Burr G. Atkinson

Biological Basis of Tissue Regression and Synthesis

The metamorphic process takes place in anuran tadpoles in an orderly fashion and with precise timing, each new development fitting into a perfectly coordinated pattern. Organs essential for an aquatic environment (tail and gills) do not begin to degenerate until the growth and development of organs essential for a terrestrial environment (legs and lungs) are nearly complete in their growth and development (Fig. 1). During the latter stages of larval development, the blood chemistry changes also manifest the functionality of the transition from gill to lung respiration.

Characterization of Rana catesbeiana HSP30 gene and its expression in the liver of this amphibian during both spontaneous and thyroid hormone-induced metamorphosis

During metamorphosis, the Rana catesbeiana tadpole undergoes developmental changes in almost every tissue/organ. These changes prepare the ammonotelic, swimming larva for its transition to a ureotelic, terrestrial adult, and involve dramatic remodeling. The postembryonic changes in this tadpole are initiated by the thyroid hormones (TH) and result in the extensive degradation of proteins and degeneration of tissues characteristic of the larval phenotype and in the de novo synthesis of proteins characteristic of the adult phenotype. We questioned whether the drastic nature and abruptness of the TH-dependent, postembryonic changes occurring in the tissues of this tadpole might be perceived by the cells in some tissues as stressful and, therefore, cause them to express heat shock and/or stress-like proteins. To address this question, we isolated and characterized a Rana catesbeiana hsp30 gene and used sequences from it to determine if mRNAs encoded from it, or other members of this gene family, are expressed in tissues of tadpoles undergoing metamorphosis. Our results demonstrate that the liver of metamorphosing Rana catesbeiana tadpoles accumulate hsp30 mRNAs and express the heat shock proteins they encode. The fact that the expression of these hsp30s in the liver of these tadpoles is coincidental with the TH-induced expression of genes encoding the liver-specific urea cycle enzymes suggests that TH may influence, directly or indirectly, the expression of these hsp30 genes and, moreover, implies that the presence of one or more of these heat shock proteins may be necessary for the developmental transitions occurring in this organ.

Developmental changes in the synthesis of haemolymph polypeptides and their sequestration by the prepupal fat body in Calpodes ethlius stöll (Lepidoptera:Hesperiidae)

Abstract Developmental changes in haemolymph protein and in its synthesis were examined in Calpodes ethlius from late in the fourth stadium to early pupation. The major haemolymph proteins have mol. wts of 470,000, 580,000 and 720,000. Upon dissociation, the 470 K and 580 K proteins each resolve into single polypeptides with mol. wts of 82,000 and 86,000 respectively. The 720 K protein separates into two distinct polypeptides with mol. wts of 86,000 and 90,000. The 82,000 polypeptide is present in the haemolymph at all times but not synthesized either very early in the fifth stadium or after the second critical period. The 86,000 and 90,000 polypeptides are present and synthesized only between the first and second critical periods. Disappearance of the 86,000 and 90,000 polypeptides from C. ethlius haemolymph is correlated with a decrease in haemolymph protein concentration and an increase in fat body protein granules. Injection of [ 14 C]-labelled haemolymph proteins into larvae after the second critical period results in the fat body acquiring radioactive polypeptides having M r s of 86,000 and 90,000. These results suggest that the prepupal fat body of C. ethlius sequesters at least one and probably two (the 720 K and 580 K proteins) of the three major fifth stadium haemolymph proteins.

Schistosoma mansoni: One- and two-dimensional electrophoresis of proteins synthesized in vitro by males, females, and juveniles

Abstract Polyacrylamide gel electrophoresis coupled with fluorography is a sensitive method for visualizing individual gene products synthesized in vitro by Schistosoma mansoni (K. Atkinson and B. G. Atkinson 1980, Nature (London) 283 , 478–479). In vitro labelling with radioactive amino acids ensures that the proteins are of parasite origin and fluorography permits detection of minute amounts of newly synthesized, electrophoretically separated gene products. One-dimensional electrophoretic separation in polyacrylamide gels with sodium dodecyl sulphate and fluorography of juvenile and adult proteins reveal that juveniles produce most adult proteins. Although similar studies with proteins from sexed adults imply that analogous gene products are elaborated by both sexes, a number of sex-specific gene products are resolvable by more rigorous two-dimensional electrophoretic separations. The homogametic male produces 5 polypeptides not produced by the heterogametic female. Three outstanding male-specific gene products include a polypeptide with a molecular weight (MW) of 88 kilodaltons (kd) and an isoelectric point (p I ) of 5.65, one with an MW of 66 kd and a p I of 5.25, and one with an MW of 58 kd and a p I of 5.25. Other, readily detectable male-specific polypeptides include one which coelectrophoreses with β-actin and one which coelectrophoreses with β-tropomyosin. The female synthesizes 4 specific polypeptides which have isoelectric points between 4.3 and 4.7, are of low molecular weight, and are resolvable only with 12% acrylamide gels. Two-dimensional electrophoresis resolves 74 major polypeptides synthesized by adult worms, and a code is presented which identifies each polypeptide by sex specificity, isoelectric point, and molecular weight.

Biochemical and histological changes in the respiratory system of Rana catesbeiana larvae during normal and induced metamorphosis

Abstract The incorporation of amino acids and thymidine into lungs and gills of bullfrog tadpoles were studied during normal and thyroid hormone-induced metamorphosis. During normal metamorphosis both amino acid and thymidine incorporation into gill tissue decrease during the late metamorphic stages. During this time period, histological studies show a reduction in gill vascularity and an increase in the number of melanocytes relative to gill epithelium cells. The rates of amino acid and thymidine incorporation into the lungs in the late metamorphic stages are much higher than in young tadpoles. The lung histology shows an increase in the potential respiratory epithelium by an increase in the number and size of septa during the later metamorphic stages. Thyroid hormone administration to young tadpoles mimics the biochemical changes observed during normal metamorphosis but does not lead to the same morphological changes as observed during normal metamorphosis. The discrepancies between normal and induced metamorphosis are discussed.

Sequence, identification and characterization of cDNAs encoding two different members of the 18 kDa heat shock family of Zea mays L.

Heat-shocked maize seedlings (cv. Oh43) synthesize a characteristic set of heat-shock proteins (hsps) which include an 18 kDa family containing at least six major isoelectric variants. A cDNA library was constructed from poly(A)+ RNAs isolated from the radicles of heat-shocked maize seedlings and screened with a DNA fragment from the theoretical open reading frame of a putative Black Mexican Sweet maize hsp 18 genomic clone. Two clones, cMHSP18-3 and cMHSP18-9, were isolated, and the RNA transcripts generated from them were translated into proteins which immunoreact with antibodies directed against the maize 18 kDa hsps and exhibit the same electrophoretic characteristics as two different members of the 18 kDa hsp family. Nucleotide sequence analyses of the cDNAs in these clones reveal that their 5′ and 3′ untranslated regions exhibit 33–34% identity and that their protein encoding regions share 93% identity. The deduced amino acid sequences of these clones show 90% identity, and the apparent molecular masses and isoelectric points of these proteins agree with those established for two different 18 kDa hsps, numbered 3 and 6. This report substantiates that at least two of the 18 kDa hsps in maize are products of different but related genes. Moreover, it establishes that transcripts for these proteins accumulate during heat shock and that both their nucleotide and deduced amino acid sequences share extensive similarities with the class VI small hsps in soybean and with transcripts expressed during meiosis in Lilium.

Thyroid-hormone-induced differentiation and development of anuran tadpole hind limbs: Detection and quantitation of M-line protein and α-actinin synthesis

Abstract Administration of triiodothyronine (T 3 ) to Stage VII Rana catesbeiana tadpoles induces precocious hind limb development. Muscle fiber formation in undifferentiated hind limb tissue is first evident by phase microscopy in the thigh region 48 hr after T 3 treatment. Indirect immunofluorescence studies, using antibodies to frog skeletal muscle M-line protein and α-actinin, demonstrate that these proteins are initially detectable in the thigh region within 24 hr after hormone administration. With increasing time after hormone treatment, myofibrillar protein and muscle fiber formation are enhanced in the thigh region and progress from the thigh to the distal areas of the limb. Quantitative immunochemical determinations of the synthesis of α-actinin and M-line protein indicate that their rates of synthesis increase markedly and synchronously in the 24 hr following hormone treatment. These quantitative data, in conjunction with the immunofluorescence and cytological results, provide evidence for thyroid hormone involvement in amphibian limb muscle differentiation and development.

Role for theRana catesbeiana homologue of C/EBP α in the reprogramming of gene expression in the liver of metamorphosing tadpoles

During the spontaneous or thyroid hormone (TH)-induced metamorphosis of Rana catesbeiana, developmental changes occur in its liver that are necessary for the transition of this organism from an ammonotelic larva to a ureotelic adult. These changes include the coordinated expression of genes encoding the urea cycle enzymes carbamyl phosphate synthetase (CPS-I) and arnithine transcarbamylase (OTC). Although the expression of these genes is dependent on TH, the mechanisms(s) by which TH initiates this tissue-specific response is thought to be indirect and to involve early TH-induced upregulation of a gene(s), which, in turn, upregulates the coordinated expression of these urea-cycle enzyme genes. Herein, we demonstrate that mRNAs encoding the Rana homologue of the mammalian transcription factor C/EBP alpha (designated RcC/EBP-1) accumulate early in response to TH and that the product of these mRNAs can bind to and transactivate the promoters of both the Rana CPS-1 and OTC genes. These results support the contention that the reprogramming of gene expression in the liver of metamorphosing tadpoles involves a TH-induced cascade of gene activity in which RcC/EBP-1 and, perhaps, other transcription factors coordinate the expression of genes, such as those encoding CPS-I and OTC, whose products are characteristic of the adult liver phenotype.

Synthesis of heat shock proteins in quail red blood cells following brief, physiologically relevant increases in whole body temperature

Cultured RBCs from quail respond to thermal stress (heat shock) by a rapid and dramatic change in gene expression. This change in gene expression includes the new and/or enhanced non-coordinate synthesis of a small group of heat shock polypeptides (HSPs) having molecular masses of 90,000, 70,000 and 26,000. RBCs obtained from hyperthermic quail exhibit a change in gene expression similar to that observed in RBCs heat-shocked in vitro. Since in vitro studies have linked the synthesis of HSPs in heat-stressed cells with thermotolerance, the similar change in gene expression in RBCs from hyperthermic quail suggests that, here too, this cellular response may be an important homeostatic mechanism by which avian RBCs cope with and/or survive hyperthermic conditions.

Expression of the myosin heavy chain genes in the tail muscle of thyroid hormone-induced metamorphosing Rana catesbeiana tadpoles.

In tadpoles of the North American bullfrog, Rana catesbeiana, spontaneous and thyroid hormone (T3)-induced metamorphosis is characterized by regression of the tail, which is preceded by a decrease in total protein synthesis in tail tissues. We have demonstrated that thyroid hormone treatment of a tadpole does not affect the synthesis of all proteins equally in the tadpole tail muscle. For example, the synthesis of myosin heavy chains (MHCs) is depressed within 1 day and decreases to 45% of control values after 5 days of T3 treatment, whereas the decreased synthesis of soluble muscle proteins is transient and returns to above control levels by day 5. To determine whether the hormone-induced decrease in MHC synthesis is the result of changes in the transcription of translation of MHC mRNAs, we isolated cDNAs complementary to five different MHC mRNAs from a tail muscle cDNA library and used them to examine the levels of each MHC mRNA in the tail muscle of T3-treated tadpoles. mRNAs that recognize the cDNAs for these five different MHCs are all expressed in the tadpole tail and limb muscles, as well as in the adult leg muscles. MHC mRNAs unique to tadpole tail were not detected. Interestingly, the relative amounts of mRNA for four of the five MHCs increase in tail muscle after T3 treatment of the tadpole, suggesting that repression of MHC gene expression at the protein level does not result from a decrease in the amount of MHC mRNAs. Rather, these results support the contention that the decreased synthesis of MHCs in the tail muscle of T3-treated tadpoles is caused by this hormone, either directly or indirectly, depressing the translation of the MHC mRNAs in this tissue. These results, coupled with the observation that the synthesis of soluble muscle proteins is depressed only in a transient fashion, suggest that T3 may be initiating the expression of a gene(s) that encodes a protein(s) responsible for inhibiting the translation of the MHCs and, perhaps, other structural proteins in the tadpole tail muscle. Whatever the case, the translational regulation of MHC synthesis occurs well before any degradation of the tail tissue is evident and appears to be one of the earliest events in the hormone-induced cell death program of the tadpole tail muscle.