Actinomycin D, cycloheximide, and tunicamycin inhibit vitamin A induced proximodistal duplication during limb regeneration in the axolotl Ambystoma mexicanum

1988 ◽  
Vol 66 (4) ◽  
pp. 879-884 ◽  
Author(s):  
Steven R. Scadding
Keyword(s):  
Protein Synthesis ◽  
Vitamin A ◽  
Actinomycin D ◽  
Limb Regeneration ◽  
Ambystoma Mexicanum ◽  
Glycoprotein Synthesis ◽  
Left Limb ◽  
Site Of Action ◽  
The Right

Vitamin A is known to cause proximodistal duplication of parts of the limb during limb regeneration in amphibians. The objective of this study was to investigate the cellular site of action of vitamin A when it causes this duplication in the axolotl Ambystoma mexicanum. Both forelimbs of larval axolotls were amputated through the radius–ulna, and treated with retinol palmitate by immersion for 10 days. Actinomycin D, cycloheximide, and tunicamycin were each applied to the right forelimb by implantation of a silastin block containing one of these antibiotics on days 2 and 6 postamputation. The left limb received a control silastin block at the same time. Actinomycin D and cycloheximide completely blocked the proximodistal duplication caused by retinol palmitate, and tunicamycin substantially inhibited the development of these duplications. These results suggest that retinol palmitate (i) induces duplication by switching on a gene (as it is antagonized by actinomycin D), (ii) requires protein synthesis (as it is antagonized by cycloheximide), and (iii) may effect glycoprotein synthesis (as it is antagonized by tunicamycin).

Skeletal patterns in the autopodium of native and regenerated limbs of the larval axolotl, Ambystoma mexicanum

1991 ◽  
Vol 69 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Steven R. Scadding
Keyword(s):  
Vitamin A ◽  
Significant Variation ◽  
Limb Regeneration ◽  
Ambystoma Mexicanum ◽  
Skeletal Patterning ◽  
Skeletal Pattern ◽  
Limb Pattern ◽  
Salamander Species

The purpose of this investigation was to study the autopodial skeletal patterns that are observed in native (never regenerated) and regenerated limbs of the larval axolotl, Ambystoma mexicanum. The axolotl is used widely in limb regeneration studies, and in the regenerating axolotl limb mesopodial patterns can be modified by such factors as vitamin A administration. It is also known that other salamander species show significant variation in autopodial skeletal patterning. Hence, it seemed important to determine the type and frequency of autopodial variants in both native limbs and those that have regenerated after amputation at either the stylopodial and zeugopodial levels. The results showed that native limbs exhibited a complete skeletal pattern in the majority of cases, but that variants involving loss of a phalange or reduction in the number of carpals or tarsals occurred frequently. Regenerated limb patterns were more variable than those seen in native limbs, and limbs regenerating from zeugopodial level amputations were more variable than those regenerating from stylopodial level amputation. The significance of these observations for the development and regeneration of limb pattern is discussed.

Histological effects of vitamin A on limb regeneration in the larval axolotl, Ambystoma mexicanum

1990 ◽  
Vol 68 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Steven R. Scadding
Keyword(s):  
Electron Microscope ◽  
Vitamin A ◽  
Limb Regeneration ◽  
Ambystoma Mexicanum ◽  
Ciliated Cells ◽  
Posterior Edge ◽  
Histological Changes ◽  
Transmission Electron ◽  
Retinoid Treatment ◽  
Scanning Electron

Vitamin A causes profound changes in the development of pattern during amphibian limb regeneration. Vitamin A effects include the induction of duplications of skeletal structures in the anteroposterior, proximodistal, and dorsoventral axes. The purpose of this investigation was to study the underlying histological changes that are induced in the regenerating limb of the larval axolotl, Ambystoma mexicanum, by treatment with vitamin A. Axolotl larvae (7–10 cm in length) had forelimbs amputated through the radius and ulna and were then immersed in 75 IU/mL retinol palmitate for 14 days. Limbs were removed and fixed at intervals over the period of regeneration, both during and beyond the period of retinoid treatment. They were then examined in the light microscope, scanning electron microscope, or transmission electron microscope. Compared with the controls, the retinol palmitate treated regenerating limbs exhibited the development of an eccentric epidermal cap which was always displaced towards the posterior edge of the limb. Beneath this epidermal cap, the density of the cells of the blastema was greater than that of the cells towards the anterior edge of the developing blastema where the cells were much less densely arranged. Epidermal changes induced by retinol palmitate included the development of a very uneven and creviced surface, with a great deal of variation in cell size, and the development of ciliated cells in the surface layer of the epidermis. The significance of these observations for pattern modification by vitamin A are discussed.

Vitamin A modification of the positional information of blastema cells during limb regeneration in the axolotl Ambystoma mexicanum

1988 ◽  
Vol 66 (9) ◽  
pp. 2065-2070 ◽  
Author(s):  
Steven R. Scadding
Keyword(s):  
Vitamin A ◽  
Limb Regeneration ◽  
Normal Structure ◽  
Positional Information ◽  
Ambystoma Mexicanum ◽  
Limb Amputation ◽  
Temporary Change ◽  
Cellular Changes ◽  
Regeneration Blastema

Vitamin A is known to cause proximodistal duplication of parts of the limb during limb regeneration in amphibians. The objective of this study was to investigate the nature and location of the cellular changes induced by vitamin A when it causes this duplication in the axolotl Ambystoma mexicanum. When axolotls were treated with retinol palmitate by immersion for 14 days before limb amputation, proximodistal duplications were still observed in subsequent regenerates of limbs amputated after vitamin A treatment was discontinued. This observation suggests that some characteristic of the cells is changed by vitamin A and that exogenous vitamin A need not be present while the limb is regenerating. When a limb that was induced to undergo proximodistal duplication by vitamin A was reamputated 49 days later through the original mid radius–ulna amputation plane, it regenerated a limb of normal structure. A regeneration blastema transplanted from a vitamin A treated axolotl to an untreated axolotl regenerated on the host limb stump, producing a limb with proximodistal duplication; this indicates that the blastema cells underwent some change by the early to mid cone stage, which was expressed later when the blastema redifferentiated into a new limb. Conversely, when an untreated blastema was transplanted onto a vitamin A treated axolotl from which the forelimb blastema had been removed, proximodistal duplications developed. This result is interpreted to mean that the stump cells, although morphologically of the radius–ulna level, were proximalized by the prior vitamin A treatment and still displayed proximal positional values, leading to intercalation of missing proximodistal structures. These results are consistent with the hypothesis that vitamin A brings about a temporary change in the positional information of the limb stump and blastema cells, and that when the vitamin A treatment is discontinued, there is a gradual return to normal positional values over a period of several weeks.

Comparison of the effects of vitamin A on limb development and regeneration in the axolotl, Ambystoma mexicanum

Development ◽  
1986 ◽  
Vol 91 (1) ◽  
pp. 19-34
Author(s):  
S. R. Scadding ◽  
M. Maden
Keyword(s):  
Vitamin A ◽  
Limb Development ◽  
Species Differences ◽  
Limb Regeneration ◽  
Ambystoma Mexicanum

The objective of this investigation was to compare the effect of vitamin A on limb development and limb regeneration in the same animal, at the same time, thus eliminating the possibility that species differences or different rates of uptake between animals would influence the results. Axolotl larvae had both right limbs amputated and then were treated with retinol palmitate by immersion at 60 or 300 mg 1−1 for 4 or 10 days. Intact left developing limbs at the cone, two-digit, or four-digit stages responded to the treatment by deletion of skeletal elements producing hypomorphic limbs. Severity of the deletions was correlated with higher dose, longer times, and earlier stages of limb development. In contralateral right regenerating limbs, the effect of the same treatment was to cause various degrees of proximodistal duplication as well as occasional hypomorphic regenerates. Thus, there is a marked difference in response to vitamin A between developing and regenerating limbs. The implications of this observation are discussed especially with respect to the underlying morphogenetic mechanisms.

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.

Lipopolysaccharide-induced sensitization of adenylyl cyclase activity in murine macrophages

2006 ◽  
Vol 290 (1) ◽  
pp. C143-C151 ◽  
Author(s):  
Y. Osawa ◽  
H. T. Lee ◽  
C. A. Hirshman ◽  
D. Xu ◽  
C. W. Emala
Keyword(s):  
Protein Synthesis ◽  
Adenylyl Cyclase ◽  
Actinomycin D ◽  
Inhibitory Effect ◽  
Cytokine Release ◽  
Adenylyl Cyclase Activity ◽  
Phosphorylation State ◽  
Murine Macrophages ◽  
Dependent Pathway ◽  
New Protein

LPS is known to modulate macrophage responses during sepsis, including cytokine release, phagocytosis, and proliferation. Although agents that elevate cAMP reverse LPS-induced macrophage functions, whether LPS itself modulates cAMP and whether LPS-induced decreases in proliferation are modulated via a cAMP-dependent pathway are not known. Murine macrophages (RAW264.7 cells) were treated with LPS in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, CaM, Giproteins, and NF-κB translocation or transcription/translation. LPS effects on CaMKII phosphorylation and the expression of relevant adenylyl cyclase (AC) isoforms were measured. LPS caused a significant dose (5–10,000 ng/ml)- and time (1–8 h)-dependent increase in forskolin-stimulated AC activity that was abrogated by pretreatment with SN50 (an NF-κB inhibitor), actinomycin D, or cycloheximide, indicating that the effect is mediated via NF-κB-dependent transcription and new protein synthesis. Furthermore, LPS decreased the phosphorylation state of CaMKII, and pretreatment with a CaM antagonist attenuated the LPS-induced sensitization of AC. LPS, cAMP, or PKA activation each independently decreased macrophage proliferation. However, inhibition of NF-κB had no effect on LPS-induced decreased proliferation, indicating that LPS-induced decreased macrophage proliferation can proceed via PKA-independent signaling pathways. Taken together, these findings indicate that LPS induces sensitization of AC activity by augmenting the stimulatory effect of CaM and attenuating the inhibitory effect of CaMKII on isoforms of AC that are CaMK sensitive.

Citral, an inhibitor of retinoic acid synthesis, modifies pattern formation during limb regeneration in the axolotl Ambystoma mexicanum

1999 ◽  
Vol 77 (11) ◽  
pp. 1835-1837 ◽  
Author(s):  
Steven R Scadding
Keyword(s):  
Retinoic Acid ◽  
Pattern Formation ◽  
Limb Regeneration ◽  
Acid Synthesis ◽  
Ambystoma Mexicanum

While the effects of exogenous retinoids on amphibian limb regeneration have been studied extensively, the role of endogenous retinoids is not clear. Hence, I wished to investigate the role of endogenous retinoic acid during axolotl limb regeneration. Citral is a known inhibitor of retinoic acid synthesis. Thus, I treated regenerating limbs of the larval axolotl Ambystoma mexicanum with citral. The result of this inhibition of retinoic acid synthesis was that limb regeneration became extremely irregular and hypomorphic, with serious pattern defects, or was inhibited altogether. I conclude that endogenous retinoic acid plays an important role in pattern formation during limb regeneration.

Symmetrical vascularization patterns in normal and retinoic acid treated limb regenerates of the axolotl, Ambystoma mexicanum

1998 ◽  
Vol 76 (9) ◽  
pp. 1795-1796 ◽  
Author(s):  
Steven R Scadding ◽  
Andrew Burns
Keyword(s):  
Retinoic Acid ◽  
Pattern Formation ◽  
Vascular System ◽  
Limb Regeneration ◽  
Ambystoma Mexicanum ◽  
Limb Pattern ◽  
Regeneration Blastema

The purpose of this investigation was to determine whether there were any asymmetries in the vascularization of the limb-regeneration blastema in the axolotl, Ambystoma mexicanum, that might be related to pattern formation, and to determine if retinoic acid could modify the vascular patterns of the blastema. We used acrylic casts of the vascular system of the limbs to assess the pattern of vascularization. We observed a very regular symmetrical arrangement of capillaries in the limb-regeneration blastema that did not appear to be modified by doses of retinoic acid sufficient to modify the limb pattern.

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