DNA-relaxing activity and endonuclease activity in Xenopus laevis oocytes.

Complex simian virus 40 DNA produced by a soluble cell-free extract derived from stage 6 oocytes of Xenopus laevis consists of fully relaxed circles (i.e., with no superhelical turns). An endonuclease and a DNA-relaxing protein, either or both of which could be responsible for the relaxation of the complex DNA, have been purified from the extract. The endonuclease(s) produces nicked circles (having a single-strand scission) and linear full-size molecules. The DNA-relaxing protein is in the nucleus, has a molecular weight of apporximately 70,000, and is able to remove both negative and positive superhelical turns.     

Circadian rhythm of interrenal activity in Xenopus laevis

Young specimens of Xenopus laevis were kept under constant environmental conditions (artifical light from 600 to 1800 hr, feeding between 800 and 900 hr) and the concentrations of aldosterone and corticosterone in the serum were measured every 3 hr. Furthermore, the kidneys containing the interrenals were removed and their corticosteroid release under stimulation by the pars distalis and mammalian ACTH was determined. Under these conditions, the corticosteroid levels in the serum were maximal from 900 to 1200 hr (corticosterone, 7.7 +/- 0.47 ng/ml; aldosterone, 2.8 +/- 0.27 ng/ml) and minimal during the night (corticosterone, 5.2 +/- 0.43 ng/ml; aldosterone, 1.7 +/- 0.18 ng/ml). The basal secretion rate of the interrenals in vitro showed the opposite course (corticosterone, 24 +/- 3 to 72 +/- 8 pg/min/tissue; aldosterone, 46 +/- 5 to 68 +/- 9 pg/min/tissue). Stimulation by the pars distalis and mammalian ACTH clearly increased the secretion rate. After both types of stimulation the original rhythm was lost for aldosterone but still present for corticosterone. The ratio of the amounts of corticosterone/aldosterone was greater than 1.0 in the serum but less than 1.0 in the incubation fluid. It decreased significantly after stimulation in vitro by pars distalis or ACTH.     

Myogenesis in Xenopus laevis

The amphibian embryo provides a convenient experimental system with which to study myogenesis. The earliest steps in the formation of axial and cardiac muscle are accessible for investigation using both embryological and molecular approaches. We review the origins of skeletal and cardiac muscle in the Xenopus embryo, the molecular markers available to detect muscle differentiation, and the use of embryo explants to investigate the regulation of myogenesis.     

Deiodinase activity is present in Xenopus laevis during early embryogenesis

Thyroid hormones orchestrate amphibian metamorphosis. The type 2 and type 3 deiodinases make vital contributions to this process by controlling levels of the thyroid hormones T(4) and T(3) available to different tissues. Because the tadpole thyroid gland is not functional until stage NF44, it has been widely assumed that thyroid signaling is absent during amphibian early development, thyroid hormone only becoming a major regulator during premetamorphic stages. Similarly, in mammals, thyroid function is known to be essential to neuronal development, especially during the perinatal stages, but again little is known about early stages of development. Here we demonstrate that key elements of thyroid hormone signaling are present during early development of Xenopus. In particular, we find functional thyroid hormone-activating deiodinases and significant levels of their substrates, T(4) and T(3), during early embryogenesis. Furthermore, we have further characterized a recently identified deiodinase in amphibians, homologous to mammalian type 1 deiodinase (D1). This enzyme is expressed in marked, spatially defined patterns during embryogenesis. The patterns of expression of type 1 deiodinase are distinct from those of type 2 and type 3 deiodinases. Deiodinase expression is found in neurogenic areas from stage NF30 onward, both in the central and peripheral nervous systems. We conclude that both activating and inactivating deiodinases show dynamic patterns of expression during early embryogenesis in amphibians, particularly in neurogenic areas. These findings suggest that thyroid hormone signaling is a key component of early neuronal development in vertebrates.     

Controlling transgene expression to study Xenopus laevis metamorphosis

Sperm-mediated transgenesis of Xenopus laevis is the first application of genetic methodology to an amphibian. However, some transgenes are lethal when they are expressed constitutively. To study the influence of these genes on amphibian metamorphosis and to generate F1 progeny from mature transgenic adults, these transgenes must be placed under the control of an inducible system so that they can be activated at specific times in development. We show that two well known binary inducible gene expression systems supplement transgenesis for the study of X. laevis metamorphosis, one system controlled by the progesterone analogue RU-486 and the other controlled by the tetracycline derivative doxycycline. By inducing a dominant negative form of the thyroid hormone receptor under the control of doxycycline specifically in the nervous system we have delimited the developmental periods within which thyroid hormone controls innervation of the developing limb from the spinal cord.     

The distribution of interrenal stimulating activity in the brain of Xenopus laevis

The stimulation of corticosteroid release by the interrenal tissue of Xenopus laevis was investigated using an in vitro preparation of the interrenals and a radioimmunoassay for determinations of corticosterone and aldosterone in the perifusate. The stimulatory activity of four different areas was tested, the pars distalis, the post- and preoptic hypothalamus, and the cerebrum. It was found that besides the pars distalis, the post- and preoptic hypothalamus exhibit stimulatory activity on the interrenals. No such activity was found in the cerebrum. There was a decrease of activity found from the pars distalis to the postoptic and then to the preoptic part of the hypothalamus. The release of both steroids had different sensitivity. Aldosterone release was significantly elevated by all three different tissues, while the increase of the release of corticosterone was only found significant in the case of stimulation by the pars distalis. The results also make clear that the increase of the release of corticosterone is less than that of aldosterone. The ratio of corticosterone to aldosterone decreased significantly after stimulation. This points to the fact that the rate of conversion of corticosterone to aldosterone (18-hydroxylation) is stimulated more than the production of corticosterone from progesterone.     

Exonucleolytic degradation of double-stranded RNA by an activity in Xenopus laevis germinal vesicles

We have identified, in extracts from Xenopus laevis germinal vesicles, a 5' exonuclease activity that cleaves double-stranded RNA (dsRNA). Features of the 5' ends of dsRNAs determine whether the strands are symmetrically or asymmetrically degraded. The activity hydrolyzes in the 5' to 3' direction, releasing 5'-mononucleotides processively, favoring strands with 5'-monophosphate termini; molecules with capped ends are resistant to digestion. Because of its ability to processively digest dsRNA to mononucleotides, we have named the exonuclease Chipper, which could cooperate or compete with Dicer (an endonuclease that produces molecules with a 5'-phosphate) in the processing of dsRNA.     

In vitro progesterone binding to Xenopus laevis oocytes.

The melanosome fraction of Xenopus laevis oocytes was found to bind progesterone specifically. The binding is of high affinity and low capacity and displays a mean dissociation constant K_d = 5.8 ± 3.2 × 10^?8M at 4°C. Preliminary characterization of the receptor was performed and experiments attempting to assign a biological role in relation to oocyte maturation are discussed.     

Aerobic and anaerobic microbiology of paronychia

Pus specimens from 28 patients with paronychia of the finger yielded bacterial growth by techniques for cultivation of aerobic and anaerobic bacteria. Anaerobic and aerobic organisms only were isolated in pure culture in five (18%) and eight patients (29%), respectively; mixed aerobic and anaerobic flora were present in 15 patients (54%). Seventy-two isolates were recovered, or 2.6 isolates per specimen. The predominant anaerobic organisms were Gram-positive anaerobic cocci, Bacteroides species, and Fusobacterium species. The predominant aerobic organisms were Staphylococcus aureus, gamma-hemolytic streptococci, Eikenella corrodens, group A beta-hemolytic streptococci, alpha-hemolytic streptococci, and Klebsiella pneumoniae. Candida albicans was recovered in four cases. This study demonstrates the mixed aerobic and anaerobic bacteriology of paronychia.     

Germ-line transmission of transgenes in Xenopus laevis

Adult Xenopus laevis frogs made transgenic by restriction enzyme-mediated integration were bred to test the feasibility of establishing lines of frogs that express transgenes. All of the 19 animals raised to sexual maturity generated progeny that expressed the transgene(s). The patterns and levels of expression of green fluorescent protein transgenes driven by a viral promoter, rat promoter, and four X. laevis promoters were all unaffected by passage through the germ line. These results demonstrate the ease of establishing transgenic lines in X. laevis.     

Comparative analysis and expression of neuroserpin in Xenopus laevis

Serine protease inhibitors form a diverse family of proteins of which most members inhibit target serine proteases. Neuroserpin is a member of this family. Here, we have characterized neuroserpin in the nonmammalian species Xenopus laevis and found a high degree of aminoacid sequence conservation, especially of the reactive center loop, of the Xenopus protein compared to mammalian and chicken neuroserpin sequences, suggesting a conserved target specificity. Neuroserpin mRNA and protein were expressed throughout Xenopus development, while in the adult frog high mRNA expression was found in neuronal and neuroendocrine tissues, and the reproductive organs, and the neuroserpin protein was detected mainly in brain and pituitary. More specifically, in Xenopus pituitary neuroserpin mRNA was expressed higher in the neurointermediate lobe than in the pars distalis. At the protein level, we detected a 55-kDa neuroserpin protein in the pars nervosa, two neuroserpin proteins of 44- and 50-kDa in the melanotrope cells of the pars intermedia, and a 46-kDa product in the pars distalis. On the basis of its relatively high degree of sequence conservation and its expression pattern, we conclude that Xenopus neuroserpin may play an important physiological role, e.g. as a serine protease inhibitor during development, and for proper neuronal and neuroendocrine cell functioning.     

DNA primase activity associated with DNA polymerase alpha from Xenopus laevis ovaries.

One of the two forms of DNA polymerase alpha from ovaries of the frog Xenopus laevis catalyzed ribonucleoside triphosphate-dependent DNA synthesis on single-stranded circular fd phage DNA templates. DNA synthesis was dependent on ATP and added template. CTP, GTP, and UTP stimulated DNA synthesis but were not required and could not substitute for ATP. DNA synthesis was not inhibited by alpha-amanitin. Neither poly(dT) nor double-stranded DNA served as template. Analysis of [32P]-dTMP-labeled product by neutral and alkaline agarose gel electrophoresis showed that 0.1- to 1-kilobase DNA fragments (average size of approximately equal to 0.25 kilobase) were synthesized. The fragments were not covalently linked to the template. Either [alpha-32P]NMP, [gamma-32P]ATP, or [gamma-32P]GTP were incorporated also into the product. Analysis of the product after hydrolysis by KOH, alkaline phosphatase, or bacteriophage T4 3' leads to 5' exonuclease showed the presence of a small oligoribonucleotide primer at the 5' end of the newly synthesized DNA. NTP-dependent DNA-synthesizing activity copurified on six columns and cosedimented during glycerol gradient centrifugation with one form of DNA polymerase alpha activity but not with the other form. These results suggest that DNA primase activity is associated with one of the two forms of X. laevis DNA polymerase alpha.     

Major DNA Polymerases Common to Different Xenopus laevis Cell Types

DNA polymerases from Xenopus laevis oocytes, unfertilized eggs, and kidney cells grown in culture have been characterized. The same three major DNA polymerase activities are present in all cell types examined. We attempt to relate the characteristics of amphibian enzymes to those of mammalian polymerases.     

Cerulein mRNA and peptide alpha-amidation activity in the skin of Xenopus laevis: stimulation by norepinephrine

The skin of Xenopus laevis, because of its high concentrations of amidated peptides, is an excellent model for the study of neuropeptide biosynthesis. In this paper we determined levels of the mRNAs which encode cerulin and characterized the enzymatic activity which likely produces the carboxyl terminal alpha-amide moiety of cerulein. This enzyme is stimulated by the addition of CuSO4 and ascorbate and requires the presence of molecular oxygen and is thus similar to the peptidyl-glycine alpha-amidating monooxygenase (PAM) described in mammals. Hybridization analysis showed that three cerulein mRNAs of different molecular sizes are present in varying proportions in the skin of individual frogs. Injection of norepinephrine into the dorsal lymph sac of Xenopus caused immediate secretion and depletion from skin of PAM activity but did not cause a comparable immediate decrease in skin levels of cerulein mRNA or total RNA. By 2 weeks after norepinephrine injection, cerulein mRNA levels increased to fourfold over control levels. By 4 weeks, skin PAM activity was almost restored to control levels.     

Aerobic and anaerobic bacterial interactions in the development of anaerobic bacterial pneumonia.

The interactions between aerobic and anaerobic bacteria in the development of anaerobic bacterial pneumonia were studied by introducing Bacteroides fragilis and Escherichia coli alone or in combination into guinea pigs by tracheal infusion. The lung lesions induced by B. fragilis were mainly located near the pleura, unlike those induced by E. coli, and were accompanied by pneumonia, lung abscess, and pleuritis. The lung lesions produced by mixed infection with B. fragilis (10(9) cfu) and E. coli (10(7) cfu) were significantly more severe than those induced by either microbe alone, and the redox potentials at the foci of inflammation were markedly reduced (max: -330 mV). Analysis of lung lesions after treatment with aztreonam and clindamycin and neutrophil phagocytic activity suggested that E. coli was primarily responsible for the lung lesions and that B. fragilis promoted the accompanying inflammation, resulting in increased pathogenicity of the mixed infections.