Purification of chicken intestinal receptor for 1,25-dihydroxyvitamin D. The 3.3S chicken intestinal receptor for 1,25-dihydroxyvitamin D [1,25-(OH)2D] has been purified approximately 86,000-fold from the cytosolic fraction. The receptor was selectively precipitated with Polymin P from high-speed supernatants derived from 800 g of intestinal mucosa and then sequentially chromatographed on DNA-cellulose. Sephacryl, blue dextran-Sepharose, DNA-cellulose, and heparin-Sepharose. Polyacrylamide gel electrophoresis of 8-10 microgram of the purified receptor in sodium dodecyl sulfate indicated the presence of one major and three minor protein bands of molecular weights 50,000-65,000. Sucrose gradient analysis of the purified material in 0.3 M KCl suggested that a fraction of the receptor remained complexed to the 1,25-(OH)2D and that its sedimentation property of 3.3S remained unchanged. These results represent a major purification of the chick intestinal receptor for 1,25-(OH)2D, an extremely rare and labile protein whose isolation is estimated to require a 200,000-fold purification. Of primary importance is the observation tht affinity ligands such as DNA and blue dextran can effect major purification of this protein, lending credence to the hypothesis that the 1,25-(OH)2D receptor functions within the cell nucleus by altering the expression of specific genes.


Regulation of synthesis of a major outer membrane protein: cyclic AMP represses Escherichia coli protein III synthesis. Cyclic AMP is the effector molecule for both positive and negative control of synthesis of several Escherichia coli proteins. Among the latter is the major outer membrane protein III. The control mechanism occurs at the level of transcription and involves the cyclic AMP receptor protein. The repressing system is saturated at lower concentrations of cyclic AMP than is the positive control system.


Complementation of the temperature-sensitive defect in H5ts125 adenovirus DNA replication in vitro. Soluble extracts of adenovirus-infected HeLa cell nuclei support DNA replication on exogenous adenovirus DNA templates. Conditions of synthesis using both wild-type and temperature-sensitive extracts have been defined. Nuclear extracts prepared from cells permissively infected with the adenovirus mutant H5ts125 expressed the temperature-sensitive phenotype and could be inactivated at 37 degrees C in vitro. These extracts were completely complemented by the addition of wild-type adenovirus DNA binding protein but not by H5ts125 DNA binding protein. Enhancement by binding protein in the mutant extracts represents replication, as demonstrated by the production of full-sized products and orderly chain elongation originating, as in vivo, at both ends of the linear DNA. Replicative synthesis required the 5'-terminal protein bound covalently to template DNA and could be inhibited by denaturation of this 55,000-dalton protein. Various inhibitors of eukaryotic DNA polymerases, such as aphidicolin and 2',3'-dideoxythymidine triphosphate, inhibited replication of exogenous adenovirus templates in this system as they do in previously reported systems that only elongate endogenous replicating intermediates.


Spliced adenovirus-associated virus RNA. We describe the structure of cytoplasmic RNA species transcribed from the DNA of adenovirus-associated virus, a defective parvovirus. The RNA was hybridized with minus strand template DNA and visualized in the electron microscope. Alternatively, the DNA.RNA duplex molecules were digested with nuclease S1 or Escherichia coli exonuclease VII and analyzed by agarose gel electrophoresis. A set of RNA species was observed with 5' terminal at map positions 5, 13, 19, or 39 and a 3' terminus and poly(A) tail at position 96 (one map unit is equivalent to 1% of genome length). Most of these RNAs are spliced and lack sequences approximately between positions 40 and 49. Some RNA preparations also contained unspliced molecules with 5' and 3' terminal at positions similar to those in the spliced RNA.


Solubilization of the low density lipoprotein receptor. The low density lipoprotein (LDL) receptor was solubilized from membranes of bovine adrenal cortex and cultured human cells by incubation with the nonionic detergent octyl-beta-D-glucoside. Receptor activity released into the 100,000 x g supernatant was assayed by a solid-phase procedure: an aliquot of the soluble extract was removed, the detergent was diluted below its critical micellar concentration, causing the receptor to precipitate as a lipid-protein aggregate; the precipitate was collected by centrifugation and incubated with (125)I-labeled LDL ((125)I-LDL); and the receptor-bound (125)I-LDL was separated from free (125)I-LDL by filtration. The (125)I-LDL binding site that was precipitated from the soluble extract of bovine adrenocortical membranes appeared to be the same as the functional LDL receptor of cultured bovine adrenocortical cells and human fibroblasts. It exhibited high affinity and specificity (affinity for LDL more than 200-fold greater than for acetylated LDL, methylated LDL, or high density lipoprotein), dependence on calcium, and susceptibility to destruction by Pronase. The amount of (125)I-LDL binding activity in solubilized membranes from cultured cells was proportional to the number of receptors on the surface of the intact cells. Thus, the number of solubilized receptors was 1/20th of normal in mutant fibroblasts from a subject with homozygous familial hypercholesterolemia and was 1/4th of normal in human epithelioid carcinoma A-431 cells when they were grown in the presence of 25-hydroxycholesterol plus cholesterol. While in the soluble form in the presence of octyl-beta-D-glucoside, the LDL receptor can be carried through several steps of purification.


Synthesis of simian virus 40 t antigen in Escherichia coli. Plasmids are constructed by using recombination in vitro according to Roberts, T.M., Kacich, R. & Ptashne, M. (1979) Proc. Natl. Acad. Sci. USA 76, 760-764 in which the t antigen gene of simian virus 40 is fused to a promoter of the Escherichia coli lac operon. In the fusions, transcription commences at the lac promoter, and, in some of the fusions, translation begins at the ATG initiator codon of the t gene. This translation is directed most efficiently by those plasmids in which the lac sequences abut the t gene such that a hybrid ribosome binding is encoded. In this case, the Shine-Dalgarno sequence is of lac origin but the ATG derives from the t gene. translation from this initiator codon is greatly decreased if the lac sequences are separated from the ATG by 17 base pairs and is abolished if the AT of this triplet is deleted. Cells bearing the productive fusions synthesize a 20,000-dalton protein with t antigenic determinants. This protein has an isoelectric point(s) indistinguishable from that of t antigen isolated from simian virus 40-transformed cells. Moreover, a partial sequence of the amino-terminal region of the bacterial product is that predicted for authentic t antigen. We conclude that these bacteria are for authentic t antigen. We conclude that these bacteria are producing a protein, the sequence of which is identical to that of authentic t antigen unfused to other polypeptides.


Differentiation as a requirement for simian virus 40 gene expression in F-9 embryonal carcinoma cells. Infection of differentiated mouse embryo cells by simian virus 40 (SV40) leads to the production of the early mRNAs and the tumor (T) antigens that they encode. In contrast, undifferentiated F-9 murine teratocarcinoma cells do not support these early stages of the SV40 cycle. This block results from the inability to accumulate stable processed early SV40 mRNAs. It has recently been shown that vitamin A and its derivatives can induce in vitro differentiation of stem cells. Undifferentiated F-9 cells, upon treatment with a low concentration of retinoic acid, exhibited pronounced morphologic changes as well as the appearance of the H-2 surface antigens. After differentiation, the susceptibility of F9 cells to SV40 infection could be demonstrated by the appearance of large T and small T antigens, as shown by immunofluorescence and immunoprecipitation. Furthermore, SI nuclease mapping of early SV40 transcripts confirmed the presence of the two spliced early mRNAs. These results indicate that the undifferentiated F-9 stem cells contain the genetic information needed for generating stable processed early SV40 mRNAs but are blocked in the production of functional species.


Glycoproteins of Sendai virus are transmembrane proteins. Radiolabeled Sendai viral envelope proteins were incorporated into human erythrocyte membranes by the process of fusion of the viral envelope with the erythrocyte membrane. Inside-out (IO) vesicles were prepared from the erythrocyte membranes containing viral proteins, and the presence of viral proteins was assessed by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. Proteolysis with trypsin, chymotrypsin, or Pronase, which digests only the external surface of the IO vesicles (the cytoplasmic surface of the erythrocyte membrane) revealed that the viral nucleocapsid and the nonglycosylated inner-envelope (M) proteins were present on the external surface. In addition, small segments of the viral envelope glycoproteins (HN and F1) were removed by these proteases, while the major portions of the glycoproteins were protected from digestion, indicating that in the erythrocyte membrane they are transmembrane proteins. Results of experiments carried out on right side-out membranes, unsealed membranes, and membranes containing attached virus that was unable to fuse indicated that the results obtained with IO membranes could not be accounted for by contamination with these membrane species. The identify of the proteolysis products was confirmed by peptide mapping. The selective exposure of the cytoplasmic surface, and hence the internal components of the virus, in IO vesicles makes this membrane system an attractive model for studying the interactions involved in virus maturation at host cell membranes.


Coupling of opiate receptors to adenylate cyclase: requirement for Na+ and GTP. Inhibition of the adenylate cyclase activity in homogenates of mouse neuroblastoma-glioma hybrid cells (NG108-15) by the opioid peptide [D-Ala2,Met5]enkephalin amide (AMEA) requires the presence of Na+ and GTP. In this process, the selectivity for monovalent cations is Na+ greater than or equal Li+ greater than K+ greater than choline+; ITP will replace GTP but ATP, UTP, or CTP will not. The apparent Km for Na+ is 20 mM and for GTP it is 1 microM. Under saturating Na+ and GTP conditions, the apparent Ki for AMEA-directed inhibition is 20 nM for basal and 100 nM for prostaglandin E1-activated adenylate cyclase activity. For both cyclase activities, maximal inhibition is only partial (i.e., approximately 55% of control in each case). In intact viable NG108-15 cells, the decrease in basal and prostaglandin E1-stimulated intracellular cyclic AMP concentrations by AMEA is also dependent upon extracellular Na+. The enkephalin-directed reductions in cyclic AMP concentrations are at least 75%. The specificity of the monovalent cation requirement for enkephalin action on intact cells is the same as for enkephalin regulation of homogenate adenylate cyclase activity. Based on these data, a model is presented in which the transfer of information from opiate receptors to adenylate cyclase requires active separate membrane components, which correspond to the sites of action of Na+ and GTP in this process.


Rotational diffusion of cell surface components by time-resolved phosphorescence anisotropy. The rotational diffusion of concanavalin A receptors of viable Friend erythroleukemia cells and the band 3 anion transport system of human erythrocytes has been measured via the time-dependent phosphorescence emission intensity and anisotropy of triplet probes excited by a 5-ns laser pulse. High-quality phosphorescence decay curves with a 10-microseconds time resolution were obtained at concentrations of the eosin probe down to 20 nM and in aqueous media at temperatures of 4-38 degrees C. A strong temperature dependence in the rotational behavior was observed for the band 3 anion transport protein, but the lectin receptors of the Friend erythroleukemia cells were found to be immobile on the time scale of 1-4000 microseconds at either 4 degrees C or 37 degrees C. The technique is applicable to other triplet probes and membrane components of living cells under conditions that do not destroy viability.


Interaction of physalaemin, substance P, and eledoisin with specific membrane receptors on pancreatic acinar cells. We have prepared 125I-labeled physalaemin and have examined the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini from guinea pig pancreas. Binding of 125I-labeled physalaemin was saturable, temperature-dependent, and reversible and reflected interaction of the labeled peptide with a single class of binding sites on the plasma membrane of pancreatic acinar cells. Each acinar cell possessed approximately 500 binding sites, and binding of the tracer to these sites could be inhibited by physalaemin [concentration for half-maximal effect (Kd), 2 nM], substance P (Kd, 5 nM), or eledoisin (Kd, 300 nM) but not by cholecystokinin, caerulein, bombesin, litorin, gastrin, secretin, vasoactive intestinal peptide, glucagon, somatostatin, neurotensin, bovine pancreatic polypeptide, leucine-enkephalin, methionine-enkephalin, atropine, or carbamylcholine. With physalaemin, substance P, and eledoisin, there was a close correlation between the relative potency for inhibition of binding of labeled physalaemin and that for stimulation of amylase secretion. For a given peptide, however, a 3-fold higher concentration was required for half-maximal inhibition of binding than for half-maximal stimulation of amylase secretion, calcium outflux, or cyclic GMP accumulation. These results indicate that dispersed acini from guinea pig pancreas possess a single class of receptors that interact with physalaemin, substance P, and eledoisin and that occupation of 45% of these receptors will cause a maximal biological response.


Hormone receptor topology and dynamics: morphological analysis using ferritin-labeled epidermal growth factor. Previous studies using a biologically active 1:1 conjugate of EGF and ferritin (F-EGF) have traced the binding and internalization of the hormone molecules. In the present report, we develop ultrastructural criteria for identification of the F-EGF.receptor complex, and, thereby, enable utilization of the F-EGF as an indirect marker to localize the receptor for this peptide hormone. The ferritin cores of bound F-EGF are situated 4-6 nm from the extracellular surface of the membrane. When cells were incubated for up to 30 min at 37 degrees C, this characteristic spatial relationship was observed in all uptake stages (surface clustering, endocytosis, and incorporation into multivesicular bodies), indicating that the hormone.receptor complex remains intact through these steps. However, when incubation was continued for periods sufficient to allow hormone degradation (30-60 min), pools of free ferritin were observed in lysosomes. In the presence of various amine inhibitors of hormone degradation, internalization and multivesicular body incorporation proceeded, but hormone.receptor degradation was blocked as evidenced by preservation of the ferritin-membrane relationship; i.e., no pools of free ferritin were seen after 60 min. These data provide morphological support for the hypothesis that down-regulation of surface receptors involves internalization of intact hormone.receptor complexes. In addition, we have developed a method for viewing the surface of intact cells en face, allowing closer scrutiny of the clustering of F-EGF.receptor complexes in the plane of the membrane prior to internalization. The particles in the F-EGF clusters observed by this method are spaced at 12 nm center-to-center, serving to set upper limits on the packing dimensions of the EGF.receptor complex.


Passage of phenotypes of chemically transformed cells via transfection of DNA and chromatin. DNA was prepared from 15 different mouse and rat cell lines transformed by chemical carcinogens in vitro and in vivo. These DNAs were applied to NIH3T3 mouse fibroblast cultures by using the calcium phosphate transfection technique. DNAs of five donor lines were able to induce foci on the recipient monolayers. Ten other donor DNAs yielded few or no foci. DNAs from control, nontransformed parental cell lines induced few or no foci. Chromosomes were transfected from one donor whose naked DNA was unable to induce foci, and morphologic transformation of recipients was observed. These experiments prove that in five of these cell lines the chemically induced phenotype is encoded in DNA, and the sequences specifying the transformed phenotype behave as a dominant allele in the NIH3T3 recipient cells. The sequences encoding the transformation are likely found on a single fragment of DNA.


Antibodies to Epstein-Barr virus-determined antigens in normal subjects and in patients with seropositive rheumatoid arthritis. Prior studies have shown that patients with seropositive rheumatoid arthritis (RA) have an increased frequency of precipitating antibody against a nuclear antigen, the RA nuclear antigen, detected in human B lymphoblastoid cell lines infected by Epstein-Barr virus. The present investigations demonstrate that patients with seropositive RA also have specifically elevated titers of antibodies to another, better-characterized Epstein-Barr virus-associated B cell antigen, the Epstein-Barr nuclear antigen, which is detected by anti-complement immunofluorescence. Titers of these two antibodies were not affected by absorption of rheumatoid factor from serum. Furthermore, patients with RA did not have elevated titers of antibodies against the Epstein-Barr virus capsid antigen or to three other species of human herpesviruses: herpes simplex type 1, varicella-zoster virus, and cytomegalovirus. In both normal individuals and RA patients there was a significant association between the presence of antibodies to RA nuclear antigen and the titers of antibody to Epstein-Barr nuclear antigen. Thus, normal subjects with antibody to RA nuclear antigen had titers of antibody to Epstein-Barr nuclear antigen equivalent to those of patients with RA and significantly higher than normal subjects lacking antibody to RA nuclear antigen. One interpretation of these results is that patients with seropositive RA derive from a larger population with enhanced immune responsiveness to B lymphocyte nuclear antigens determined by the Epstein-Barr virus.


Triene prostaglandins: prostaglandin D3 and icosapentaenoic acid as potential antithrombotic substances. Addition of the 3-series fatty acid precursor (icosapentaenoic acid, IPA), its endoperoxide [prostaglandin (PG)H(3)], or thromboxane A(3) to human platelet-rich plasma (PRP) does not result in aggregation of the platelets. In fact, preincubation of human PRP with exogenous PGH(3) actually inhibited aggregation by increasing platelet cyclic AMP concentrations. PGH(3) undergoes rapid spontaneous degradation to PGD(3) in human PRP. The PGD(3) so formed is adequate to account for the increase of platelet cAMP and inhibition of aggregation. Furthermore, addition of PGD-specific antisera to human PRP blocked the platelet inhibitory activity of exogenous PGH(3). PGD(3) has considerable potential as a circulating antithrombotic agent. Pretreatment of human PRP with the adenylate cyclase inhibitor 2',5'-dideoxyadenosine blocked the increase of platelet cyclic AMP and the inhibition of aggregation normally produced by PGI(2), PGE(1), PGD(2), PGH(3), and PGD(3). Furthermore, the dideoxyadenosine unmasked a direct but moderate reversible aggregatory effect in response to the subsequent addition of PGH(3). Similarly, the dideoxyadenosine markedly enhanced the aggregation produced by exogenous PGH(2). IPA is readily incorporated into tissue lipids but proved to be a poor substrate for kidney, blood vessel, or heart cyclooxygenase. IPA was previously shown to be a poor substrate for platelet cyclooxygenase. IPA is readily deacylated from the renal phospholipid pool in response to bradykinin, a substance that also stimulates the release of arachidonic acid. A diet that relies primarily on cold-water fish, as in the case of the Greenland Eskimos, lowers endogenous arachidonic acid and markedly increases the IPA content of tissue lipids. Thus, because IPA has the potential to act as an antagonist with arachidonic acid for platelet cyclooxygenase and lipoxygenase, the simultaneous release of IPA could suppress any residual arachidonic acid conversion to its aggregatory metabolites.


Changes in plasma lipoprotein distribution and formation of two unusual particles after heparin-induced lipolysis in hypertriglyceridemic subjects. Lipoprotein morphology and distribution were studied in three moderately to severely hypertriglyceridemic patients after heparin-induced lipolysis. Lipoproteins with a flotation rate of Sf degrees 12-20 in rho 1.063-g/ml NaCl solution increased substantially in mass 2.5-10 min after heparin administration. This fraction contained 40- to 120-nm flattened particles and increased concentrations of phospholipid and free cholesterol. At 2.5 and 10 min after heparin, the high density lipoproteins (HDL) of rho, 1.126-1.21 g/ml contained small (5.8-nm diameter) spherical particles. Both types of particles disappeared 30-60 min after heparin. Results indicated that with lipolysis HDL3 may be transformed into HDL2a by incorporation of chylomicron and very low density lipoprotein constituents. It is suggested that the 40- to 120-nm particles represent surface fragments of chylomicrons and very low density lipoproteins generated during lipolysis, whereas the 5.8-nm particles are produced either by fragmentation of the large surface constituents or by loss of lipid from existing HDL3.


Reactivation of herpes simplex virus type 2 from a quiescent state by human cytomegalovirus. The ability of human cytomegalovirus to stimulate replication of herpes simplex virus type 2 (HSV-2) was examined. The system used involved HSV-2-infected human embryonic lung cells under conditions (39.5-40 degrees C) in which HSV-2 remains undetectable. Reactivation of HSV-2 was maximal and persisted for the longest duration when cultures were superinfected with 0.02 plaque-forming unit of human cytomegalovirus per cell. Infectious HSV-2 appeared 2 days after superinfection with human cytomegalovirus and ranged from 10(2) to 10(6) plaque-forming units per culture. Virus reactivated from these cultures was neutralized by rabbit immune serum produced against HSV-2. The specificity of this interaction was demonstrated by various criteria: production of HSV-2 was not observed in cultures treated with mock infecting fluid, and inactivation of human cytomegalovirus by heat, ultraviolet irradiation, or immune serum prior to superinfection eliminated its ability to induce HSV-2 replication. These results sugges that interaction between these two human herpesviruses may be of importance in herpesvirus latency in vivo.


Apolipoprotein is responsible for neutralization of xenotropic type C virus by mouse serum. We have shown that the circulating lipoproteins of the mouse contain a potent inhibitor of infectivity of the xenotropic type C virus. This virus neutralization does not involve immunoglobulins or complement. After fractionation of the lipoproteins on the basis of particle size, flotation properties, and electrostatic charge, virus neutralizing activity is found primarily in the triglyceride-rich lipoproteins (predominantly the chylomicrons) and in the HDL(2) subfraction of the high density lipoproteins. In fasted animals, activity resides chiefly in the high density lipoproteins. Neutralization titers increase strikingly during alimentary lipemia in both the lipoproteins of the rho < 1.006 g/cm(3) fraction and the high density lipoproteins. Increased activity persists in the high density lipoproteins after the lipemia recedes. Virus neutralizing activity is completely eliminated in all fractions by antiserum against high density lipoproteins and, in the triglyceride-rich fractions, by antiserum to murine apolipoprotein B. Complete removal of lipids markedly reduces the neutralizing activity of both classes of lipoproteins. Apolipoproteins delipidated with tetramethylurea retain some activity, which is enhanced by binding to a phospholipid-stabilized triglyceride emulsion and which is abolished by proteolytic digestion. We have demonstrated in vitro transfer of activity between high density and very low density lipoproteins of the mouse. These data indicate that xenotropic virus neutralization by normal mouse serum depends upon a protein that transfers among lipoprotein particles in a fashion analogous to the C apolipoproteins of other mammalian species.


Chronic treatment with lithium or desipramine alters discharge frequency and norepinephrine responsiveness of cerebellar Purkinje cells. Cerebellar Purkinje cells were studied by electrophysiological techniques in rats treated chronically with either desipramine (DMI) or lithium chloride given intragastrically. A striking decrement occurred in discharge frequencies of simple spikes and climbing fiber bursts in both groups of animals, similar to the depression produced by iontophoresis of these agents. Chronic treatment with DMI markedly decreased responsiveness to iontophoretically applied norepinephrine (NE), whereas long-term LiCl therapy slightly enhanced response to NE; responses to gamma-aminobutyric acid were unchanged by these treatments. The inhibitory responses to locus ceruleus stimulation were unaffected by chronic LiCl treatment. The effects of these chronic treatments on responsiveness to NE are opposite to the effects these same drugs produce when administered by acute iontophoresis to single cells: DMI then potentiates and LiCl antagonizes noradrenergic responses. These results provide electrophysiological evidence for reciprocal adaptive changes in NE sensitivity, supporting results of biochemical studies.


Protein and phospholipid methylation during chemotaxis in Dictyostelium discoideum and its relationship to calcium movements. Suspensions of cyclic AMP sensitive cells of Dictyostelium discoideum responded to a cyclic AMP pulse with increased methylation of a protein of molecular weight about 120,000 and increased phospholipid demethylation. Protein methylation reached its peak 15-30 sec after cyclic AMP addition. Phospholipid demethylation reached its maximum within 2 min and basal levels were recovered in 3 min. S-Adenosyl-L-methionine is probably the methyl donor. In vitro addition of 0.25 mM and 25 microM S-adenosyl-L-methionine to sonicated D. discoideum cells inhibited ATP-dependent 45Ca2+ uptake by 70% and 25%, respectively. Based on these lines of evidence we propose that protein and phospholipid methylation are involved in D. discoideum chemotaxis probably by regulation of intracellular Ca2+ movements.


A topoisomerase from Escherichia coli related to DNA gyrase. We have identified a topoisomerase activity from Escherichia coli related to DNA gyrase (topoisomerase II): we designate it topoisomerase II'. It was constructed of two subunits, which were purified separately. One is the product of the gyrA (formerly nalA) gene and is identical to subunit A of DNA gyrase. The other is a 50,000-dalton protein, which we have purified to homogeneity and call v. v may be a processed form of the much larger gyrase subunit B or may be derived from a transcript of part of the subunit B structural gene, because preliminary peptide maps of the two subunits are similar. Topoisomerase II' relaxes negatively supercoiled DNA and, uniquely among E. coli topoisomerases, relaxes positive supercoils efficiently. It is the only topoisomerase that can introduce positive supercoils; these are stoichiometric with enzyme molecules. Topoisomerase II' resembles gyrase in its sensitivity to oxolinic acid, the wrapping of DNA in an apparent positive supercoil around the enzyme, and the introduction in an aborted reaction of site-specific double-strand breaks in the DNA with concomitant covalent attachment of protein to both newly created 5' ends. Unlike DNA gyrase, topoisomerase II' has no negative supercoiling activity. Functional chimeric topoisomerases were constructed with the alpha subunit of the Micrococcus luteus gyrase and v or gyrase subunit B from E. coli. We discuss the implications of the dual of the gyrA gene product.


Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin. The circular genome of simian virus 40 is a model mammalian replicon, containing a unique origin of replication (ori) and coding for a protein (SV40 T antigen) known to be involved in initiation of viral DNA replication and to bind in vitro to the origin region. Mutations within the ori sequence lead to defective viral DNA replication and the formation of small viral plaques after infection of a cell monolayer. Second-site revertants (pseudorevertants) of ori mutants were isolated by random local mutagenesis of mutant DNA followed by transfection of cultured cells and the selection of large plaques. In each case, reversion of the plaque phenotype was associated with an increased rate of viral DNA replication. The second-site mutations that suppressed the replication defects were localized by in vitro recombination or marker rescue experiments to the gene for T antigen. Their map positions differ from those of previously described T antigen mutants, possibly reflecting a specific ori-binding domain of T antigen. From these results we infer that T antigen interacts with the ori signal during virus development as it does in vitro and that this interaction regulates the rate of viral DNA replication.


Electron-nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase: identification of the free radical site with a methionyl cluster. The results of electron-nuclear double resonance and electron paramagnetic resonance (EPR) studies on the hydrogen peroxide compound of yeast cytochrome c peroxidase are inconsistent with previous proposals for the source of the EPR signal in this compound, in particular with its identification with an aromatic amino acid radical such as would arise by oxidation of a tryptophanyl side chain. The present observations lead us to propose that the EPR signal is associated with a cluster containing at least one methionine and in which proximate side chains share the charge created by loss of one electron.


Internal genome deletions in two distinct classes of defective interfering particles of vesicular stomatitis virus. We have characterized the genome sequences represented in two defective interfering particles derived from the heat-resistant strain of vesicular stomatitis virus by means of end-labeling and hybridization techniques. Both defective particle RNAs, which differ slightly in size, contain 5'-end sequences identical to each other and to that of the standard infectious virus genome, for at least the first approximately 55 bases. In contrast, the 3'-end sequences of these two RNAs are different. The 3'-end sequence of the smaller RNA is identical to that of the standard genome for at least the first 48 bases. The 3'-end sequence of the larger RNA is an inverted complement of its 5' end for approximately 65 bases. The bulk of the sequences in both RNAs is derived from the 3' half of the standard genome. We also show that the two defective particles differ in vitro transcription and in vivo replication properties. These results provide direct evidence for the presence of internal genome deletions in defective interfering particles of negative-stranded RNA animal viruses and demonstrate the existence of at least two distinct classes of these particles.


Metal-coordinating substrate analogs as inhibitors of metalloenzymes. A group of active-site metal coordinating inhibitors of zinc proteases (carboxypeptidase A, thermolysin, Bacillus cereus neutral protease, and angiotensin-converting enzyme) have been synthesized and their properties investigated. Their general structures are R-SH and R-NH-PO2(O phi)H, where-S- or -O- serve as metal ligands and R refers to an amino acid or peptide group designed to interact with substrate recognition sites. These inhibitors can be extremely potent; thus, N-(2-mercaptoacetyl)-D-phenylalanine, e.g., inhibits carboxypeptidase A with a Kiapp of 2.2 x 10(-7) M. The spectral response of cobalt(II)-substituted thermolysin or carboxypeptidase A to the sulfur-containing inhibitors signals the direct interaction of the mercaptan with the metal. An S leads to Co(II) charge transfer band is generated near 340 nm and is detected by absorption, circular dichroism, and magnetic circular dichroism. The cobalt(II) spectra indicate both inner sphere coordination with sulfur and 4-coordination in the enzyme-inhibitor complex. Thus, the metal undergoes a simple substitution reaction, the inhibitor most likely displacing water at the fourth coordination site.


Prostaglandin D2, a neuromodulator.

Prostaglandin D2, a neuromodulator. The distribution of prostaglandin D synthetase activity was determined in various tissues of rat by using the supernatant fraction (10,000 x g, 20 min) of the homogenates. The highest activity was found in brain, spinal cord, and alimentary tract. The activity was uniquitously distributed in all parts of brain, and the highest specific activity was found in hypothalamus and thalamus. Homogenates of two neuroblastoma cell lines were found to produce prostaglandin D2, whereas a glioma cell line was almost inactive. Prostaglandin D2 is a potent and specific activator of the adenylate cyclase system of cultured neuroblastoma cells, suggesting the possibility that it may act as a neuromodulator in the central nervous system.


Evidence that the avian sarcoma virus transforming gene product is a cyclic AMP-independent protein kinase. The avian sarcoma virus transforming gene product pp60src has been partially purified by using ion exchange or immunoaffinity chromatography. These preparations contain a cyclic AMP-independent protein kinase activity capable of transferring radiolabel from [gamma-32P]ATP to immune rabbit IgG, casein, and the heavy chain purified from normal rabbit IgG. The casein kinase activity is specifically inhibited by anti-pp60src IgG. Comparison of the pp60src-protein kinase isolated from cells transformed by a wild-type ASV or by an ASV temperature-sensitive transformation mutant revealed that the latter product had a half-life of 3 min at 41 degrees C whereas that of the wild-type product was 20 min.


DNA gyrase: purification and catalytic properties of a fragment of gyrase B protein. A protein isolated from Escherichia coli complements the DNA gyrase A (NalA) protein to generate an activity that relaxes supercoiled DNA. Oxolinic acid, a known inhibitor of DNA gyrase, blocks this activity and causes double-strand cleavage of DNA at the same sites as are attacked by DNA gyrase. The protein, of molecular weight 50,000, appears to be fragment of the DNA gyrase B (Cou) protein (molecular weight, 90,000) as judged by the identical sizes of numerous peptides produced by partial proteolytic digestion. The complex of this fragment and the gyrase A protein lacks both the DNA-supercoiling and DNA-dependent ATPase activities of DNA gyrase.


Single-strand binding protein enhances fidelity of DNA synthesis in vitro. The effect of Escherichia coli single-strand binding protein on the accuracy of in vitro DNA synthesis has been determined by using two independent methods. By using the synthetic polynucleotide poly[d(A-T)] and measuring dGTP misincorporation or by using phi X174 DNA and measuring nucleotide substitutions, we found that binding protein increases the fidelity of DNA synthesis by as much as 10-fold. This increase is observed with DNA polymerases of divergent sources and is progressive with increasing concentration of binding protein. The increased accuracy observed with DNA polymerases lacking a 3' leads to 5' exonuclease points to a mechanism other than augmented proofreading. In accord with the properties of single-strand binding proteins, it is suggested that increased fidelity is a result of enhanced base selection by the DNA polymerase, resulting from increased rigidity of the template due to its interaction with binding protein.


Enhanced phosphorylation of many endogenous protein substrates in human fibroblasts transformed by simian virus 40. Protein phosphorylation in normal and in simian virus 40-transformed human skin fibroblasts was assessed by two different methods: incubation of whole-cell homogenates with [gamma-(32)P]ATP or labeling of intact cells with Na(2)H(32)PO(4). Phosphorylated proteins were detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. With both methods, the Coomassie-blue-stained protein patterns of the three transformed cell lines studied were similar to the patterns of the nontransformed normal human cells. However, although the phosphoprotein autoradiograms of the three transformed cell lines were nearly identical, their patterns were strikingly different from those of the nontransformed cells. Each of the three transformed lines tested showed approximately 25-30 phosphoprotein bands that were significantly enhanced when compared to the patterns of the nontransformed cells. Quantitation of 12 of the enhanced phosphoprotein bands in one of the transformed cell lines showed an average of 4.4 times as much phosphorylation as in the normal cells. The enhanced phosphorylation observed in the transformed cell lines was not dependent on the growth rate of the cells or on cyclic AMP. Furthermore, when homogenates of transformed and nontransformed cells were mixed prior to incubation with [gamma-(32)P]ATP, the resultant phosphoprotein patterns resembed those obtained with transformed cells alone. In addition, an evaluation of the time course of protein phosphorylation revealed that the initial reaction rate was greater in the transformed than in the normal cells, although in both cell types the reaction was complete after 1 min. The results suggest that the simian virus 40-transformed human fibroblasts possess an increased ability to phosphorylate proteins rather than that the normal cells possess a diffusible inhibitor. There appear to be many endogenous cellular substrates for this increased activity.


Identification of transferrin receptors on the surface of human cultured cells. We have examined the binding of human transferrin to cultured human choriocarcinoma cell lines and to detergent extracts of such cells. The results indicate the presence of a high-affinity saturable binding site (Ka = 4.25 x 10(8) M-1) that is specific for transferrin. This receptor has also been detected on three other human cell lines of different phenotypic origin, including Wil-2 (splenic lymphocytes of B-cell origin), RPMI-2650 (a quasi-diploid nasopharyngeal carcinoma), and WI-38 (embryonic lung fibroblasts). By using anti-human transferrin antiserum to immunoprecipitate the receptor-transferrin complex from detergent extracts of cells containing saturating levels of transferrin followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single polypeptide of 90,000 daltons has been identified as a subunit of the putative transferrin receptor. The protein shows immunochemical identity and coelectrophoreses in sodium dodecyl sulfate gels with a cell surface glycoprotein subunit, previously identified in placental brush border membrane preparations, on all human cultured cell lines examined. These results suggest that the recent demonstration of transferrin dependence of maximal cell growth in culture [e.g., Hutchings, S.E. & Sato, G.H. (1978) Proc. Natl. Acad. Sci. USA 75, 901-904] is mediated through expression of this glycoprotein receptor.


Induction of erythroid differentiation of murine erythroleukemia cells by nicotinamide and related compounds. Nicotinamide and its analogues were evaluated for their activity as inducers of differentiation of murine erythroleukemia cells in culture. N'-Methylnicotinamide was the most effective of the compounds tested; at its optimal concentration it was more effective than dimethyl sulfoxide. With 8-10 mM N'-methylnicotinamide, almost all the cells contained hemoglobin (benzidine-reactive) after a 60-hr culture. Commitment to differentiate, assayed by transfer of the cells to semisolid medium without inducers, occurred much earlier and was more extensive with N'-methylnicotinamide than that with dimethyl sulfoxide or nicotinamide. Increase in globin mRNA was greater in the cells cultured with N'-methylnicotinamide than in cells cultured with dimethyl sulfoxide or nicotinamide. The relationship between the inducing activities of nicotinamide analogues and their effect on poly(ADP-ribose) polymerase in vitro was studied. All the compounds studied that had strong inhibitory effects on poly(ADP-ribose) polymerase in vitro induced differentiation of erythroleukemia cells in culture. This property is not a prerequisite of inducers; N'-methylnicotinamide did not inhibit the enzyme in vitro.


Polarity of influenza and vesicular stomatitis virus maturation in MDCK cells: lack of a requirement for glycosylation of viral glycoproteins. We have investigated whether glycosylation of membrane glycoproteins is a determinant of the site of maturation of enveloped viruses in Madin-Darby canine kidney (MDCK) cells. In MDCK cell monolayers, vesicular stomatitis virus buds exclusively from the basal or lateral plasma membranes and contains a sialylated glycoprotein, whereas influenza virus buds exclusively from the apical plasma membrane and lacks neuraminic acid. In order to study the possible relationship between glycosylation of viral glycoproteins and the budding site, infected MDCK cells were treated with tunicamycin at a concentration that completely inhibits glycosylation of viral glycoproteins and the site of virus maturation was examined by electron microscopy. When tunicamycin-treated monolayers were compared to controls, the polarity in the maturation sites of both viruses was maintained. These results indicate that glycosylation of viral glycoproteins is not required for the determination of the cellular maturation site of these enveloped viruses.


Microenvironmental influences on the in vivo behavior of neoplastic lymphocytes. A transplantable hamster lymphocytic neoplasma of probable monoclonal derivation, induced by the oncogenic DNA simian virus 40, has been adapted to grow in the allogeneic host either as leukemia (characterized by dissemination and poor prognosis) or as lymphoma (characterized by localization and favorable prognosis) [Diamandopoulos, G. Th. (1978) Proc. Natl. Acad. Sci. USA 75, 2011-2015]. In the present experiments the circumstances under which neoplastic lymphocytes that are transplanted in allogeneic animals retain, lose, or regain the capacity for dissemination or localization are assessed. Results indicate that the in vivo behavior of neoplastic lymphocytes is not a stable, irreversible characteristic that is transmitted to the cell progeny. On the contrary, it can be altered by the origin/tissue microenvironment in which the cells proliferate. It is suggested that, whereas neoplastic cell mutation followed by host selection could be responsible for changes in cell behavior, a more likely explanation is that the proliferating neoplastic lymphocytes acquire reversible nonmutational phenotypic characteristics during their interaction with the host microenvironment, which modify their behavior and, as a result, the prognosis of the neoplastic process.


Chromosome 1 contains the endogenous RAV-0 retrovirus sequences in chicken cells. We have developed a structurally unique probe which can be used to determine the chromosomal location of nonreiterated genes in vertebrate organisms by the method of in situ hybridization. The probe consists of several specific RNA molecules attached by means of poly(A) . poly(BrdUrd) hybrids to 125I-labeled DNA of high molecular weight. The probe can be synthesized with a variety of RNA molecules, giving it versatility for detecting a variety of genes irrespective of gene size, copy frequency, and host genome complexity. Using this probe prepared with retrovirus genomic RNAs, we have physically mapped all three detectable endogenous genomes of Rous-associated virus type 0 (RAV-0) in Spafas gs- chf- (group-specific antigen negative, chicken helper factor negative) chicken fibroblasts to specific sites on chromosome 1. This finding suggests that these multiple nontranscribed RAV-0 genomes evolved through gene duplication of an original RAV-0 genome. The endogenous src gene coding for a 60,000-dalton protein also has been localized to one of the small macrochromosomes, 10, 11, or 12, in both chicken and Japanese quail cells. The results presented here are consistent with and greatly extend previously reported data obtained by using both chromosome fractionation and restriction endonuclease techniques and thus support the soundness of this hybridization approach.


Five TGA "stop" codons occur within the translated sequence of the yeast mitochondrial gene for cytochrome c oxidase subunit II. A mitochondrial mutation that genetically maps in the middle of the gene coding cytochrome c oxidase subunit II has been found to be a single-base-pair deletion. Three independently isolated spontaneous revertants of this mutant have different single-base-pair insertions within 15 nucleotides of the mutation. These findings clearly identify the location of the gene and suggest that the mutation causes a frame-shift. The sequence of about 900 base pairs surrounding the mutation has been determined and found to have several chain termination codons in every possible reading frame. The sequence can, however, be translated in one frame by assuming that the codon TGA does not cause chain termination in yeast mitochondira, as was recently suggested for the human organelle [Barrell, B. G., Bankier, A. T. & Drouin, J. (1979) Nature (London), in press]. If TGA codes for tryptophan residues, as is apparently the case in human mitochondria, a polypeptide can be read from the yeast mtDNA that is identical to bovine cytochrome oxidase subunit II at 37.8% of its residues. Furthermore, the DNA sequences of the frame-shift revertants discussed above predict relative isolectric point differences between the wild-type and various revertant forms of the polypeptide. The detection of these isolectric point differences by two-dimensional electrophoresis of subunit II from the various strains independently confirms the presumed reading frame of the gene. It is concluded that TGA is translated in yeast mitochondria, most probably as tryptophan.


Inhibition of cis-platinum nephrotoxicity by diethyldithiocarbamate rescue in a rat model. The nephrotoxic effects of cis-dichlorodiammineplatinum(II) (NSC-119875) administered to male F344 rats at the median lethal dose (LD50; 7.5 mg/kg) were inhibited by treatment with sodium diethyldithiocarbamate (500 or 750 mg/kg) between 1 and 4 hr after cis-platinum administration. Those animals receiving cis-platinum alone had mean serum blood urea nitrogen levels of 234 mg/dl at the time of maximal toxicity (day 5); kidney sections revealed large areas of degeneration and necrosis. When dithiocarbamate rescue was carried out after cis-platinum treatment, mean blood urea nitrogen levels were in the range 56-95 mg/dl; kidney sections were grossly normal with a barely discernible band of degeneration at the corticomedullary junction. Gastrointestinal toxicity was observed in greater than 95% of the cis-platinum-treated rats but was totally absent in those receiving subsequent rescue treatment. A significant decrease in weight loss was also observed in the dithiocarbamate-rescued rats. Based on the chemistry of platinum-sulfur interactions and the observed time-dependence of the rescue treatment, it is suggested that dithiocarbamate exerts its effects via competitive chelation and removal of platinum coordinated to protein-bound sulfhydryl groups of the kidney tubule cells.


Defect in 3'-phosphoadenosine 5'-phosphosulfate formation in brachymorphic mice. Incorporation of 35SO42- into adenosine 5'-phosphosulfate (APS), 3'-phosphoadenosine 5'-phosphosulfate (PAPS), and chondroitin sulfate was simultaneously assessed with extracts prepared from epiphyseal cartilage of neonatal normal or homozygous brachymorphic mice. Radioactivity measured in APS, PAPS, and chondroitin sulfate of extracts from brachymorphic cartilage was approximately 300%, 9%, and 13% of the normal levels, respectively. Even though more APS accumulated in the mutant cartilage extracts, APS actually synthesized (total 35SO42- incorporated into APS, PAPS, and macromolecular products) was only 17% of that in the normal. However, of the amount synthesized, 90% and 55% of newly synthesized APS were converted to PAPS by cartilage extracts of normal and brachymorphic mice, respectively. Specific assays for ATP sulfurylase (sulfate adenylyltransferase; ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (adenylylsulfate kinase; ATP:adenylylsulfate 3'-phosphotransferase, EC 2.7.1.25) showed that the sulfurylase enzyme activity is reduced to approximately 1/2 and the kinase to approxomately 1/14 in brachymorphic mice. These results suggest that the production of an undersulfated proteoglycan in brachymorphic cartilage results from a defective conversion of APS to PAPS.


Production of choriogonadotropin-like factor by a microorganism. Extracts from an acetone powder preparation of a culture of a microorganism tentatively named Progenitor cryptocides contain choriogonadotropin (CG)-like factor as determined by radioimmunoassay with antiserum to human (h)CG beta subunit COOH-terminal peptide and radioreceptor assay with bovine corpus luteum membranes. Possible interference by proteases in the extracts was excluded. Immunoreactive materials reacting with antisera to hCG beta subunit and hCG beta subunit COOH-terminal peptide were also found in the extracts. No free alpha subunit was detected. The CG-like factor was purified by chromatography on Sephadex G-100, concanavalin A-Sepharose, and DEAE-Sephadex A-50. The factor was adsorbed by concanavalin A-Sepharose, suggesting that it contains mannose and glucose moieties. The factor was eluted at the same position as standard hCG on Sephadex G-100. It dissociated into two bands when subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis; the bands corresponded to the respective standard hCG subunits. The biological activity of the purified factor as determined by the rat uterine weight assay and the ovarian weight assay was equivalent to 380 (95% confidence limits: 320-490) and 880 (780-1020) international units/mg, respectively. It can be concluded from the present results that a microorganism produces a glycoprotein that is biologically active and has physicochemical properties similar to those of hCG.


Human cell lines containing Epstein-Barr virus but distinct from the common B cell lymphoblastoid lines. A group of very similar cell lines was established from peripheral blood or bone marrow of 12 patients with a variety of disorders. The cells in these cell lines were uniform and round in shape. They grew as single-cell suspensions or as aggregates of small numbers of cells in stationary culture. The most striking characteristic of these lines was the lack of cells with surface immunoglobulin or with demonstrable immunoglobulin synthesis. This lack of immunoglobulin synthesis and their special growth characteristics distinguished them from the lymphoblastoid B cell lines previously described. The cells of these unusual cell lines had strong Fc receptors and C3 receptors and expressed Ia antigens. They did not form rosettes with sheep erythrocytes and did not have detectable levels of terminal deoxynucleotidyltransferase. They did not secrete lysozyme and failed to stain for peroxidase. The presence of the Epstein-Barr virus nuclear antigen in the cells indicated the presence of Epstein-Barr viral genome. The possibility that these cells represent some type of precursor cell in the B cell lineage is discussed, but the exact cellular origin remains to be ascertained.


Immunofluorescent localization of angiotensin converting enzyme in epithelioid and giant cells of sarcoidosis granulomas. Angiotensin converting enzyme (ACE) (dipeptidyl carboxypeptidase; peptidyldipeptide hydrolase, EC 3.4.15.1) was localized in epithelioid and giant cells within ACE-rich sarcoidosis granulomas, but not control granulomas, by immunofluorescence by using a rabbit anti-human ACE immunoglobulin for localization and fluorescein-labeled goat anti-rabbit immunoglobulin for detection. ACE was frequently not detected in the very central epithelioid cells, perhaps due to a paucity of cellular or molecular stimulatory interaction in this region. The results suggest that marked elevation of ACE in the granulomas and often in the serum in sarcoidosis may be due to intense induction of ACE in most epithelioid and giant cells of the sarcoidosis granulomas.


Dynorphin-(1-13), an extraordinarily potent opioid peptide. We describe the opioid properties of a tridecapeptide, the sequence of which corresponds to the NH2-terminal sequence of dynorphin, a novel porcine pituitary endorphin. It contains [Leu]enkephalin. In the guinea pig ileum longitudinal muscle preparation it is about 700 times more potent than [Leu]enkephalin. Its effects in this tissue are blocked completely by naloxone, but the apparent affinity of naloxone is 1/13th that for blockade of [Leu]enkephalin or normorphine. In the mouse vas deferens, this peptide is 3 times more potent than [Leu]enkephalin. Well-washed rat brain membranes degrade the peptide rapidly, suggesting the presence of a membrane-bound degradative enzyme. The peptide displays considerable immunoreactivity in assays with antisera that have been used for the immunohistochemical localization of [Leu]enkephalin. The remarkable enhancement of the potency of [Leu]enkephalin by the COOH-terminal extension -Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-OH suggests new interpretations concerning the structure of opiate receptors and the function of the enkephalin pentapeptides.


Comparison of the effects of ouabain and high tissue sodium on the calcium pools involved in excitation-contraction coupling. According to one hypothesis for the mechanism of action of cardiac glycosides, inhibition of cardiac Na,K-ATPase elevates [Na]i and by increasing Na:Ca exchange, increases [Ca]i to ultimately produce the positive inotropic effect (PIE). Therapeutic concentrations of ouabain specifically increase the Ca content of a superficial pool involved in excitation-contraction coupling. Thus, for this hypothesis to be correct, equivalent increases in total Na by means other than Na,K-ATPase inhibition must also specifically increase the Ca content of this pool and produce a PIE. However, unlike the effects produced by ouabain, contracture, an elevation of tissue K and a nonspecific increase in Ca uptake occurred in Na-loaded hearts. Although Na loading and ouabain treatment increased total tissue Na to the same extent, the results suggest that (a) the increased tissue Na may not reflect an equivalent increase in [Na]i; (b) the Na-loading procedure was not a valid model for Na,K-ATPase inhibition, or (c) the hypothesis for cardiac glycoside action is incorrect.


Selective deprivation of paradoxical sleep and consolidation of shuttle-box avoidance. Investigated whether paradoxical sleep is implicated in the storage of information acquired during shuttle-box avoidance. Wistar rats were given 5 brief training sessions distributed over the light period of the diurnal cycle. During the intervals between sessions the animals were selectively deprived of paradoxical sleep by awakening them every time they showed this type of sleep. The onset of paradoxical sleep was identified when hippocampal theta rhythm occurred during behavioural sleep. Yoked control animals got the same treatment irrespective of their sleep-waking behaviour, whereas free sleep rats were allowed to sleep undisturbed. In spite of large differences in the amount of paradoxical sleep during the intersession intervals no differences in learning performances were found among the groups. A tendency toward more intertrial crossings was noted in the paradoxical sleep deprived group at the end of training. It is concluded that storage of information acquired during distributed shuttle-box avoidance is not dependent on the presence of paradoxical sleep immediately following learning. Some possibilities are considered that paradoxical sleep may still be involved in memory storage processes.


REM sleep deprivation increases preference for novelty in rats. As a further test of the hypothesis that REM deprivation decreases fear, the behavior of 40 male rats was measured in a Y-maze adapted to test for preference for novelty. Prior to this test, the animals were exposed for 4 days to one of four treatments, i.e., either a dry environment control, a wet environment control, a 2-day REM deprivation period or a 4-day REM deprivation period. During the test both number of grid crossing in the novel and non-novel arms of the Y-maze and three indices of emotionality were recorded. The results were congruent with data we had reported earlier and offered convincing evidence in support of the hypothesis.


The effect of cortisol on the excitability of the rat muscle fibre membrane and neuromuscular transmission. The present experiments show that cortisol when applied in vitro, exerted two different effects on the electrical excitability of the diaphragm muscle fibre membrane and on the neuromuscular transmission depending on the concentration used. At low concentrations (2.5X10(-6) mol.l-1) it potentiated action potentials, increased resting membrane polarization by 3--4 mV and did not affect neuromuscular transmission. Higher concentrations (10(-2) mol.l-1) suppressed the action potential to a certain extent, depolarized the muscle fibre membrane by 6 mV and reduced the amplitudes of m.e.p.p.s and e.p.p.s as well as those of iontophoretically evoked acetylcholine potentials. It was concluded that the effect of low concentrations of cortisol is primary and is probably due to the enhancement of resting membrane permeability for K+ ions and to the changes in ion channels. Cortisol in high doses increased muscle oxygen consumption, so that its suppressing effect might be due to inhibition of energy metabolism.


The effect of prostaglandin E2 infusion in the fetal lamb on fetal plasma ACTH, prolactin and cortisol concentrations. PGE2 (2 micrograms/min) has been infused for 1h into the fetal jugular vein of 8 chronically catheterized fetuses on 13 occasions from 112 to 138 days gestation. Infusion of ethanol vehicle alone was conducted in fetuses from 111-139 days gestation. PGE2 administration produced a significant increase in fetal plasma cortisol after 30 min. No significant change was observed in fetal plasma prolactin concentration. Fetal plasma ACTH concentration was significantly elevated above resting concentration after 30 min. of PGE2 infusion. Metabolic clearance rate of PGE2 was 860 ml/min or 350 ml/kg/min. Intrauterine pressure was not changes during the infusion at any gestational age.


Unsaturated fatty acid effect on cyclic AMP levels in human embryo lung fibroblasts. The addition of arachidonic acid at 250 muM to cultures of human embryo lung fibroblasts (IMR-90) increases cellular cyclic AMP levels within 5 minutes to approximately 15-fold over basal. Other unsaturated fatty acids, 11, 14, 17-eicosatrienoic, linoleic, 8, 11, 14-eicosatrienoic and oleic also cause similar rapid elevation of cellular cyclic AMP. During this time interval, no detectable conversion of the added linoleic or arachidonic acids to prostaglandin is observed. These cells produce prostaglandins at measurable concentrations in response to treatment with ascorbic acid or bradykinin. Saturated fatty acids have no influence on cyclic AMP levels in these cells. This effect of unsaturated fatty acids on cellular cyclic AMP levels varies with the cell type. For example, smooth muscle and endothelial cells obtained from the calf pulmonary artery show very little or no increase in cellular cyclic AMP upon exposure to arachidonic acid.


PGF2 alpha, thromboxane B2 and HETE levels in gerbil brain cortex after ligation of common carotid arteries and decapitation. The effects of ligation of both common carotid arteries in the gerbil on the levels of PGF2 alpha, TXB2, HETE and of energy metabolites in brain cortex, have been investigated. Also, in the same experimental conditions the changes of cyclic AMP in brain cortex, cerebellum, striatum and hippocampus have been monitored. ATP, glycogen, glucose and phosphocreatine decrease whereas, lactate and cyclic AMP are enhanced in the ischemic brain, as previously reported. In contrast, levels of arachidonic acid metabolites are not modified. During ischemia following decapitation, instead, PGF2 alpha, and TXB2, show considerable increase.


Failure of prostaglandins E1 and E2 to alter canine gastric mucosal cyclic nucleotides. The action of prostaglandins and indomethacin on gastric mucosal cyclic nucleotide concentrations was evaluated in 18 anesthetized mongrel dogs. Prostaglandins E1 (PGE1) and E2 (PGE2) (25 microgram/kg bolus, then 2 micrograms/kg/min) were administered both intravenously (4 experiments; femoral vein) and directly into the gastric mucosal circulation (10 experiments; superior mesenteric artery). The possible synergistic effect of pre-treatment and continuous arterial infusion of indomethacin (5 mg/kg bolus for 5 min, then 5 mg/min), a prostaglandin synthetase inhibitor, with PGE2 was studied in 4 experiments. Antral and fundic mucosa were biopsied and measured by radioimmunoassay for cyclic nucleotides. Doses of PGE1 and PGE2 which inhibited histamine-stimulated canine gastric acid secretion did not significantly alter antral or fundic mucosal cyclic nucleotide concentrations. Concomitant infusion of PGE2 with indomethacin did not potentiate the mucosal nucleotide response compared to PGE2 alone. These studies fail to implicate cyclic nucleotides as mediators of the inhibitory acid response response induced by PGE1 or PGE2 in intact dog stomach.


The effects of synthetic prostaglandin analogs on canine hepatic bile flow. The synthetic prostaglandin analogs 16, 16-dimethyl PGF2 alpha and 16, 16-dimethyl PGE2 were administered to dogs with chronic biliary and gastric fistulas. The effects of 16, 16 diMePGF2 alpha and 16, 16 diMePGE2 were evaluated on bile flow and composition and bile adenosine 3', 5' monophosphate (cyclic AMP) secretion. 16, 16 diMePGF2 alpha in doses of 0.125 and 0.25 microgram-kg-min significantly increased hepatic bile flow. The choleresis was characterized by increased chloride and bicarbonate secretion. Measurement by radioimmunoassay of bile cyclic AMP concentration demonstrated no evident increase in bile cyclic AMP secretion associated with the choleresis produced by 16, 16 diMePGF2 alpha. The administration of 16, 16 diMePGE2 in a dose range 0.01 to 1.0 microgram-kg-min did not significantly alter bile flow rates or composition. Bile erythritol-14C clearance, a measure of canalicular bile flow, was significantly increased by PGF2 alpha but not by 16, 16-dimethyl PGF2 alpha, suggesting that the mechanism of action of PGF2 alpha in stimulating hepatic bile flow may be different from that involved in 16, 16-dimethyl PGF2 alpha choleresis. The results of this study indicate that the synthetic PGF2 alpha analog produces a choleretic response not mediated by adenylate cyclase and associated with increased chloride and bicarbonate secretion.


I - Prostaglandin hyperalgesia, a cAMP/Ca2+ dependent process. Prostaglandins stimulate cAMP increase in several biological systems including CNS. The possible participation of a cAMP/Ca2+ related mechanism in prostaglandin induced hyperalgesia in the rat paw, as measured by a modification of the Randall-Selitto method was investigated. A serie of agents was administered in the paw in an attempt to change either Ca2+ or cyclic AMP concentration at the nociceptive terminations. PGE2, dibutyryl cyclic AMP, isoprenaline, noradrenaline, adrenaline, Ca2+ionophore (A23187), BaCl2 caused a dose dependent hyperalgesia. The hyperalgesic effect of these substances was enhanced by methyl-xanthines. Cyclic GMP as well as agents which interfere with Ca2+ influx (verapamil and lanthanum) were local analgesics in normal and hyperalgesic paws.


II - Prostaglandin hyperalgesia: the peripheral analgesic activity of morphine, enkephalins and opioid antagonists. Morphine, enkephalins, nalorphine, naloxone and pentazocine are shown to have a peripheral analgesic effect. In our modification of the Randall-Selitto test these substances were 50--100 times more potent than a standard local anaesthetic, lidocaine. At this peripheral site, naloxone did not antagonize the effect of morphine. Morphine had a marked analgesic effect on the hyperalgesia induced by PGE2 and PGI2, BaCl2, Ca2+ ionophore A23187, isoprenaline but not on that induced by dibutyryl cyclic AMP. It was suggested that the peripheral analgesic effect of morphine is due to an inhibition of adenylate-cyclase activity.


Evidence for separate PGD2 and PGF2 alpha receptors in the canine mesenteric vascular bed. Potential interactions between PGD2 and PGF2 alpha in the mesenteric and renal vascular beds were investigated in the anesthetized dog. Regional blood flows were measured with electromagnetic flow probes. PGD2, PGF2 alpha and Norepinephrine (NE) were injected as a bolus directly into the appropriate artery, and responses to these agents were obtained before, during and after infusion of either PGD2 or PGF2 alpha into the left ventricle. In each case, the infused prostaglandin caused vascular effects of its own. Left ventricular infusion of PGD2 reduced responses to local injections of PGD2 in the intestine, and a similar effect was observed for PGF2 alpha, suggesting significant receptor or receptor-like interactions for each of the prostanoids. However, systemic infusion of prostaglandin F2 alpha (20--100 ng/kg/min) had no effect on renal or mesenteric vascular responses to local injection of prostaglandin D2. Similarly, PGD2 administration (100 ng/kg/min) did not affect responses to PGF2 alpha in the intestine. The present results therefore suggest that these prostaglandins, i.e., D2 and F2 alpha, act through separate receptors in the mesenteric and renal vascular beds. In addition, increased prostaglandin F2 alpha levels produced by infusion of F2 alpha reduced mesenteric but not renal blood flow, suggesting that redistribution of cardiac output might participate in side effects often observed with clinical use of this prostaglandin, such as nausea and abdominal pain.


Receptor binding in various tissues of PGE2, PGF2 alpha and sulprostone, a novel PGE2-derivative. Sulprostone is a tissue-specific PGE2-derivative with high abortifacient activity in various species including man. The dissociation constant KD of the receptor binding of this compound was compared with PGE2 and PGF2 alpha in various tissue preparations of different species. A structure-binding relationship was developed from competition curves after a logit/log transformation. It is demonstrated that the relative affinities of Sulprostone, PGE2 and PGF2 alpha remain essentially constant in all the tissues investigated. It is concluded that the tissue-specificity of Sulprostone cannot be ascribed to structural differences of the receptor molecule.


(3,4-Dihydroxybenzyl)-2-imidazoline (DHBI): an analogue of dopamine. (3,4-Dihydroxybenzyl)-2-imidazoline (DHBI) was prepared and evaluated for dopamine receptor activity by studying its relaxant effects in the rabbit isolated renal and ear arteries and by measuring its ability to enhance dopamine sensitive adenylate cyclase in the rat striatum. DHBI relaxed both the rabbit isolated renal and ear arteries in the presence of phenoxybenzamine (2. 9 X 10(-5)M) and propranolol (3.4 X 10(-7) M) while dopamine and the specific renal dopamine agonist 6,7-dihydroxy-2-aminotetrahydronaphthalene (6,7-ADTN) demonstrated a ratio of specificity for the renal versus the less responsive ear artery. DHBI did not cause a typical increase in adenylate cyclase activity, however DHBI did attenuate the stimulatory a


Effect of ascorbic acid on prolyl hydroxylase activity, collagen hydroxylation and collagen synthesis in human synovial cells in culture. Synovial cells derived from patients with either rheumatoid arthritis, or simple joint-trauma were grown in tissue culture. The rheumatoid osteoarthritic and non-arthritic synovial cells in cultured all had similar levels of prolyl hydroxylase activity. Following a 3 hour incubation with ascorbate (10(-4)M), prolyl hydroxylase activity was elevated to a similar extent in all synovial cell cultures examined. The activation of prolyl hydroxylase by ascorbate (10(-4)M) was accompanied by increased radioactive hydroxyproline formation and secretion into the media. Increased amounts of collagenase degradable radioactive protein were also secreted into the media, but no changes in total collagen synthesis (media plus cell layer) were observed as a result of ascorbate supplementation using this assay system.


Potentiation of ethyl para-nitrophenyl phenyl-phosphonothioate (EPN)-induced inhibition of liver microsomal carboxylesterase by NAD in vitro in rats. The higher inhibition of liver microsomal carboxylesterase (CEase) by EPN, as compared to that of acetylcholinesterase (AchE) may be, at least in part, explained by the present findings that NAD potentiated the anti-CEase, but not anti-AchE, action of EPN. This phenomenon was referred to as "NAD-effect" in this paper. NAD-effect was not due to the increased formation of oxygen analog of EPN (EPN=O) by NAD addition through liver microsomal cytochrome P-450 catalyzed monoxygenase, because the amounts of EPN=O formed during incubation in the presence and absence of NAD were not significantly changed as shown by gaschromatography-mass spectrometric estimations. In addition, HAD-effect could be observed in the experiments even under carbon monoxide atmosphere. Such NAD-effect was observed only when NAD, EPN and an unidentified component bound to liver microsomal membrane were co-existent in the incubation mixture.


[Sleep polygraphy with recording of intracranial pressure and measure of cerebral blood volume in hydrocephalic infants (author's transl)]. This study was carried with the intention of explaining the causes of modifications in intracranial pressure (ICP) during paradoxical sleep (PS) in normal and hydrocephalic infants, and establishing relationships between these modifications and cerebral blood volume (CBV). All tests (conventional sleep polygraphy, ICP measured by a transducer on the fontanel, CBV measured by isotopic labelling of red blood cells in vivo) were carried out without use of surgical procedures. During paradoxical sleep there was a sustained wave of increased intracranial pressure lasting from 10 to 20 minutes, as well as phase-type variations lasting no longer than 1 minute. Increased ICP was also observed in the normal subjects. Recordings show that there is well-defined correlation between the sustained wave of high intracranial pressure and an increase in CBV.


[Experimental hepatic encephalopathy. Central nervous system excitability in rats following portacaval anastomosis]. The excitability of reticulo-thalamo-cortical and reticulo-cortical system (RtCS) was studied by electrical stimulation of reticular formation during waking, slow wave sleep and paradoxical sleep during five weeks in control rats and in rats with portocaval shunt. In control rats, no modification of the excitability of RtCS was observed during the experimentation. In rats with portocaval shunt and from the fourteenth post-operative day, the excitability of RtCS was decreased during waking. All along the post-operative period, the excitability of RtCS was increased during slow wave sleep. No modification of the excitability of RtCS was observed postoperatively during paradoxical sleep. These modifications of the excitability of RtCS during waking and slow wave sleep could be related to quantitative variation of the waking-sleep cycle that have been described in rat with portocaval shunt. Their responsability in the disorganization of the waking-sleep states during the experimental hepatic encephalopathy may be hypothetized.


Effect of chronic ethanol administration on serum high density lipoprotein cholesterol in rat. In rats chronic ethanol administration was found in fasting state not to induce hypertriglyceridemia but moderate hypercholesterolemia through an increased level of high density lipoprotein cholesterol. In the liver, cholesterol contents slightly and triglyceride centents significantly increased. The result suggests that cholesterol accumulation in the liver leads to an increased level of high density lipoprotein cholesterol.


Introduction and multiplication of bovine Babesia in human cells. Bovine erythrocytes parasitised with Babesia major were fused with human HeLa cells by means of Sendai virus. B major parasites entered the HeLa cell cytoplasm and in some cases underwent multiplication over a period of three days.


Measurement of immunosuppression in chickens caused by infectious bursal disease vaccines using Brucella abortus strain 19. The serological response of chicks to Brucella abortus strain 19 was monitored over a period of seven weeks to assess the degree of immunosuppression caused by vaccination at one day of age with two infectious bursal disease vaccines. One of the vacy. This vaccine caused severe immunosuppression judged by the minimal serological response following B abortus inoculation. The test also detected a significant delay caused by the other vaccine in the development of the serological response but the maximum titre was not significantly different from that in chicks which had received no infectious bursal disease vaccine.


Distributions of VA/Q in dog lungs obtained with the 50 compartment and the log normal approach. From the lung wash-out ratios of six inert gases that were intravenously administered to 26 anaesthetized normal dogs, 120 distributions of ventilation--perfusion ratios (VA/Q) were determined, using two different techniques: the 50 compartment approach developed by Wagner et al. (1974a) and a least squares curve fitting method, comparing observed and calculated inert gas data corresponding to a single log normal distribution. The latter is defined by three parameters, the (geometric) mean VA/Q, the standard deviation of log VA/Q and a shunt fraction. A unimodal log normal fit was almost always obtained, but in 14 cases the 50 compartment approach yielded a bimodal distribution, which gave a better fit to the data in 7 cases. In most other cases the log normal distribution fitted better; the mean VA/Q's were about equal, but the standard deviations obtained from the log normal fit were often much less. It appears that, whereas in the log normal approach distributions of any sharpness can be fitted, the computer technique used in the 50 compartment approach, involving a smoothing algorithm, shortens and broadens any sharp and narrow peak in them. The effect of different smoothing factors in the 50 compartment approach was investigated. The PaO2 predicted from either distribution was about the same and somewhat less than the actual PaO2, so that one model is not superior to the other in describing oxygen transfer in the lung.


Voluntary changes in ventilation distribution in the lateral posture. To determine whether voluntary changes in the pattern of inspiratory muscle contraction influence topographical distribution of ventilation in the lateral decubitus posture during tidal breathing, we studied 4 normal subjects who breathed either naturally (N) or preferentially with intercostal and accessory muscles (IC), or with enhanced motion of the diaphragm and abdomen (Ab). We performed N2 as well as 133Xe washouts (after equilibration) which were measured at the mouth while recording regional count rates by external scintillation detectors. Ventilation per unit volume (delta V/Vo) in the nondependent lung regions was 0.55 +/- 0.05 (mean +/- 1 SD) and 0.42 +/- 0.02 of that in the dependent regions during natural and sustained Ab breathing, respectively. In contrast, during IC breathing this ratio was 0.99 +/- 0.17. Although N2 washout curves obtained during IC breathing more closely approached a monoexponential than did those from N and Ab runs, a two compartment analysis of washouts at the mouth did not demonstrate significant differences between breathing patterns. We conclude that in the lateral posture voluntary relaxation of the diaphragm during tidal breathing distributes the gas preferentially to the nondependent lung regions. Conversely, during N and Ab breathing the preferential ventilation of dependent regions is due to contraction of the diaphragm.


Effect of SiO2-liberated macrophage factor on protein synthesis in connective tissue in vitro. 1. To elucidate the role of macrophages in fibrosis the effect of the SiO2-liberated macrophage factor was assessed on protein synthesis in granulation-tissue slices. SiO2 could be replaced by chrysotile asbestos. The active factor is found in the 45/55% sucrose interface of macrophage homogenate. 2. There is no evidence of the involvement of collagenase. 3. The SiO2 effect is not influenced by the addition of yeast RNA to the incubation medium. 4. At a small concentration of polyvinylpyridine-N-oxide (PVNO) protein synthesis in the granulation tissue slices is stimulated, but at higher concentrations PVNO prevented the liberation of the fibrogenic factor from macrophages by SiO2. 5. Phospholipase C and trypsin inhibited the effect of SiO2, which was partially abolished also by heating, and by repeated freezing and thawing. 6. The macrophage RNAse, its inhibitors and fibroblast mRNA are suggested as key factors in the development of fibrosis.


Separation of two serum very low density lipoprotein fractions using starch block electrophoresis. A quantitative electrophoretic method has been developed in order to differentiate very low density (VLDL) pre-beta lipoproteins from late pre-beta lipoproteins using starch as a supporting medium. It was possible to obtain a bimodal distribution of lipoprotein lipids from VLDL which on agarose gel electrophoresis had a pre-beta band and a late pre-beta band. Optimal conditions were: ammonium carbonate buffer, mu = 0.025, dialysis prior to electrophoresis. Agarose gel electrophoresis demonstrated that the fast and slow components obtained on starch block electrophoresis corresponded to the pre-beta and late pre-beta band respectively. With increasing migration towards the anode the ratio of cholesterol to triglycerides decreased continuously. It is suggested that the fast triglyceride rich component represent newly secreted VLDL species and the slower component mainly postlipolytic particles. The pre-beta band on agarose gel electrophoresis might represent more newly secreted VLDL than the late pre-beta band. However, it cannot be excluded that part of late pre-beta lipoproteins may be secreted de novo.


Cyclic AMP, renal function and dihydralazine-stimulated renin secretion in hypertensive patients. The concentrations of plasma cAMP and plasma renin activity were determined in arterial and renal venous plasma in nineteen patients investigated for renin-mediated hypertension. The cAMP measurements were performed in two different situations (1) under basal conditions and (2) after i.v. dihydralazine administration, a potent renin stimulation procedure. Thirteen patients had a lateralization of the renin secretion in the basal state and the administration of dihydralazine caused a further marked renin-secretion. The cAMP concentration was higher in the renal veins draining renin-positive kidneys than in the contralateral renal veins. No significant change was observed between the arterial cAMP concentration and the cAMP concentration in either of the renal veins during dihydralazine-stimulated renin secretion. There was no correlation between the cAMP extraction and the renin secretion of the individual kidneys, but the cAMP extraction correlated with the extraction ratio of PAH. These results show that cAMP values are mainly influenced by the renal function and are not related to the state of renin secretion. Increased cAMP levels in renovascular patients and urameic patients are therefore mainly due to defective elimination of the nucleotide by the kidneys.


Injury to human endothelial cells in culture induced by low density lipoproteins. Low density lipoproteins (LDL) have been shown to injure culture endothelial cells derived from the human umbilical cord. During a 48 h incubation period LDL significantly increased 51Cr release from prelabelled cells and induced marked cellular injury if the ratio between the LDL cholesterol and the infranatant proteins was kept above 0.1-0.12 mmol/g protein. Actually, an injurious effect of a fixed concentration of LDL could be completely prevented by increasing the concentration of infranatant proteins. High density lipoproteins within physiological concentration ranges had no effect when tested in the presence of infranatant proteins. The effects of LDL were not cell specific because normal as well as LDL receptor negative human skin fibroblasts were injured by LDL.


Injury to cultured endothelial cells induced by low density lipoproteins: protection by high density lipoproteins. Cultured human endothelial cells derived from umbilical cord veins were injured when exposed to low density lipoproteins (LDL). Addition of high density lipoproteins (HDL), together with LDL, inhibited the cellular injury induced by LDL as demonstrated by lowered 51Cr release and prevention of morphological changes. Serum albumin had a similar, but far weaker effect. Preincubation of the cells with HDL did not reduce injury inflicted during a subsequent incubation with LDL, while preincubation with LDL aggravated later damage. The protective effect of HDL could be overcome by increasing the DLD concentration.


Diseases of the human breast: selective isolation and exposure of epithelia and their correlative surface features and histopathology. Tissues from seventy-one patients were obtained within one-half hour of biopsy, mastectomy, or reduction mammoplasty and processed for scanning electron microscopy (SEM). Epithelial structures were detected by supravital staining with methylene blue and isolated by microdissection. Mammary lobules and ductules required bacterial collagenase digestion to remove the covering stroma for optimal visualization. Major histopathological categories were compared and correlated with the characteristics observed with methylene blue supravital staining and light microscopy and/or transmission electron microscopy as a function of surface features revealed by SEM. Mammary ducts can be readily distinguished from ductules and lobules by their characteristic surface epithelium. Duct epithelium undergoes a variety of changes in fibrocystic disease (FCD) and in carcinoma. Both apocrine and merocrine secretory activity was observed in the surface epithelium. Proliferation of both epithelial and stromal elements was observed in benign fibroadenomata. Intraductal carcinomas were distinguished by their relative lack of surface microvilli. SEM offers a practical tool in the potential identification of premalignant cells of the human breast epithelium.


Prospective on cell surface labeling using the hapten-sandwich method: an integrated approach. Specific molecular units on the surface membranes of cells were tagged with markers visible in fluorescence, transmission and scanning electron microscopy. The Hapten-sandwich method does this by the sequential application of hapten-modified anti-cell-surface antibody, anti-hapten antibody and a hapten-modified marker. Details are given for the preparation and testing of the various reagents used in this method. The specificity and efficiency of Hapten-sandwich coupling of hemocyanin to mammary tumor virus (MTV) budding from breast tumor cells was demonstrated by quantitative transmission electron microscopy. Con A and anti-saccharide antibodies were used to demonstrate the presence of various sugars on the surfaces of the mammary tumor cells using fluorescence, transmission and scannings electron microscopy. Some samples were doubly labeled for the simultaneous presence of two different sugars. Light and electron microscopy were used to show that several hormones (insulin(I), hydrocortisone(F), and prolactin) greatly affect the expression of tumor virus and the kind and density of cell surface sugars on mammary tumor cells. Individual cells produced virus and expressed surface sugars independently. A micro-scanning densitometer was used to quantitate the results from fluorescence microscopy. Future advances in cell labeling including other applications of the Hapten-sandwich method, and problems such as the quantitation of electron microscopy labeling are discussed.


Electron probe studies of Na+ - K+-ATPase. Use of appropriate reaction conditions allows direct quantitative measurements of the activity of Na-K-ATPase to be made with the electron probe microanalyzer. In addition, the demonstration by microanalysis of the linearity of the complete cytochemical reaction sequence is the first detailed analysis of the elemental sequence in a histochemical method and provides the basis for a visual semi-quantitative analysis of enzyme activity in tissue sections. This technique indicates the potential of electron probe microanalysis as a basic tool in enzymology.


Establishment of anti-TNP antibody-producing human lymphoid lines by preselection for hapten binding followed by EBV transformation. Human lymphoblastoid cell lines that produce specific antibody against the hapten trinitrophenyl (TNP) have been established by selecting TNP-binding human B lymphocytes by TNP-rosetting and Ficoll-Isopaque separation, followed by Epstein-Barr virus (EBV) immortalization. Derived lines secreted polyclonal anti-TNP antibodies and contained relatively small numbers of specific rosette- and plaque-forming cells against TNP-RBC. Following rerosetting with TNP-RBC, the frequency of rosette-forming cells increased from 2% to 75%. In parallel, the frequency of plaque-forming cells increased from 0.4% to 30%. The antibody titres in the supernatants increased from 64 to 512 and from 48 to 192, as measured by TNP agglutination and haemolytic assays, respectively. The antibodies were 19S, IgM. The specificity of the anti-TNP antibody was confirmed by the hapten inhibition test, in comparison and cross-reactivity tests with the supernatant of the previously established, EBV-transformed anti-4-hydroxy-3,5-dinitrophenacetic acid (NNP) antibody-producing cell line. Both antibodies were specific: the homologous hapten inhibited them but the heterologous hapten did not.


Rat memory T lymphocytes. II. Differences in macrophage-dependent activation shown by Actinomyces viscosus antigens and by mitogens, using silica in vitro. Protein-coated silica, a macrophage toxin, was used to assess the requirement for accessory cells in the induction of an in vitro proliferative response to (i) antigens from Actinomyces viscosus and (ii) the mitogens conconavalin A (Con A) and phytohaemagglutinin (PHA). T cells were obtained from RIC-Sprague-Dawley rats primed in vivo with A. viscosus Nyl by splenectomy and filtering the spleen cell suspensions through Degalan Ig-anti-IgG columns. In the presence of 100 microgram silica/ml during 4 days of culture, the proliferative response of T lymphocytes was not diminished. In contrast, when the T cells were precultured with silica for 24 h, washed, and subsequently cultured with the antigen fractions, antigen-induced proliferation was abolished. This procedure, however, had no influence on mitogen-induced proliferation was abolished. This procedure, however, had no influence on mitogen-induced T-cell activation. It is therefore concluded that the antigen-dependent anamnestic in vitro response (but not activation by mitogens) of rat T lymphocytes needs help from silica-sensitive macrophages.


[Effect of menopausal syndromes on symptoms of denture intolerance]. 19 female patients suffering from incompatibility to their dentures with a suspected lack of estrogen were examined gynecologically, endocrinologically and cytologically. Substitution with conjugated estrogen caused in 14 patients improvement of general condition, in 12 persons reduction of symptoms in the oral cavity. Endocrinologically and cytologically no clear differences before and after treatment could be observed.


[Clinical aspect and actual frequency of bilateral squamous cell bronchial carcinoma with simultaneous revelation (author's transl)]. Clinical and physiopathological data about 10 cases of bilateral squamous cell bronchial carcinoma with simultaneous revelation, are reviewed. This clinical form is not rare, usually found by a systematic bilateral endoscopy. In these 10 cases, the clinical and radiological data did not allow to predict the diagnosis before the endoscopy. Physio-pathological mechanisms are not well established. Various hypotheses are propounded : air-way, blood or lymphatic metastases, simultaneous tumoral blossoming, superficial propagation, genetic factors intervention. For these 10 cases, the authors develop arguments for one or another of these mechanisms.


[Unusual tumours of the digestive tract (author's transl)]. The authors summarize the difficulties in diagnosis some tumour-like lesions or digestive tumours. They rely upon thirty years experience in digestive pathology and upon a survey of relevant literature. They recall the difficulties arising from the inflammatory tumour-like lesions (infiltrating sclerosis or granulomas, colitis cystica profunda, inflammatory polyps). They mention the circumstances which bring about errors or incomplete diagnosis among these very tumours. Some difficulties are due to the circumstances in which these tumours appear : the digestive cancers of the child, the assocations of some family cancers (the ill-forming tumours and the dysembryoplasic cancers, and lastly the connections that exist between some inflammatory or dystrophic diseases and tumours. Other difficulties depend upon the topography or the tumorous structure. Paneth-cell carcinomas, goblet cell carcinoid are chosen as instances among epithelial tumours. In the group of non-epithelial tumours, digestive localizations of angiomatosis and of lymphomatosis may cause errors. Likewise this can be applied to some secondary digestive tumours. Regarding these difficulties, the authors stress the necessity of a close cooperation between gastro-enterologists, surgeons and pathologists. They also stress the strict observance of techniques in fixation and preparation of specimens and biopsies, and also the necessity of completing, if need be, the habitual microscopy through more elaborate explorations (histochemistry, electron microscopy...).


[Transient osteoporosis of the hip in pregnancy. Report of three cases (author's transl)]. Three cases of transient osteoporosis of the hip in pregnant women are reported, and compared with 13 similar observations, published in world literature. The disease occurs during second half of pregnancy, revealed byrnotion hip pain, and slight limitation of mobility. On radiography, the femoral head and acetabulum are radiolucent and heterogenous. These modifications may be impressive but careful analysis of X rays rules out coxitis or malignant osteolysis. Major functional disturbance is common but the course of pregnancy and delivery is not impaired. Healing, without sequelae, occurs in a few months. Before delivery, attention should be paid to the fetal risk when prescribing X rays examinations or drug treatment. Pathogeny is unclear; the role of nerve or vein compression by the pregnant womb is discussed.


[Severe forms of imported malaria concerning three pernicious attacks and a black water fever (hemoglobinuria) (author's transl)]. Four severe cases of imported malaria are reported here. Three of them are pernicious and the fourth is a black water fever (hemoglobinuria). The difficulties of initial diagnosis are exposed. There was no mortality in these four cases which represent the annual recruiting of a multipurpose intensive care unit. But the complications are severe in contradiction with easy effective chemoprophylaxis. The principles of treatment and prophylaxis are resumed in the light of these four observations.


[Gammacisternography and fronto-basal head injuries (author's transl)]. The authors give the results of 65 radioisotopic cisternographies for suspected rhinorrheas in frontal basal head injuries. Indium 111-DTPA lombar injection allows the investigation at least for 48 hours. After localisating the CSF fistulae, the gammacisternography permits us to study associated CSF circulation anomalies. Results and discussion give the correlation between positive tests and surgical findings. The best successful conditions of exploration are enough activity and a fluent clinical leakage of CSF. Nasal coton pledgets improve the success of the exploration.


[Clinical and bacteriological evaluation of sisomicin in sixteen cases of severe bronchopulmonary infection (author's transl)]. Sisomicin, an aminoglycoside antibiotic, was used as sole bactericidal therapy in sixteen cases of severe bronchopulmonary infection for an average of 11,5 days (range 7 to 14) with a dose of 3,5 mg/kg per day in three intramuscular injections. Fifteen of these hospitalized patients presented with chronic airway obstruction which resulted in lowered O2 saturation (SaO2 congruent to 86,1%, range 74 to 93) and, in twelve patients, hypercapnia (PaCO2 = 55,7 torr, range 33--73). Two of these patients were under continuous assisted ventilation. Two patients had a lower lobe infiltrate and a lung abscess respectively. Proteus (n = 3), Pseudomonas (n = 9), K. pneumoniae (n = 2), E. Coli (n = 1), Enterobacter (n = 1), S. aureus (n = 1) and D. pneumoniae (n = 1) were the dominant organisms in the bronchial secretions isolated by Mulder's method; P. aeruginosa was associated with K. pneumoniae, P. rettgeri and E. coli respectively in three cases. In thirteen cases, either a clinical cure (n = 2) or a definite improvement was observed (sputum volume and purulence, auscultatory signs, temperature). However, among these patients were three cases of superinfection/colonization and two cases of persistent organisms. In addition, in two of the three cases showing no improvement colonization occured. The favorable results of this study appear to be due to vigorous antibiotic therapy combined with intensive adjunct therapy. No adverse local or systemic reactions clearly attributable to the drug were observed.


[Congenital coronary artery malformations and associated clinical syndromes (author's transl)]. In 34 cases, corresponding to 3,7% of all patients underwent coronary arteriography for evaluation of anginal syndrome in our laboratory, it was observed a congenital coronary anomaly on their angiograms. The purpose of this paper is to verify the frequency of coronary anomalies and to evaluate relations existing between these anomalies and anginal syndrome. Coronary arteriograms, in 22 cases (2,44%), revealed exclusively; the presence of a coronary anomaly without occlusive coronary disease, which might per se justify angina. They were distributed as follows : 5 with coronary fistula, 2 with coronary aneurysms, 2 cases with single ostium and finally, 13 subjects with hypoplasia of one of the three major coronary arteries. One infant, 14 months old, had a hyperplasic left discending artery (LDA) draining in right ventricle cavity. Her ECG revealed signs of right ventricle overload. All the other cases had a typical angina syndrome with positive stress test. Left cineventriculography demonstrated left ventricle asynergy in 16 patients. We concluded that typical angina syndrome in our 22 patients, may be attributable to coronary anomalies observed at their coronary arteriograms.


[Chronic neurogenic hypernatremia with arhinencephaly (author's transl)]. A case of chronic neurogenic hypernatremia with arhinencephaly has been studied. The clinical and biological picture of neurogenic hypernatremia is reviewed. Hormonal studies have been performed, to try to clear up the physiopathology of this syndrom.


[Colonic Crohn's disease and oral contraceptive (author's transl)]. One colonic Crohn's disease case is reported in a young woman under pill. Stopping it, recovery was observed. Oral contraceptive responsability is pointed out.


[Muscular metastases. A case report (author's transl)]. Muscular metastases are exceptionally reported. The authors present a case of crural neuralgia in relation with a localisation in the psoas iliacus of a gastric carcinoma. A review of literature defines the rarity of this facts (156 cases) the etiology (carcinome more often) and the explication who is still obscur.


[Double spontaneous viscero-renal fistula. A report of one case (author's transl)]. The authors report one case of double spontaneous viscerorenal fistula on the right kidney. After consultation of the world literature, they expose the main character of this lesion. They insist on the transparietal pyelography in the diagnosis and on the surgical treatment.


[Anterior perinephritic phlegmon (author's transl)]. A case is reported of an unsuspected anterior perinephritic phlegmon which had caused peritonitis of pyelonephritic origin thought to be due to a cholecystitis. The lesion was discovered during laparotomy and intravenous urography at operation demonstrated the condition of the underlying kidney and determined the therapeutic approach and the prognosis.