cyclops encodes a nodal-related factor involved in midline signaling.

Ventral structures in the central nervous system are patterned by signals emanating from the underlying mesoderm as well as originating within the neuroectoderm. Mutations in the zebrafish, Danio rerio, are proving instrumental in dissecting these midline signals. The cyclops mutation leads to a loss of medial floor plate and to severe deficits in ventral forebrain development, leading to cyclopia. Here, we report that the cyclops locus encodes the nodal-related protein Ndr2, a member of the transforming growth factor type beta superfamily of factors. The evidence includes identification of a missense mutation in the initiation codon and rescue of the cyclops phenotype by expression of ndr2 RNA, here renamed "cyclops." Thus, in interaction with other molecules implicated in these processes such as sonic hedgehog and one-eyed-pinhead, cyclops is required for ventral midline patterning of the embryonic central nervous system.

Zebrafish nodal-related genes are implicated in axial patterning and establishing left-right asymmetry.

Nodal-related 1 (ndr1) and nodal-related 2 (ndr2) genes in zebrafish encode members of the nodal subgroup of the transforming growth factor-beta superfamily. We report the expression patterns and functional characteristics of these factors, implicating them in the establishment of dorsal-ventral polarity and left-right asymmetry. Ndr1 is expressed maternally, and ndr1 and ndr2 are expressed during blastula stage in the blastoderm margin. During gastrulation, ndr expression subdivides the shield into two domains: a small group of noninvoluting cells, the dorsal forerunner cells, express ndr1, while ndr2 RNA is found in the hypoblast layer of the shield and later in notochord, prechordal plate, and overlying anterior neurectoderm. During somitogenesis, ndr2 is expressed asymmetrically in the lateral plate as are nodal-related genes of other organisms, and in a small domain in the left diencephalon, providing the first observation of asymmetric gene expression in the embryonic forebrain. RNA injections into Xenopus animal caps showed that Ndr1 acts as a mesoderm inducer, whereas Ndr2 is an efficient neural but very inefficient mesoderm inducer. We suggest that Ndr1 has a role in mesoderm induction, while Ndr2 is involved in subsequent specification and patterning of the nervous system and establishment of laterality.

Expression of activin transcripts in follicle cells and oocytes of Xenopus laevis.

Activin is a potent inducer of axial mesoderm in vitro and may have a similar role in vivo. Xenopus laevis eggs contain significant amounts of activin or activin-like factors, but maternal activin transcripts have not been detected in Xenopus eggs. The maternal activin protein might be translated from activin beta A or beta B mRNAs that are transiently expressed during oogenesis, or activin polypeptides might be transferred from follicle cells to oocytes. To assess these possibilities we studied activin mRNAs in follicle cells and oocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) and RNA blotting. Activin beta A, beta B1, and beta B2 transcripts occur in follicle cells; among them, beta A mRNA is by far the most abundant. Activin beta A and beta B1 mRNAs were not detected by RT-PCR in the corresponding stage IV oocytes, but activin beta B2 transcripts were found at low levels. These observations are consistent with synthesis of activin beta A and possibly beta B polypeptides in follicle cells followed by their secretion and uptake by oocytes, although synthesis of activin beta B2 in the oocytes could make a contribution to the maternal activin pool.

[Elevation of arterial pressure during surgery with a pneumatic tourniquet: an independent action of renin?]. / Elévation de la tension artérielle lors de chirurgie sous garrot pneumatique: un mécanisme indépendant de la rénine?

The aim of this study was to evaluate the effectiveness of captopril for the prevention of the increase of arterial pressure during orthopaedic surgery requiring the application of lower limb tourniquets with balanced anaesthesia. Twenty consecutive patients were included in the study and were randomly divided into two groups. The first (n = 10) received 50 mg captopril orally together with the preanaesthetic medication, the second (n = 10) received a placebo at the same time. The different variables studied (arterial pressure, heart rate) were continuously measured. This study demonstrated that the pretreatment with captopril did not prevent an increase of the arterial pressure during the application of a tourniquet. The means of the systolic and diastolic arterial pressures at the end of the application of the tourniquet were 128/86 and 128/81 in the captopril group and the placebo group, respectively. This result shows that the renin-angiotensin system does not significantly contribute to the increase of the arterial pressure induced by a tourniquet.

The role of growth factors in embryonic induction in Xenopus laevis.

Establishment of the body pattern in all animals, and especially in vertebrate embryos, depends on cell interactions. During the cleavage and blastula stages in amphibians, signal(s) from the vegetal region induce the equatorial region to become mesoderm. Two types of peptide growth factors have been shown by explant culture experiments to be active in mesoderm induction. First, there are several isoforms of fibroblast growth factor (FGF), including aFGF, bFGF, and hst/kFGF. FGF induces ventral, but not the most dorsal, levels of mesodermal tissue; bFGF and its mRNA, and an FGF receptor and its mRNA, are present in the embryo. Thus, FGF probably has a role in mesoderm induction, but is unlikely to be the sole inducing agent in vivo. Second, members of the transforming growth factor-beta (TGF-beta) family. TGF-beta 2 and TGF-beta 3 are active in induction, but the most powerful inducing factors are the distant relatives of TGF-beta named activin A and activin B, which are capable of inducing all types of mesoderm. An important question relates to the establishment of polarity during the induction of mesoderm. While all regions of the animal hemisphere of frog embryos are competent to respond to activins by mesoderm differentiation, only explants that include cells close to the equator form structures with some organization along dorsoventral and anteroposterior axes. These observations suggest that cells in the blastula animal hemisphere are already polarized to some extent, although inducers are required to make this polarity explicit.(ABSTRACT TRUNCATED AT 250 WORDS)

The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos.

The properties of antisense phosphorothioate and unmodified oligodeoxynucleotides have been studied in Xenopus oocytes and embryos. We find that phosphorothioates, like unmodified oligodeoxynucleotides, can degrade Vg1 mRNA in oocytes via an endogenous RNase H-like activity. In oocytes, phosphorothioate oligodeoxynucleotides are more stable than unmodified oligodeoxynucleotides and are more effective in degrading Vg1 mRNA. In embryos, neither unmodified nor phosphorothioate deoxyoligonucleotides were effective in degrading Vg1 message at sub-toxic doses.

Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity.

Previous experiments have shown that mRNA translation in frog oocytes can be inhibited by the injection of a complementary antisense RNA. Here we explore the use of antisense RNAs to study the functions of localized maternal mRNAs during postfertilization development. While developmental abnormalities were observed in injected fertilized eggs, these abnormalities could not be attributed to the antisense RNA since they were induced at a similar frequency in control embryos. Biochemical tests show that the injected antisense RNA does not form stable hybrids in vivo with its complementary endogenous mRNA. In addition, a novel activity that unwinds RNA:RNA duplexes was found. This activity exists at high levels in eggs and early embryos and is absent or very much diminished in oocytes and late blastula embryos. These results suggest that antisense RNAs may be of limited use in studying the functions of maternal RNAs in Xenopus.

Effect of beta-blockers on plasma lipids.

Forty-five hypertensive patients (I-II WHO), after two weeks wash out, were randomly allocated to receive 100 mg/day atenolol, 200 mg/day metoprolol and 10 mg/day mepindolol for three months, in order to evaluate their possible effect on lipid metabolism. Plasma triglyceride levels were increased by the three drug treatments; the increase was, however, greater after mepindolol. Total cholesterol was unchanged by atenolol, increased by metoprolol and decreased by mepindolol. HDL-cholesterol was unchanged by atenolol, decreased by metoprolol and increased by mepindolol, whereas LDL-cholesterol was increased by atenolol, unchanged by metoprolol and decreased by mepindolol. Therefore, the LDL/HDL ratio was decreased by mepindolol (from 3.15 +/- 1.71 to 2.92 +/- 1.17) and increased by atenolol and metoprolol. The results show that the treatment with atenolol, metoprolol and mepindolol does not significantly affect the lipid levels suggesting that cardioselective beta-blockers as well as those with ISA have no untoward effect on lipid metabolism.

Identification and cloning of localized maternal RNAs from Xenopus eggs.

A central question in developmental biology is to explain how cells in different regions of an embryo acquire different developmental fates. We have begun to address this question by investigating whether specific RNAs are localized within a frog egg. Differential screening of a cDNA library shows that most maternal RNAs are uniformly distributed along the animal-vegetal axis. However, we find that a rare class of maternal RNAs is localized. cDNA clones of four localized RNAs have been characterized. Three of these cDNAs are derived from maternal RNAs that are concentrated in the animal hemisphere of unfertilized eggs and remain localized through the early blastula stage. One cDNA is derived from a maternal RNA found almost exclusively in the vegetal hemisphere at both stages. These studies show that some informational molecules, specifically RNAs, are localized in eggs and are inherited by particular blastomeres.

Computer-assisted mechanistic structure-activity studies: application to diverse classes of chemical carcinogens.

In the first part of this paper we have indicated how the techniques and capabilities of theoretical chemistry, together with experimental results, can be used in a mechanistic approach to structure-activity studies of toxicity. In the second part, we have illustrated how this computer-assisted approach has been used to identify and calculate causally related molecular indicators of relative carcinogenic activity in five classes of chemical carcinogens: polycyclic aromatic hydrocarbons and their methyl derivatives, aromatic amines, chloroethanes, chloroalkenes and dialkyl nitrosamines. In each class of chemicals studied, candidate molecular indicators have been identified that could be useful in predictive screening of unknown compounds. In addition, further insights into some mechanistic aspects of chemical carcinogenesis have been obtained. Finally, experiments have been suggested to both verify the usefulness of the indicators and test their mechanistic implications.

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Metabolism and relative carcinogenic potency of chloroethanes: a quantum chemical structure-activity study.

Using the all-valence electron, semiempirical molecular orbital method, MNDO, properties have been identified and calculated for eight chloroethanes which can serve as indicators of their extent of transformation to alcohols by cytochrome P450 and the subsequent formation of aldehydes by loss of HCl from these alcohols. The assumption was made that these aldehydes are the active carcinogens of the chloroethanes and that they act as electrophiles in adduct formation with DNA bases. Electrophilic properties of these putative ultimate carcinogens have been calculated which are indicators of the rank order of carcinogenic activity of the parent compounds in susceptible species. Particularly relevant in this respect are (a) the electron affinity of aldehydes as measured by the energy of their electron accepting (lowest energy virtual) orbital, and (b) the net charge on the C alpha carbon, adjacent to the carbonyl carbon, which can participate in electrophilic attack on nucleophilic sites of DNA bases. The molecular properties identified in this study as indicators of rank order or carcinogenic activity of the parent chloroethanes are consistent with the importance of cytochrome P450 in transforming halohydrocarbons to active carcinogens and of acylchlorides and chloroaldehydes as the active form. Their validity and usefulness can be further tested in screening unknown and more complex chlorohydrocarbons for carcinogenic activity.

Metabolism and relative carcinogenic potency of chloroethylenes: a quantum chemical structure-activity study.

Properties of six chloroethylenes which could serve as indicators of their relative metabolic behavior and carcinogenic activity have been calculated using Modified Neglect of Diatomic Overlap (MNDO), a semiempirical, all valence electron, molecular orbital method. Possible pathways of transformation of parent compounds to acylchlorides, chloroaldehydes and epoxides--their putative ultimate carcinogens--were considered, and heats of formation and relative stabilities of intermediates were calculated. Our results indicate that carbonyl compounds could be formed with and without the intermediacy of epoxides, suggesting the possibility of more than one pathway in activation of parent compounds. Electronic properties of carbonyl products and epoxide carbocations, putative ultimate carcinogens which could serve as indicators of their relative electrophilicities, were also calculated. The results obtained indicated that the relative extent of metabolism to carbonyl products, rather than their electrophilicity, is a determinant of the relative carcinogenic activity of the parent compound. Of the various thermodynamic criteria investigated, four were found to be indicators of both relative metabolic behavior and carcinogenic activity.