of 6 h with gentamicin achieving individual steady-state serum levels ranging from 0.5 to 20 μg/mL. Pyelonephritis was induced by a direct inoculation of the left kidney with a suspension ofE faecalis.This model resulted in severe infection of the left kidney and mild infection of the right kidney. Gentamicin cortical concentrations were analyzed as a function of serum levels by linear regression (least squares regression analysis). Steady-state elevation of serum concentrations of gentamicin was associated with a linear increase of cortical concentrations in normal, and in both left and right infected kidneys. No significant difference in the kinetics of gentamicin uptake was observed between normal and right and left infected kidneys. Therefore,E faecalispyelonephritis does not modify the intracortical accumulation kinetics of gentamicin.

scholarly journals Fluoride at Mitogenic Doses Induces a Sustained Activation of p44mapk, but Not p42mapk, in Human TE85 Osteosarcoma Cells*

1997 ◽  
Vol 82 (4) ◽  
pp. 1126-1135
Author(s):  
Li-Wha Wu ◽  
Hyun Koo Yoon ◽  
David J. Baylink ◽  
Lee M. Graves ◽  
K.-H. William Lau
Keyword(s):  
Cell Proliferation ◽  
Steady State ◽  
Specific Activity ◽  
Bone Cell ◽  
Time Dependent ◽  
Dependent Manner ◽  
Osteosarcoma Cells ◽  
Fluoride Treatment ◽  
Phosphorylation Level ◽  
Tyrosyl Phosphorylation

Abstract Fluoride, at micromolar concentrations, stimulates bone cell proliferation in vitro. In this study, we sought to test whether fluoride at mitogenic doses increases the tyrosyl phosphorylation level and specific activity of a mitogen-activated protein kinase (MAPK) in human TE85 osteosarcoma cells. Analysis by immunoprecipitation with antiphosphotyrosine antibody followed by Western analysis using an anti-pan extracellular signal-regulated kinase antibody revealed that fluoride at the optimal mitogenic dose (i.e. 100 μmol/L) induced a time-dependent increase in the steady state tyrosyl phosphorylation level of p44mapk, but not p42mapk, with the maximal increase (4- to 13-fold) after 1–3 h fluoride treatment. The effect was sustained in that a 9-fold increase was seen after 12 h of the fluoride treatment. The sustained nature of the effect is consistent with an inhibition of dephosphorylation rather than a direct stimulation of phosphorylation. The fluoride effect on the tyrosyl phosphorylation level of p44mapk was dose dependent, with the optimal dose being 100μ mol/L fluoride. The mitogenic dose of fluoride also increased the specific activity and the in-gel kinase activity of p44mapk, but not that of p42mapk, in a time-dependent manner similar to the effect on the p44mapk tyrosyl phosphorylation level. Fluoride at the same micromolar doses did not increase cell proliferation, tyrosyl phosphorylation, or specific activity of any MAPK in human skin foreskin fibroblasts, which are fluoride-nonresponsive cells. Consistent with the interpretation that the effect of fluoride on the steady state tyrosyl phosphorylation level of p44mapk is a consequence of an inhibition of a phosphotyrosyl phosphatase (PTP), mitogenic doses of orthovanadate, a bone cell mitogen and a PTP inhibitor, also increased the steady state tyrosyl phosphorylation level of p44mapk, but not p42mapk, in a time-dependent sustained manner similar to that observed with fluoride. Together, these findings support the concept that inhibition of a PTP activity in bone cells could lead to an activation of MAPK activity.

scholarly journals Desensitization and recovery of muscarinic and histaminergic Ca2+ mobilization in 1321N1 astrocytoma cells

1988 ◽  
Vol 249 (1) ◽  
pp. 135-141 ◽  
Author(s):  
P M McDonough ◽  
J H Eubanks ◽  
J H Brown
Keyword(s):  
Steady State ◽  
Extracellular Calcium ◽  
Time Dependent ◽  
Initial Increase ◽  
Dependent Manner ◽  
Half Time ◽  
Fura 2 ◽  
Astrocytoma Cells ◽  
Heterologous Desensitization ◽  
1321N1 Astrocytoma

Intracellular free Ca2+ was monitored in suspensions of 1321N1 astrocytoma cells by using the Ca2+ indicator fura-2. The cytoplasmic Ca2+ concentration increased from 237 +/- 6 nM to 1580 +/- 170 nM within 3-5 s of addition of 300 microM-carbachol. After the peak in response, the Ca2+ concentration diminished, establishing a new steady state in about 1 min that was approx. 150 nM above the previous baseline. Histamine increased cytoplasmic Ca2+ to about 40% of the maximal value seen with carbachol. In Ca2+-free buffer each agonist elicited a normal initial increase in cytoplasmic Ca2+, but the sustained portion of the response was abolished. The increase in Ca2+ in response to either carbachol or histamine could be completely inhibited by pretreating the cells with carbachol; the response to carbachol could be partially inhibited by pretreating the cells with histamine. The Ca2+ responses did not recover in the continued presence of carbachol. However, if the carbachol was washed out or if atropine was added after carbachol, the responses to agonist recovered in a time-dependent manner (half-time 3-4 min), and recovery depended on the presence of extracellular calcium. The results indicate that carbachol and histamine stimulate release of Ca2+ from the same intracellular Ca2+ store, that depletion of this store is responsible for heterologous desensitization between these two agonists, and that repletion of the agonist-sensitive Ca2+ pool does not occur in the continued presence of agonist or in the absence of extracellular Ca2+.

scholarly journals Transient kinetics of a cGMP-dependent cGMP-specific phosphodiesterase from Dictyostelium discoideum.

1984 ◽  
Vol 98 (2) ◽  
pp. 709-716 ◽  
Author(s):  
P J Van Haastert ◽  
M M Van Lookeren Campagne
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Binding Sites ◽  
Catalytic Site ◽  
Transient Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Rate Of Activation ◽  
Stimulation Of

Chemotactic stimulation of Dictyostelium discoideum cells induces a fast transient increase of cGMP levels which reach a peak at 10 s. Prestimulation levels are recovered in approximately 30 s, which is achieved mainly by the action of a guanosine 3',5'-monophosphate cGMP-specific phosphodiesterase. This enzyme is activated about fourfold by low cGMP concentrations. The phosphodiesterase has two distinct cGMP-binding sites: a catalytic site and an activator site. cAMP does not bind to either site; inosine 3',5'-monophosphate (cIMP) binds only to the catalytic site, whereas 8-bromoguanosine 3',5'-monophosphate (c-b8-GMP) preferentially binds to the activator site. For detailed kinetical measurements we have used [3H]cIMP as the substrate and c-b8-GMP as the activator. c-b8-GMP activated the hydrolysis of [3H]cIMP by reducing the Km, whereas the Vmax was not altered. The hydrolysis of [3H]cIMP was measured at 5-s intervals by using a new method for the separation of 5'-nucleotides from cyclic nucleotides. The hydrolysis of [3H]cIMP by nonactivated enzyme or by preactivated enzyme was linear with time, which indicates that a steady state is reached at the catalytic site within 5 s after addition of the substrate. In contrast, the hydrolysis of [3H]cIMP immediately after activation by 0.1 microM c-b8-GMP was not linear with time, but increased in a quasi-exponential manner with a time constant of 21 s. This suggests that a steady state at the activator site is only reached in 30-45 s after addition of the activator. The on-rate of activation (k1) was 3 X 10(5) M-1s-1 for c-b8-GMP and 1.4 X 10(5) M-1s-1 for cGMP. The off-rate of activation (k-1) was 0.03 s-1 for both c-b8-GMP and cGMP. The significance of these kinetic constants for the chemoattractant-mediated cGMP response in vivo is discussed.

Sign in / Sign up

Export Citation Format

Share Document