Affinity labeling of the catalytic subunit of cyclic AMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethyl ketone.

The catalytic subunit of cyclic AMP-dependent protein kinase (from rabbit skeletal muscle; ATP:protein phosphotransferase, EC 2.7.1.37) was found to be irreversibly inactivated by chloromethyl ketone derivatives of lysine and phenylalanine, chemical reagents originally designed for labeling the active sites of the proteolytic enzymes trypsin and chymotrypsin. This inactivation was shown to occur at pH 7.5 and 22 degrees C, conditions under which chemically related alkylating reagents such as chloroacetamide and chloroacetic acid (which do not possess the amino acid side chain) fail to inactivate the enzyme. In the case of the chloromethyl ketone derivative of N alpha-tosyl-L-lysine, the enzyme could be protected by its nucleotide substrate (MgATP), by one of its protein substrates (histone H2b), and by its regulatory subunit which, upon binding, shields the active site of the catalytic subunit. Differential labeling experiments, together with kinetic studies of the rates of modification of the sulfhydryl groups in the enzyme before and after inactivation with the chloromethyl ketone, suggest that the loss of activity is associated with one (kinetically characterized) sulfhydryl group present either at the active site of the enzyme or at a site intimately associated with it. The general implications of these results regarding the interpretation of affinity labeling experiments carried out in complex mixtures of proteins or under in vivo conditions are discussed.


Sequence heterogeneity at the 5' termini of late simian virus 40 19S and 16S mRNAs.

The 5'-cap-containing leader sequence of the most abundant 19S and 16S mRNAs of simian virus 40 (SV40) was previously mapped between 0.67 and 0.76 map units. We now find that the two late mRNA species contain multiple 5' ends. Eight different RNase T2-resistant cap structures were identified:m7GpppmAmpU (47%); m7GpppmAmpUmpU (19%); m7GpppmAmpC (16%); m7GpppmAmpCmpA (5%); m7GpppmAmpG (6%); m7GpppGmpC (3%); m7GpppmAmGmpA (2%); m7GpppGmpCmpG (2%). Capped T1 oligonucleotides of 19S and 16S mRNAs have been isolated by two different procedures: (i) chromatography on a DEAE-cellulose column followed by paper electrophoresis and (ii) two-dimensional electrophoresis/homochromatography. Cap structures of the isolated 5' oligonucleotides were identified. Each of the major caps was found to be associated with a few differential 5' oligonucleotides, implying a vast heterogeneity at the termini of SV40 late mRNAs. The results suggest that on SV40 DNA, RNA polymerase II has a reportoire of initiation points. In most of the cases, initiation takes place with adenosine triphosphate followed by a pyrimidine. Alternatively, transcription may start at one specific point but a unique mechanism of processing generates heterogeneous populations of termini with a common 5' adenosine triphosphate.


Cellular information in the genome of recovered avian sarcoma virus directs the synthesis of transforming protein.

Recovered avian sarcoma viruses, whose sarcomagenic information is largely derived from cellular sequences [Wang, L.-H., Halpern, C.C., Nadel, M. & Hanafusa, H. (1978) Proc. Natl. Acad. Sci. USA 75, 5812-5816], produce the transforming protein p60src in infected cells, in amounts comparable to the amount found in cells transformed by standard strains of avian sarcoma virus. Though displaying some virus-specific differences in electrophoretic mobility, p60srcs from these viruses are similar to those of other avian sarcoma virus strains by the criteria of (i) antigenicity, (ii) partial proteolysis mapping, and (iii) association with protein kinase activity. We also find that p60sarc, a protein present in normal cells at a low level, is associated with a protein kinase activity, and thus it too is similar by the above criteria to p60src of avian sarcoma virus. Possible causes for the pathogenicity of p60src are discussed in light of these similarities.


A normal cell protein similar in structure and function to the avian sarcoma virus transforming gene product.

This report extends our previous studies concerning the identification and characterization of a protein from normal cells that is closely related to the avian sarcoma virus (ASV) transforming gene product pp60src. This normal cellular protein, which we have found in both avian and mammalian cells and have tentatively designated pp60sarc, was detected by immunoprecipitation of radiolabeled cell extracts with serum derived from both mice and rabbits bearing ASV-induced tumors. The normal cell pp60sarc is a 60,000-dalton phosphoprotein that is structurally similar, but not identical, to viral pp60src. The phosphorylation patterns of the normal cell and viral proteins are also similar: both contain two major phosphorylated residues, a phosphoserine located on the NH2-terminal 60% of the polypeptide and a phosphothreonine present on the COOH-terminal 40% of the molecule. In addition, the normal cell pp60sarc from both chicken and mammalian cells appears to have an associated protein kinase activity analogous to that previously described for the viral pp60src. The possible roles played by the normal cell protein pp60sarc and the ASV transforming protein pp60src in normal cellular growth and neoplastic disease, respectively, are discussed.


Cell-free translation and thyroxine induction of carbamyl phosphate synthetase I messenger RNA in tadpole liver.

Total RNA of tadpole and frog (Rana catesbeiana) liver was isolated by either 7 or 8 M guanidine . HCl extraction and translated in a cell-free protein-synthesizing system derived from rabbit reticulocytes. The identity of carbamyl phosphate synthetase I[carbamoyl-phosphate synthase (ammonia); ATP:carbamate phosphotransferase (dephosphorylating), EC 2.7.2.5] synthesized in vitro with the purified enzyme was established as follows: (i) immunoprecipitation by a specific antibody; (i) comigration with purified carrier enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis; (iii) copurification with carrier enzyme by affinity chromatography on Cibacron Blue F3GA-coupled agarose; and (iv) formation of identical proteolytic cleavage products. Inclusion of protease inhibitors in the system resulted in no apparent change in the polypeptide molecular weight. These results indicate that carbamyl phosphate synthetase I is synthesized as a polypeptide that is indistinguishable from the mature enzyme by the analytical methods used and that it is not grossly modified during its transport into mitochondria. The level of translatable mRNA for carbamyl phosphate synthetase-I in tadpole liver was increased about 2-fold 1 day after thyroxine treatment and did not change significantly through 4 subsequent days of treatment. Thus the thyroxine-induced synthesis of carbamyl phosphate synthetase I in tadpole liver is at least partly due to an increase of translatable mRNA for this enzyme.


Coupling of the glucagon receptor to adenylyl cyclase by GDP: evidence for two levels of regulation of adenylyl cyclase.

In rat liver plasma membranes preactivated with guanosine 5'-[beta,gamma-imido[triphosphate (GuoPP[NH]P), GDP promoted coupling of occupied glucagon receptor to adenylyl cyclase [adenylate cyclase; ATP, pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with an apparent association constant Ka of 0.1-0.15 microM. The apparent Ka for the same effect of GTP was 0.2 microM. The effect of GDP was shown not to be due to GTP formed by putative transphosphorylation reaction(s) when ATP was present in the assay as substrate. In membranes not preactivated with GuoPP[NH]P, GDP both competitively inhibited GuoPP[NH]P stimulation of adenylyl cyclase (Ki 0.10 microM) and supported stimulation of cyclizing activity (apparent Ka 0.10 microM) by glucagon. These effects of GDP occurred in the absence of added GTP and in the absence of sufficient formation of GTP by putative transphosphorylation reaction(s) to account for them. It is concluded that two levels of regulation of liver adenylyl cyclase (cyclizing) activity must exit. One level is termed "receptor regulation"; it depends on occupancy of a receptor-related R site by nucleotide and is specific for either GDP or GTP. The second level of regulation is termed "GTPase regulation"; it is inhibited by GDP, depends on both GTP and GTPase, and accounts for activation of cyclizing activity by nonhydrolyzable analogs of GTP. The data suggest that both levels of regulation coexist and may synergize, one mediating responses to stimuli external to the cell (receptor regulation) and the other mediating stimuli of intracellular origin (GTPase regulation).


Cyclic AMP as a modulator of polarity in polycistronic transcriptional units.

The degree of natural polarity in the lactose and galactose operons of Escherichia coli is affected by adenosine 3',5'-cyclic monophosphate (cAMP). This effect, mediated by the cAMP receptor protein, is exerted at sites distinct from the promoter. Experiments performed with a mutant bearing a thermosensitive rho factor activity indicate that cAMP relieves polarity by interfering with transcription termination. Conflicting results in the literature concerning the role of cAMP receptor protein and cAMP in galactose operon expression can be reconciled by the findings that cAMP stimulates the expression of operator distal genes without significantly affecting the proximal genes. Therefore, it appears necessary to reevaluate the classification of the galactose operon as exhibiting cAMP-mediated catabolite repression at the level of transcription initiation.


Structure of the antithrombin-binding site in heparin.

Heparin preparations from pig intestinal mucosa and from bovine lung were separated by chromatography on antithrombin-Sepharose into a high-affinity fraction (with high anticoagulant activity) and a low-affinity fraction (with low anticoagulant). Antithrombin-binding heparin fragments (12-16 monosaccharide units) were prepared, either by digesting a high-affinity heparin-antithrombin complex with bacterial heparinase or by partial deaminative cleavage of the unfractionated polysaccharide with nitrous acid followed by affinity chromatography on immobilized antithrombin. Compositional analysis based on separation and identification of deamination products reduced with sodium boro[3H]hydride showed that nonsulfated L-iduronic acid occurred in larger amounts in high-affinity heparin than in low-affinity heparin; furthermore, this component was concentrated in the antithrombin-binding regions of the high-affinity heparin molecules, amounting to approximately one residue per binding site. It is suggested that nonsulfated L-iduronic acid is essential for the anticoagulant activity of heparin. The location of the non-sulfated uronic acid in the antithrombin-binding site was determined by periodate oxidation of antithrombin-binding fragments containing a terminal 2,5-anhydro-D-[1-3H]mannitol unit. Tentative structures for antithrombin-binding sequences in heparin are proposed, including some structural variants believed to be compatible with, but not required for, activity.


Theoretical methods for study of kinetics of models of the mitochondrial respiratory chain.

In earlier work, Hill and Chance obtained exact steady-state kinetic properties for partial models of the mitochondrial respiratory chain with two isopotential pools and one four-state "site enzyme" between the two pools. That work is extended here to full models of the respiratory chain with four isopotential pools and three four-state site enzymes between pairs of pools. Because of the complexity of the model, exact calculations are no longer possible. Instead, we show, by means of some examples, the feasibility of using Monte Carlo calculations on all cases and numerical solution of thousands of kinetic differential equations in many cases.


Kinetics of the induction of three translation-regulatory enzymes by interferon.

Three enzymes that cause inhibition of mRNA translation, eukaryotic initiation factor 2 protein kinase PK-i, oligoisoadenylate synthetase E, and phosphodiesterase 2'-PDi, have been recently isolated from interferon-treated cells. We show that the rise in these three enzyme activities may be used to study the response of uninfected cells to interferon. For each enzyme, a specific microassay that can be carried out on extracts from 2-5 x 10(4) monolayer cells from mouse, monkey, or man was developed. With these assays, the kinetics of induction of the three enzymes in mouse L cells are compared. The dose dependence for protein kinase PK-i induction is shown to be similar to that for the development of the antiviral state. Actinomycin D and anti-interferon serum block enzyme induction if added to the cells early after interferon treatment. The quantitative measurements of the intracellular level of these enzymes provide a new and convenient model to study the cell's response to interferon.


Binding characteristics and apparent molecular size of detergent solubilized nerve growth factor receptor of sympathetic ganglia.

Nerve growth factor (NGF), a hormone-like regulator of sympathetic neuron ontogeny and metabolism affects its target cells initially by associating with specific plasma membrane receptors. We have solubilized the NGF receptor of adult rabbit superior cervical ganglia (SCG) with the nonionic detergent Triton X-100. The high-affinity equilibrium binding constant of the detergent-extracted receptor is 2-8 x 10(-10) M. Gel chromatography of the receptor or the 125I-labeled NGF receptor complex on a column of Sepharose 6B indicated, in both cases, a single component of an apparent hydrodynamic radius of 71 +/- 5 A. In parallel investigations, we have confirmed the similarity between the hydrodynamic size of the NGF receptor of rabbit SCG and that of the insulin receptor of IM-9 lymphocytes evaluated by similar methods.


Presence of free cyclic AMP receptor protein and regulation of its level by cyclic AMP in neuroblastoma-glioma hybrid cells.

Neuroblastoma-glioma hybrid cells of line 108CC-5 were found to contain high levels of soluble adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase activity and high levels of two specific cAMP receptor proteins, RI and RII. Treatment of the hybrid cells with dibutyryl cAMP increased the level of RI but did not significantly affect the level either of RII or of cAMP-dependent protein kinase activity. The effect of dibutyryl cAMP could be mimicked by prostaglandin E1 and 3-isobutyl-1-methylxanthine, both of which are known to raise cAMP levels in neuroblastoma-glioma hybrid cells. Both in control as well as in dibutyryl cAMP-treated cells, RII but not RI was associated with cAMP-dependent protein kinase. Several lines of evidence suggest that RI represents the free regulatory subunit of type I cAMP-dependent protein kinase. The presence of this regulatory subunit as free cAMP receptor protein in neuroblastoma-glioma hybrid cells may be of significance with respect to the regulation of growth and differentiation in tumor cells.


beta-Endorphin: synthesis of analogs modified at the carboxyl terminus with increased activites.

Three analogs of human beta-endorphin (beta h-EP) have been synthesized: [Gly31]beta h-EP, [Gly31]beta h-endorphinamide, and [Gly31]beta h-endorphinylglycine. All are more active than beta h-EP in both the guinea pig ileum bioassay and the opiate receptor binding assay. The last two analogs are about twice as active as beta h-EP in an assay for analgesia. Modification at position 31 and extension at the COOH terminus may afford a route toward analogs with even greater biological activity.


Differential sensitivity of gene expression in vitro to inhibitors of DNA gyrase.

We have used the antibiotics coumermycin A1, novobiocin, and oxolinic acid, which are specific inhibitors of DNA gyrase, to study the coupled transcription and translation of several bacterial and plasmid genes in a DNA-directed cell-free system. The expression of different genes is reduced to different extents by inhibition of DNA gyrase activity. Among the genes tested, the lac operon, an rRNA gene, and the colicin gene of colicin E1 plasmid were found to be most sensitive, while the trp operon and some other genes in colicin E1 plasmid were relatively unaffected by the inhibitors. These results, together with earlier work on the transcription of circular DNA templates, indicate that DNA supercoiling can significantly enhance transcription from certain promoters.


Structure of cytochrome a3-Cua3 couple in cytochrome c oxidase as revealed by nitric oxide binding studies.

The addition of NO to oxidized cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) causes the appearance of a high-spin heme electron paramagnetic resonance (EPR) signal due to cytochrome a3. This suggests that NO coordinates to Cu+2a3 and breaks the antiferromagnetic couple by forming a cytochrome a+33-Cu+2a3-NO complex. The intensity of the high-spin cytochrome a3 signal depends on the method of preparation of the enzyme and maximally accounts for 58% of one heme. The effect of N-3 on the cytochrome a+33-Cu+2a3-NO complex is to reduce cytochrome a3 to the ferrous state, and this is followed by formation of a new complex that exhibits EPR signals characteristic of a triplet species. On the basis of optical and EPR results, a NO bridge between cytochrome a+23 and Cu+2a3 is proposed--i.e., cytochrome a+23-NO-Cu+2a3. The half-field transition observed at g = 4.34 in the EPR spectrum of this triplet species exhibits resolved copper hyperfine splittings with [A+2] = 0.020 cm-1, indicating that the Cu+2a3 in the cytochrome a+23-NO-Cu+2a3 complex is similar to a type 2 copper site.


Receptor-mediated endocytosis: insights from the lipoprotein receptor system.

The low density lipoprotein (LDL) receptor system coordinates the metabolism of cholesterol, an essential component of the plasma membrane of all mammalian cells. Study of this system has led to an enhanced understanding of the cellular basis of cholesterol homeostasis. It has also brought into focus an important mechanism of metabolic regulation--the process of receptor-mediated endocytosis. In this article, we first describe the receptor-mediated endocytosis of LDL, a sequence of events in which receptor binding and internalization are coupled in specialized regions of the plasma membrane called coated pits. Second, we trace the cellular functions of the cholesterol derived from internalized LDL. Third, genetic evidence is presented to indicate that both the binding and internalization of LDL are mediated by a single receptor molecule that contains two active sites, one mediating binding and the other internalization. Finally, the characteristics of the LDL receptor system are used to suggest models for receptor systems in general.


Increase of normal myeloblast viability and multiplication without blocking differentiation by type C RNA virus from myeloid leukemic cells.

Clones of mouse myeloid leukemic cells that differ in their competence to be induced for normal cell differentiation by the protein inducer MGI produce type C virus. These viruses have been studied for their effect on the viability, multiplication, and differentiation of normal bone marrow cells either with or without the addition of MGI. Virus from leukemic clones that can differentiate normally to mature macrophages and granulocytes (MGI+D+ clones) induced some multiplication of myeloblasts in the bone marrow, but the cells did not differentiate without adding MGI. In the presence of MGI, this virus then induced an increased number of colonies whose cells differentiated to mature macrophages or granulocytes as in colonies of uninfected cells. Virus infection also resulted in a decrease in the amount of MGI and fetal calf serum that was required for colony formation. Virus from MGI+D+ clones, in the presence of MGI, was 500-fold more effective in increasing colony formation than virus from the differentiation-defective MGI-D- clones, although both types of virus replicated with equal efficiency in the normal bone marrow cells. No such increase was obtained after infection with the Friend leukemic virus complex or the Moloney murine leukemia virus. Infection with virus from a MGI+D+ clone that was differentiated by MGI mainly to macrophages induced a higher percentage of macrophage colonies than virus from MGI+D+ clones that were differentiated by MGI to granulocytes and macrophages. Studies with isolated myeloblast colony-forming cells from the bone marrow have indicated that these are the target cells for the virus. Infections of these isolated myeloblasts with virus from MGI+D+ clones induced some multiplication without differentiation in the absence of MGI, and increased the viability and multiplication of the myeloblasts without inhibiting their ability to differentiate in the presence of MGI. The results, therefore, indicate that virus from MGI+D+ cells can increase the viability and multiplication of normal myeloblasts in the bone marrow without blocking the ability of these cells to be induced to differentiate by MGI, and that this effect was directly related to the competence of the leukemic host cells to be induced for normal differentiation. It is suggested that the difference between the effect of virus from MGI+D+ and MGI-D- cells may be due to a difference in their integration sites in relation to the genes that control cell viability, multiplication, and differentiation.


Different rat-derived transforming retroviruses code for an immunologically related intracellular phosphoprotein.

Kirsten sarcoma virus (Ki-MSV) and Harvey sarcoma virus (Ha-MSV) are mouse-rat recombinant viruses that were originally isolated by experimental inoculation of rats with helper-independent mouse type C viruses. We have recently identified in cells transformed by Ki-MSV or Ha-MSV, a phosphoprotein, p21, coded for by Ki-MSV and Ha-MSV [Shih, T.Y., Weeks, M.O., Young, H.A. & Scolnick, E.M. (1979) Virology 95, in press]. The p21, which is not a virion structural protein, was identified with antisera prepared by transplantation in rats of syngeneic Ha-MSV- or Ki-MSV-transformed nonproducer cells. In this study, we have applied the same methodology to examine a purely rat sarcoma virus (RaSV), which was isolated in cell culture by using helper-independent rat type C viruses [Rasheed, S., Gardner, M.B. & Huebner, R.J. (1978) Proc. Natl. Acad. Sci. USA 75, 2972-2976]. We report here that this new, purely rat sarcoma virus apparently codes for a p29, which shares immunological determinants and common V-8 protease-generated peptides with the p21 of Ha-MSV. The data suggest that the RaSV has acquired genetic information with similar coding capacity to some rat genetic information with similar coding combinant viruses, Ki-MSV and Ha-MSV. Based on data obtained on the p21 of a mutant of Ki-MSV temperature-sensitive for the maintenance of transformation, we suggest that the gene in RaSV that codes for the p29 is also required for the maintenance of RaSV-induced fibroblast transformation.


Thermolabile protein kinase molecules in a temperature-sensitive murine sarcoma virus pseudotype.

Murine sarcoma virus-associated protein kinases that bind to actin have been purified by affinity chromatography on actin coupled to Sepharose. Heat inactivation studies showed the presence of thermolabile enzyme activity in pseudotypes containing a temperature-sensitivity mutant of murine sarcoma virus (MSV) but not in two independent wild-type MSV pseudotypes. Studies with Sephadex G-75 column fractions showed that a low molecular weight form, approximately 15,000, is the major thermolabile kinase in the temperature-sensitive MSV virions. Antibodies raised against the MSV-coded p60 protein, when added to the in vitro reaction mixtures, showed specific phosphorylation of the IgG heavy chain and a simultaneous reduction in the extent of phosvitin phosphorylation catalyzed by the various MSV pseudotype kinases. Thus a transforming retrovirus-coded enzyme activity that interacts directly with a major cytoskeletal protein and whose activity parallels the transforming ability of a conditional MSV mutant has now been identified.


Transcriptional regulation of the yeast cytochrome c gene.

DNA from the cloned yeast iso-1-cytochrome c, cycl, gene was used in a hybridization assay to measure levels and rates of synthesis of cycl RNA. Derepressed cells synthesized cycl RNA at 6 times the rate of that of glucose-repressed cells. Upon glucose addition to a derepressed culture, the transcription of the cycl gene was repressed within 2.5 min. The half-life of hybridizable cycl RNA was determined to be 12-13.5 min under repressed and derepressed conditions and during repression. The results demonstrate that the expression of the cycl gene is subject to transcriptional regulation.


beta-Endorphin: formation of alpha-helix in lipid solutions.

Human beta-endorphin adopts a partial helical conformation in aqueous solutions of cerebroside sulfate, ganglioside GM1, phosphatidylserine, and phosphatidic acid, but not of cerebroside and phosphatidylcholine, as evidenced by circular dichroic spectra. Addition of Ca2+ to the peptide in cerebroside sulfate solution can break up the helix; at 10 mM Ca2+ the peptide (12 microM) essentially exists in an unordered form. For comparison, sheep beta-lipotropin in acidic cerebroside sulfate solution (pH less than 4) also has a partial helical conformation of the complex between human beta-endorphin and lipids may be related to the opiatelike function of this peptide hormone.


Protein kinase activity associated with the D2 hybrid protein related to simian virus 40 T antigen: some characteristics of the reaction products.

Protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) has been found associated with the D2 hybrid protein, a highly purified protein of 107,000 daltons specified by the adenovirus-simian virus 40 (SV40) hybrid Ad2(+)D2, which has many properties associated with authentic SV40 T antigen [Tjian, R. & Robbins, A. (1979) Proc. Natl. Acad. Sci. USA 76, 610-614]. We have now examined some of the biochemical characteristics of the reaction products. Acceptors for the terminal phosphoryl group of [gamma-(32)P]ATP are the purified protein itself and at least four proteins extracted from nuclei of uninfected cells. Purified histones do not serve as substrate for the enzyme. Phosphorylation is markedly reduced by heating the D2 hybrid protein to 50 degrees C for 30 min. The products of phosphorylation are stable to treatment with ethanol/ether, DNase, and RNase, but completely degraded by digestion with Pronase, demonstrating their protein nature. The phosphate bonds are liable to hot alkali and sensitive to digestion with alkaline phosphatase but stable to treatment with hot acid or hydroxylamine. These results provide evidence that (32)P is incorporated into O-phosphoserine or O-phosphothreonine residues of acceptor proteins, indicating that the enzymatic activity is characteristic for protein kinase, and that cell-specified nuclear proteins other than histones may serve as substrates for the enzyme.


Interrupting the early region of polyoma virus DNA enhances tumorigenicity.

The tumorigenicity of DNA from polyoma virus after cleavage with a variety of restriction enzymes was evaluated in suckling hamsters. Cleavage with enzymes that interrupt the region of the genome coding for the large tumor (T) antigen of polyoma virus markedly enhanced the tumorigenicity above that observed with DNA I of the virus. Cell lines established in vitro from tumors induced by polyoma virions, polyoma virus DNA I, or polyoma virus DNA that had been cleaved with restriction endonucleases in the early region all contain the polyoma virus middle and small T antigens but not the large T antigen of polyoma virus is not required for maintenance of the transformed state and probably not for initiation of tumorigenesis by viral DNA.


T4 DNA-delay proteins, required for specific DNA replication, form a complex that has ATP-dependent DNA topoisomerase activity.

Under some conditions, T4 DNA replication requires the products of the DNA-delay genes, genes 39, 52, 58, and 60. By using an in vitro complementation assay that stimulates DNA replication in T4 39(-)-infected cell extracts, T4 gene 39 protein has been purified. The purified fraction also contains complementing activities for T4 genes 52 and 60. On sodium dodecyl sulfate/polyacrylamide gel analysis the purified preparation exhibits three protein components: a 51,000-dalton protein corresponding to the product of gene 52, a 64,000-dalton protein corresponding to the product of gene 39, and a 110,000-dalton protein. This purified fraction shows a DNA topoisomerase activity that untwists superhelical DNA in an ATP- and Mg2+-dependent reaction. The analogs adenylyl imidodiphosphate and adenyl [beta, gamma-methylene]diphosphonate cannot be used to replace ATP. The topoisomerase activity is not sensitive to the antibiotics oxolinic acid and novobiocin, known antagonists of Escherichia coli DNA gyrase. The possible relationship among the three polypeptides and their biological activities is discussed.


Synthesis of a small RNA in cells coinfected by standard and defective interfering particles of vesicular stomatitis virus.

A small RNA, containing approximately 50 nucleotides, is synthesized by cells coinfected with standard vesicular stomatitis virus and its defective interfering (DI) particles. Infection of cells by standard virus or DI particles alone does not lead to synthesis of significant amounts of small RNA. The RNA is initiated at its 5' end with (p)ppXp and is not polyadenylylated at the 3' end despite a content of 51% adenosine. It has sequences complementary to the genome of a DI particle. The synthesis of the small RNA correlates with the replication of the genome of DI particles with molar ratio small RNA/genome RNA of DI particles greater than 50. When replication of DI genomes is prevented by the addition of cycloheximide or prior UV irradiation of DI particles, small RNA is not synthesized in coinfected cells. These results indicate that the small RNA is not the result of transcriptional initiation and that it may relate to interference mediated by DI particles.


Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12.

A herpes simplex virus DNA fragment that is produced by digestion with BamHI endonuclease and carries the thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been cloned in Escherichia coli. A recombinat plasmid, pFG5, has been analyzed extensively and a detailed restriction map is presented. pFG5 DNA efficiently transforms TK- mouse L cells. The TK coding sequence in the cloned fragment has been localized and a smaller recombinant plasmid, pAG0, also carrying an active TK gene, has been constructed to serve as a more convenient vector for transfer, into TK- cells, of genes previously cloned in E. coli.


Nicking-closing activity associated with bacteriophage lambda int gene product.

Integrative recombination of bacteriophage lambda requires the action of the protein Int, the product of the phage int gene. In this paper we show that highly purified Int relaxes supercoiled DNA. The association of this nicking-closing activity with Int is shown by: (i) the cosedimentation of nicking-closing and recombination activities of purified Int, (ii) the parallel inactivation of the two activities in purified Int by both heat and a specific antiserum, and (iii) the alteration of both activities in crude extracts of a strain expressing a mutant int gene. The nicking-closing activity of Int functions in the absence of divalent cations and in the absence of an apparent source of chemical energy. The activity displays no obvious sequence specificity and is inhibited by Mg2+, spermidine, and single-stranded DNA. Int relaxes positive as well as negative supercoils. We present a model for the mechanism of strand exchange that describes how the nicking-closing activity of Int might be used during recombination.


Nicking-closing enzyme assembles nucleosome-like structures in vitro.

The four core histones (H2A, H2B, H3, and H4) and DNA were assembled into nucleosome-like particles at physiological ionic strengths either by an extract of chromatin rich in nicking-closing activity or by the purified nicking-closing enzyme itself. When histone-DNA complexes were assembled in vitro from relaxed circular DNA, nearly physiological numbers of superhelical turns were induced in the DNA molecule. Electron microscopy of the complexes assembled by the chromatin extract revealed a beaded structure and a reduction of the contour length compared to free DNA. Micrococcal nuclease digestion of the histone-DNA complexes yielded 145-base-pair DNA fragments typical of nucleosome core particles and shorter subnucleosomal DNA fragments of discrete length.


Use of the UGA terminator as a tryptophan codon in yeast mitochondria.

We propose that the UGA terminator regularly occurs as a tryptophan codon in yeast mitochondrial DNA. This conclusion is based on the sequence analysis of mitochondrial DNA regions coding for structural genes of cytochrome b, cytochrome oxidase, and the ATPase.


Characterization of the iron-sulfur centers in succinate dehydrogenase.

Two techniques have been applied to the determination of the number and type (2-Fe, 4-Fe) of iron-sulfur centers in the iron-sulfur flavoprotein succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1]. One procedure uses p-CF3C6H4SH as an extrusion reagent and Fourier transform 19F nuclear magentic resonance as the method of detection and quantitation of extruded cores of these centers in the form of [Fe2S2(SRF)4]2- and [Fe4S4(SRF)4]2- (RF = p-C6H4CF3). The second procedure, interprotein core transfer, involves thiol displacement of iron-sulfur cores followed by specific core transfer to the apoproteins of Bacillus polymyxa ferredoxin and adrenodoxin. Detection and quantitation are accomplished by electron paramagnetic resonance of reduced proteins at low temperatures. Both procedures clearly show that succinate dehydrogenase contains two dimeric (Fe2S2) and one tetrameric (Fe4S4) centers per mole of histidyl flavin, accounting for all eight nonheme iron and eight labile sulfur atoms found by chemical analysis. These results remove uncertainties created by the less than stoichiometric amounts of binuclear centers detected by electron paramagnetic resonance after dithionite reduction and provide secure characterization of the iron-sulfur centers in this enzyme.


Methylation of Herpesvirus saimiri DNA in lymphoid tumor cell lines.

Several continuous lymphoid cell lines have been established from tumors induced by Herpesvirus saimiri. At least a portion of the viral DNA in the marmoset lymphoid cell line 1670, which does not produce detectable virus, is present as covalently closed circular episomal DNA. The use of restriction endonuclease digestion, transfer to nitrocellulose filters, and hybridization of the virus-specific DNA has produced strong evidence that viral DNA sequences present in total 1670 cell DNA and in isolated episomes are extensively methylated. The restriction endonuclease Hpa II has the same recognition sequence as Msp I but, unlike Msp I, fails to cleave when the C of the C-G dinucleotide is methylated. Viral DNA sequences of 1670 cells are refractory to cleavage by Hpa II but not Msp I; greater than 80% of the Hpa II cleavage sites appear to be methylated. Similarly, viral DNA sequences of 1670 cells are refractory to cleavage by Sma I (C-C-C-G-G-G) and Sac II (C-C-G-C-G-G) but not Sac I, Pvu II, or Pst I, which lack the dinucleotide C-G in their recognition sequences. Methylation of mammalian DNA has been previously found exclusively at C residues in the dinucleotide C-G. H. saimiri DNA sequences of another nonproducer cell line, 70N2, also appeared to be extensively methylated, but analysis of total cell DNA extracted from three virus-producing lymphoid lines revealed no evidence of methylation of viral DNA sequences. It remains to be seen if methylation of viral DNA plays a role in the lack of complete expression of H. saimiri genome information in nonproducing lymphoid cell lines.


Protein influence on the heme in cytochrome c: evidence from Raman difference spectroscopy.

Raman difference spectra have been obtained for the cytochromes c of a number of species by simultaneous data acquisition from two samples. Frequency differences as small as 0.1 cm-1 can be measured reproducibly by the technique we have developed. In comparisons between cytochromes c isolated from two different species, the frequency differences in the heme vibrational modes range from 0 to 6 cm-1. The vibrational frequencies of the heme are sensitive to the electronic charge density on the porphyrin macrocycle. The frequency differences are interpreted in terms of the influence of the heme-packed aromatic and highly electronegative amino acid side chains on the pi* charge density and distribution on the heme. Such a control of the electronic properties of the heme by the protein may be important for the function of cytochrome c.


Torsional stress and local denaturation in supercoiled DNA.

It is shown that local denaturation can be a natural consequence of supercoiling, even in environments where base pairing of linear DNA is energetically favored. Any change in the molecular total twist from its unstressed value is partitioned between local denaturation and smooth twisting in both the native and coil regions so as to minimize the total conformational free energy involved. Threshold degrees of torsional deformation are found for the existence of stable, locally melted conformations. As these thresholds are surpassed, the number of denatured bases increase smoothly from zero. Existing experimental evidence regarding denaturation in supercoiled DNA is in good agreement with the predictions of this theory. In addition, from existing data one can estimate the partitioning of superhelicity between twisting and writhing. Possible consequences of stress-induced strand separation on the accessibility of the DNA to enzyme attack are discussed. Control of local melting by DNA topoisomerases and DNA gyrases could regulate diverse events involved in transcription, replication, recombination, and repair.


Reactivation of silent rRNA genes by simian virus 40 in human-mouse hybrid cells.

Mouse-human hybrid cells were used to study the ability of simian virus 40 to regulate the expression of rRNA genes in vivo. In these hybrid cells, only the rRNA genes of the dominant species are expressed; the genes for the rRNA of the recessive species are silent. Simian virus 40 infection of these hybrids led to the production of two distinct 28S rRNA species as analyzed by agarose/2.4% polyacrylamide gel electrophoresis. These species were identified as human and mouse rRNAs. This result was confirmed by histochemical studies which indicated that the nucleolus organizer regions of both mouse and human chromosomes were actively synthesizing rRNA in the virus-infected hybrid cells. These results indicate that simian virus 40 infection can induce the expression of otherwise silent rRNA genes.


Effect of interferon on concentrations of cyclic nucleotides in cultured cells.

Constant intracellular concentrations of both adenosine 3',5'-cyclic-monophosphate (cyclic AMP) and guanosine 3',5'-cyclic-monophosphate (cyclic GMP) were obtained when leukemia L1210 cells were cultivated under steady-state conditions in the chemostat. In this sensitive and controlled system addition of mouse interferon resulted in a rapid (5-10 min) increase in the intracellular concentration of cyclic GMP, which preceded by several hours an increase in the intracellular concentration of cyclic AMP. In contrast to the effect of interferon, addition of prostaglandin E1 induced a rapid increase in the intracellular concentration of cyclic AMP without markedly affecting the intracellular concentration of cyclic GMP. It is suggested that the rapid effect of interferon on cyclic GMP plays a role in mediating some of the effects of interferon on cells.


Morphological revertants of an avian sarcoma virus-transformed mammalian cell line exhibit tumorigenicity and contain pp60src.

The biological and biochemical properties of Rous sarcoma virus-transformed and revertant field vole cells were investigated. Revertant vole cells appear morphologically similar to normal, uninfected cells, yet, like transformed vole cells, they are fully capable of growing in agar suspension and producing tumors in athymic nude mice. These highly tumorigenic, yet morphologically normal appearing, vole cells express viral-specific antigens such as the gag gene product (Pr76) but lack the env gene protein (gp85). Moreover, they contain the src gene protein, pp60src. These results support the concept of the pleiotropic nature of the src gene product and in addition suggest that pp60src may have multiply mechanisms of action. With this revertant cell system it may be feasible to distinguish between those biochemical functions of the src gene product that are important for tumorigenicity in vivo and those that are related to in vitro morphological transformation.


Proliferation of Rous sarcoma virus-infected, but not of normal, chicken fibroblasts in a medium of reduced calcium and magnesium concentration.

Both normal and Rous sarcoma virus-infected chicken fibroblasts proliferate actively in a culture medium containing physiological concentrations of calcium (1.2 mM) and magnesium (0.7 mM). In the presence of a physiological concentration of magnesium, reduction of the calcium concentration to 0.125 mM resulted in a significant decrease in the proliferation of the normal, but not of the neoplastic, fibroblasts. Reduction of the magnesium concentration to 0.05 mM in the presence of a physiological concentration of calcium had a similar effect. In a culture medium containing reduced concentrations of both calcium (0.20 mM) and magnesium (0.05 mM), the normal fibroblasts were maintained without proliferation, whereas the Rous sarcoma virus-infected fibroblasts continued to proliferate actively. The cytosol concentrations of ionized calcium and magnesium are known to be regulated by a balance between net passive influx and active extrusion and sequestration. On the basis of this consideration and the findings described above it can be hypothesized that: (i) Fibroblast replication is initiated when cytosolic concentrations of calcium, magnesium, or both rise above a critical level. (ii) Autonomous initiation of replication of neoplastic fibroblasts is a result of failure of cytoplasmic divalent cation homeostasis; alternatively, sarcoma virus infection may endow cells with a divalent cation-independent mechanism that bypasses an initiation mechanism that is, normally, divalent cation-dependent. (iii) Proliferation of normal fibroblasts is controlled by extracellular matrix components that interact with cell surfaces in a manner that limits the permeability of plasma membranes to divalent cations or otherwise functions to lower cytosol divalent cation concentrations.


Loss of epidermal growth factor requirement and malignant transformation.

Serum provides growth factors that regulate and limit the growth of normal cells in tissue culture. Animal cells that are malignantly transformed usually exhibit diminished serum requirements for growth in culture. We have used a defined, serum-free medium to determine which of these growth factors becomes dispensable for the growth of transformed Syrian and Chinese hamster fibroblast cells. The medium's four growth factors-epidermal growth factor (EGF), insulin, fibroblast growth factor, and transferrin-were added or omitted as desired. A decreased requirement for EGF was most closely related to tumorigenicity of chemically (ethyl methanesulfonate) transformed cells in nude mice. All lines examined retained their requirement for transferrin, which is needed throughout the growth cycle, in contrast to the other factors, which are needed primarily in G(1) phase. Lines that had lost their EGF requirement but had retained their insulin requirement were arrested in G(1) by insulin deficiency, indicating that their growth control system remained. Mutagenesis with ethyl methanesulfonate can also create requirements of the transformed cells for unknown factors in serum. We conclude that an initial step that reduces the serum requirement in culture, and in tumorigenesis, is relaxation of the growth-regulatory function of EGF.


Chinese hamster ovary cell population density affects intracellular concentrations of calcium-dependent regulator and ability of regulator to inhibit adenylate cyclase activity.

The adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of crude Chinese hamster ovary cell membranes was inhibited 30-40% by low concentrations (6-600 ng/ml) of calcium-dependent regulator (CDR). This inhibitory effect was lost at concentrations of CDR above 600 ng/ml. The adenylate cyclase activity of membranes prepared from low population density Chinese hamster ovary cells was not appreciably altered by CDR. However, with increasing cell population density there was a significant increase in the ability of CDR to inhibit cyclic AMP formation. Further, the intracellular levels of CDR determined in the 12,000 x g supernatant and particulate fractions varied inversely with increasing cell population density. As cell number increased from 2 x 10(6) to 10 x 10(6) cells per dish the CDR concentration present in the supernatant fraction increased from 0.4 to 0.8 mug of CDR per mg of protein, while the amount of endogenous CDR associated with the particulate fraction decreased from 0.6 to 0.4 mug of CDR per mg of protein. This suggests that possible changes in the distribution of CDR between the supernatant and membrane fractions might serve as a regulatory mechanism for activities under CDR control.


Stereospecific hexose transport by membrane vesicles from mouse fibroblasts: membrane vesicles retain increased hexose transport associated with viral transformation.

Membrane vesicles isolated from nontransformed BALB/c 3T3 mouse fibroblasts (3T3) and from those cells transformed by simian virus 40 (SV3T3) displayed carrier-mediated and stereospecific uptake of hexose as measured by the difference between D-[(14)C]glucose or its analogues and L-[(3)H]glucose uptake. Stereospecific uptake appeared to be linear for 5 sec and reached a maximum at 5-10 min. Stereospecific D-[(14)C]glucose uptake, osmotically sensitive and temperature dependent, was inhibited by unlabeled D-glucose or its analogues and was stimulated by the countertransport of accumulated unlabeled D-glucose. As with whole cells, the initial rate of stereospecific uptake by SV3T3 membrane vesicles was approximately 2.5-fold greater than that by 3T3 vesicles. Efflux of preloaded D-[(14)C]glucose was also faster from SV3T3 than from 3T3 membrane vesicles. The K(m) value was 5 mM for both the 3T3 and the SV3T3 membrane vesicles, but the V(max) values were 36 and 86 nmol/mg of protein per min, respectively, suggesting an increase in the number or availability of hexose carriers in transformed cell membranes. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicles. The binding of cytochalasin B to the SV3T3 membrane vesicles was significantly greater than that to 3T3 vesicles. Thus, the membrane vesicles retained many of the features of the altered hexose transport observed in whole cells in association with viral transformation.


Molecular analysis of chromosome-mediated gene transfer.

Metaphase chromosomes isolated from a cell line carrying the thymidine kinase (TK) gene of herpes simplex virus type I were used to transform the TK-deficient cell line LMTK- to the TK+ phenotype. Four independent transformants were isolated, all of which expressed virus-specific TK. Each of the four transformant cell lines initially became TK- at a rate of 12% per day. All four transformants possessed multiple copies of the TK gene and in one of the four a rearrangement occurred adjacent to the TK sequences. Stable TK+ derivatives of each line, isolated after prolonged cultivation, retained fewer copies of the TK gene than did their unstable parents. The transferred chromosomal fragment was larger than 17 kilobases in each line.


Amyloid protein SAA is an apoprotein of mouse plasma high density lipoprotein.

Mouse plasma contains a protein antigenically related to mouse protein AA, the principal protein derived from tissue deposits of amyloid substance in mice. In this work the plasma antigen, SAA, was found mainly as a high molecular weight form (2 x 10(5)) residing for the most part in the density interval 1.063-1.21 g/cm3 (high density lipoprotein, HDL); the largest amount of SAA, absolute and relative to total protein, was found in the density interval 1.125-12.1 g/cm3 (HDL3). When apoproteins of the mouse HDL obtained by delipidation of the lipoprotein particles were chromatographed in acid/urea, the antigenic activity appeared in the 10,000- to 15,000-dalton portion of the apoprotein complex. In these characteristics mouse SAA closely resembles human SAA and the behavior of the protein related to amyloid protein AA indicates that is one of the apoproteins of the HDL complex in both species. Therefore we suggest that it be named apoSAA.


In vitro infectivity assay for mouse mammary tumor virus.

Studies of mouse mammary tumor virus (MMTV) have been impeded by the lack of an in vitro infectivity assay. We have developed a rapid, quantitative in vitro assay for MMTV infectivity based on the detection of positively staining foci by immunoperoxidase. This assay and a 50% end-point titration of MMTV infectivity gave identical virus titers. Infection of a rat hepatoma cell line, a feline kidney cell line, and a normal murine mammary gland cell line by virus from the mouse mammary tumor GR3A cell line was linear with respect to virus concentration. The infectious titers obtained in both homologous and heterologous cell lines were not significantly different, demonstrating a lack of host range specificity. Virus infectivity was inactivated by heating at 55 degrees C and by ultraviolet irradiation. Rabbit anti-MMTV serum neutralized the infectivity with a 50% neutralization end point of 1:5000. Applications of this assay to the study of the immunological, biological, and biochemical characteristics of MMTV are discussed.


Biogenesis of poxviruses: role for the DNA-dependent RNA polymerase II of the host during expression of late functions.

The participation of host RNA polymerase II in the vaccinia life cycle was examined by comparing efficiency of multiplication after treating the Ama+ sensitive and Ama 102 drug resistant lines with alpha-amanitin. In the latter, resistance is due to a mutation in RNA polymerase II. The toxin profoundly reduces synthesis of virus-specified polypeptides and morphopoeisis in Ama+ but not in Ama 102 rat myoblasts without appreciably altering vaccinia DNA replication in either cell type. This implicates RNA polymerase II in the expression of late virus functions. Circumstantial evidence from a model system indicates that gamma irradiation of the host prior to infection might disrupt transcription into functional mRNA from the nucleus. Irradiation does not, however, alter the capability of the host to support vaccinia multiplication fully. Therefore, ongoing host nuclear transcription may not be required by this virus. The above results are consistent with the ability of cytoplasts to produce small quantities of mature progeny. Our studies lead us to hypothesize that RNA polymerase II or a subunit of the host enzyme may participate directly in late transcription of the vaccinia genome.


Cholinergic activity regulates muscarinic receptors in central nervous system cultures.

Muscarinic acetylcholine receptor activation induces a loss of muscarinic receptors from cultured neuroblastoma and embryonic chicken cerebrum cells. As measured by specific binding of [3H]quinuclidinyl benzilate, steady-state receptor concentrations decrease 75% in response to receptor-saturating concentrations of cholinomimetic drugs. Both the degree and duration of activation determine the extent of receptor loss. A method for analyzing receptor turnover, which does not rely upon protein synthesis inhibitors, shows that activated receptors have a half-life of 1.6 hr. The regulated rate of receptor disappearance begins as soon as activators are added, and the rate is maintained as long as activators are present. The receptor blocker atropine causes an increase in receptor levels in central nervous system cultures but has no effect on receptors in cultures of adrenergic neuroblastoma cells. Because spontaneous cholinergic activity is expected only in the central nervous system cultures, the increase likely reflects blockade of endogenous regulation. Cytochalasin B blocks receptor regulation, suggesting that regulation may be mediated by a process involving microfilaments.


Toad urinary bladder epithelial cells in culture: maintenance of epithelial structure, sodium transport, and response to hormones.

Epithelial cells from the toad urinary bladder have been grown in continuous culture. Many of the cells resemble the granular cell type of the urinary bladder. They form an epithelium with typical tight junctions and gap junctions. The transport properties of two cell lines have been examined. When cells of the line designated TB-M or of line TB-6c are grown on collagen-coated nucleopore filters, epithelia are formed that have transepithelial potential differences of 40 and 20 mV, resistances of 5000 and 10,000 omega-cm2, and short-circuit currents (ISC) of 8.5 and 2.5 muA/cm2, respectively. Net mucosa to serosa sodium transport accounts for all of ISC in line TB-M and for 70% of ISC in line TB-6c. Vasopressin, which stimulates adenylate cylase and ISC in the intact bladder, has no effect on the cells in culture. Cyclic AMP stimulates ISC and lowers resistance in both lines. Aldosterone stimulates ISC in both lines. This is accompanied by a fall in resistance in line TB-M and no change in resistance in line TB-6c. Amiloride inhibits ISC in TB-M cells under basal conditions and after stimulation by aldosterone. In line TB-6c amiloride has no effect under basal conditions but lowers ISC of aldosterone-treated cells to the basal level. Thus, the cells have retained the ability to form oriented, high-resistance epithelial membranes that manifest hormone-sensitive transepithelial sodium transport.


Alteration of cyclic nucleotide levels in brain following intracranial self-stimulation in the rat.

In a first experiment, 14 rats were implanted with an electrode in the ventral tegmental area and trained to self-stimulate. On the experimental day only half of the rats were allowed to self-stimulate for about one hour. All rats were then sacrificed by immersion in liquid nitrogen. Seven brain regions were dissected and assayed for the endogenous concentration of cyclic nucleotides. Self-stimulation induced significant changes in striatum and hippocampus. However, a subsequent experiment showed that the same pattern of changes in the striatum can be produced by motor activity. On the other hand, changes in the hippocampus were specific to the self-stimulation group suggesting that this structure is associated with the brain reward system.


Effect of adrenaline and triamcinolone on cytochrome oxidase activity in the brain and liver of young and adult rats.

The effect of adrenaline (0.15 i.p./kg b.w.) and of the synthetic glucocorticoid triamcinolone (40 mg i.p./kg b.w.) on cytochrome oxidase activity, the terminal enzyme of the cytochrome system, was studied in homogenates of the cerebral cortex, subcortical formations (including the basal ganglia, the thalamus and the hypothalamus), the medulla oblongata and the liver of 5-day-old and adult rats. Activity in the above mentioned homogenates was measured polarographically 15 and 30 min after administering adrenaline or 48 h after administering triamcinolone. Fifteen minutes after its injection, adrenaline caused a statistically significant drop in cytochrome oxidase activity in the cerebral cortex, subcortical formations and liver of 5-day-old rats. The decrease still persisted 30 min after administration of the hormone, but was intensified only in the liver. In adult rats, on the other hand, a significant increase in activity was observed in the cerebral cortex and liver after adrenaline. Triamcinolone had no effect on cytochrome oxidase activity in any of the given parts of the brain in either young or adult rats. It significantly stimulated cytochrome oxidase activity in the liver of 5-day-old rats, but severely inhibited it in the liver of adult rats.


Emergency hepatic arterial embolization for secondary hypercalcemia in hepatocellular carcinoma.

Two successive hepatic arterial embolizations were performed in a patient with hypercalcemia secondary to hepatocellular carcinoma. The first procedure was performed on an emergency basis due to a cardiovascular episode and was immediately beneficial. The second procedure, performed five months later for a recurrence, was effective in 3 days; 13 months later, there had been no recurrence.


Preoperative diagnosis and localization of aldosteronomas by measurement of corticosteroids in adrenal venous blood.

Adrenal venous aldosterone/cortisol ratios were determined in 14 patients with primary aldosteronism. Of 12 patients diagnosed as having an adrenal adenoma, ten underwent surgery in which an adenoma was found. Bilateral adrenal hyperplasia was thought to have developed in two other patients. Measurement of adrenal vein cortisol and establishment of aldosterone/cortisol ratios were done to correct for catheter placement and dilute adrenal efflux. Determination of steroids after ACTH stimulation was helpful in distinguishing quiescent from suppressed adrenal in two patients and in confirming the diagnosis of the others.


The computed tomographic appearance of the normal pituitary gland and pituitary microadenomas.

With the use of axial and coronal computed tomography (CT), the authors compared the density, contrast enhancement, and dimensions of normal pituitary glands and pituitary microadenomas. The normal gland appears homogeneous, nearly isodense with brain tissue, and it enhances uniformly. Its upper surface is concave downward or flat and its height 2-7 mm. The cavernous sinuses, the third, fourth, and sixth cranial nerves, the infundibulum, and the adjacent carotid arteries are well demonstrated by CT. Abnormal height and upward convexity of the gland are reliable signs of prolactinoma; abnormal density and enhancement are suggestive signs. CT findings in prolactin- and ACTH-secreting tumors may differ. CT is more sensitive and more specific than polytomography in the diagnosis of pituitary adenoma.


Early imaging of experimental intestinal infarction with 99mTc-pyrophosphate.

Extensive mucosal small-bowel infarction was produced in 8 dogs by occluding the cranial mesenteric artery. After one hour of reperfusion, 15 mCi (555 MBq) of 99mTc-pyrophosphate was intra-arterially injected into 4 dogs and venously into the other 4. Positive images were obtained in all dogs except 1 which had received an intravenous injection. Diagnostic images were obtained consistently as early as 15 minutes after injection, and the infarcted bowel could still be visualized two hours later. The average tracer content in infarcted small bowel was 0.015% I.D./g. This was about eight times the uptake found in normal dogs. The results show that experimental small-bowel infarction can be detected as early as five hours after the onset of ischemia.


Evidence for the existence of alpha-adrenoceptors in the rat lung.

Isolated rat lung parenchymal strips responded to carbachol, 5-HT and phenylephrine (PE) with contractions. Bradykinin (BK), histamine, PGF2 alpha, PGE1, PGE2 and dimaprit were either inactive or produced weak relaxations. Isoproterenol, noradrenaline (NA) and epinephrine (E) relaxed lung strips. The further increase in their concentrations produced contractions of varying magnitudes. The lung strips contracted to 5-HT or carbachol, responded to isoproterenol and E and NA with relaxations. Metiamide and cimetidine (selective histamine H2-receptor antagonists) did not alter responses to histamine and carbachol. Propranolol (a beta-adrenoceptor blocker) antagnoized or reversed relaxations to E and isoproterenol, and markedly enhanced contractions to NA, PE and E. Atropine and phentolamine or dibenzyline antagonized contractions to carbachol and sympathomimetic agents, respectively. From study it may be concluded that there are alpha- and beta-adrenergic receptors mediating contractions and relaxations in the peripheral airways of rat.


Effect of paraquat treatment on prolyl hydroxylase activity and collagen synthesis of rat lung and kidney.

Morphological evidence has shown that the herbicide paraquat produces severe lung fibrosis in experimental animals. In order to investigate the biochemical basis for this fibrosis, prolyl hydroxylase activity, total hydroxyproline content and collagen and protein synthesis were estimated in rats (200 gm) which were pretreated with paraquat (25 mg/Kg; I.P.) nine days before sacrifice. Prolyl hydroxylase activity in the treated rats was significantly elevated in the lungs. However, no such increase was seen in the kidney. There was no significant change in the total hydroxyproline content or collagen formation in the treated animals. When paraquat was added to the culture medium and incubated with lung slices, there was a marked decrease in collagen synthesis along with a decrease in protein synthesis. When paraquat was added to lung cells in culture, it was found that lung explants were more sensitive to paraquat toxicity than cells obtained by trypsinization. In explants which were sensitive to paraquat, there was a marked decrease in both protein synthesis and collagen synthesis.


Modification of a commercial microwave oven for animal sacrifice in cyclic GMP assays.

After sacrifice with a modified Litton Minutemaster(R) commercial oven rat cerebellar tissues were assayed for cyclic guanosine 3',5'-monophosphate (cGMP) concentrations. The results were found to be comparable to recent literature reports in which microwave sacrifice employed considerably more intense and more costly energy sources.


[Fatal varicella pneumonia in adults. Report of two cases and review of literature (author's transl)].

Two mortal cases of varicella pneumonitis are described. The patients were respectively 41 and 34 years-old men of good health. They caught the disease from their children. The disease lasted one week, and death was due in both cases to respiratory failure. The autopsy confirmed massive lung involvement with varicella pneumonia, and also showed other organs involvement (liver, spleen, pancreas, lymph nodes, oesophagus). In both cases, human fibroblast cultures infected by material obtained from cutaneous lesions (in the second case also from the spleen) revealed the presence of Herpesvirus varicellae. A review of 41 cases of adult mortal varicella in the literature demonstrates that death is practically always due to pneumonia and consequent respiratory failure. The dissemination of the virus in other organs is constant. Circumstances, such as pregnancy, immunosuppression, etc., which aggravate the prognosis of varicella in adults, are discussed.


Respiratory and hemodynamic study during wakefulness and sleep in myotonic dystrophy.

Six young male patients with grade I (mild) myotonic dystrophy and a complaint of excessive daytime sleepiness were studied during wakefulness and sleep. Pulmonary function tests during wakefulness showed evidence of mild abnormality related to respiratory muscle weakness. During sleep, some patients developed a sleep apnea syndrome with high sleep Apnea Indices. There was no relation between hypoxic and hypercapnic ventilatory responses during wakefulness and sleep Apnea Indices. But hypoxemia and hypercapnia worsened considerably during REM sleep. Myotonic dystrophy patients with sleep apnea presented increased pulmonary and systemic arterial pressures during sleep. It was also during sleep that arrhythmias were observed.


Development of sinus arrhythmia during sleeping and waking states in normal infants.

The development of variability in heart rate (HR) due to respiration (sinus arrhythmia; SA) has been examined in normal infants from birth through the first 6 months of life. Two aspects of HR variation were examined: the absolute variation at the median respiratory frequency, or extent of sinus arrhythmia (XSA), and the degree to which HR follows respiration regardless of the absolute amount of variation, or coherence of sinus arrhythmia (CSA). Extent of sinus arrhythmia tended to be highest in quiet sleep (QS), lower in active or REM sleep (AS), and lowest in waking (AW), especially after 2 months of age. Extent declined at 1 month of age in QS, but rose over the first 6-month period in all states. During this same period, CSA was also highest in QS, lower in AS, and lowest in AW. Coherence in QS also declined at 1 month and rose between 1 and 6 months; however, no age effects were found in other states. Heart rate was negatively correlated with XSA, but less so with CSA. Sleep state appears to have a significant effect on cardiorespiratory coupling, and this coupling undergoes dramatic changes at 1 month in QS.


Upper airway function during sleep and wakefulness: experimental studies on normal and anesthetized cats.

Normal (N = 6) and anesthetized (N = 70) cats were used to study the laryngeal abductors, the posterior cricoarytenoid (PCA) muscles, during sleep and wakefulness and to investigate sites within the brainstem that influenced PCA and diaphragmatic activity. The findings were as follows: 1. During wakefulness, PCA activity occurred throughout the respiratory cycle but was most intense during inspiration. Both expiratory and inspiratory PCA activity declined during sleep--the former more so than the latter. The decline in abductor activity was maximal in REM sleep. 2. Barbiturate anesthesia, according to the dosage, produced PCA activity patterns characteristic of either wakefulness or sleep. 3. The brainstem between A4 and P14 was mapped with stimulating electrodes. Rostral brainstem sites showed predominantly facilitatory effects of PCA activity; caudal sites produced predominantly blocking effects. 4. PCA facilitation consisted of (a) an increase in the duration of the PCA burst, (b) and increase in the discharge frequency of the PCA motor units, and (c) a recruitment of larger motor units. PCA blocking effects were the opposite, i.e., burst durations were shortened and motor units were decruited. 5. Facilitatory sites produced clear change in intensity and duration of PCA activity at stimulation intensities below those necessary to obtain changes in the intensity of diaphragmatic activity. 6. Stimulation of facilitatory sites during expiration caused phase switching to inspiration. In some cases, stimulation during inspiration caused phase switching to expiration. The results are discussed in terms of their implications for the obstructive apneas of sleep and in terms of the neural control of breathing.


Induction and resetting of REM sleep rhythm in normal man by arecholine: blockade by scopolamine.

Arecholine, a cholinergic muscarinic agonist, was administered intravenously 35 min after sleep onset in eight normal volunteers who had been pretreated with either methscopolamine or scopolamine, muscarinic receptor blockers that do not and do cross the blood--brain barrier, respectively. Following pretreatment with methscopolamine, arecholine shortened the REM latency and increased the number of REM periods without altering the duration of individual REM periods or altering the REM--REM intervals. Pretreatment with scopolamine blocked the effects of arecholine. The results support the hypothesis that cholinergic muscarinic mechanisms are involved in the timing but not the duration of REM sleep.


The single-mindedness and isolation of dreams.

Dreams are descirbed as "single-minded," meaning that they tend to be unaccompanied by other, simultaneous streams of thought and imagery. Four manifestations of single-mindedness are discussed: (1) the absence of a reflective awareness that one is dreaming while the dream is in progress; (2) the absence of alternative images and thoughts while attending to the primary dream content; (3) the tendency for dream content to stay on a single thematic track; (4) the absence of a set to remember the dream while it is in progress. This isolation of dream content, from other thought systems is then considered as but one manifestation of a more generalized relative isolation of dream content, which includes isolation from presleep stimuli, contemporaneous stimuli, organismic state, and autonomic and motor activity. Some of the implications of dream isolation for dream psychophysiology and theories of dreaming are outlined.


Chronic primary insomnia: possible physiopathological involvement of slow wave sleep deficiency.

This study was designed to compare the sleep of a carefully selected group of patients with chronic primary insomnia toage- and sex-matched controls, in order to investigate the possible physiopathological role of slow wave sleep deficiency in this disturbance. In addition, the effect of age on normal sleep was studied in a group of 40 normal subjects. Sleep was recorded in the laboratory and automatically scored with an electronic system already described. The general trends of sleep stages were computed. Aging in normal subjects was characterized by a sharp decrease of stage 4, but a good stability of stage 3. Insomniacs' sleep showed a sleep-waking imbalance and a marked deficiency in stages 3 and 4. This deficiency seems to be similar to the age effect in normals, but more accentuated; it cannot be attributed merely to increased pressure of wakefulness. We suggest that slow wave sleep is involved in sleep induction and maintenance, and that its deficiency is linked to the fragility of sleep in chronic primary insomnia.


Amitriptyline and EEG sleep in depressed patients: I. Drug effect.

The paucity of studies on the tricyclic antidepressants led us to conduct an investigation on 30 drug-free patients treated with amitryptyline in a double-blind protocol over a 4-week period. Consistent with previous reports, there was an immediate REM suppression on 50 mg, which was more pronounced at the 100-mg level. However, no significant incremental changes were noticed on 150 or 200 mg with regard to REM sleep suppression. Drug administration was associated with changes in other sleep variables such as stage 1 percent, stage 2 percent, and various measures of sleep continuity. The incomplete tolerance of the drug on certain REM sleep variables enhances our understanding of amitryptyline effects on the EEG sleep of depressed patients.


Relationship of arousal threshold to sleep stage distribution and subjective estimates of depth and quality of sleep.

The relationship of arousal threshold, amounts of various sleep stages, and subjective rating of sleep was studied in two separate experiments involving 7 and 26 young adult subjects, respectively. Electroencephalographic sleep stage data, subjective response data, and arousal threshold data were collected over a series of nights in the sleep laboratory. Only nonsignificant relationships were found between magnitude of arousal threshold and amounts of various sleep stages or subjective rating of sleep quality in between-subjects analyses. However, when the nights representing extremes in arousal threshold were examined within-subject, it was found that nights of high threshold were accompanied by subjective reports of significantly better sleep in both studies and by a significant decrease in the amount of stage W in one study. Arousal threshold was not related to subjective depth of sleep in either study. Rather, subjective ratings of depth of sleep were significantly related to the amount of EEG-defined wakefulness and stage 1.


The forty-eight hour day.

Four normal young adult male subjects were evaluated in a systematically imposed regime of 32 hr of wakefulness and 16 hr of sleep time in an environment free from time cues. Electroencephalographic and electrooculographic recordings were made continuously during the experiment, which lasted for 10 complete cycles. Sleep efficiency was assessed by determining the percentage of sleep time during the assigned sleep period. The average sleep efficiency for the experimental period was 77%. Results, in general, conformed to earlier findings of non-24 hr schedules of sleep and waking: the overall sleep system remains relatively stable across a variety of scheduled variations; however, utilization of the sleep period becomes less efficient as the schedule increasingly deviates from the normal approximately 16 hr wakefulness/8 hr sleep schedule.


Membrane potential of spinal motoneurons during natural sleep in cats.

The membrane potential of spinal motoneurons was recorded during wakefulness, NREM sleep, and REM sleep in minimally restrained, behaving cats. At the onset of sleep, the membrane potential generally increased in polarization in rough proportion to time spent asleep. During the postural atonia of REM sleep, the membrane potential of all motoneurons was tonically hyperpolarized. Antecedents of NREM sleep electromyographic suppressions, and REM sleep myoclonic twitches were seen as transient hyperpolarizations and depolarizations, respectively.


The sleep of conjoined twins.

Conjoined twins with a common heart system and circulatory system were observed during the 14th and 15th days for 11 hr. Sleep, Waking, and Quiet and Active Sleep were recorded in minute intervals. Clear independence of sleep and waking was manifest, and total independence of Quiet and Active sleep was noted.


[Evaluation of initial supervised and integrated treatment of tuberculosis].

The operational and technical efficiencies were evaluated, of the initial, intermittently administered chemotherapy, supervised and integrated by the general medical network. The treatment was applied in 160 patients detected in 53 rural medical districts. It was noted that under the operational aspect the treatment was applied in 100% of the eligible patients, and administration of all the necessary doses was achieved in 91,6% of the patients. Bacteriological negativation by the direct examination was obtained after three months and negative cultures were obtained in 93,3% of the patients at three months and in 97,4% of all patients after 9 and 12 months. Closure (filling) of the cavities was obtained in 75,4% of the cases after three months of treatment, and in 87,7% of all cases after 9 and 12 months. Finally the technical efficiency was completed by an assessment of the socio-professional recovery, that was achieved in 91,8% of the cases.


[Strictly supervised antitubercular chemotherapy in district and plant medical dispensaries in the city of Craiova].

The mode of administration of chemotherapy is evaluated, in conditions of integration, and under strict supervision, in tuberculosis patients in 12 medical dispensaries and in 6 enterprise dispensaries from Craiova over a period of one year. A total of 88,54 +/- 17,99 per cent of the number of programmed doses was administered in the territorial medical dispensary, as compared with 81,9 +/- 21,3 per cent in the tuberculosis dispensary. In enterprise dispensaries 97,69 +/- 16,17 per cent of the doses was given effectively, as compared with 78,17 +/- 29,39 per cent of doses given to the same patients in the tuberculosis dispensary. During the sick-leave 92,14 +/- 9,19 per cent of the doses were given, in the medical territorial dispensary as against 86,98 +/- 17,58 per cent in the tuberculosis dispensary. The difference is not significant. After reinsertion 77,53 +/- 24,84 per cent of the programmed doses were administered in the tuberculosis dispensary, 88,2 +/- 19,84 per cent in the territorial medical dispensary and 97,12 +/- 5,66 per cent in the enterprise dispensary. The differences were not significant. The authors conclude that administration of anti-tuberculosis chemotherapy can be perfectly well carried out in integrated conditions in the urban environment, especially in interprise medical units, after reinsertion. The phtysiologist should make a periodical survey in these units in view of securing a strict administration of the treatment regimens.


Plasma and synovial fluid cAMP in patients with rheumatoid arthritis.

cAMP was measured in plasma and synovial fluid from 11 patients suffering from rheumatoid arthritis with a specific protein-binding assay. Plasma and synovial fluid values were 17.0 +/- 6.8 pmol/ml and 8.3 +/- 3.7 pmol/ml, range 5-28 pmol/ml and 5-16 pmol/ml, respectively. No correlation could be established between plasma and synovial fluid levels, plasma and disease activity, or synovial fluid and disease activity, as judged by Lansbury's index. It is concluded that it seems unlikely that synovial fluid cAMP is derived solely from plasma and that no simple relation exists between cAMP in plasma and synovial fluid and total disease activity.


Pyrophosphate arthropathy.

Pyrophosphate arthropathy has a wide spectrum of clinical symptoms and is a common cause of synovitis in the elderly. The acute "pseudogout" attack is the most commonly recognized, but chronic synovitis might be just as prevalent. Familial pyrophosphate arthropathy with a dominant autosomal heredity is a rare form, with an earlier debut and a more severe prognosis. Another rare expression of pyrophosphate arthropathy is severe joint destruction. The cause of pyrophosphate arthropathy is still unknown. Both individual reactivity and crystal properties determine the intensity of the inflammatory response to crystals.


[Considerations on pleural puncturo biopsy, based on the results of an analysis of 100 samples].

Results are given on 100 cases in which pleural bioptic puncture (PBP) has been performed for diagnosis purposes. The data given in the study show the large possibilities to apply this method, readily acceptable by patients. The high diagnosis value of PBP method in based on strictly objective criteria. Stress is laid upon the contribution of the method in precising the etiology of pleural fluids, which, up to the application of PBP, were labelled as having an "undetermined" etiology. The right therapeutic measures to be taken on precise diagnosis are also stressed in the study.


Effect of hydralazine and dihydralazine on connective tissue and binding to serum protein.

Hydralazine and dihydralazine, chelators of Fe2+, Fe3+ and Mn2+ ions, inhibited collagen biosynthesis in organ culture of chicken embryo tibiae. Both substances inhibited the in vitro hydroxylation of [14C]Pro-labelled protocollagen by protocollagen proline hydroxylase. A decreased incorporation of [14C]-D-glucosamine into glyco- and/or muco-proteins was found under the effect of the two drugs. Dihydralazine, a cationic compound, was bound to DNA and acid mucopolysaccharides (glycosaminoglycans), forming an insoluble complex. Both substances were bound to serum alpha 2-globulin. Subcutaneous injections of dihydralazine in rats produced a local as well as a general toxic effect.


[Aspects of tuberculosis prevention in the industrial environment].

The dynamics were studied of the tuberculous endemy in a limited area of the Cluj-Napoca District over a period of 5--16 years. An industrial environment was considered in comparison with a non-industrial one in the vicinity. The results show a marked decrease in the incidence of tuberculosis in the industrial environment (from 710 0/0000 in 1961 to 102 3/0000 in 1976), but, at the same time, it was demonstrated that morbidity due to tuberculosis is higher as compared with the non-industrial area (318 0/0000 as compared with 49 0/0000). The importance is discussed, of some particular factors of the industrial environment, such as pneumoconioses -- of which the most important are those due to silica -- which are significant risk factors for tuberculosis. A close cooperation is recommended, between enterprise physicians and pneumophtysiologists working in territorial polyclinics.


[Prevention of arteriosclerosis. Current basis].

Despite the advances in therapy, the high incidence, high mortality, and prematurity of coronary heart disease demonstrate the need for prevention. Measurement of a series of easily determined risk factors permits the early recognition of subjects at risk with remarkable reliability. However, reduction of risk factors affords protection against the illness only if they are causally connected with the disease mechanisms. The major evidence for linking atherosclerosis and its consequences with risk factors is reviewed. Particular attention is focused on serum lipids and the "lipid theory", smoking, elevated blood pressure, and physical inactivity, which are, on the basis of current knowledge, not only the most important factors but those most readily influenced by changes in daily living habits. Among the multiple risk factors mention is also made of obesity, diabetes, psychosocial stress, and hereditary predisposition. The probability of a causal relationship between risk factors and disease mechanisms justifies every effort to prevent the development of these precursors, or to treat them prophylactically if already present.


[The atopy syndrome].

Atopy is defined as that familial form of increased reactivity which is characterized by a probably genetically determined, spontaneously occurring preferential production of IgE as a response to the natural exposure to allergens normally present in the environment, and whose clinical manifestation as a disease is additionally dependent on a non-immunologically conditioned increase in reagibility of the tissues. An attempt is made to show how many different factors must cooperate to produce atopic disease, and how these factors are mutually influenced by their actions. The implications of this definition of atopy are discussed with reference to prophylaxis and treatment in clinical practice.


[Bartter's syndrome, chondrocalcinosis and hypomagnesemia].

In two cases of Bartter's syndrome with hypomagnesaemia the authors report radiological findings typical of chondrocalcinosis associated with joint symptoms corresponding to this condition. In Bartter's syndrome there is, in addition to hypokalaemic alkalosis, often concomitant magnesium depletion which has marked repercussions with respect to both physiopathology and symptomatology. In particular, hypomagnesaemia could well be important in the pathogenesis of chondrocalcinosis through two simultaneous mechanisms: by reducing the activity of pyrophosphatases and by facilitating the crystallisation of pyrophosphates. These mechanisms could explain the association of Bartter's syndrome and chondrocalcinosis, which is described here for the first time.


[Epidemic of tuberculosis in a maternity hospital triggered by congenital tuberculosis in a newborn infant].

The development of several cases of miliary tuberculosis in infants aged 3--4 months, born in the same maternity led to an epidemiological investigation. On this occasion it was discovered that one of the patients that had been discharged immediately after delivery had pulmonary granulia a monthlater. The immature newborn was left in the maternity and died after one month with a diagnosis of "pulmonary microabscesses". The histopathologic examination of the lungs showed the presence of necrotic foci without any cellular reaction. Five months after the death of the infant a new examination of a pulmonary fragment was performed and Ziehl-Neelsen staining evidenced a large number of acido-resistant bacilli filling the lung alveoles. It was shown in this way that the infant with tuberculosis was the primary source of infection. All the 8 children that had developed the disease had been hospitalized in the same ward with the sick child. The transmission of the bacilli was done indirectly, through the feeding tubes that had not been sufficiently serilised, and the penetration ronte was the digestive tract. In two cases an otic primary focus was discovered and in one case an intestinal one was found on necropsy.


Acute reactions to mega ascorbic acid therapy in malignant disease.

Three cases are described, 2 of Hodgkin's disease and a further case of bronchial carcinoma, where high dosage ascorbic acid treatment appeared to be associated with the development of potentially dangerous symptoms. It is suggested that mega ascorbic acid therapy should be given with caution in malignant disease, with a slow build-up over several days to high levels of dosage.


Hazards of hepatic artery infusion of streptozocin.

Three patients with metastatic malignancy received streptozocin by hepatic artery infusion through a percutaneous transfemoral catheter. Two patients developed major arterial thrombosis, from which one died. The other suffered renal damage following displacement of the catheter tip. Possible avoidable risk factors are identified.


Phase advance of the circadian sleep-wake cycle as an antidepressant.

Sleep in depressed patients resembles sleep in normal subjects whose circadian rhythms of temperature and rapid-eye-movement sleep are phase-advanced (shifted earlier) relative to their sleep schedules. If this analogy is relevant to the pathophysiology of depressive illness, advancing the time of sleep and awakening should temporarily compensate for the abnormal timing of depressed patients' circadian rhythms. Four of seven manic-depressive patients studied longitudinally spontaneously advanced their times of awakening (activity onset) as they emerged from the depressive phase of their illness. In a phase-shift experiment, a depressed manic-depressive woman was twice brought out of depression for 2 weeks by advancing her sleep period so that she went to sleep and arose 6 hours earlier than usual. The antidepressant effect of the procedure was temporary and similar in duration to circadian desynchronization induced by jet lag in healthy subjects. This result supports the hypothesis that abnormalities of sleep patterns in some types of depression are due to abnormal internal phase relationships of circadian rhythms.


Opiate (Enkephalin) receptors of neuroblastoma cells: occurrence in clusters on the cell surface.

A bioactive, fluorescent derivative of enkephalin, Tyr-D-Ala-Gly-Phe-Leu-Lys-rhodamine, was used to determine the distribution of opiate receptors in living neuroblastoma cells. The receptors appeared in clusters on the cell surface, and no internalization was detected. No specific fluorescence or clusters were observed in the presence of [D-Ala2, Leu5]enkephalin or at 4 degrees C, and the clusters were much reduced under ionic conditions (that is, with 100 millimolars sodium) that specifically decrease the binding of opiate agonists.


A sign inversion mechanism for enzymatic supercoiling of DNA.

Both the introduction and the removal of supertwists by DNA gyrase change the linking number of DNA in steps of two. This surprising finding provides strong evidence that gyrase acts by a mechanism, called sign inversion, whereby a positive supercoil is directly inverted into a negative one via a transient double-strand break.


Cerebral vessels have the capacity to transport sodium and potassium.

The activity of Na+, K+-activated adenosinetriphosphatase and the uptake of a potassium analog, rubidium, were found to be similar in cerebral microvessels and choroid plexus when measured in vitro. This similarity suggests that sodium and potassium concentrations in the nascent brain extracellular fluid are determined by the same active process that regulates their concentration in nascent cerebrospinal fluid. The brain microvessels may thereby play on active role in brain potassium homeostasis and brain extracellular fluid formation.


Physostigmine and recent memory: effects in young and aged nonhuman primates.

The effect of physostigmine on recent memory was evaluated in young and aged rhesus monkeys. All aged monkeys had previously shown impaired memory. The performance of the young monkeys treated with physostigmine was similar to that recently reported for young humans--no effects at low doses, some improvement at a restricted range of doses, and deficits at the highest dose. Although the aged subjects also improved at the same general doses, their overall response as a group was much more variable than that of the younger subjects. The performance of some aged monkeys was impaired by low doses that did not affect young monkeys. Continued improvement was observed in some aged monkeys at the highest dose, which typically impaired young monkeys. These variable effects across aged subjects suggest that physostigmine cannot easily or reliably be used as an agent for treating geriatric cognition. Nevertheless, the differential age-related effects suggest that appropriate manipulation of the cholinergic system may eventually be developed to alleviate some of the cognitive impairments suffered by aged subjects.


[The fight against alcoholism and its consequences in general practice (author's transl)].

Alcoholism is the number-one social scourge in France today and any means that become available for combating its effects are of the utmost importance. The author used an original approach to the problem by obtaining the co-operation of the family doctors in order to study 60 cases, including 52 men and 8 women, with alcoholism. Clinical symptoms, drinking habits, and in some cases biological test results using Gamma GT were recorded. The patients were then treated with tiapride. Daily family, professional and social activities were always better after treatment with extension of ideation and improved relations with others. Tremor was also greatly diminished. Distate for alcohol was not observed constantly but in some rare cases. Total abstinence was only noted in one case, but reduced alcohol consumption was reported in 50% of the patients. It is true that the product was not associated systematically with psychotherapy which might have made the results more demonstrative, but the objective of the study was to evaluate whether tiapride could be of value by assisting in the fight against alcoholism, and this was confirmed.


[Anatomic pathological aspects of chronic obliterating arteriopathies of the extremities (author's transl)].

It appears that the classical concept of atherosclerosis cannot be applied indiscriminately to all cases and that it is essentially valid for the aorta and its large branches of predominantly elastic structure. When more peripheral arterial trunks are considered, those of muscular type, the lesions are in great part characterized by a dystrophic fibrosis of the media, associated with a diffuse intimal thickening and with stratified fibro-hyaline plaques narrowing the lumen; the latter are responsible for the circulatory insufficiency and gangrene. Comparative analysis of 50 surgical specimens allow us to conclude that those fibro-hyaline plaques represent progressively stratified parietal thrombi that are populated by myocytes, at the contact of which ultrastructural investigation reveals important phenomena of elastogenesis and elastolysis. Most probably, the myocytes originate from the media, through fenestration within the inner elastic membrane. These facts have more than just a purely speculative interest: they express the great plasticity of the arterial walls and their capacity of adaptation to new hemodynamic and biologic conditions. It is interesting to note that this lesion constantly presents signs of metabolic activity and structural remodeling despite its long standing caracter.


[Renal lesions in progressive systemic sclerosis. A report on five cases (author's transl)].

Renal lesions are one of the must serious visceral complications of progressive systemic sclerosis: three of the five cases reported died in anuria within a few weeks. A part from this very severe fulminating form, however, cases in which lesions can be demonstrated by pathological examination may produce much milder clinical and biological signs, though still having the same prognostic significance. They can become worse at any moment, either spontaneously, or more often because of other factors such as corticotherapy or pregnancy. A review of the published literature has shown the exact frequency of these lesions and their two principal modes of progression, in both of which the same basic histological changes of proliferation of the intima of the interlobular and preglomerular arteries are found. Kidney function tests can be used to guage the severity and extent of these vascular lesions. It is much more difficult, however, to determine the nature of the initial mechanism causing these lesions. The vascular lesion could be a primary one or may be secondary to an immunological disorder, the exact nature of which has still to be determined.


[The role of arterial wall proteoglycan in arteriosclerosis (author's transl)].

Arteriosclerosis is the consequence of numerous factors which may be studied by inspecting; the composition of blood (cholesterol) and haemostasis, as well as, modifications in the composition and structure of aging arterial-wall tissue. Arterial-tunica cells synthesise in particular, elastin and proteoglycan (mucopolysaccharide-protein complexes) which account for the artery's mechanical and elastic properties. We have shown by biochemical separation and characterization that proteoglycan synthesis is considerably modified in aging arterial tissue. The metabolism of these macromolecules declines. Large aggregates of proteoglycan form. Alterations are observed in the distribution of the different protein-associated long chain polysaccharides, in fatty acids and in calcium bound to these proteoglycans favorising the formation of arteriosclerotic plaques. Studies on the aging of arterial cells in culture also demonstrate these same alterations in proteoglycans.


[Uretral cytology patterns during pregnancy and post partum period (author's transl)].

During pregnancy and post partum period, uretral cytology shows qualitative and quantitative changes which may be compared to those of vaginal and urinary cytology. However, as the cellular types or uretral cytology are most numerous, the different patterns are not so uniform and seem to offer a more various picture according to the different hormonal status. The relation between glandular cells and trigonal looking epidermoid cells is essential. The trigonal cells lowering, in favour of glandular cells, is rather linked to hormonal deficiency. These different facts may let us hope a better detection of pregnancy and post-maturity hormonal deficiency.


[A controlled double-blind clinical trial of a new association of a wide-spectrum antifungal preparation and a corticoid (author's transl)].

In a controlled double-blind study, the author has comparatively studied the effectiveness and the safety of econazole and of a combination econazole-triamcinolon topically applied, on inflammatory dermatomycoses. Results on 58 patients are reported. The respective well-known properties of econazole and of triamcinolon are maintained in the combination. Clinical symptoms statistically faster disappear with the combined preparation, during the first days of treatment. In addition, the average treatment duration is shortened when econazole-triamcinolon is applied. The new preparation allows security to the physician as well it makes the patient more comfortable.


[Therapeutical evaluation on neoplasia-related pain (author's transl)].

The antalgic effects of tiapride by IM route are evaluated in a randomized study on 30 patients with different kinds of neoplasia-related pain. Using an average dose of 300 mg/day, the effectiveness was considered as excellent or good in 60% and equivalent to the reference drug. This effect was obtained after 30 minutes and lasted about 4 hours. The results compared closely with the reference drug. Tolerance was good with the rare problem of somnolence.


[Remarks on a satellited Y chromosome (author's transl)].

A previous report in 1967 on the observation of a satellited Y chromosome found in a french canadian family line is confirmed by the use of the ammoniacal silver procedure which stains selectively the nucleolus organizer regions (NORs) in acrocentric human chromosomes. There is evidence that this peculiar chromosome results from the translocation to the distal end of the Y chromosome long arms of a satellited segment from a D or G autosome.


[The surgical treatment of stomach cancers. A review of 759 operations (author's transl)].

759 patients were studied, deriving from the Statistics of the Department from the last twenty years and the result of an inquiry sent out to the French members of the Collegium Internationale Chirurgiae Digestival. For 602 tumors of the cardia, the long term results of the upper gastrectomies were, inspite of area of non-invaded lymph nodes for 1/3 of the patients, the same for the two sources of statistics, with almost no survival after the fourth year. On the contrary, however, the percentages of survival of total gastrectomies "by choice" are 40 and 31% after five years. As for the cancers of the lower part, the result was not as precise and the apparent or proved non invaded state of lymph nodes during the operation make us rather tend towards a partial gastrectomy. (Combined with a mortality rate inferior to those of total gastrectomies). As for the total of gastric cancers, the total gastrectomies "by necessity" (eith by tumor growth or lymph tive therapy in a specialized center permit fifty per cent after five years, for the patients who would have been condemned by any other therapeutical act. The nutritional consequences of the total gastrectomies were studied and permitted to conclude that post operative therapy in a specialized center permit fifty per cent of the patients of work age to take up their professional activities again and, on the whole, sixty five per cent of them to have completely satisfactory independance.