Granular cell myoblastoma of the biliary tree. A granular cell myoblastoma of the common bile duct is described which caused episodes of jaundice in a young woman. Histological examination demonstrated a close association of the tumour with nerve fibres, tending to support the theory of origin from Schwann cells. The lesion is benign and after excision carries an excellent prognosis.


Cellular transformation and differentiation. Effect of Rous sarcoma virus transformation on sulfated proteoglycan synthesis by chicken chondrocytes. Incorporation of sulfate into sulfated proteoglycans by isolated chicken chondrocytes was inhibited up to 74% by transformation with the Rous sarcoma virus, and a similar inhibitory effect was observed on acetate incorporation into chondroitin sulfate. Slower sedimenting sulfated proteoglycans appear after the viral transformation. The ratio of chondroitin 4-sulfate to chondroitin 6-sulfate in these slower sedimenting sulfated proteoglycans was different from that of normal chondrocytes, but the chain lengths of sulfated glycosaminoglycans produced by normal chondrocytes and transformed chondrocytes were not significantly different. Chondrocytes were also infected with a temperature-sensitive mutant of RSV, ts LA24, which has a temperature-sensitive lesion in the transforming gene. Hyaluronic acid production by these cells was increased, and the slower sedimenting sulfated proteoglycan was produced only at the permissive temperature.


Location of protein(s) involved in oligomycin-induced inhibition of mitochondrial adenosinetriphosphatase near the outer surface of the inner membrane. Mitoplasts, that is, mitochondria freed from their outer membranes, were prepared from pig heart. Sonication induced an inversion of these mitoplasts, giving inside-out vesicles. Added cytochrome c can be bound much better to mitoplasts than to sonicated vesicles; addition of trypsin increased adenosinetriphosphatase (ATPase) (ATP phosphohydrolase; EC 3.6.1.3) activity of sonicated vesicles without significantly affecting that of the mitoplasts. Since the site of fixation of cytochrome c was located on the outer side of the inner mitochondrial membrane and since the protein inhibitor of the mitochondrial ATPase is present on the inner face of the inner membrane and is very sensitive to trypsin, it can be concluded that mitoplasts are mainly oriented as normal mitochondria while sonicated vesicles are mainly inverted. Trypsin treatment can abolish the oligomycin sensitivity of ATPase activity of either mitoplasts or sonicated vesicles. However, trypsin induced the solubilization of the soluble F(1)-ATPase of sonicated vesicles while the ATPase activity remained with the mitoplasts after trypsin action. Therefore, trypsin destroyed the oligomycin effect by rupturing the liaison between F(1) and the membrane in sonicated vesicles. On the other hand, the effect of trypsin on mitoplasts must be attributed to the hydrolysis of a protein located near the outer surface of the inner membrane that is at least structurally involved in the oligomycin sensitivity of the ATPase complex.


[Mechanism of stimulation of protein synthesis in the thyroid gland with cyclic nucleotides]. A study was made of cyclic guanosine-3+, 5+-monophosphate (cGMP) on protein synthesis in the cell-free system of the thyroid gland. Preincubation of S30 fraction with cGMP led to increase of labeled amino acid incorporation into proteins (by 56% with the cGMP concentration of 1.73 X 10(-5) M, and by 160% with the concentration of 2 X 10(-4)). No stimulating effect was observed on addition of cGMP directly into the incubation medium. The results obtained suggested that protein synthesis was stimulated under the effect of cGMP at the translation level. The same protein fractions [27S, 19S, 12S, 3-8S (I) and 3-8S (II)] were revealed by disc-electrophoresis of soluble proteins synthesized in the cell-free system in which polyribosomes preincubated with cAMP (1.73 X 10(-5) M) were used as in the system devoid of cAMP. Specific radioactivity of thyroglobulin subunits corresponded in experiments with cAMP to that in experiments without cAMP. At the same time specific radioactivity of 19S thyroglobulin was more than doubled. It can be supposed that thyroglobulin subunit synthesis was accelerated under the effect of cAMP, and, as a result, labeled thyroglobulin increased in the system. The data obtained confirmed a hypothesis put forward earlier that cAMP had a direct influence upon the translation process.


Elimination of size and charge heterogeneities of human leukocyte interferons by chemical cleavage. Human leukocyte interferon (HuLeIF) preparations contain distinct molecular forms of interferon exhibiting significant heterogeneties in sizes when analyzed by electrophoresis in sodium dodecyl sulfate (NaDodSO(4))/polyacrylamide gels, migrating in two broad bands of activity with peaks at about 21,000 and 15,000 daltons. HuLeIFs exhibit extensive charge heterogeneities when analyzed by isoelectric focusing, resolving into several major peaks of approximately equal activity distributed from pH 5.7 to 7.0. When HuLeIF preparations are treated with 0.01 M sodium periodate buffer, pH 4.5, at 4 degrees , both the size and charge heterogeneities rapidly disappear: periodate-treated HuLeIF migrates as a single, narrow band at 15,000 daltons in NaDodSO(4)/polyacrylamide gels and focuses as a single, narrow band at pH 5.7. Quantitative considerations suggest that either the larger, heterogeneously charged HuLeIFs are converted to the smaller, size- and charge-homogeneous interferon by extensive chemical deglycosylation, or, alternatively, the smaller, 15,000 dalton, pH 5.7 interferon is much more stable to periodate treatment than are the other interferon forms. However, the activity of each of the variously charged forms of HuLeIF isolated from focusing gels exhibited the same stability as the pH 5.7 component; similarly, the activity of each of the size-forms of HuLeIF isolated from NaDodSO(4)/polyacrylamide gels exhibited the same stability as the 15,000-dalton interferon.


Acyl group and electron pair relay system: a network of interacting lipoyl moieties in the pyruvate and alpha-ketoglutarate dehydrogenase complexes from Escherichia coli. The dihydrolipoyl transacetylase component of the Escherichia coli pyruvate dehydrogenase complex [pyruvate:lipoate oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1] bears two sites on each of its 24 polypeptide chains that undergo reductive acetylation by [2-(14)C]pyruvate and thiamin pyrophosphate, acetylation by [1-(14)C]acetyl-CoA in the presence of DPNH, and reaction with N-ethyl[2,3-(14)C]maleimide in the presence of pyruvate and thiamin pyrophosphate. The data strongly imply that these sites are covalently bound lipoyl moieties. The results of similar experiments with the E. coli alpha-ketoglutarate dehydrogenase complex [2-oxoglutarate:lipoate oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2] indicate that its dihydrolipoyl transsuccinylase component bears only one lipoyl moiety on each of its 24 chains. Charging of the 48 acetyl acceptor sites on the transacetylase or the 24 succinyl acceptor sites on the transsuccinylase by pyruvate or alpha-ketoglutarate, respectively, and thiamin pyrophosphate was observed in the presence of only a few functionally active pyruvate dehydrogenase or alpha-ketoglutarate dehydrogenase chains. Extensive crosslinking of the transacetylase chains was observed when the pyruvate dehydrogenase complex was treated with pyruvate and thiamin pyrophosphate or with DPNH in the presence of N,N'-o- or N,N'-p-phenylenedimaleimide, respectively. When the alpha-ketoglutarate dehydrogenase complex was treated with DPNH in the presence of N,N'-p-phenylenedimaleimide, only transsuccinylase monomers and crosslinked transsuccinylase dimers were detected. It appears that the 48 lipoyl moieties in the transacetylase and the 24 lipoyl moieties in the transsuccinylase comprise an interacting network that functions as an acyl group and electron pair relay system through thiol-disulfide and acyl-transfer reactions among all of the lipoyl moieties.


A light-activated GTPase in vertebrate photoreceptors: regulation of light-activated cyclic GMP phosphodiesterase. We have been studying the mechanism by which light and nucleoside triphosphates activate the discmembrane phosphodiesterase (oligonucleate 5'-nucleotidohydrolase; EC 3.1.4.1) in frog rod outer segments. GTP is orders of magnitude more effective than ATP as a cofactor in the light-dependent activation step. GTP and the analogue guanylyl-imidodiphosphate function equally as allosteric activators of photoreceptor phosphodiesterase rather than participating in the formation of a phosphorylated activator. Moreover, we have found a light-activated (5-fold) GTPase which participates in the modulation of photoreceptor phosphodiesterase. This GTPase activity appears necessary for the reversal of phosphodiesterase activation in vitro and may play a critical role in the in vivo regulation of light-sensitive phosphodiesterase. The K(m) for GTP in the light-activated GTPase reaction is <1 muM. The light sensitivity of this GTPase (number of photons required for half-maximal activation) is identical to that of light-activated phosphodiesterase. The GTPase action spectrum corresponds to the absorption spectrum of rhodopsin. There is, in addition, a light-insensitive GTPase activity with a K(m) for GTP of 90 muM. At GTP concentrations above 5 muM, there is no appreciable activation of GTPase activity by light. The substrate K(m) values for guanylate cyclase, light-activated GTPase, and light-activated phosphodiesterase order an enzyme array that might permit light to simultaneously cause the hydrolysis of both the substrate and product of guanylate cyclase. These findings reveal yet another facet of light regulation of photoreceptor/cyclic GMP levels and also provide a striking analogy to the GTP regulation of nonphotoreceptor, hormone-sensitive adenylate cyclase.


Semisynthetic cytochrome c.

Semisynthetic cytochrome c. Horse heart cytochrome c can be split with cyanogen bromide into a heme peptide (residues 1-65) and a nonheme peptide (residues 66-104). In a process involving (i) complex formation between the two fragments and (ii) restoration of the severed peptide linkage, a fully active cytochrome c preparation can be re-formed. Use has been made of this process to couple the heme peptide to peptide 66-104 synthesized by the Merrifield solid-phase procedure. The semisynthetic product formed in this manner is indistinguishable from reconstituted cytochrome c prepared with nonsynthetic peptide 66-104.


Studies on the mechanism of phosphorylation of synthetic polypeptides by a calf thymus cyclic AMP-dependent protein kinase. Synthetic polypeptides were employed as substrates in kinetic analyses of the reaction mechanism for the catalytic subunit of a cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from calf thymus. This enzyme preparation was shown to catalyze the transfer of phosphate from ATP to histone H1 from calf thymus, as well as to two synthetic polypeptides, Arg-Lys-Ala-Ser-Gly-Pro (H1-6) and Arg-Arg-Lys-Ala-Ser-Gly-Pro (H1-7), corresponding to the amino acid sequence about serine-38 in calf H1. A related, basic heptapeptide corresponding to a sequence from pig liver pyruvate kinase, Leu-Arg-Arg-Ala-Ser-Leu-Gly (K), was also a substrate. The stoichiometry of peptide phosphorylation was established in each case as the transfer of 1 mol of phosphate from the gamma position of MgATP to the serine hydroxyl of 1 mol of the peptide. Steady-state, initial-velocity, kinetic parameters were determined for each substrate, using various concentrations of ATP. Under the conditions used, all synthetic peptides reacted with greater maximum velocities than whole histone H1. Nevertheless, the K(m) for H1, 54 muM, was lower than the K(m) values of the synthetic substrates. The most efficient substrate was peptide K, which had a V(max) of 50.6 mumol/min per mg of kinase and a K(m) of 63 muM. In the absence of peptide substrate no ATPase activity was detectable at a sensitivity of 0.05% of the rate of peptide phosphorylation, suggesting that ATP is not cleaved to form an unstable phosphoenzyme complex. The data are consistent with a sequential reaction mechanism involving a ternary complex between enzyme, polypeptide substrate, and ATP.


Integration of avian sarcoma virus DNA sequences in transformed mammalian cells. DNA from six avian sarcoma virus (ASV)-transformed mammalian cell lines was digested with the restriction endonucleases EcoRI, Xho I, or Sal I, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filter strips, and hybridized with specific ASV [32P]cDNA probes. DNA from all of the ASV-transformed cell lines yielded three common virus-specific DNA fragments (2.4, 1.8, and 1.3 X 10(6) daltons) upon cleavage with EcoRI. Xho I appeared to cleave at least once within the integrated provirus and yielded a common fragment of 3.3 X 10(6) daltons as well as a second virus-specific DNA fragment whose size varied from 4.0 to 5.0 X 10(6) daltons in the different transformed cell lines. Sal I did not cleave within the provirus and yielded a single major virus-specific fragment of about 11 X 10(6) daltons in all transformed lines examined. Using specific cDNA probes, we show that the 1.8 X 10(6)-dalton EcoRI fragment contains sequences homologous to the 3' end of the viral RNA as well as to the src region of the viral genome. These studies clearly demonstrate that the same region on the ASV genome is utilized for provirus integration in different ASV-transformed cell lines.


The RNA of avian acute leukemia virus MC29. The RNA of myelocytoma virus MC29, a replication-defective avian acute leukemia virus, was investigated. Sedimentation and electrophoretic analyses indicated that the virus contains a distinct 28S RNA with about 5700 nucleotides. It is the smallest avian tumor virus RNA detected to date. The small size of the RNA suggests that the defectiveness of the virus is due to deletions in replicative genes. The RNA shared 3 to 5 of 30 large RNase T1-resistant oligonucleotides with the RNA of other avian leukosis and sarcoma and may represent the transforming information of the virus. Sequences of the conserved transforming gene of avian sarcoma viruses were not detected in MC29 RNA. It was concluded that the transforming sequences of MC29 RNA define a new class of avian tumor viral transforming genes.


Steroidogenic activity of fragments of adrenocorticotropin with arginine in residue positions 3 and 5. The maximal steroidogenic response (Smax) to adrenocorticotropin (ACTH) is enhanced by the substitution of arginine in position 3. Though the Smax of adrenocorticotropin-(3-10)-octapeptide [ACTH-(3-10)] is only 20% of ACTH-(1-24) in isolated rabbit adrenal cells and 10% in rat cells, [Arg3]ACTH-(3-10) has an Smax comparable to that of the longer peptide. Activity determined from the midpoints of dose-response curves (A50), however, is several orders of magnitude less. The Smax of ACTH-(4-10) is not significantly different from that of the 3-10 analog. Substitution of arginine in the 5 position of ACTH-(4-10) decreases activity for rabbit cells, but does not affect activity for rat cells. The substitution of arginine in the 5 position of ACTH-(1-20) markedly decreases Smax in rabbit but not rat cells. The Smax of [Arg 3,5)]ACTH-(3-10), containing both substitutions, is greater than that of ACTH-(3-10), but much less than that of [Arg3]acth-(3-10) in both species studied. These findings contrast with those in the rabbit adipocyte, where both arginine-containing ACTH analogs have enhanced activity and the effects of both substitutions in a single peptide are additive.


Replication of viral RNA by a defective interfering vesicular stomatitis virus particle in the absence of helper virus. The genome of a defective interfering particle (DILT) derived from the heat-resistant strain of vesicular stomatitis virus is expressed in vivo without the assistance of infectious helper virus. The rates of RNA synthesis in the presence of cycloheximide (primary transcription) are the same when infections are with equal numbers of physical particles of DILT or virus. With this treatment, DILT synthesizes only 12-17S mRNAs as characterized by size, polarity, and polyadenylylation. In the absence of cycloheximide, DILT-infected cells produce not only these mRNAs but also a 28S RNA species. This RNA, which represents one half of the viral specific RNA, contains newly synthesized full-length (+) and (-) strand DILT RNA. Both strands are found intracellularly as ribonucleoprotein complexes. Without cycloheximide present, the rate of RNA synthesis by DILT was less than that by virus. This curtailment is most likely due to the inability of DILT to synthesize L protein mRNA. An expanded role for defective interfering particles in infection is discussed.


Choleragen activation of solubilized adenylate cyclase: requirement for GTP and protein activator for demonstration of enzymatic activity. The requirements for choleragen activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] were investigated by using an enzyme preparation solubilized with Triton X-100 from an extensively washed brain particulate fraction and partially purified with DEAE-cellulose. Unlike the particulate enzyme, this preparation was not activated after incubation with choleragen plus dithiothreitol, ATP, and NAD. Addition of the purified protein activator of cyclic nucleotide phosphodiesterase and calcium to the partially purified enzyme increased basal activity somewhat, but choleragen activation was minimal. When cyclase was incubated with GTP plus the protein activator (and calcium), choleragen markedly increased the activity 3- to 6-fold. When GppNHp and protein activator were incubated with the cyclase prior to assay, activity was elevated but no effect of choleragen was observed. GTP and GppNHp had relatively small effects on cyclase activity in the absence of protein activator or if they were added directly to the assay. Boiled brain supernatant was consistently more effective than protein activator (plus calcium) and GTP, suggesting that other factors are required for maximal cyclase activity after choleragen treatment. It appears that the cyclase system is dissociable into several components, all of which may be necessary for optimal regulation of activity. It is probable that one of these is the heat-stable calcium-dependent protein activator of cyclic nucleotide phosphodiesterase and adenylate cyclase that we have found is required along with GTP for demonstration of choleragen activation of partially purified brain adenylate cyclase.


Homology between a prolyl hydroxylase subunit and a tissue protein that crossreacts immunologically with the enzyme. A protein, enzymatically inactive but immunologically related to prolyl hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase; EC 1.14.11.2) (cross-reacting protein), has been purified to near homogeneity from skin of newborn rats. The purified protein has a molecular weight of 60,000 on gel filtration and sodium dodecyl sulfate gel electrophoresis, corresponding to that of the smaller of the two dissimilar subunits of the enzyme. The two subunits of prolyl hydroxylase differ markedly from one another in their amino acid compositions, but crossreating protein and the smaller subunit are very similar in composition. On antibody-affinity chromatography both subunits reacted with the antibody developed against the intact enzyme. Neither crossreacting protein nor the 60,000 molecular weight subunit was adsorbed onto concanavalin A, which adsorbed the intact enzyme as well as the larger subunit. It would appear that crossreacting protein is identical to one of the subunits of prolyl hydroxylase or metabolically related to it.


Cyclic AMP, the microtubule-microfilament system, and cancer. Additional evidence is presented for the previously proposed existence in normal fibroblasts of a cyclic AMP-dependent network of microtubules and microfilaments, which is connected with cell membrane elements on one end and with nuclear structures on the other and whose disorganization leads to malignant transformation. In the presence of cyclic AMP derivatives sufficient to promote integrity of this network, cell growth limitation in suspension, increased transport of alpha-[14C]aminobutyrate, and the relatively tranquilized membrane of the normal fibroblast are also achieved. A pattern of distribution of actin and tubulin has been demonstrated showing aggregated actin deposits which are presumably responsible for the oscillatory knob activity of cells with the transformed habitus. Specific orientations of microtubular and filamentous elements with respect to the nucleus can be demonstrated. The hypothesis that the microtubular-microfilamentous structure conveys growth-regulatory information from the cell membrane to the nucleus and that its disorganization can lead to malignancy has been extended to explain various cellular manifestations.


Norepinephrine induces glial-specific enzyme activity in cultured plasma glioma cells. Addition of 1 micronM 1-norepinephrine to cultures of C6TK- rat glioma cells caused a 2-fold increase in specific activity of the glial-specific enzyme 2':3'-cyclic nucleotide 3'-phosphohydrolase (nucleoside-2':3'-cyclic-phosphate 3'-nucleotidohydrolase, EC 3.1.4.16). Specific activity could also be stimulated by analogues of 3':5'-cyclic AMP, and the effect of norepinephrine could be blocked by beta-adrenergic receptor antagonists but not by alpha-adrenergic antagonists. Norepinephrine or cyclic AMP analogues also increased the specific activity of this enzyme in other clones of glioma and Schwannoma cells and in glioma X neuroblastoma cell hybrids. These results show that the stimulatory effect of norepinephrine on cyclic AMP concentrations in glioma cells leads ultimately to a stimulation of glial-specific cell funtion.


Tritium suicide selection of mammalian cell mutants defective in the transport of neutral amino acids. Mouse lymphocytic cells of the established line GF-14 were allowed to accumulate intracellular 3H-labeled aminoisobutyric acid (AIB), frozen, and stored over liquid N2. After internal radiation had reduced survival to 1 in 10(4), survivors were plated and tested for their ability to transport AIB. Out of 200 clones tested, two (designated GF-17 and GF-18) were found to have reductions to 13-35% of the parent in the rate of transport of AIB, L-alanine, L-proline, and L-serine; GF-18 also showed significant reductions in the rate of transport of L-glutamate and DL-cysteine. Little or no change was observed for 10 other amino acids or for thymidine. Kinetic analyses revealed that the mutants were not altered in Km for AIB uptake, but had Vmax values approximately 20% the value of the parent strain, GF-14, suggesting that either the number of AIB transport sites or the turnover rate of the sites has been reduced in the two mutants.


Identification of amber and ochre mutants of the human virus Ad2+ND1. Although human adenoviruses grow poorly in monkey cells, this defect can be overcome either by coinfection of cells with simian virus 40 (SV40) or by insertion of the relevant portion of the SV40 genome into the adenovirus genome to form an adenovirus-SV40 hybrid virus. The nondefective adenovirus-2-SV40 hybrid virus, Ad2+ND1, contains an insertion of 17% of the SV40 genome, which codes for at least part of a 30,000 dalton protein. A set of Ad2+ND1 host-range mutants that have lost the ability to grow in monkey cells and behave as point mutants fail to synthesize the 30,000 dalton ND1 protein. Translation in vitro of SV40-specific mRNA from mutant-infected cells produces unique short polypeptides instead of the 30,000 dalton protein. Here we show that this set of host-range mutants includes both ochre and amber nonsense mutations. When the SV40-specific mRNA from the host-range mutants is translated in vitro to produce the polypeptide fragments, yeast suppressor tRNA can partially restore synthesis of wild-type-size 30,000 dalton protein. By this assay, one mutant is ochre and two are amber.


In vivo and in vitro phosphorylation of rat liver fructose-1,6-bisphosphatase. Incorporation of 32P from [gamma-32P]ATP into a homogenous preparation of rat hepatic fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphatase 1-phosphohydrolase, EC 3.1.3.11) was catalyzed by a homogeneous preparation of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine liver. Approximately 4 mol of phosphate were incorporated per mol of the tetrameric enzyme. This phosphorylation was associated with an increase in enzyme activity. In addition, in vivo phosphorylation of the enzyme was observed after injection of radioactive inorganic phosphate into rats and subsequent isolation of the enzyme by conventional purification methods and by immunoprecipitation. All of the labeled phosphate incorporation into the enzyme, both in vitro and in vivo, was precipitated by antibody specific for the enzyme. Furthermore, the 32Pi counts were coincident with the enzyme subunit band when the immunoprecipitates were examined by sodium dodecyl sulfate/disc gel electrophoresis. Acid hydrolysis of the immunoprecipitated enzyme that was phosphorylated in vitro revealed that only seryl residues were labeled. On the basis of the concentration of protein kinase (0.2-1.0 muM) necessary to phosphorylate physiological amounts of fructose-1,6-bisphosphatase (1.0-4.0 muM), it is suggested that cyclic AMP-dependent protein kinase may catalyze the phosphorylation of fructose-1,6-bisphosphatase in vivo.


Enzyme replacement therapy in Gaucher's disease: preliminary clinical trial of a new enzyme preparation. A patient with far-advanced adult type Gaucher's disease was treated with solubilized, highly purified placental glucocerebrosidase administered after entrapment in human erythrocytes or by direct intravenous injection. In some instances the enzyme-containing erythrocytes were coated with gamma globulin. No toxic side effects were observed after enzyme infusion. There were suggestive, but not conclusive, findings that enzyme infusion may have been beneficial. After therapy, there was a decrease in transfusion requirement, some improvement of liver function, possible decrease in liver size, and relief of subjective symptoms. Erythrocyte and plasma glucocerebroside levels were unchanged during therapy, but there was a possibly significant decrease in leukocyte and platelet levels of the glycolipid. No changes occurred in serum acid phosphatase or angiotensin-converting enzyme activity.


Recessive dystrophic epidermolysis bullosa: evidence for an altered collagenase in fibroblast cultures. Collagenase has been implicated in the pathogenesis of blister formation in recessive dystrophic epidermolysis bullosa. In order to examine whether aberrations in this enzyme are important in the disease, fibroblast cultures from two patients were used to compare the properties of the collagenases from the mutant cells with those from control fibroblast lines. Purified procollagenase preparations from the mutant fibroblasts were significantly more thermolabile at low Ca2+ concentration than control enzymes. They also showed a decrease in affinity for Ca2+, a cofactor required both for enzyme activity and thermal stability. In addition, the collagenase from each mutant line displayed diminished specific activity, expressed as activity per unit of immunoreactive protein, with a mean value of 39% of control for one patient's enzyme and 16% for the other. The data support the postulate that, in these two patients, the altered collagenase is the result of a structural gene mutation, a defect in the post-translational modification of the enzyme, or a mutation in a gene regulating the normal degradation of collagenase.


Comparison of Epstein-Barr viral DNAs in Burkitt lymphoma biopsy cells and in cells clonally transformed in vitro. The nucleotide sequences of intracellular Epstein-Barr viral DNAs in tumor biopsy cells of clones independently transformed in vitro were generally compared by determing the electrophoretic mobilities of restriction endonuclease cleavage fragments of the viral DNA. To carry out this comparison, cleaved cell DNAs were electrophoresed in agarose gels and transferred to nitrocellulose paper, and the immobilized viral species were identified by hybridization with purified, viral DNA labeled in vitro. These studies lead to three findings: (i) The complexities of all of the intracellular viral DNAs are similar to one another and to that of purified virion DNA. (II) There are small differences in the cleavage patterns of some viral DNAs, but the differences of the cleavage patterns of the viral DNA resident in the tumor cells and in the cells transformed in vitro are not more pronounced than those found between the different clones of the cells transformed in vitro. (iii) All of the viral DNA species contain a repeated sequence. The first two conclusions indicate that the Epstein-Barr virus strain studied in the laboratory appears indistinguishable from that associated with Burkitt lymphoma.


Translation of 35S and of subgenomic regions of avian sarcoma virus RNA. Rabbit antiserum monospecific for an internal structural protein, p27, of avian sarcoma viruses (ASV) was found to immunoprecipitate polypeptides with molecular weights (Mr) of 180,000 and 76,000 from cell-free reticulocyte lysates programmed by ASV 35S RNA and also from lysates of ASV-infected cells. In addition, the Mr 180,000 protein was also precipitated by antiserum raised against virion DNA polymerase, suggesting that is a product of the two genes nearest the 5' end of virion 35S RNA. We have also investigated the ability of subgenomic portions of virion RNA to program cell-free protein synthesis. A 10-12S poly(A)-containing fragment of RNA from both nondefective and transformation-defective ASV directed the synthesis of a polypeptide of Mr 29,000 immunologically unrelated to the gs antigens; 20-24S poly(A)-containing RNA from nondefective ASV directed the synthesis of a polypeptide of Mr 60,000 not found when a similar RNA preparation from transformation-defective ASV was translated, suggesting that it is the product of the ASV src gene. These results indicate that internal initiation sites for protein synthesis exist on the 35S RNA genome.


Friend strain of spleen focus-forming virus is a recombinant between ecotropic murine type C virus and the env gene region of xenotropic type C virus. The spleen focus-forming virus (SFFV), a replication-defective murine leukemia virus that causes the rapid transformation of certain hematopoietic target cells, has acquired specific xenotropic viral genetic information not contained in Friend helper virus. In the current studies, it is shown that a cDNA that represents a xenotropic virus portion of SFFV detects genetic sequences derived from the env gene region of murine xenotropic virus. The significance of the acquisition of these xenotropic viral sequences by SFFV is discussed with regard to their possible role in the rapid leukemogenicity of SFFV, and an analogy is drawn between the formation of SFFV and the formation of the Kirsten and Harvey sarcoma viruses.


Biochemical evidence that MCF murine leukemia viruses are envelope (env) gene recombinants. Recently, a novel class of murine type C virus (MCF), some strains of which are highly oncogenic in the AKR acceleration test, has been isolated from premalignant and malignant thymuses of AKR mice. The biology of these viruses suggested that MCFs are the product of recombination between endogenous ecotropic and xenotropic viruses and, further, that the recombination has taken place within the envelope (env) gene which encodes the surface glycoprotein (gp70) of the virion. We have compared by tryptic peptide analysis, the gp70s of four MCF isolates with the gp70s of various possible parental viruses. In addition, we have compared the tryptic peptides of the gag gene products p30 and p15 from several of these viruses. The results allow the following conclusions: (i) the gp70s of the MCF viruses are not identical to one another and are different from the gp70s of the possible parental viruses tested; (ii) the MCF virus gp70s have tryptic peptides in common with xenotropic virus gp70s as well as with ecotropic virus gp70s; and (iii) the gap region protein, p30, of the MCFs tested is identical to p30 of AKR ecotropic virus (Akv-1 or Akv-2) and distinct from p30 of xenotropic viruses, suggesting that the 5' end of the recombinant viruses is of Akv origin. The findings are discussed with respect to the possible role a recombinant virus might play in leukemogenesis in AKR mice.


Nerve growth factor increases activity of ornithine decarboxylase in superior cervical ganglia of young rats. Nerve growth factor produces a rapid increase in the activity of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) in superior cervical ganglia of young rats in vivo and in vitro. Maximum activity occurs 6-7 hr after the addition of nerve growth factor. The nerve growth factor-mediated increase in ornithine decarboxylase activity in vitro can be prevented by the addition of cycloheximide, actinomycin D, or antibody to nerve growth factor. A number of other agents were tested for their ability to increase ornithine decarboxylase were tested for their ability to increase ornithine decarboxylase activity in the ganglia; only nerve growth factor, and, to a slight extent, insulin were able to raise the activity of the enzyme. High concentrations of dibutyryl cyclic AMP (10 mM) were able to mimic the effect of nerve growth factor.


Mechanism of action of nalidixic acid: purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. A target protein for nalidixic and oxolinic acids in Escherichia coli, the nalA gene product (Pnal), was purified to homogeneity as judged by gel electrophoresis, using an in vitro complementation assay. It is a dimer of identical 110,000-dalton subunits. A polypeptide of this molecular weight is uniquely induced by a lambda nalA transducing phage, thereby showing that the purified Pnal is a product of the nalA gene. Nalidixic and oxolinic acids inhibit DNA gyrase activity and induce formation of a relaxation complex analogue. Treatment of the complex with sodium dodecyl sulfate causes a doublestrand break in the DNA substrate and the resulting linear molecule seems covalently bound to protein. Complex formation, unlike the introduction of supertwists, does not require ATP or relaxed circular DNA and is insensitive to novobiocin. DNA gyrase from a strain with a nalA mutation conferring drug resistance (nalA(r)) is 1/100 as sensitive to oxolinic and nalidixic acids with respect to inhibition of supertwisting and induction of the pre-linearization complex. Addition of Pnal restores drug sensitivity and stimulates DNA gyrase activity. DNA gyrase preparations and Pnal catalyze a third reaction sensitive to nalidixic and oxolinic acids, the ATP-independent relaxation of supertwister DNA. Relaxation by gyrase from nalA(r) cells is drug resistant. The nicking-closing activity is distinct from E. coli omega protein in several properties, including the ability to relax positively supertwisted DNA. We postulate that the nalA gene product occurs in two molecular forms, as Pnal and as a gyrase component. Both forms catalyze nicking-closing, and inhibition of this activity by nalidixic and oxolinic acids may account for the inhibition of DNA synthesis by these drugs.


Occurrence of partial deletion and substitution of the src gene in the RNA genome of avian sarcoma virus. The genome size of 20 transformation-defective (td) viruses derived from different strains of Rous sarcoma viruses [Prague (subgroups A and C), Schmidt-Ruppin (subgroups A and D) (SR-D), Bratislava 77, and Carr-Zilber subgroup D)] was examined by polyacrylamide gel electrophoresis. All of the td viruses except td SR-D have 35S RNA of the same size-i.e., class b RNA. Two of five td SR-D viruses examined have a slightly larger RNA, corresponding to a td deletion that is about 25% smaller than that of class b RNA. However, the RNase T(1)-oligonucleotide fingerprints of all the td SR-D viruses are identical, lacking two sarcoma-specific oligonucleotides. The fingerprints of these viruses also showed a minor oligonucleotide present at very low concentration. A study of heteroduplex molecules formed between genome-length cDNA made from wild-type SR-D and 35S RNA of td SR-D showed a deletion loop of 2.0 and 1.5 kilobases, respectively, at the map position of the src gene for these two classes of td SR-D viruses, confirming the results of polyacrylamide gel electrophoresis. In addition, some heteroduplex molecules with a substitution loop of 0.6-0.7 kilobase at the same site as the deletion loop were observed in all five of the td SR-D viruses. We conclude that some of the td SR-D viruses have a partially deleted src gene and that all of the td SR-D viruses have incorporated heterologous sequences of distinct length in some RNA molecules at the position of the src gene. The nature and origin of these heterologous sequences are discussed.


Immunologic selection against simian virus-40 transformed cells: concomitnat loss of viral antigens and early viral gene sequences. A clonal line of highly oncogenic "spontaneously transformed" mouse cells (T AL/N clone 3) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned. The clone of SV40-transformed cells (subclone 1) expressed SV40-specific T (nuclear) and transplantation antigens but was 100 times less tumorigenic than the parent T AL/N clone 3 cells. When large numbers of subclone 1 cells (10(4)-10(5)) were injected into syngeneic AL/N mice, tumors were produced. From the tumors, cell lines were established in culture, all of which were consistently negative for T antigen. Tumor lines tested were found not to contain SV40-specific transplantation antigen and had again become highly tumorigenic. The original subclone 1 cells contained about one copy of SV40 DNA per diploid amount of cell DNA, as well as RNA complementary to the early region of the SV40 genome. The T antigen-negative cells from tumor line 124 contained approximately 0.5 copy of SV40 DNA per diploid equivalent and did not synthesize any detectable virus-specific RNA. Reassociation kinetic analysis with restriction enzyme fragments of viral DNA demonstrated that the cells from tumor line 124 (and also the clones of this line) had lost DNA sequences predominantly from the early region of the SV40 genome. The results indicate that a set of stably integrated SV40 DNA sequences can be present in a cell without the expression of viral antigens.


Glucocorticoids increase the responsiveness of cells in culture to prostaglandin E1. The influence of steroid hormones on the response of human astrocytoma cells (1321N1) to prostaglandin E(1) (PGE(1)) has been investigated. Responsiveness to PGE(1) was determined by measuring the conversion of [(3)H]ATP to cyclic [(3)H]AMP in cells prelabeled with [(3)H]adenine. After incubation of the cells with dexamethasone, a marked increase in both the maximal effect (2- to 3-fold) and the potency (5-fold) of PGE(1) was observed. The effect was specific for the action of PGE(1) in that no change in the response of the cells to isoproterenol was observed. The EC(50) for dexamethasone was 0.001 muM and the effect was dependent on the presence of serum. The effect of dexamethasone was first observed after a 30- to 60-min lag and was maximal by 6-8 hr. Preconfluent cultures (3 days after seeding) exhibited optimal responsiveness to glucocorticoids. Both hydrocortisone and corticosterone mimicked the effect of dexamethasone but both were less potent. The action of dexamethasone was blocked by progesterone, testosterone, and 17alpha-methyltestosterone. Cycloheximide, at a concentration (1.0 mug/ml) that blocked protein synthesis (>90%) in 1321N1 cells, totally prevented the effect of dexamethasone on the response of the cells to PGE(1). Upon removal of dexamethasone from cells treated for 16 hr, responsiveness to PGE(1) returned to control levels with a half-time of 4 hr. Dexamethasone also was found to increase the response to PGE(1) of a Rous sarcoma virus-transformed human astrocytoma cell line and the WI-38 human fibroblast line. The most obvious interpretation of our findings is that glucocorticoids induce the synthesis of a protein that selectively modifies the sensitivity of adenylate cyclase to PGE(1).


Characterization of a common precursor to corticotropin and beta-lipotropin: cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule. mRNA was isolated from cultures of AtT-20/D-16v tumor cells and translated in a mRNA-dependent reticulocyte cell-free system. The corticotropin (ACTH) product was purified by a double-antibody immunoprecipitation procedure using antisera specific for the alpha(1-24) sequence of ACTH. The product is shown by sodium dodecyl sulfate/gel electrophoresis and gel filtration on guanidine-HCl columns to be homogeneous with an apparent molecular weight (Mr) of 28,500. A product with the same molecular weight is synthesized when membrane-bound polysomes from D-16v cells are allowed to complete their nascent chains in a reticulocyte cell-free system. Mr 31,000 ACTH isolated from tumor cells has been separated into three proteins of different apparent Mr:29,000, 32,000, and 34,000. The cell-free product contains the same lysine-, methionine-, and phenylalanine-labeled tryptic peptides as the Mr 29,000 ACTH synthesized in the tumor cells. Tryptic peptide analysis also reveals the presence of the alpha(1-39) sequence in the Mr 28,500 cell-free product and suggests that there is only one copy of this sequence in the Mr 28,500 molecule.


Regulatory function of simian virus 40 DNA replication for late viral gene expression. Inhibition of simian virus 40 (SV40) DNA synthesis prevents late but not early viral gene expression in infected cells. To test whether the late SV40 template specificity is elicited by replicative intermediate DNA molecules (RI-DNA), we isolated subcellular fractions containing RI-DNA from SV-40-infected monkey cells and microinjected these preparations into various cell lines under conditions in which viral DNA synthesis was blocked. Late SV40 gene expression (V-antigen synthesis) was obtained by microinjection of wild-type RI-DNA or temperature-sensitive mutant A7 RI-DNA preparations at 37 degrees or 41.5 degrees, respectively, while SV40 native superhelical DNA (DNA I) at a 10-fold higher concentration failed to induce V-antigen synthesis under the restrictive conditions. V-antigen synthesis was also obtained by microinjection of partially denatured SV40 DNA or a high number of randomly nicked DNA molecules (DNA II) in the absence of DNA synthesis; both single-stranded regions and nicks are specific features of RI-DNA molecules.


Dinucleoside pyrophosphate are substrates for T4-induced RNA ligase. RNA ligase isolated from bacteriophage T4-infected Escherichia coli will utilize a number of different compounds with the general structure Ado-5'PP-X as substrates in an ATP-independent reaction. The P-X portions of these molecules are transferred to the 3'-hydroxyl of an oligoribonucleotide to form a phosphodiester bond, and the Ado-5'P (AMP) portion is released. AMP, CMP, GMP, UMP, dTMP, NMN, alphaNMN, reduced NMN, FMN, Rib-5P, phosphopantetheine, and cyanoethylphosphate all have been added to [Cyd-3H](Ap)3C from their corresponding AMP adducts. Contrary to the relative lack of specificity of RNA ligase for the P-X -group added, the failure of NADP+, deamino-NAD+, epsilonNCD+, epsilon NAD and CoA to react indicates that the enzyme shows a high degree of selectivity for the AMP portion of the substrate. The diversity of chemical groups that can be efficiently added suggests that this reaction of RNA ligase will prove useful for the modification of the 3' ends of RNA molecules.


Activation of hormone-sensitive lipase and phosphorylase kinase by purified cyclic GMP-dependent protein kinase. Cyclic GMP-dependent protein kinase, purified to homogeneity from bovine lung, was shown to activate hormone-sensitive lipase partially purified from chicken adipose tissue. The degree of activation was the same as that effected by cyclic AMP-dependent protein kinase although higher concentrations of the cyclic GMP-dependent enzyme were required (relative activities expressed in terms of histone H2b phosphorylation units). Activation by cyclic AMP-dependent protein kinase was completely blocked by the heat-stable protein kinase inhibitor protein from skeletal muscle but activation by the cyclic GMP enzyme was not inhibited. Lipase fully activated by cyclic AMP-dependent protein kinase showed no further change in activity when treated with cyclic GMP-dependent protein kinase. Lipase activated by cyclic GMP-dependent protein kinase was reversibly deactivated by purified phosphorylase phosphatase (from bovine heart); full activity was restored by reincubation with cyclic GMP and cyclic GMP-dependent protein kinase. Cholesterol esterase activity in the chicken adipose tissue fraction, previously shown to be activated along with the triglyceride lipase by cyclic AMP-dependent protein kinase, was also activated by cyclic GMP-dependent protein kinase. Crude preparations of hormone-sensitive triglyceride lipase from human or rat adipose tissue and cholesterol esterase from rat adrenal were also activated by cyclic GMP-dependent protein kinase. Purified phosphorylase kinase (rabbit skeletal muscle) was also shown to be activated by cyclic GMP-dependent protein kinase. The present results, together with those of other workers on histone phosphorylation, suggest that the substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinase may be similar. This is discussed in the light of a model recently proposed with regard to the relationship between the subunit structures of the two kinases. The physiologic significance of the findings remains to be established.


Role of nucleotides in tubulin polymerization: effect of guanylyl 5'-methylenediphosphonate. Incubation of 48,000 X g rat brain supernatants for 30 min at 37 degrees with 1-2 mM guanylyl 5'-methylenediphosphonate [Gmp(CH2)pp] results in polymerization of 95-98% of the tubulin present. This is considerably more than the 50% polymerization that can be achieved with the natural nucleotide, GTP, under optimal conditions. Gmp(CH2)pp is also much more effective than GTP in inducing polymerization of purified tubulin. Assembly of microtubules with Gmp(CH2)pp occurs at tubulin concentrations one-third of those possible with GTP. Furthermore, with Gmp(CH2)pp, microtubule assembly does not require the high molecular weight basic proteins needed with GTP. Polymerization of tubulin by Gmp(CH2)pp is neither prevented nor reversed by concentrations of calcium (2 mM) that can either prevent microtubule assembly or disrupt already formed microtubules if the nucleotide used is GTP or guanylyl imidodiphosphate. When Ca2+ is added before or after microtubule assembly, electron microscopy of the Gmp(CH2)pp preparations reveals normal microtubules turning into twisted ribbons. Low temperature (4 degrees) can both prevent and disrupt the tubulin assembled Gmp(CH2)pp although disruption proceeds much more slowly when GTP is used.


Binding of the endogenous nonpeptide morphine-like compound to opiate receptors. The endogenous central nervous tissue substance called MLC (morphine-like compound) is shown to bind to the opiate receptors of the mouse neuroblastoma X glioma hybrid cell line NG108-15. The interaction of MLC with these opiate receptors is noncooperative, as is the interaction of morphine, naloxone, and Leu-enkephalin with these receptors. A specific antibody to morphine will bind MLC but will not bind beta-endorphin, Leu-enkephalin, or Met-enkephalin. It would appear, therefore, that MLC can be considered to be a different type of endogenous ligand for the opiate receptor.


Cell-free synthesis of the precursor polypeptide for avian myeloblastosis virus DNA polymerase. High molecular weight RNA (35S) isolated from avian myeloblastosis virus directs the cell-free synthesis of two prominent polypeptides of 180,000 and 76,000 molecular weight. The latter polypeptide has previously been identified as the precursor to the group-specific antigens of the virus ("gag" proteins) [Vogt, V. M., Eisenman, R. & Diggelmann, H. (1975) J. Mol. Biol. 96, 471-493]. Two-dimensional tryptic peptide analyses of the [35S]methionine-labeled peptides demonstrate that the 180,000-dalton product is a polyprotein that can account for all the peptides of the avian myeloblastosis virus DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7) and those of the gag viral proteins. This is direct confirmation of the genomic order of the viral structural genes, placing the polymerase gene adjacent to the 5'-proximal gag gene of the virus. Furthermore, our findings suggest that the primary polymerase gene product is the beta subunit of the enzyme. These results are discussed in relation to the proposed structural gene map for the avian retraviruses and suggest a model for the in vivo processing of the viral polymerase.


Prostaglandin regulation of macrophage collagenase production. The production of collagenase (EC 3.4.24.3) by endotoxin-stimulated macrophages was significantly inhibited by indomethacin, indicating that prostaglandins (PGs) mediate this effect. Inhibitions of collagenase production by indomethacin was overcome by addition of exogenous PGE2 at 10 nM whereas the addition of 0.1 and 1.0 micrometer PGE2 increased the enzyme production to 3 times that achieved by endotoxin. Although the addition of prostaglandin alone to macrophage cultures did not stimulate collagenase production, the simultaneous addition of PGE1 or PGE2 and endotoxin enhanced collagenase activity 2- to 10-fold. This increase was detectable at PGE concentrations of 10 nM and was optimal at 0.1-1.0 micrometer. PGF2alpha had little effect on either the enhancement of collagenase production by endotoxin-stimulated macrophages or its restoration in cultures inhibited by indomethacin. Radioimmunoassay of prostaglandins in the culture media revealed that macrophages exposed to endotoxin secreted 40-fold more PGE2 than did unstimulated cells. The increase in PGE2 was detected 4 hr after exposure to endotoxin and was maximal at 14 hr. The peak PGE2 concentrations in the culture media were similar to those of exogenous PGE2 (about 10 nM) needed to restore collagenase production in indomethacin-treated cultures. These findings demonstrate the involvement of PGE in the endotoxin-activation of macrophages with resultant production of collagenase.


Sequence arrangement of the 5' ends of simian virus 40 16S and 19S mRNAs. Electron microscopic examination of molecular hybrids between simian virus 40 DNA, that had been cleaved with EcoRI and then digested with exonuclease III, and either 16S or 19S mRNA produced late during the viral infection cycle indicated that each of these mRNAs contained a 5'-terminal "leader" sequence that was encoded in the viral genome at about map position 0.71-0.75. Hybridization of each of these mRNAs to viral DNA immobilized on nitrocellulose filters supported the electron microscopic observations.


Complexes of inorganic pyrophosphate, orthophosphate, and calcium as stimulants of 3T3 cell multiplication. Addition of 0.1-0.5 mM sodium PP(i) for 17 hr to confluent cultures of BALB/c 3T3 cells in low serum concentrations stimulated the incorporation of [(3)H]thymidine into DNA to an extent equal to that produced by high serum concentration. PP(i) prevented much but not all of the cell detachment that accompanies decreasing the serum concentration of confluent cultures and it increased the saturation density of cultures in high serum concentrations. The stimulation had a sharp concentration dependence and was associated with the appearance in the medium of a flocculent precipitate. Stimulation and precipitate formation were dependent on Ca(2+) and inorganic orthophosphate (HPO(4) (2-)) and were inhibited by Mg(2+). More than half the Ca(2+) requirement could be met with Sr(2+). In the absence of PP(i), supranormal concentrations of either Ca(2+) or HPO(4) (2-) caused graded increases in [(3)H]thymidine incorporation and total cell yield. The effect of supranormal [Ca(2+)] depended on [HPO(4) (2-)] and vice versa, and the Ca(2+) requirement could be partially met by Sr(2+). The stimulation was associated with increasing turbidity of the medium. Various other complexing agents of Ca(2+), including the divalent cation ionophore A 23187, failed to produce stimulation of 3T3 cells. We conclude that water insoluble complexes of PP(i), HPO(4) (2-), and Ca(2+) or, at much higher concentrations, the latter two together, stimulate 3T3 cells and we speculate that this is brought about by the association of these complexes with the cell membrane.


Hexamethylene bisacetamide induces morphologic changes and increased synthesis of procollagen in cell line from glioblastoma multiforme. Addition to hexamethylene bisacetamide (diacetyldiaminohexane) to cultures of a malignant mesenchymal cell line derived from a human glioblastoma multiforme induces morphological changes and stimulates the synthesis of procollagen. The morphological changes include cell elongation, an increase of extracellular material with staining properties of collagen by light microscopy, and an increase in extracellular 220-A fibrils by electron microscopy. The rate of procollagen synthesis increased as much as 20-fold, and the ratio of type I:type III procollagen changed, with type I becoming the predominant form. The change in type I:type III ratio is similar to that seen in the maturation of normal fetal to adult connective tissue.


Simian virus 40-specific proteins in the membranes of simian virus 40-transformed hamster and mouse cells. Membranes of simian virus 40-transformed hamster lymphocytes and phagocytes, as well as of transformed mouse fibroblasts, contain two classes of antigenic virus-specific protein. The isoelectric points of these proteins, as defined by isoelectric focusing/immune electrophoresis are at pH 4.5 and 4.7. The molecular weights of the pI 4.5 and pI 4.7 components, determined by isoelectric focusing/dodecyl sulfate polyacrylamide electrophoresis, lie near 58,000 and 90,000-110,000, respectively. The pI 4.5 and pI 4.7 proteins are tentatively identified with the surface (transplantation) and U antigens, respectively.


Solubilization of apolipoprotein B  and its specific binding by the cellular receptor for low density lipoprotein. Low density lipoprotein (LDL) and very low density lipoprotein (VLDL) bind specifically to a receptor on fibroblasts, and it has been postulated that the apoprotein of LDL (apo B) confers the specificity of cellular binding. This hypothesis has been tested in the present study with a watersoluble apo B-bovine serum albumin complex. The binding of (125)I-labeled apo B to cultured fibroblasts was temperature-dependent. Specific binding ranged between 183 and 859 ng/mg of cell protein at a concentration of 5 mug/ml; at 37 degrees , 750-2199 ng/mg was bound and internalized. The binding of apo B greatly exceeded the amount of (125)I-labeled LDL bound at 4 degrees and 37 degrees in the same experiment. Fibroblasts from a subject homozygous for hyper-beta-lipoproteinemia showed minimal binding of (125)I-labeled LDL, consistent with the absence of the cellular LDL receptor. Such cells also had depressed binding of (125)I-labeled apo B. Lymphocytes grown in lipoprotein-deficient medium demonstrated specific binding of LDL; however, freshly isolated lymphocytes did not show such binding. The binding of (125)I-labeled apo B to lymphocytes paralleled the binding of (125)I-labeled LDL. Unlabeled LDL and apo B-albumin complex both competitively inhibited the binding of (125)I-labeled apo B and (125)I-labeled LDL to fibroblasts. When labeled LDL was incubated with fibroblasts for 6 hr at 37 degrees , it underwent cellular internalization and degradation, as measured by the release of (125)I-labeled fragments into the medium. This degradation was inhibited by unlabeled apo B. Conversely, (125)I-labeled apo B also was internalized and degraded by fibroblasts, and this process was inhibited by LDL. These findings demonstrate that apo B binds specifically to the LDL receptor and that the cellular binding of LDL is determined by this apoprotein.


Arterial uptake and synthesis of low density lipoproteins. The accumulation of cholesterol in atherosclerotic lesions is associated with an increased uptake of plasma cholesterol and LDL by the arterial wall. During the regression of atherosclerosis, the uptake of these macromolecules returns to or below normal, suggesting that the retention of cholesterol in regressed lesions is due to a defect in the removal of cholesterol from the arterial wall rather than to an abnormality in vascular permeability. Although increased amounts of 125I-LDL were detected in atherosclerotic vessels, the percent distribution and fractional degradation rate of 125I-LDL appeared similar in normal and diseased vessels. The present studies in support of earlier findings in human vessels indicate that LDL in the artery is contained in a number of different cellular and extracellular pools in close association with AMPS. These lipoproteins appeared to be derived not only from the lipoproteins contained in the plasma but also from lipoproteins synthesized by the arterial wall.


Structural correlates of arterial endothelial permeability in the Evans blue model. Normal young pigs were injected intravenously with azo dye Evans blue, which is preferentially and consistently taken up by focal areas in the aorta known to be areas of enhanced permeability. Ferritin was intravenously injected into these animals, and its uptake was quantitated at the electron microscope level at 1, 5 and 15 min after administration. The number of vesicles, large inclusions or vacuoles, and junctions, both labelled and unlabelled, were counted in areas of normal and enhanced permeability. The results have revealed that the number of ferritin grains per vesicle is the same in both areas. However, the percentage of vesicles containing ferritin is significantly higher in areas of enhanced permeability.


Studies on the passage of plasma proteins across arterial endothelium in relation to atherogenesis. Previous studies using isotopically labelled lipoproteins or immuno-histological characterisation of the apolipoproteins present in atherolsclerotic lesions have suggested that lipoproteins varying in size from LDL to VLDL gain entry to the lesions. It has also been shown that a fraction of these lipoproteins is firmly bound to various constituents of the arterial wall. Using the immunoperoxidase technique at the electron microscopic level, it is now confirmed that spherical molecules specifically interacting with labelled anti-LDL (anti-apolipoprotein B) and of a range of molecular diameters corresponding to those of LDL to VLDL are indentifiable in the intersitial matrix of lesions. The labelled antibodies also identified lipoprotein bound to elastin, within smooth muscle cells, and bound to collagen fibres. In advanced lesions lipoprotein was found to be bound not only to "normal" (native) collagen fibrils but also with a characteristic periodicity to fibrous long-spacing collagen. The occurrence of this "abnormal" form of collagen in plaques is now reported for the first time but its possible significance in relation to atherogenesis is discussed.


Ultrastructural changes in the lipid inclusions of arterial smooth muscle cells after reduction of high serum cholesterol levels. Intimal smooth muscle cells overloaded with lipid droplets can, when the serum cholesterol is reduced to a sufficiently low level over a sufficiently long period, reduce the size of droplets and at least some can completely clear inclusions.


Smooth muscle cells of intimal cushions and the localization of atherosclerotic lesions. Atherosclerosis results from a precise sequence of endothelial interaction with platelets or thrombocytes and mononuclear cells, uptake of specific lipoproteins, cholesteryl ester removal, production of collagen, elastin ad proteoglycans by activated smooth muscle cells. Thus, both the filtration hypothesis and the thrombogenic hypothesis for the pathogenesis of atherosclerosis appear to be correct.


Evidence for interactions between central noradrenergic neurons and adrenal hormones in learning and memory. The role of ascending noradrenergic projections in the acquisition and retention of a passive avoidance step-down response was evaluated by means of bilateral stereotaxic 6-hydroxydopamine-induced lesions of these systems. Lesions of the dorsal NA bundle alone, or in combination with lesions of the ventral NA bundle, failed to influence either the acquisition or retention of the passive avoidance response. In contrast, animals subjected to dorsal and ventral NA bundle lesions and adrenalectomy exhibited severe deficits in both the acquisition and retention of this response, and this effect was of the same magnitude as was observed after posttrial injections of diethyldithiocarbamate (DDC, 300 mg/kg). Adrenalectomy by itself had a small but significant effect on retention but did not influence acquisition of the response. The results are discussed with reference to the possibility that interactions between adrenal hormones and central NA mechanisms may serve important roles in learning and memory. However, the data provide no support for the hypothesis that central NA neurons are, by themselves, critically involved in these phenomena.


Drugs affecting dopamine neurons and yawning behavior. Drugs stimulating the dopamine (DA) neurons in different ways (apomorphine, piribedil, amphetamine, nomifensine, L-DOPA) given in low doses (not producing behavioral excitation) induced yawning in rats. Blockade of DA receptors with neuroleptics counteracted DA-agonists induced yawning which may indicate a dopaminergic component of this behavior.


Effect of elevated brain GABA concentrations on the actions of bicuculline and picrotoxin in mice. gamma-Acetylenic GABA and gamma-vinyl GABA, two catalytic irreversible inhibitors of mammalian brain GABA transaminase that produce several-fold increases in brain GABA concentrations were tested for their effects on bicuculline and picrotoxin-induced seizures and mortality in mice. Neither inhibitor influenced the frequency of seizures or death produced by either bicuculline or picrotoxin. Both inhibitors, however, produced a dose-dependent prolongation of the time to onset of seizures and death induced by picrotoxin but by bicuculline. These results suggest differences in the antagonism by bicuculline and picrotoxin of GABA-mediated neural functions.


Stimulus effects of delta(9)-THC and its interaction with naltrexone and catecholamine blockers in rats. Rats trained in a T-shaped maze to discriminate the effects of i.p. injections of delta(9)-tetrahydrocannabinol (delta(9)-THC, 4 mg/kg) and the effects of the vehicle were tested for antagonism and generalization to the delta(9)-THC stimulus by naltrexone (4 mg/kg), haloperidol (0.32 mg/kg), propranolol (20 mg/kg), and phenoxybenzamine (10 mg/kg). None of these drugs blocked the delta(9)-THC stimulus, nor were they found to generalize to delta(9)-THC.


[A case of varices caused by angiodysplasia of the upper limb]. The authors recall that the syndrome of Klippel and Trenaunay, which in its typical form most commonly involves a lower limb, constitutes the best known example of the angiodysplastic disorders, and go on to describe the case of a patient with extensive congenital varicose veins of the left upper limb, with slight bone hypertrophy and changes in the limb skeleton. In the varicose veins were numerous phleboliths, and phlebography revealed agenesis of the deep veins of the forearm. In discussion they try to demonstrate the diagnostic difficulties of this disorder which may suggest a phlebo-angiomatosis, a "pure" phlebodysplasia or an atypical Klippel and Trenaunay syndrome.


Computed tomographic demonstration of hepatic tumor with the aid of intravenous iodinated fat emulsion. An experimental study. Computed tomography showed improved visualization of carcinogen-induced hepatoma in monkeys following intravenous injection of an experimental contrast material AG 60-99; selective opacification of the liver and spleen was accomplished with a dose of greater than or equal to 0.1 ml/kg, a fraction of the dose used in conventional x-ray tomography. This marked reduction was made possible by the improved density resolution of the CT scanner. This may be the first organ-specific contrast material intended primarily for use with CT scanners.


Comparison of laboratory performance with blind and mail-distributed proficiency testing samples. Simulated addict urine samples containing drugs were sent to collaborating hospital administrators and officials of methadone centers, who then forwarded the samples to their supporting laboratories as though they were ordinary specimens from patients. The laboratories, which were already participating in the proficiency testing program of the Center for Disease Control, received the identical test samples in the mail as part of a regular Center for Disease Control proficiency testing program. Most of the laboratories performed acceptably with the mail-distributed samples, but many performed poorly when the identical samples were sent to them as if they were specimens from patients. Because of the limitations of proficiency testing involving mail-distribution samples and the impracticality of extensive testing with blind samples on a national level, the Center for Disease Control proposes to compliment its regular proficiency testing program with a monitored, onsite program of performance evaluation.


Viral attributes and host factors in carcinogenesis: herpesviruses. This paper describes two different experiments of nature: 1) the persistence of unusual virus strains of Epstein-Barr virus (EBV) (which proved oncogenic in vitro) and cytomegalovirus (CMV) in lymphoid cells following congenital or early acquired infection; 2) the occurence of multiple cases of Burkitt's lymphoma and nasopharyngeal carcinoma in one family. All the members of this family were EBV viral capsid antigen (VCA) and nuclear antigen (EBNA) antibody positive. The two patients with nasopharyngeal carcinoma had high titers of EBV-VCA, EA, and EBNA antibodies. The only member of this family having EBV early antigen (EA antibodies in addition to the patients with tumors was the mother. Borderline IgA deficiency was documented in 3 members of this family. These findings illustrate the importance of host factors (intracellular resistance to transformation and secondarily, immunological surveillance) in the outcome of the host-virus challenge whether cancer or infectious disease is the outcome. Extensive studies of these cases may provide the best insight into the mysteries of viral oncogenesis.


Retinal structure in Synbranchus marmoratus with observations on two new cone myoid inclusions. The retina of a South American swamp eel, Synbranchus marmoratus (Synbranchidae), was studied by Golgi impregnation, light and electron microscopy. Its principal features include (1) the presence of a dense matrix, possibly a new type of tapetum lucidum, in the pigment epithelium, (2) a well developed photoreceptor layer containing large rods, single, double and triple cones, and (3) well developped inner nuclear and plexiform layers, with the exception of horizontal cells which are few and relatively small. These and other observations are discussed in relation to the photic environment and habits of this fish. The presence of microfilament bundles and two unusual features, microtubuleladen dense bodies and paracrystalline inclusions, in cone myoids are discussed in terms of their possible involvement in retinomotor responses.


[Comparison between two types of pertussis vaccines]. We have produced pertussis vaccines with laboratory and industrial methods. The characteristic of laboratory cultivation of microorganisms is, in this context, growth on Hornibrook medium in low form flask and in stationary culture. Industrial cultivation is done in homogenous culture on a B-2 medium in fermentor. The strains utilized were isolated from whooping-cough cases in the Montreal region. The yield (org. x 10(9)/ml) obtained with an industrial cultivation of B. pertussis was 4 to 7 times higher than that reached with a laboratory cultivation of this microorganism. The non-toxicity as expressed in weight gain of mice was shown for both types of vaccine. The vaccines produced in fermentor were less histamino sensibilizing for mice than the one produced in stationary flash culture. The quality of the vaccines achieved by industrial method is easily reproducible due to the fact that enough variables can be measured.


[Pharmacological suppression of paradoxical sleep in the newborn rat (author's transl)]. In an attempt to suppress paradoxical sleep (PS) an intracerebral injection of 6 0HDA was carried out on rat faetuses at a gestational age of 16-20 days. In these animals a delay in maturation occurred compared to controls, the only modification of sleep being observed however, related to the circadian rhythmn of PS. The possible reasons for this are discussed within the framework of a concept relating paradoxical sleep to cerebral maturation.


Comparisons of antidotal efficacy of sodium diethyldithiocarbamate, D-penicillamine and triethylenetetramine upon acute toxicity of nickel carbonyl in rats. Sodium diethyldithiocarbamate, D-penicillamine, and triethylene-tetramine were administered to rats by im injection in dosages equivalent to 0.6 times their respective LD50 values in order to compare their relative effectiveness in prevention of death caused by exposure for 15 min to inhalation of nickel carbonyl (1.4 or 4.2 mg Ni (CO)4/liter of air). When the three drugs were administered to groups of rats at 10 min before or after the exposure to nickel carbonyl, sodium diethyldithiocarbamate was the most effective antidote. In contrast, then the drugs were administered at 6 hr after exposure to nickel carbonyl, D-penicillamine was the most effective antidote. Based upon the combined results of 4 sets of experiments, sodium diethyldithiocarbamate and D-penicillamine were significantly more effective than triethylenetetramine. The authors recommend that sodium diethyldithio-carbamate should remain the chelating agent of choice for therapy of nickel carbonyl poisoning. If sodium diethyldithiocarbamate is not available or if its use is contraindicated, D-penicillamine might be considered as an alternative chelating agent.


Angiotensinase activity in red blood cell membranes and intact adrenal cells. The present study was undertaken to characterize angiotensinase activity in human red blood cells (RBCs), RBC ghosts and rabbit adrenal cells. It was found, by the use of paper chromatography, that each of these preparations possess enzymes capable of converting angiotensin II to its heptapeptide derivative, [des=Asp1] angiotensin II (angiotensin III). Further characterization of these enzymes by the use of a chromogenic assay indicates that although intact RBCs do not split sarcosine-beta-naphthylamide, RBC ghosts do. Intact rabbit adrenal cells from the zona glomerulosa, however, do show activity against sarcosine-beta-naphthylamide. This is interpreted to indicate the presence of non-specific angiotensinases on the inside of the RBC membrane and the outside of the adrenal cell membrane.


[The normal E.E.G. of the neonate (author's transl)]. This study is the statement of the present knowledge of the normal EEG activity of the full term and the premature neonates. The criteria for normality of the records are based on transversal studies of normal newborn babies, born after a normal pregnancy and a normal delivery (Apgar 9 and 10). Follow up studies of children over of period of 3 years or more have allowed to appreciate the variability of the neonatal EEG, especially in premature babies having a later normal psycho-motor development. The variability of bioelectric activity is considerable from child to child and in the same child from one moment to another especially during quiet sleep. EEG activity varies according to the states of vigilance and sleep. Organisation of sleep is independent of the recording methods, as shown by a comparative study using different technics of electrode application.


Cyclic AMP-dependent protein kinase activation in hearts from euthyroid and hyperthyroid rats. The degree of cyclic AMP-dependent protein kinase activation in response to dibutyryl cyclic AMP and norepinephrine was determined in samples of rat heart obtained from hyperthyroid and euthyroid animals. The data indicate that thyroid hormone-induced supersensitivity to the metabolic effects of the catecholamines cannot be explained on the basis of a change in activity of supernatant protein kinase.


[Normal children sleep E.E.Gs from 5 months to 3 years old (author's transl)]. The sleep E.E.G. of the child under 3 years old is characteristic from several points of view: Drowsiness: The hypersynchrony is always observed. It is more continuous in younger children, than in the older one, where only bursts of hypersynchrony are seen. The fast rhythms of low amplitude may exist, during stage 2 sleep, but they are rare. Calm Sleep (C.S.): The vertex humps are more and more acute and sharper from 18 months. The sleep spindles are of high amplitude and frequent at 6 and 8 months. They diminish later. It may be for that reason that III and IV sleep stages seem to be more important after 12 months than before. From 21 months, we have observed an unusual pattern during C.S.: rhythmic and diffuse thêta rhythms of about 6 c/s. Paradoxal sleep (P.S.): If it is possible to observe P.S. during day-naps 8 or 9 months, it is rare later and for recording P.S., it is necessary to record E.E.G.s at night. The E.E.Gs are either a little like stage I E.E.G, or consistent of high and monomorphic delta waves. If it is possible to observe at least 3 complete sleep cycles, the proportion of P.S. is about 20 p. 100 of the total length of the sleep. At least, it is very important, even in medical practice, to record not only E.E.G. but also polygraphic leads: breathing, electro-myogram, electrocardiogram, and to notice also the rapid eyes movements.


Human fat cell adenylate cyclase. Modulation of parathyroid hormone action by guanine nucleotides. The effects of guanine nucleotides on basal and parathyroid hormone-stimulated adenylate cyclase of human fat cell ghosts were studied. GTP (10(-7)-10(-3) M) caused a dose-dependent inhibition of basal enzyme activity, but it had no significant effect on PTH-stimulated rates of cAMP-formation. The guanine nucleotide analogue 5'-guanylyl-imidodiphosphate GMP (PNP) when applied in the same concentration range, stimulated basal as well as PTH-activated adenylate cyclase activity up to 300%. GMP (PNP) activation was non-linear with time. PTH-activated the human fat cell adenylate cyclase via an individual receptor distinct from beta-adrenergic receptor sites.


The occurrence of antibody to Babesia and to the virus of louping-ill in deer in Scotland. Sera of wild red deer from 16 localities in Scotland were tested by the indirect fluorescent antibody technique for antibody to Babesia and by the haemagglutination inhibition test for antibody to the virus of louping-ill. Babesial antibody was detected in sera from all localities in proportions ranging from 22 to 100 per cent. Antibody to louping-ill virus could not be demonstrated in sera from five of the localities and in the other 11 was found less frequently than was antibody to Babesia. Sera from male and female deer were positive for louping-ill in almost equal proportions whereas the incidence of babesial antibody was significantly lower in females than in mature males. This difference could be explained by the habits of the deer. The variable occurrence of louping-ill antibody suggested that red deer are tangential hosts for the virus.


Identification of the receptor involved in adrenaline mediated sweating in the horse. Using adrenergic agonists and antagonists this study has demonstrated that adrenaline induced sweating is mediated via beta2-adrenoreceptors in the horse.


Phosphatase activity of placental extracts and pregnant mare's plasma. Equine placental extracts show phosphatase activity with a pH optimum between 4.5 and 6. The enzyme shows heat stability to 45 degrees C and electrophoresis on cellulose acetate demonstrates the presence of two bands of activity. Histochemistry confirms the presence of phosphatase activity in the placental villi. Assay of plasma samples from pregnant mares showed no increase in phosphatase activity through pregnancy under conditions of test.


[Meningitis caused by enteroviruses. Clinical and virological study of several sporadic cases in adults]. In the course of seven years (1968--1974) viral investigations were carried out in 212 casese of meningitis; predominantly Coxsackie viruses and sometimes ECHO viruses were isolated from 80 cases (37.73%). No polioviruses were isolated. Analysis of the clinical data (onset, general conidtion, evolution, complications, psychical, renal and cardiac disturbances etc.) revealed the severe aspect of these cases. The aspect and evolution of the cerebrospinal fluid showed the presence in almost 25% of the cases of an increased number of cellular elements of more than 1 000/mm3 and albumin sometimes far above normal values with a slow return (3--4 weeks). The complex therapy applied in more than half the cases demonstrates the necessity of attentive follow up of these cases of viral meningitis.


[Quantitative evaluation of the dynamics of tubercular mediastinal adenopathy treated with rifampicin and ethambutol]. The authors have followed for one year the rhythm of involution of adenopathy in 114 patients aged between 0 and 16 years, with patent primary infection, in which four therapeutic regimens have been randomly applied, in continuous or intermittent administration, with INH-RMP; RMP-EMB; INH-EMB; INH-SM. Quantified data were obtained with the aid of a planimeter, measuring the surface of the adenopathy on trimestrial seriotomographies. The rhythm of involution was evaluated by the variable halving time of the surface of the adenopathy under treatment. The percentage, the data of the physical model, showed that in the first 3--4 months, detection of the curves is more rapid for RMP and the 7/7 formula, but with almost similar results (8--14%) at 12 months. By processing the data in the computer, depending on the mathematical model, it was estimated that the halving time is by 90,4 days shorter for RMP and similar for EMB and SM.


[Clinical, radiological and bacteriological study of pulmonary tuberculosis with bacteria showing drug resistance]. The authors have investigated two groups of patients of young age suffering from tuberculosis with germs showing primary resistance to chemotherapeutic agents (61 cases), and with germs displaying sensitivity to these agents (64 cases). The following conclusions have been reached: tuberculosis with germs showing primary drug resistance did not display onset modalities, clinically and radiologically, that differed significantly from other types of tuberculosis; sputum conversion is more slow in patients with resistant germs than in those with sensitive germs in the first two months of treatment, but following application of the treatment according to the data resulting from the antibiogram, this differences quickly disappeared. The presence of a smaller number of complete recoveries and of a surplus of doubtful recoveries can be explained in the same way in this group of patients: the final results are similar in both groups of patients, evidencing that the treatment of pulmonary tuberculosis due to germs showing parimary drug resistance does not give rise to particular problems.


[Bronchopulmonary carcinosarcomas. Therapeutic indications]. The authors make an analysis of 11 cases of carcinosarcoma that have underwent surgery in the Clinic of Thoracic Surgery. The authors stress the rarity of these cases in their statistics. Only 11 carcinosarcomas have been identified out of a total of 2.500 cases of bronchopulmonary tumors operated in the Clinic. The difficulty of pre-operatory diagnosis is also stressed. Indications for surgical treatment are mandatory in all cases, even when the expected effects are palliative.


[Prevalence of chronic productive cough and obstructive ventilatory dysfunction in a rural community with minimal air pollution]. Results obtained during an epidemiological investigation on chronic cough and obstructive ventilatory dysfunction in subjects without bronchopulmonary affections of known etiopathogeny, carried out in a rural environment with minimal air pollution on 282 men aged between 40 and 69 years, indicate the following: --there was a lower prevalence of chronic cough in the rural environment as compared with the urban one (12.8:21.0). The percentage of heavy smokers being approximately similar in both environments, the difference could be explained by the variable intensity of air pollution, the impact of which was exerted on the large airways (hypertrophy of the serous-mucous glands resulting in hypercrinia with dyscrinia); --similar prevalence in the two environments of the obstructive ventilatory dysfunction (11,0:8,7) suggests that this abnormality, an expression of the narrowing of the small airways, could be due to other pathogenic factors. Functional pulmonary tests with sensitivity higher than the ratio between FEV 1.0 and VC (as, for instance VEmx 50% VC) could provide higher percentages of obstructive disturbances both among chronic coughing and non-coughing subjects, revealing the latent form of the obstruction, as opposed to the manifest one as evidenced by a decrease of the FEV 1.0/VC ratio.


[Correlations between culture methods, double diffusion and immunofluorescence in the laboratory diagnosis of mycoses (candidiasis and aspergillosis)]. Of a total of 1950 patients with various pneumologic affections 115 cases were selected, on the basis of positive mycologic results on examination of the bronchial aspirate. In these patients immunodiffusion and immunofluorescence serological tests were carried out. Confrontation with the clinical and histopathologic diagnosis showed that the simultaneous use of mycological studies (of the bronchial aspirate) and of complex serologic investigations (immunodiffusion, immunofluorescence) allow to make a correcs and aspergilloses.


[Angiography of the liver (author's transl)]. Angiography is a decisive technique of diagnosis of space-occupying processes in the liver. In contrast to laboratory-techniques, scintigraphy and ultrasound which ought to precede it, angiography permits a diagnosis of the type of lesion and is a guide to the chances of surgery on the basis of vascular topography. Angiographic findings in primary and secondary benign and malignant tumors of the liver, of cysts, abscesses and trauma are described and demonstrated on cases. The various forms of portal hypertension are a further field for angiographic diagnosis, explained on examples. The techniques of showing the hepatic arteries, the portal circulation and hepatic veins including direct portography are described and their usefulness for the various diseases discussed. The still little tested therapeutic use of liver angiography in hemorrhage and tumors is mentioned.


[Idiopathic pulmonary hemosiderosis (2 clinical cases)]. The authors present two children with idiopathic pulmonary hemosiderosis, in whom the diagnosis was very difficult on the basis of evolution by stages, hypochromic anemia, hemoptoic sputum containing siderophages, cloudy pulmonary shadow and microscopical skin and lung examination that evidenced marked alterations of the elastic system and siderophages on pulmonary tissue preparations. All the clinical, hematologic and radiological signs were considerably improved by corticoid therapy. The necessity is stressed of such investigations in children with anemia.


Isozymes of creatine phosphokinase and myokinase in human heart and skeletal muscle. The isozyme patterns of creatine phosphokinase (CPK) and myokinase (MK) were investigated in biopsy material from human heart and skeletal muscle. The protein separation was performed on crude muscle homogenates by means of flat-bed isoelectric focusing. Two isozymes were demonstrated for each enzyme, irrespective of the sampling site. The pI values were, on the average, 9.8 (MK-1) and 8.9 (MK-2), 7.2 (CPK-1) and 6.9 (CPK-2), respectively. MK-1 and CPK-1 constituted an average of 81% and 70% of total staining density for each enzyme, respectively. The relative contribution of MK-1 differed, however. Heart muscle showed the highest values (mean 91%) and vastus lateralis the lowest (mean 74%). The mean value for soleus was 86% MK-1. Furthermore, the percentage of MK-1 present was negatively correlated with the percentage of fast twitch fibres in m. vastus lateralis (r = -0.67). No corresponding differences could be demonstrated for CPK isozyme distribution. In conclusion, it was demonstrated that MK and CPK each occurred as two isozymes in human heart and skeletal muscle, and that the relative distribution of MK isozymes, in contrast to CPK, was related to muscle fibre type composition, and thus to the metabolic profile of the muscle.


Increased plasma cyclic AMP concentrations in fasting man. Plasma concentrations of adenosine 3', 5' monophosphate (cyclic AMP), free fatty acids (FFA), glycerol and glucose were measured after 24, 48 and 72 h fasting in healthy male students. There was a marked rise in plasma concentrations of cyclic AMP, FFA and glycerol from 24 to 48 h fasting with no further changes from 48 to 72 h fasting. Plasma glucose concentrations fell. After 72 h fasting, inhibition of adipose tissue lipolysis with a nicotinic acid analogue effected no significant change in plasma cyclic AMP concentrations, indicating that the elevated plasma nucleotide concentrations during fasting were not related to stimulation of lipolysis.


Free radical increases in cancer: evidence that there is not a real increase. The controversial finding of an increase in free radicals with the development of cancer was restudied with Walker 256 carcinosarcoma cells. It was confirmed that such an increase appears to occur, but it was also demonstrated that it is not a real increase. With growth of the tumor, the electron spin resonance lines for lyophilized samples became narrower, resulting in an increase in peak-to-peak height measurements, but there was no change in the total number of spins. The signals for lyophilized tumor samples were different from those for the same samples before lyophilization. Changes in line shape indicated that lyophilization. Changes in line shape indicated that lyophilized tumor samples contain a different mixture of radicals than lyophilized samples of normal tissue.


Regions of cerebral ischemia located by pyridine nucleotide fluorescence. The fluorescence of the reduced form of the endogenous pyridine nucleotide nicotinamide adenine dinucleotide was used to map regions of ischemia in cat brain. A remarkably microheterogeneous pattern of increased fluorescence resulted from a critical level of incomplete cerebral ischemia. The fluorescence pattern suggests that ischemia occurs initially in microwatershed zones between penetrating cerebral arteries.


Phosphoribosylpyrophosphate synthesis in cultured human cells. Phosphoribosylpyrophosphate (PRPP) concentrations and PRPP synthetase activity were studied in cultured human fibroblasts with control and mutant hypoxanthine-guanine phosphoribosylpyrophosphate (HPRT) activity. The PRPP concentrations increased 20- to 50-fold and PRPP synthetase activity 3-fold in cells from patients with the Lesch-Nyham syndrome when aminopterin, an inhibitor of de novo purine synthesis, was added to the medium. Concentrations of PRPP and PRPP synthetase activity did not increase in control cells in medium containing aminopterin unless hypoxanthine was removed from the medium. Exposure of cells to cycloheximide, a protein synthesis inhibitor, prevented the induction of PRPP synthetase and the formation of high PRPP concentrations. Cells from a patient with a mutant HPRT with a high Michaelis constant for PRPP increased PRPP levels and PRPP synthetase activity to a new steady state when they were grown in medium containing aminopterin. Inhibitors of de novo purine synthesis, the presence of hypoxanthine in the medium, and mutant HPRT activity affect the regulation of PRPP levels and PRPP synthetase activity.


Structural basis for on-and off-center responses in retinal bipolar cells. Electron microscopy of Golgi preparations of goldfish retina shows that dendrites of type a (hyperpolarizing, off-center) bipolar cells make wide cleft junctions unassociated with synaptic ribbons, while those of type b (depolarizing, on-center) bioplar cells make narrow cleft junctions and synaptic ribbon contacts, with rods and cones. This suggests that wide cleft junctions are the site of sign-conserving, and narrow cleft junctions or ribbon contacts (or both) are the site of sign-inverting synaptic transmission from photoreceptors to bipolars.


[Muscular and nervous system manifestations of Graves' disease]. A study of 30 cases of Grave's disease confirmed the frequency of hyperthyroid myopathy, whether obvious or revealed by E.M.G. More than by the association of various symptoms (pretibial myxoedema, antithyroid antibodies), we were struck by the multiple symptoms of Grave's disease with myopathy, which confirms the anglo-saxon concept of the polysystemic nature of Grave's disease. This symptomatic richness is not necessarily proportional to the thyroid hypersecretion, and one may suppose that these patients have a greater peripheral sensitivity to the effects of hormone excess, which would explain the multiple clinical signs. The frequency of neuropathy seemed to us more important than usually believed. The clinical signs are often, or absent, or may be masked by the predominance of muscle wasting. If E.M.G. of the distal muscle and measurement of conduction rates were carried out more often as a routine, this would no doubt permit better assessment of the true frequency of neural involvement.


[Action of bromocriptine on glucose metabolism in diabetics]. 24 diabetics, (9 thin diabetics, 14 cases of maturity onset diabetes, 6 intermediate forms and one case of partial pancreatectomy) received 7.5 to 20 mg of bromocriptine (CB 154) per 24 hours. A definite improvement in glucose metabolism was noted in 6 cases, a definite aggravation in 4 cases; 14 results were not significant (p-0.05). The efficacy of CB 154 did not depend on the clinical type nor on the patient's age. It appeared related to duration of the diabetes (2 years and 3 months in improved patients, 11 years in aggravated patients) and perhaps the degree of retinopathy (more frequent and severe in aggravated patients). The improvement seems to be linked to the existence in certain diabetics of a paradoxical regulation of STH secretion as in acromegaly. The possibility of a peripheral effect of bromocriptine on insulin and glucagon is discussed. The CB 154 test (estimations of STH after a single dose of 2.5 mg) permits one to foresee the efficacy of the drug and perhaps the risk of diabetic retinopathy.


[Bilateral malignant exophthalmia as a manifestation of IgM multiple myeloma]. The authors report a case of IgM myeloma revealed by bilateral malignant exophtalmia, only the third case to be published in the world. This exophthalmia was due to plasma cell infiltration of the periorbital fat, and not to primary involvement of the orbit. Other rare localisations were found on autopsy: ovarian, pleuro-pulmonary and, above all, epicardiac. This was an IgM myeloma (0.5% of myelomatous disease) and not Waldenstrom's macroglobulinemia. In fact the patient had no adenopathy, no hepato or splenomegaly, and the tumour contained only plasma cells.


[Evanescent toxic thyroid adenoma. Possible role of phenylpropanolamine]. A patient with a toxic adenoma, already reduced in size by TSH, presented on the third day after treatment of a common cold by phenylpropanolomine, a severe pain in the thyroid gland. 4 weeks later, the nodule, which measured 3 x 4 cm. had clinically disappeared and the scan returned to normal. The disappearance 5 months later of the antithyroid antibodies confirmed the cure. Catecholamines, stimulating the production of thyroid hormone and producing temporary ischemia of the gland, phenylpropanolamine, a sympathomimetic drug, may have caused hemorrhagic necrosis of the adenoma and its disappearance.


The in vitro and in vivo activity of taurolin against anaerobic pathogenic organisms. An antibacterial agent, taurolin, formed by the combination of 2 molecules of taurine with 3 molecules of formaldehyde has been tested against anaerobes in vitro and in vivo. In vitro, taurolin is highly active against Bacteroides species and anaerobic Streptococci as measured by the minimum inhibitory concentration. In vivo, small dosages of taurolin given intraperitoneally increase the mortality in mice with Bacteroides peritonitis. However, by increasing the dosage of taurolin, survival is prolonged and mortality reduced. Intravenously administered taurolin protects rabbits from the effects of Bacteroides fragilis infection whether the organisms are given intravenously or intraperitoneally.


Depletion of replenishment of cellular cyclic adenosine monophosphate in hemorrhagic shock. Hemorrhagic shock in rats was produced by bleeding them to a mean arterial blood pressure of 40 millimeters of mercury, which was maintained for two hours. After sacrifice, small pieces of liver, kidney, muscle and brain were quickly removed and frozen in liquid nitrogen. A protein-free extract of tissues was prepared, and cyclic adenosine monophosphate was measured by the radioimmunoassay procedure. Analysis os liver, kidney, muscle and brain showed that there were significant reductions in cyclic adenosine monophosphate and adenosine triphosphate levels. The decrease in cyclic adenosine monophosphate levels follow the same trend in the various organs as the decreases in adenosine triphosphate levels, suggesting that these two events are related. Infusion of adenosine triphosphate-magnesium chloride, 25 micromoles each, at the end of the shock period restored the cellular cyclic adenosine monophosphate and adenosine triphosphate levels. The precise mechanism for depletion and replenishment of cyclic adenosine monophosphate levels during shock is not now known.


Patterns of combined limb malformations. Seven different limb malformations types were defined in 544 affected newborns, apparently free from other anomalies, obtained from a series of 297,299 livebirths. These seven malformation types were: polydactyly, limb reduction, brachydactyly, symphalangy, syndactyly and split hand/foot. One anomaly type was present in 472 newborns (1.58/1,000) and two or three in 72 (0.24/1,000). The observed combinations of two or three limb malformation types cannot be explained as chance association. Therefore, a common etiopathogenic mechanism has to be considered when two or more limb malformation types are combined in a given individual. The most frequent observed combinations were: reduction-brachydactyly, reduction-syndactyly, brachydactyly-syndactyly, polydactyly-syndactyly, and reduction-brachydactyly-syndactyly. Based on affected limb distribution, sex ratio, and familial recurrence rates, it is suggest that a reduction anomaly is the primary component in all tested combinations while syndactyly tends to be a secondary one when combined with any other limb anomaly type.


Studies of isolated adult rat heart cells: the surface morphology and the influence of extracellular calcium ion concentration on cellular viability. Rat cardiac muscle was dissociated into single cells by a coronary perfusion technique with collagenase and hyaluronidase in a Ca-free medium. Retention of the cylindrical shape of isolated muscle cells could be achieved by regulation of [Ca2+]0 and temperature. Cells kept at 4 degrees C, and 0-01 mM CaCl2 remained cylindrical for more than a week and contracted spontaneously upon warming at 37 degrees C. At [Ca2+]0 between 0-1-2 mM and 37 degrees C, cells underwent contracture and rounded up. Scanning (SEM) and transmission electron microscopy were used to analyze the structure of cylindrical and rounded muscle cells. The extracellular aspect of the sarcolemma at lateral cell surfaces and intercalated disc regions were clearly revealed for SEM analysis. Both the distribution and number of T-tubule openings on the surfaces can be estimated and a three-dimensional description of the intercalated disc obtained. This study reveals that isolated adult heart cells are extremely sensitive to [Ca2+]0, but with careful control of this cation, this preparation should be helpful in the analysis of both sarcolemmal structure and the pathological changes which accompany myocardial injury.