Epidermal tissue requirement for tadpole tail regression induced by thyroid hormone

In this paper we report on the requirement of the epidermal tissue for thyroid hormone-induced tadpole tail regression. The epidermis was removed by two different methods, i.e., surgically or chemically. Chemical removal included EDTA and trypsin treatment. Epidermis-free tail fin blocks were cultured in vitro according to A. Derby, 1968, J. Exp. Zool.168, 147–156. and the effect of 3,3′,5-tri-iodo-l-thyronine (T₃) was followed up for 4 days. No tissue breakdown was observed at the concentration of 10^?8M T₃, which was enough to induce tissue resorption of the epidermis-containing normal tissue blocks. Tail muscle cubes with epidermis regressed in the T₃-containing culture medium. However, the epidermis-deprived tail muscle cubes did not respond to the hormone. The tail fin mesenchymal connective tissue block deprived of the epidermis was cultured with epidermal tissues which had been removed surgically from the tail. The presence of T₃ in this reconstituted culture induced the regression of the mesenchymal connective tissue blocks. These experiments clearly show that epidermal tissue plays a critical role in T₃-induced tissue degradation.     

Modulation of fertilization by the vitellus after interaction with peptides released by hamster sperm-zona pellucida contact

Two to three minutes after hamster sperm make contact with and adhere to the surface of homologous zonae pellucidae in vitro, the first of several sets of peptides (S1 peptides) is released into the supernatant. This release occurs whether the zonae have or have not been mechanically separated from the vitellus (cellular part of the egg). Presence of the S1 peptides is detected by means of a sperm-egg assay, the premature binding assay. This assay is based on the ability of an aliquot of the medium, in which sperm are interacting with the zona surface, to induce early binding, upon addition of the aliquot to a second drop of interacting gametes. To determine if the vitellus affected the 2-min S1 peptides the ultrafiltrates of the supernatants containing them, released through sperm-egg and sperm-zona interactions, were fractionated on Biogel P-6 and their elution profiles were compared using the premature binding assay. The sperm-egg ultrafiltrates were resolved into two main domains of activity, while those of the sperm-zona formed three. The ultrafiltrates collected 2 min after the interaction of sperm with eggs or with isolated zonae were compared for their abilities to inhibit the penetration of the zona pellucida, a previously demonstrated capacity of the 2-min sperm-egg S1 peptides. The ultrafiltrate containing the sperm-zona peptides, except at a very low level, failed to inhibit penetration significantly. However, when the sperm-zona ultrafiltrate was preincubated with eggs then the resulting supernatant inhibited penetration in a dose-related manner, and the three-domain elution profile, characteristic of the sperm-zona ultrafiltrate, was converted to the egg-like two-domain profile. Taken together these data suggest that the 2-min S1 peptides consist of several subpopulations, at least one of which interacts with the vitellus. The resulting solution then acquires the ability to inhibit penetration of the egg by the sperm in a dose-related manner. Taken together these data indicate that by interacting with at least one of the components of the 2-min peptides, the vitellus is involved in regulating sperm-zona interactions.     

Differences in abundance of individual RNAs in normal and animalized sea urchin embryos

A library of cDNA clones was constructed representing polysomal polyadenylated RNA of mesenchyme blastulae of Strongylocentrotus purpuratus. Using this library, we determined whether or not individual RNA species are associated with animalization of embryos by zinc ions. Clones corresponding to the most actively synthesized RNAs during the period just prior to the mesenchyme blastula stage were selected by screening colonies with in vivo-labeled RNA. The most abundant of these were chosen for further study. Individual RNA abundance was measured as percent of mass of total polyadenylated RNA by hybridizing cDNA exhaustively with cloned DNA on filters. The RNAs in the selected, cloned sequences were present in abundances of 0.01 to 1% of the mass of polyadenylated RNA. Changes in abundance of individual RNA species occurred during normal development and departures from these developmental changes occurred in the zinc-animalized embryos. Two RNA species, which normally increase 10-fold in abundance, are drastically repressed and at least one RNA species increases in abundance dramatically in the animalized embryos. These departures from the normal program of presumptive gene expression may furnish insights into changes in the normal processes of development.     

Growth hormone receptors in lymphocytes of growth hormone-deficient children

Human growth hormone was injected intravenously into 18 growth hormone-deficient children and growth hormone binding sites in lymphocytes were investigated. Fresh circulating lymphocytes had a low initial value for the binding of growth hormone to solubilized receptors (3.45 +/- 1.46%) but after growth hormone injection, the binding rapidly increased to 14.8 +/- 4.2% at 2 1/2 h and 8.7 +/- 1.8% at 5 h. The sharp increase in binding is due to increase in the number of binding sites. Two control children who received chorionic gonadotropin had no change in lymphocyte growth hormone receptors. The methodological differences between the present study and previous attempts to identify human growth receptors in lymphocytes were (1) lymphocytes were separated and disrupted with Triton X-100 as quickly as possible (to avoid error from receptor leaking out of the cell) and (2) the receptors were assayed at 2 1/2 h after growth hormone administration (previous studies were 12-24 h later). One possible explanation for the data is that growth hormone receptor from liver is taken up by lymphocytes and rapidly released again, thus, contributing to the hormonal receptor economy in humans.     

Physical and chemical properties of lactate dehydrogenase in Homarus americanus.

Two isoenzymes of lactate dehydrogenase have been purified from Homarus americanus: One is found predominantly in the tail muscles; the other, in the walking leg muscles. This is the first demonstration of multiple forms of l-specific lactate dehydrogenase in an invertebrate organism. These proteins contain four essential sulfhydryl groups titratable by p-hydroxymercuribenzoate and 5,5′-dithiobis(2-nitrobenzoic acid). The molecular weights of these isoenzymes are dependent upon ionic strength. The native tetramer (M_r 145,000) exists in low ionic strength solutions; the active dimer (M_r 75,000), in high ionic strength solutions; this is the only example of lactate dehydrogenase disaggregation without concomitant loss in enzymatic activity. Microcomplement fixation studies suggest that there may be less than 4% difference in the primary structures of these two proteins.     

The role of superoxide anion in peroxidase-catalyzed chemiluminescence of luminol

The chemiluminescence associated with peroxidation of luminol in buffered aqueous solution is a complex process involving several intermediates. It can be inhibited by removal of oxygen from the incubation medium. Superoxide radical is both an intermediate in this reaction and an essential component in light-producing steps. The importance of O₂^? in propagating this reaction was shown by the inhibition of luminescence by superoxide dismutase. A mechanism was proposed which is consistent with the data. It appears likely that the diverse biological effects of peroxidases are largely due to the reactivities of these intermediates and products.     

Properties of human neutral bronchial mucins after modification of the peptide or the carbohydrate moieties.

Trypsin and pronase treatment of purified human neutral bronchial mucins released small fragments from the C-terminal end of these molecules and resulted in slight increases in their sedimentation coefficient presumably reflecting conformational changes. The antigenic determinant of neutral bronchial mucins which appears to be located on this C-terminal fragment is destroyed by pronase or by treatments such as periodate oxidation or galactose oxidase-bromine oxidation which modify the carbohydrate moieties. Thus, both amino acid and carbohydrate residues are involved in the structure of the antigenic determinant.     

Neuron-schwann cell interaction in basal lamina formation

The availability of tissue culture systems that allow the growth of nerve cells, Schwann cells, and fibroblasts separately or in various combinations now makes possible investigation of the role of cell interactions in the development of the peripheral nervous system. Using these systems it was earlier found that basal lamina is formed on the Schwann cell surface in cultures of sensory ganglion cells and Schwann cells without fibroblasts. It is here reported that the presence of nerve cells is required for the generation of basal lamina on the Schwann cell plasmalemma. Utilizing nerve cell-Schwann cell preparations devoid of fibroblasts, this was found in the following ways. (1) When nerve cells are removed from 3- to 5-week-old cultures, the basal lamina disappears from Schwann cells. (2) If nerve cells are added back to such Schwann cell populations, Schwann cell basal lamina reappears. (3) Removal of nerve cells from older (3–4 months) cultures does not lead to basal lamina loss; areas presumed not to have been coated with lamina before neurite degeneration remain so, suggesting that the lamina persists but is not reformed. (4) If basal lamina is removed with trypsin, it is reformed in neuron plus Schwann cell cultures but not in Schwann cell populations alone. Thus, the formation but not the persistence of Schwann cell basal lamina requires the presence of nerve cells.     

Bovine follitropin. Isolation and characterization of the native hormone and its alpha and beta subunits

1) A reproducible procedure was developed for the purification of bovine follitropin. 2) The method involved ammonium sulfate precipitation, ion exchange and adsorption chromatography, concanacaline-A-Sepharose chromatography and gel filtration. 3) A specific radioligand receptor assay was used to monitor each chromatographical step. 4) The potency of highly purified bovine follitropin as measured by Steelman and Pohley bioassay was 62 times the NIH-FSH-B1 standard preparation. 5) Contaminations of bovine follitropin by other glycoprotein hormones such as thyrotropin and lutropin amounted to 3 and 0.45 per cent by weight respectively as measured by specific radioimmunoassays and radioligand receptor assays. 6) The subunits alpha and beta of bovine follitropin were obtained by incubation in acidic urea, the chains being then separated by anion exchange chromatography. The subunits were subjitted to complete characterization. The amino-terminal residue of the alpha subunit is phenylalanine while a half cystine residue was found at the aminoterminal end of the beta chain. 8) Cross-contamination of the alpha and beta subunit preparations was measured by specific radioimmunoassays and amounted to 0.02 and 0.1 per cent by weight respectively.     

Identification of N5,N10-methylene tetrahydrofolate reductase as the enzyme involved in the 5-methyl tetrahydrofolate-dependent formation of a beta-carboline derivative of 5-hydroxytryptamine in human platelets.

An enzyme in human platelets or rat brain incubated with 5-methyl tetrahydrofolate (5MeH₄folate) yields formaldehyde (4, 13), which will combine with biogenic amines to form β-carbolines (5) or tetrahydroisoquinolines. This activity was purified 500-fold from human platelets which are the main storage site for 5-hydroxytryptamine in man. This enzyme was identical to N⁵, N¹⁰-methylene tetrahydrofolate (N⁵,N¹⁰-methylene H₄folate) reductase by the following criteria: (i) co-purification, (ii) heat denaturation, (iii) pH response, (iv) molecular weight, (5) cofactor requirements. A mechanism involving the enzymatic generation of formaldehyde followed by adduct formation with a biogenic amine is proposed.     

Studies on muscular dystrophy: a comparison by physical and chemical means of the normal human ATP-creatine transphosphorylases (creatine kinases) with those from tissues of Duchenne muscular dystrophy.

The four human Duchenne dystrophic isoenzymes (M-M, M-B, B-B, from the muscle and B-B from the brain) of ATP-creatine transphosphorylase (S. A. Kuby, H. J. Keutel, K. Okabe, H. K. Jacobs, F. Ziter, D. Gerber, and F. H. Tyler, 1977, J. Biol. Chem.252, 8382–8390) have now been compared physically and chemically with their normal human counterparts (viz., with the three isoenzymes, M-M, M-B, B-B, 2). All isoenzymes proved to be composed of two noncovalently linked polypeptide chains, by sedimentation equilibrium analyses in the presence and absence of disruptive agents. In the presence of 2-mercaptoethanol at 0.16(Γ/2), pH 7.8, the two native muscle types yielded identical values for s_20,w, concentration dependencies, and molecular weight, and similarly for the brain types (from the brain). But the human brain type proved to be slightly heavier than the muscle type (viz. 88,400 vs 85,900). All of the isoenzymes showed similar electrophoretic behavior between their several counterparts between pH 5–8, except perhaps between pH 8–10, where small differences appeared. The three native normal human isoenzymes, as well as the dystrophic human isoenzymes (M-M from the muscle and B-B from the brain) all contain 2 reactive sulfhydryl groups per mole or 1 per polypeptide chain of these two-chain proteins, which may be titrated with 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs₂); and under acidic conditions, quantitative titrations with 4,4′-dithiodipyridine yield a total of 10 -SH groups per mole of each brain type and 8 -SH groups per mole of muscle type, in the case of man, dystrophic man, calf, and rabbit. The kinetics of reactions between Nbs₂ and the sulfhydryl groups of all three normal human isoenzymes and two dystrophic human isoenzymes have been measured under several sets of denaturing conditions. A comparison of their reactive calculated second-order velocity constants reveal significant differences between these three normal human isoenzymes, but the k_{second order} values for the reactions of the sulfhydryl groups of the dystrophic M-M and B-B with Nbs₂, when compared with their normal counterparts, gave identical values in the presence of 7.3 m urea or 1.8% laurylsulfate, from which it may be inferred that very similar, if not identical, environments surround these two sets of sulfhydryl groups. A comparison of the amino acid compositions of the normal human muscle type and brain type with the human dystrophic M-M and B-B (from the brain) reveal essentially identical values for the muscle types but nearly identical values for the brain types, with a few differences. Their respective tryptic peptide maps have been compared of the S-carboxy-methylated proteins (alkylated with iodo[2-¹⁴C]acetic acid at the two exposed -SH groups per mole). Thus, the muscle types, normal and dystrophic, yield identical maps, but the brain types nearly identical maps, with a few significant differences. Isolation of the tryptic tridecapeptide from the S-carboxymethylated normal human and dystrophic human dimeric muscle-type ATP-creatine transphosphorylases, labeled at the single exposed SH group per polypeptide chain with iodo[2-¹⁴C]acetate, yielded the following sequence for both proteins: ValLeuThrCys(CH₂COOH)ProSerAsnLeuGlyThr GlyLeuArg [where Cys(CH₂COOH) is S-carboxymethyl cysteine]. This sequence showed remarkable homology with a few other equivalent peptides reported to be derived from the exposed SH group of other ATP-creatine transphosphorylases. In conclusion, there does not appear to be a mutation in the structural genes for the muscle-type creatine kinases detectable by the analyses presented here. However, the brain types warrant further investigation.     

Calcium-induced changes in thyroglobulin conformation

Polyethylene glycol solutions (10% w/v) were used to detect the effect of mono- and divalent cations on some properties of thyroglobulin. It is shown that in presence of 10% w/v polyethylene glycol in 0.01 M Tris-HCl, pH 7.5, calcium (less than 0.05 M) modifies the solubility, the sedimentation rate, and the Stokes' radius of thyroglobulin, while monovalent cations up to 0.6 M do not effect any of these properties. These findings can be explained by an increase in molecular compactness of thyroglobulin. Furthermore, it was shown that a synthetic polymer, polyethylene glycol, could be used to detect conformational changes.     

The formation of the pupal cuticle by Drosophila imaginal discs in vitro

Mass-isolated imaginal discs of Drosophila melanogaster form a chitin-containing pupal procuticle In vitro. Optimal procuticle deposition occurs when the discs are incubated for 4–6 hr with 0.5–1.0 μg/ml of 20-hydroxyecdysone and then with less than 0.05 μg/ml of 20-hydroxyecdysone. The formation of the chitin-containing procuticle is demonstrated using three independent assays: with fluorescene-conjugated cuticle proteins that bind to chitin; by electron microscopy; by incorporation of [³H]glucosamine into a chitin fraction. Synthesis and deposition of pupal cuticle proteins are also demonstrated. Incorporation of [³H]glucosamine into chitin is sensitive to inhibitors of protein, RNA and chitin synthesis, but has little sensitivity to inhibitors of DNA synthesis, and dolichol-dependent glycosylation.     

The role of 1,25-dihydroxyvitamin D3 and parathyroid hormone in the regulation of chick renal 25-hydroxyvitamin D3-24-hydroxylase.

A single 325-pmol dose of 1,25-dihydroxyvitamin D₃ given to chicks fed a vitamin D-deficient diet containing 3% calcium and 0.6% phosphorus suppresses renal mitochondrial 25-hydroxyvitamin D₃-1α-hydroxylase and stimulates the 25-hydroxyvitamin D₃-24-hydroxylase as measured by in vitro assay. This alteration in the enzymatic activity takes place over a period of hours. The administration of parathyroid hormone rapidly suppresses the 25-hydroxyvitamin D₃-24-hydroxylase. The alterations in the hydroxylases by parathyroid hormone or 1,25-dihydroxyvitamin D₃ are not related to changes in serum clacium or phosphate but could be related to changes in intracellular levels of these ions. Actinomycin D or cycloheximide given in vivo reduces the 25-hydroxyvitamin D₃-24-hydroxylase activity rapidly which suggests that the turnover of the enzyme and its messenger RNA is rapid (1- and 5-h half-life, respectively). The half-lives of the hydroxylases are sufficiently short to permit a consideration that the regulation by 1,25-dihydroxyvitamin D₃ and parathyroid hormone may involve enzyme synthesis and degradation.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.