Nodal signaling in early vertebrate embryos: themes and variations.

The nodal family of TGFbeta-related ligands have emerged as critical regulators of early vertebrate embryogenesis. Recent studies in mice, fish, and frogs of nodals and their intracellular transducers allow a comparison of how this signaling pathway is used in the patterning of early embryos of these different vertebrates.

Induction of mesoderm by a viral oncogene in early Xenopus embryos.

During frog embryogenesis, mesoderm is specified in the equatorial region of the early embryo by a signal from the vegetal hemisphere. Prospective ectodermal cells dissected from the animal hemisphere can be respecified to form mesodermal tissues by recombination with vegetal tissue or by treatment with any of several polypeptide growth factors or growth factor-like molecules. Together with the discovery that several developmental mutations in Drosophila are in genes with significant homology to mammalian mitogens and oncogenes, these observations suggest that early developmental signals may use similar transduction pathways to mitogenic signals characterized in cultured mammalian cells. Whether mesoderm can be induced by activation of intracellular signal transduction pathways implicated in mitogenesis and oncogenesis has been investigated with the viral oncogene polyoma middle T. Microinjection of middle T messenger RNA into early embryos results in the respecification of isolated prospective ectodermal tissue to form characteristic mesodermal structures. Middle T in frog blastomeres appears to associate with cellular activities similar to those observed in polyoma-transformed mouse cells, and transformation-defective middle T mutants fail to induce mesoderm. These results suggest that early inductive signals and mitogenic and oncogenic stimuli may share common signal transduction pathways.

Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo.

We have constructed a recombinant murine retrovirus which efficiently transduces avian pp60c-src into murine cells and which is easily rescued from infected cells in plasmid form. To characterize the virus, several randomly selected NIH 3T3 lines were isolated after infection with recombinant retroviral stocks. All lines overproduced avian pp60c-src and appeared morphologically normal. Immunoprecipitates made from these lines with antisera specific for pp60c-src were tested for their kinase activities in vitro. We find that both autokinase and enolase kinase activities increase proportionately with the level of pp60c-src in the immunoprecipitates. To further test the authenticity of the pp60c-src encoded by the retroviral vector, these analyses were repeated in the presence of polyomavirus middle T antigen. Avian pp60c-src was activated as a protein kinase, indicating that the virally encoded pp60c-src interacts normally with middle T antigen. Interestingly, by increasing the intracellular levels of pp60c-src 15-fold over normal endogenous levels, we were unable to obtain a proportionate increase in the amount of middle-T-antigen-pp60c-src complex. Finally, using the shuttle features designed into the vector, we have isolated the first fully processed cDNA encoding functional avian pp60c-src X pp60c-src synthesized in vitro with this cDNA had intrinsic protein kinase activity and no detectable phosphatidylinositol kinase activity.

Evidence for a novel signal transduction pathway activated by platelet-derived growth factor and by double-stranded RNA.

Platelet-derived growth factor (PDGF) and the synthetic double-stranded RNA poly(I).poly(C) [poly(I.C)] stimulate transcription of the JE gene in BALB/c-3T3 fibroblasts. The response of JE to poly(I.C) does not appear to be channeled through any known component of the PDGF receptor signal transduction apparatus. In addition, JE sequences upstream of the transcription start site are devoid of previously identified poly(I.C)-responsive elements, such as those found in the beta-interferon gene. These data suggest that a novel signal transduction pathway regulates the JE response to PDGF and double-stranded RNA. The c-myc and c-fos proto-oncogenes also respond to this pathway but with poor efficiency. However, this pathway operates very efficiently on other PDGF-inducible genes that encode the secretory proteins KC and M-CSF.

Molecular analysis of early growth-associated events during the differentiation of F9 cells into embryoid bodies.

Mouse F9 teratocarcinoma cell lines can be induced to differentiate into either parietal endoderm or embryoid bodies which contain visceral endoderm-like cells. The nature of the early molecular events involved in these two differentiation pathways has not yet been fully elucidated. Moreover, since the process of differentiation is often accompanied by changes in cell growth, it is often difficult to determine which of the events that do occur during the early stages of differentiation are a direct result of the process of differentiation and which events are indirect results that occur as a consequence of altered cell growth. In the experiments reported here we have attempted to distinguish between these two possibilities by examining the patterns of expression of a representative group of growth-associated genes (i.e., c-myc, p53, and histone H3) when F9 cell aggregates are induced to differentiate into embryoid bodies containing visceral endoderm. By analysis of the patterns of growth-associated gene expression in both retinoic acid treated and nontreated F9 cell aggregates, we were able to classify early events as differentiation-specific events (events which occurred only following retinoic acid treatment of aggregates) or nondifferentiation-specific events caused by reduction in cell growth (events which occurred even when aggregates were not treated with retinoic acid). Our results show that F9 cells differentiated into embryoid bodies containing visceral endoderm-like cell exhibit an early reduction in both growth and c-myc mRNAs which is neither retinoic acid-specific nor differentiation-specific. However, following this initial response to aggregation, constant levels of c-myc mRNA are maintained despite continued reduction in growth. Thus, it appears that alteration in c-myc expression is a differentiation-specific event along the pathway to formation of visceral endoderm. Interestingly, however, the nature and time course of this alteration in c-myc expression in F9 cells' differentiation into visceral endoderm is different from that observed in F9 cells differentiated into parietal endoderm.

Modulation of the antitumor and biochemical properties of bis(diphenylphosphine)ethane with metals.

Bis(diphenylphosphine)ethane (DPPE) and its bis[chlorogold(I)] [DPPE(Au2Cl2)], and bis[trichlorogold(III)] [DPPE(Au2Cl6)], complexes have in vivo antitumor activity. To determine if interaction with metals in situ can play a role in the antitumor activity of DPPE, we have studied the effects of DPPE, DPPE(Au2Cl2), DPPE(Au2Cl6) and mixtures of DPPE with metal salts on in vitro and in vivo biological systems. The in vitro cytotoxic potencies of the two DPPE-gold complexes were approximately 10-fold greater than that of DPPE. In addition, the cytotoxic potency of DPPE was increased when incubated with cells in the presence of Au(III) and Cu(II) salts, whereas Mg(II), Zn(II), Mn(II), Fe(II), Co(II), and Cd(II) had no effect. The effects of DPPE, DPPE(Au2Cl2) and mixtures of DPPE and metal salts on the activity of a model enzyme system, DNA polymerase alpha were measured. While DPPE did not inhibit the activity of DNA polymerase alpha, the DPPE(Au2Cl2) complex and mixtures of DPPE and Cu(II) salts inhibited the activity of the enzyme. Consistent with the effects observed in vitro, coadministration of Cu(II) or Au(III) increased the in vivo potency of DPPE in mice bearing i.p. P388 leukemia. Fifteen other DPPE analogues were evaluated for in vivo antitumor activity and for the effect of Cu(II) on their in vitro cytotoxic potency; there was a relationship between the ability of Cu(II) to potentiate the cytotoxic activities of DPPE analogues and their having in vivo antitumor activity.

A mouse homologue of FAST-1 transduces TGF beta superfamily signals and is expressed during early embryogenesis.

The transcription factor FAST-1 has recently been shown to play a key role in the specification of mesoderm by TGF beta superfamily signals in the early Xenopus embryo. We have cloned Fast1, a mouse homologue of Xenopus FAST-1, and characterized its expression during embryogenesis and function in activin/TGF beta signal transduction. In vitro, Fast1 associates with Smads in response to an activin/TGF beta signal to form a complex that recognizes the Xenopus activin responsive element (ARE) targeted by Xenopus FAST-1. In intact cells, introduction of Fast1 confers activin/TGF beta regulation of an ARE-luciferase reporter. In embryos, Fast1 is expressed predominantly throughout the epiblast before gastrulation and declines as development progresses. We propose that mouse Fast1, like Xenopus FAST-1, mediates TGF beta superfamily signals specifying developmental fate during early embryogenesis.

TGF-beta superfamily signaling and left-right asymmetry.

Despite an outwardly bilaterally symmetrical appearance, most internal organs of vertebrates display considerable left-right (LR) asymmetry in their anatomy and physiology. The orientation of LR asymmetry with respect to the dorsoventral and anteroposterior body axes is invariant such that fewer than 1 in 10,000 individuals exhibit organ reversals. The stereotypic orientation of LR asymmetry is ensured by distinct left- and right-side signal transduction pathways that are initiated by divergent members of the transforming growth factor-beta (TGF-beta) superfamily of secreted proteins. During early embryogenesis, the TGF-beta-like protein Nodal (or a Nodal-related ortholog) is expressed by the left lateral plate mesoderm and provides essential LR cues to the developing organs. In chick embryos at least, bone morphogenetic protein (BMP) signaling is active on the right side of the embryo and must be inhibited on the left in order for Nodal to be expressed. Thus, at a key point in the determination of LR asymmetry, left-sided signaling is mediated by the transcription factors Smad2 and Smad3 (regulated by Nodal), whereas signaling on the right depends on Smad1 and Smad5 (which are regulated by BMP). This review summarizes the considerable progress that has been made in recent years in understanding the complex network of feedback and feedforward circuitry that regulates both the left- and right-sided pathways. Also discussed is the problem of how signal transduction mediated by the Smad proteins can pattern LR asymmetry without interfering with coincident dorsoventral patterning, which relies on the same Smad proteins.

Homologous and heterologous desensitization mediated by vasopressin in smooth muscle cells.

Prolonged exposure of A-10 cells to Arginine Vasopressin (AVP) resulted in the following responses: (a) loss of vasopressin receptors from the cell surface (30-40%), (b) increased basal levels of inositol and inositol monophosphate, (c) decreased inositol di- and trisphosphate production and decreased intracellular calcium release in response to a second challenge with AVP, (d) attenuation of AVP-mediated inhibition of isoproterenol-stimulated cAMP and ANF-stimulated cGMP accumulation and (e) attenuation of thrombin and ATP-mediated increase in inositol di- and trisphosphate accumulation and intracellular calcium release. All the above responses depended on the time of exposure of the cells to AVP with the responses being attenuated as early as 5-10 min of exposure to AVP. The desensitization also depended on the concentration of AVP used with 50% of maximal desensitization for each response being observed at 5 nM of AVP. This concentration of AVP corresponded well with the Kd of vasopressin for binding to these sites. Desensitization of protein kinase C (PKC) by prolonged exposure of the cells to PDBu or addition of the PKC inhibitor staurosporine during pretreatment with AVP did not prevent AVP-mediated desensitization, suggesting that PKC may not be involved in AVP-mediated desensitization in smooth muscle cells. It is concluded that AVP induced both homologous and heterologous desensitization of phosphatidylinositol turnover and calcium release in smooth muscle cells. The desensitization processes did not appear to be mediated by protein kinase C. The possibility that the locus of the heterologous desensitization may be at the level of substrates such as PI, PIP and PIP2 is discussed.

The isolated major histocompatibility complex class I alpha3 domain binds beta2m and CD8alphaalpha dimers.

The MHC class I molecule plays a crucial role in cytotoxic lymphocyte function. The heavy chain of the MHC class I molecule can form many non-covalent interactions with other molecules on multiple domains and surfaces. We have generated an isolated alpha3 domain of a murine MHC class I molecule and evaluated the contribution of this domain to binding with the MHC class I light chain, beta2m, and CD8. The alpha3 domain binds beta2m at a thousand-fold higher concentration than the whole MHC, and binds CD8alphaalpha with a dependence on the alpha3 CD loop. Our results are relevant for models of MHC folding and CD8-MHC function. The study of individual domains of complex molecules is an important strategy for understanding their dynamic structure and function.

Isolation and characterization of a beta-tubulin gene from Candida albicans.

We report the isolation and nucleotide sequence determination of a beta-tubulin gene (TUB2) from the pathogenic dimorphic fungus Candida albicans. Nucleotide sequence analysis revealed that TUB2 encodes a protein of 449 amino acids (aa) with considerable sequence homology to beta-tubulins isolated from other fungal species. The nucleotide sequence of the C. albicans gene is 70% homologous to that of the Saccharomyces cerevisiae gene. The coding region for the C. albicans beta-tubulin gene is interrupted by two introns. The first intron occurs after the 4th aa and the second intron occurs after the 13th aa. A comparison with other fungal beta-tubulin genes indicates that the intron locations are highly conserved. Codon usage in the C. albicans TUB2 gene is nonrandom, as has been observed for other fungal beta-tubulin genes. The C. albicans TUB2 gene is transcribed to yield a 1.8-kb mRNA species. On the basis of genomic Southern-blot analysis, we conclude that C. albicans most likely possesses a single beta-tubulin gene.

Induction of c-fos protein by activation of vasopressin receptors in smooth muscle cells.

Stimulation of vasopressin (V1) receptors of rat aortic smooth muscle cells (A-10, ATCC CRL 1476) results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) with the mobilization of intracellular calcium. When A-10 cells are exposed to arginine vasopressin (AVP), there is an increase in the level of c-fos oncoprotein. The extent of induction of c-fos oncoprotein depends on both the time of exposure of the cells to AVP, reaching a maximum at 60 min after which there is a slow decline, and the concentration of AVP used, with an approximate EC50 of 1 nM which corresponds well with the Kd of vasopressin binding to these receptors. This vasopressin-mediated increase in c-fos protein level is inhibited by a V1/V2 antagonist (SKF 101498) suggesting that this is a receptor-mediated event. In addition dDAVP, a V2 selective agonist, is much less effective than AVP in inducing c-fos protein suggesting that AVP mediates its effect via V1 receptors. Desensitization of vasopressin receptors by prolonged exposure to AVP resulted in no additional induction of c-fos protein level in response to second challenge of AVP. In addition to AVP, phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), also stimulates the accumulation of c-fos protein although to a lesser extent than AVP. The above data suggest that c-fos protein levels in smooth muscle cells are regulated by AVP and the hormonal effect may be mediated through PI turnover and DAG, IP3 and Ca2+ signals.

PCR detection of Borrelia burgdorferi DNA in cerebrospinal fluid of Lyme neuroborreliosis patients.

We used the polymerase chain reaction (PCR), a method useful in the detection of Borrelia burgdorferi in vitro, to evaluate CSF in patients thought to have neuroborreliosis. Nested pairs of oligonucleotide primers were designed to recognize the C-terminal region of B burgdorferi OspA. CSF samples were obtained from (1) patients with immunologic evidence of systemic B burgdorferi infection and clinical manifestations suggestive of CNS dysfunction, (2) seronegative patients with clinical disorders consistent with Lyme borreliosis, and (3) patient and contamination controls; all were analyzed in a blinded fashion. PCR detected B burgdorferi OspA DNA in CSF of (1) 10 of 11 patients with Lyme encephalopathy, (2) 28 of 37 patients with inflammatory CNS disease, (3) seven of seven seronegative patients with Lyme-compatible disorders, and (4) zero of 23 patient controls. Zero of 83 additional contamination controls were PCR-positive. In eight patients from whom we obtained CSF before and after parenteral antimicrobial therapy, PCR results invariably predicted clinical outcome accurately.

Effects of coordinated gold compounds on in vitro and in situ DNA replication.

Auranofin, a coordinated gold compound, inhibits in vitro DNA synthesis and displays in vivo antitumor activity. To understand the mechanisms of inhibition of DNA replication, we have examined the effects of auranofin and other gold complexes on the activities of purified cellular and herpesvirus-induced DNA polymerases, and on in situ DNA replication in permeabilized S phase KB cells. Evaluation of the data suggests the following conclusions. (1) The gold compounds varied in their abilities to inhibit DNA polymerase activities. DNA polymerase alpha was more sensitive to inhibition by gold compounds than DNA polymerase beta; (2) Inhibition of purified DNA polymerases by gold (I) compounds was noncompetitive with both DNA template and triphosphate substrates. Inhibition by SKF 101675, a gold (III) complex was competitive with DNA. (3) None of the gold compounds tested preferentially inhibited herpesvirus-induced DNA polymerases. (4) The gold complexes that inhibited in vitro DNA replication also inhibited in situ DNA synthesis. However, the potency and order of potency of the compounds varied between the in vitro and in situ systems. (5) Auranofin and other gold compounds inhibited the clonogenic capacity of KB cells in a concentration-dependent manner. The IC50 values measured in the clonogenic assay were significantly lower than those obtained from the in vitro and in situ DNA replication assays.

Induction of functional beta-adrenergic receptors in rat aortic smooth muscle cells by sodium butyrate.

Rat aortic smooth muscle cells in culture (A-10; ATCC CRL 1476) exhibited low levels of beta-adrenergic receptors as determined by specific binding of [125I]cyanopindolol ([125I]CYP) and marginal stimulation of adenylate cyclase in plasma membranes by (-)isoproterenol. When these cells were exposed to 5 mM sodium butyrate, the number of beta-adrenergic receptors and the beta-agonist-stimulated adenylate cyclase activity increased markedly. However, basal, GTP, Gpp(NH)p, and fluoride-stimulated activities did not change. The induction of beta-adrenergic receptors and beta-agonist stimulated adenylate cyclase activity was time- and dose-dependent, and was relatively specific for sodium butyrate. Propionate and valerate were less effective than butyrate, while isobutyrate, succinate, and malonate were ineffective. The induction involved RNA and protein synthesis because induction was prevented by treatment with cycloheximide, puromycin, and actinomycin D. Butyrate did not cause a general increase in cell surface receptors, because the number of vasopressin receptors did not change. The sustained presence of butyrate appeared to be necessary for the maintenance of the induced beta-receptors. When butyrate was removed, receptor number and beta-agonist-stimulated adenylate cyclase activity were decreased by 90% over 24 hr. We conclude that the poor response of rat aortic smooth muscle cell plasma membranes to beta-adrenergic agonists is due to the presence of a low number of beta-adrenergic receptors. Butyrate markedly increased the number of beta-receptors which resulted in a proportional increase in beta-agonist-stimulated adenylate cyclase activity. The increase in receptor number was dependent on RNA and protein synthesis. Butyrate treatment did not affect the activity of the cyclase unit and the efficiency of coupling between the receptors and the guanine nucleotide regulatory protein, Ns.

Azithromycin and clarithromycin: overview and comparison with erythromycin.

Azithromycin and clarithromycin are erythromycin analogues that have recently been approved by the FDA. These drugs inhibit protein synthesis in susceptible organisms by binding to the 50S ribosomal subunit. Alteration in this binding site confers simultaneous resistance to all macrolide antibiotics. Clarithromycin is several-fold more active in vitro than erythromycin against gram-positive organisms, while azithromycin is 2- to 4-fold less potent. Azithromycin has excellent in vitro activity against H influenzae (MIC90 0.5 microgram/ml), whereas clarithromycin, although less active against H influenzae (MIC90 4.0 micrograms/ml) by standard in vitro testing, is metabolized into an active compound with twice the in vitro activity of the parent drug. Both azithromycin and clarithromycin are equivalent to standard oral therapies against respiratory tract and soft tissue infections caused by susceptible organisms, including S aureus, S pneumoniae, S pyogenes, H influenzae, and M catarrhalis. Clarithromycin is more active in vitro against the atypical respiratory pathogens (e.g., Legionella), although insufficient in vivo data are available to demonstrate a clinical difference between azithromycin and clarithromycin. Superior pharmacodynamic properties separate the new macrolides from the prototype, erythromycin. Azithromycin has a large volume of distribution, and, although serum concentrations remain low, it concentrates readily within tissues, demonstrating a tissue half-life of approximately three days. These properties allow novel dosing schemes for azithromycin, because a five-day course will provide therapeutic tissue concentrations for at least ten days. Clarithromycin has a longer serum half-life and better tissue penetration than erythromycin, allowing twice-a-day dosing for most common infections. Azithromycin pharmacokinetics permit a five-day, single daily dose regimen for respiratory tract and soft tissue infections, and a single 1 g dose of azithromycin effectively treats C trachomatis genital infections; these more convenient dosing schedules improve patient compliance. Azithromycin and clarithromycin also are active against some unexpected pathogens (e.g., B burgdorferi, T gondii, M avium complex, and M leprae). Clarithromycin, thus far, appears the most active against atypical mycobacteria, giving new hope to what has become a difficult group of infections to treat. Gastrointestinal distress, a well known and major obstacle to patient compliance with erythromycin, is relatively uncommon with the new macrolides. Further clinical data and experiences may better define and expand the role of these new macrolides in the treatment of infectious diseases.

Oncogenes and phosphatidylinositol turnover.

Products of phosphatidylinositol turnover have recently been implicated as regulators of cell growth and differentiation. Transformation of cells in culture by infection with certain viruses (Rous sarcoma virus, Kirsten sarcoma virus, and polyoma virus) or by transfection with the oncogenes carried by these viruses affect the steady-state level of intermediates in the PI turnover pathway. In addition, immunoprecipitates of the transforming gene products of Rous sarcoma virus and polyoma virus contain activities of certain enzymes in the PI turnover pathway. We have previously reported that polyoma middle T immunoprecipitates can catalyze phosphorylation of PI to phosphatidylinositol-4-phosphate (PIP). This activity is not intrinsic to middle T or c-src but is due to a cellular enzyme that specifically associates with this complex. The PI kinase is found in immunoprecipitates of the middle T protein from polyoma viruses that are capable of cell transformation but does not associate with mutants of middle T defective in transformation suggesting that this association may be important for transformation.

Normal magnetic resonance imaging and abnormal discography in lumbar disc disruption.

STUDY DESIGN: This report describes a clinical series of seven patients who had surgically proven internal disc disruption, normal magnetic resonance imaging, and abnormal discograms morphologically. SUMMARY OF BACKGROUND DATA: Numerous reports in the literature have described the utility of magnetic resonance imaging and discography in diagnosing degenerative disease within lumbar intervertebral discs. RESULTS: Discography may be useful in patients with persistent symptoms despite a normal or equivocal magnetic resonance imaging study.

Understanding the perceived need for complementary and alternative nutraceuticals: lifestyle issues.

Nutraceuticals are biological therapies used to promote wellness, prevent malignant processes, and control symptoms. The use of complementary and alternative nutraceuticals increased dramatically after passage of the Dietary Supplement and Health Education Act of 1994. Motivations for use of these products include changes in eating patterns, concerns about adequacy of consumer food supply, and interactions with conventional healthcare providers that are perceived to be insensitive, too brief, or uncaring. By becoming knowledgeable about complementary and alternative nutraceuticals and the nutritional needs of people with cancer, communicating with empathy and patience, and involving dietitians, pharmacist, and other professional providers as needed, oncology nurses can provide accurate information and support for people with cancer and their families.

The starving patient: supportive care for people with cancer.

Unintentional weight loss in people with cancer is associated with decreased quality of life and increased mortality. Addressing risk factors that lead to weight loss may improve quality of life and prevent cachexia. Specific, individualized counseling is the most beneficial and economic intervention for nutritional health. Appetite stimulants promote oral intake, and oral supplements help to meet the increased need for calories and protein during the course of the disease and its treatment. Utilizing the gut for digestion and absorption of food maintains the critical functions of the bowel lumen. Tube feeding and parenteral nutrition may be indicated for prevention of malnutrition in some disease conditions and during certain types of cancer treatment.