Carcinogenesis of N-nitrosodiethylamine (DENA) in chickens and domestic cats.

The environmental carcinogen N-nitrosodiethylamine (diethylnitrosamine, DENA) has been found to be carcinogenic in 18 animal species. In this study we demonstrate that, after intramuscular injection in chickens, it induces hepatocellular carcinomas of the liver and adenocarcinomas of the kidney. Oral administration in domestic cats causes malignant hepatomas and tumors of the esophagus and kidney. Since DENA is carcinogenic in all species in which it has been tested adequately it can be expected to be a human carcinogen, too.


Circulating immune complexes in rats bearing chemically induced tumours. II. Characterization of sera from different stages of tumour growth.

Sera from rats bearing intraperitoneal implants of an aminoazo dye-induced hepatoma were fractionated by Sephadex G200 and DEAE-cellulose ion exchange column chromatography. Isolated fractions were examined for their capacity to bind [125I]C1q as a measure of immune complex levels, and for their ability to bind soluble tumour-specific antigen as well as to react with antigens expressed at the surface of viable hepatoma cells. Elevated levels of circulating immune complexes in unfractionated serum were directly detectable during early tumour development although, following serum fractionation, immune complexes were identified at both early and late stages of tumour growth. The present findings suggest that the detection of immune complexes in unfractionated samples of late tumour-bearer serum using a C1q-binding assay is masked by the increasing production of tumour-specific antibodies and by a shift from complement fixing to non-complement-fixing tumour-specific antibodies.


Long-term T-cell-mediated immunity to Epstein-Barr virus in man. I. Complete regression of virus-induced transformation in cultures of seropositive donor leukocytes.

Peripheral blood mononuclear cells from donors of known serological status with respect to EB virus were exposed to the virus in vitro and then cultured at various cell concentrations. All cultures from nine seronegative adult and 12 foetal donors gave rise to cell lines following subculture 4 weeks post infection. In contrast, seropositive donor cultures seeded at the higher cell concentrations developed foci of proliferating EBNA-positive cells within the first 1--2 weeks but thereafter regressed completely and subcultures made after 4 weeks never gave rise to cell lines. Out of 18 seropositive donors tested, 15 showed regression in all cultures seeded at 10(6) cells/ml and above, and with the other three donors a proportion of replicate cultures regressed. T-cell depletion and reconstitution experiments showed that the effect was absolutely dependent upon the presence in the cultures of T cells from these seropositive donors. The results strongly suggest that the regression phenomenon is an in vitro expression of long-term T-cell-mediated immunity to EB virus which the large majority, if not all, infected individuals possess.


Malignant lymphoma in four of five siblings.

In one family four out of five siblings were affected with malignant lymphoma and HD has been diagnosed in two other relatives. HLA-haplotypes were identical for the three diseased siblings who were so studied, whereas for the unaffected one they were different.


Antibodies to Herpes simplex virus types 1 and 2 in patients with squamous-cell carcinoma of uterine cervix in India.

Antibody activity to Herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) was measured by the indirect hemagglutination (IHA) test in sera from 124 women with squamous-cell carcinoma of the uterine cervix, 46 women with non-cervical cancer and 116 matched normal women. The type-specific antibodies were assayed by IHA inhibition test. The proportion of cervical cancer cases positive for HSV-2 infection was significantly greater than that of non-cervical cancer cases and matched control women. No difference was observed in the percentages positive for HSV-1 infection among the three study groups. The mean titers of antibodies to HSV-1 as well as HSV-2 were significantly higher than the respective titers in the other two study groups. However, determination of type-specific antibodies did not help in establishing evidence for past infection by HSV-2 in patients with cervical cancer, as no type-specific HSV-2 antibodies were produced in 58% of the cases. The cervical cancer cases were also analysed according to clinical staging (Stages 0 to IV) and histological differentiation of squamous-cell carcinoma. Neither the percentage of positives for HSV-2 infection nor HSV-antibody titers varied, either in clinical staging or in histological differentiation of cervical cancer. These findings suggest that HSV-2 may be related to cervical cancer as an etiological agent but not as a passenger virus of carcinomatous tissue.


An e.s.r. and spin-trapping study of the reactions of the SO4- radical with protein and nucleic acid constituents.

Reactions of the SO4- radical, generated by U.V. photolysis of Na2S2O8, were studied in aqueous solutions of amino acids, dipeptides, nucleic acid bases, nucleosides and nucleotides. The transient free radicals so formed were spin-trapped by t-nitrosobutane and identified by e.s.r. spectroscopy. The amino acids primarily undergo oxidative decarboxylation. The pKs of the ammonium groups of the spin-trapped decarboxylated radicals of glycine and alanine in D2O were determined to be 8.3 +/- 0.2. An oxidation product, which is the precursor of the decarboxylated radical, is tentatively identified for alanine, valine and isoleucine. Radicals formed by hydrogen abstraction by SO-4 are identified for leucine, serine, phenylalanine and 4-hydroxyproline. In dipeptides, SO-4 produces decarboxylation of the amino acid located at the carboxylate terminal residue. For gly-ala and ala-ala, radicals generated by hydrogen abstraction from the carboxylate terminal residue alanine were also characterized. Radicals centered on the C(5) carbon were observed for uracil, cytosine and thymine. For nucleosides and nucleotides, radicals situated on the base and/or the sugar moiety were assigned.


On the e.s.r. determination of radical yields in X-irradiated amino acids.

Studies of the temperature dependence of the areas under the e.s.r. absorption spectra of X-ray-induced free radicals in amino acids at various power levels and of power saturation at different temperatures are reported. They indicate that power saturation is responsible for the anomalous Curie--Weiss behaviour previously reported. The consequences of power saturation to the e.s.r. determination of radical yields are discussed. The effect of impurity e.s.r. signals in amino acids on quantitative e.s.r. determinations is also discussed.


[Classification of vascular neoplasms].

Neoplasms of blood and lymph vessels differ from angiectatic and angiokeratotic nevi by real proliferating growth. According to their features of growth and their wall structures, they are classified into three main groups: angiomas, glomangiomas and malignant vascular tumors. Within the angiomas on the one hand, capillary angiomas are classified into: planotuberous and tuberonodous angiomas of childhood and Kasabach-Merritt syndrome, multilocular hemangiomatosis, progressive multiple angiomas, tardive ("senile") angiomas, eruptive angiomas (granulomata pediculata), papular angioplasia, gemmangioma, and benign juvenile hemangioendothelioma. On the other hand, cavernous angiomas, i.e. arterial and venous cavernomas, as well as blue rubberbleb nevus, Mafucci's syndrome, angioleiomyoma, benign juvenile hemangiopericytoma and cavernous lymphangioma, form thick walled structures without involution. Glomangiomas occur as solitary, multiple systematized, and multiple disseminated and familiar forms. Within the group of malignant vascular tumors--Kaposi sarcoma, lymphangiosarcoma in lymphedema, hemangioendothelioma and angioplastic reticulosarcoma, hemangio- or lymphangiosarcoma, angioendotheliomastosis proliferans, rarity and increasing loss of characteristic differentiated structures give rise to difficulties in nosologic classification.


[Multinodular reticulohistiocytosis in the adult. Clinical picture, histology and electron microscopy].

Multiple nodular infiltrates of reticulohistiocytic cells may progressively develop in human adult skin showing intracellular lipid storage (neutral lipids, phospholipids, free cholesterin), without changes of the lipid levels in blood. These are obviously benign reactive lesions, which may spontaneously disappear. Concomitant skin or systemic diseases may occur. According to the individual data of each case several names are given to the skin lesions which seem to represent variants of the same entity. The cell infiltrate may predominantly consist of histiocytes, giant cells or lipid storing (foam) cells. Ultrastructural examination did not reveal specific types of lipids or specific types of intracellular storage; however, it seems necessary, in order to differentiate this entity from histiocytosis X: dermal Langerhans cells are lacking in this disease.


Histoenzymological analysis of mesencephalic auditory, tegmental and cranial nerve nuclei in the frog (Rana tigrina).

The distributions of acid and alkaline phosphatases, 5-nucleotidase, ATPase, non-specific esterase, specific cholinesterase, succinic dehydrogenase and beta-galactosidase are described in the mesencephalic auditory, tegmental and cranial nerve nuclei of the frog (Rana tigrina). The main results of the study are as follows: The laminar, principal, and magnocellular nuclei of the torus semicircularis, which are associated with auditory functions, show intense activity of specific cholinesterase. On the other hand, the commissural and subependymal mid-line nuclei, whose functions are doubtful, show a complete lack of this enzyme. The nucleus isthmi shows intense acid phosphatase, ATPase, non-specific esterase, specific cholinesterase and succinic dehydrogenase activities. Non-specific esterase is virtually absent from all the areas studied except the nucleus isthmi and the 3rd and 4th cranial nerve nuclei. Most of the commissures and fibre tracts show intense activity for beta-galactosidase and 5-nucleotidase. The possible roles of these enzymes in glycolipid and myelin metabolism are discussed.


Isolation and characterization of a membrane-bound, low-potential c-type cytochrome from purple photosynthetic bacteria, with special reference to Rhodospirillum rubrum.

Other investigators have isolated soluble, low-potential, c-type cytochromes (cytochrome c3) from a few photosynthetic procaryotes, i.e., a cyanobacterium and two species of purple nonsulfur bacteria. However, such cytochromes appeared to be absent from other purple bacteria, including Rhodospirillum rubrum and Chromatium vinosum. We now report evidence for the presence of low-potential c-type cytochromes in these two species, in which they were found to be bound to the photosynthetic membranes. Evidence for a membrane-bound, low-potential c-type cytochrome was also found in Rhodopseudomonas sphaeoides. The low-potential c-type cytochrome of R. rubrum was solubilized by a Triton X-100 treatment of chromatophores and was partly purified. It was found to have a molecular weight of about 17,000, a midpoint oxidation-reduction potential of -192 mV, and an alpha-absorption peak at 552 nm. It appears that low-potential c-type cytochromes may be present in all purple photosynthetic bacteria, of both the sulfur and the nonsulfur types.


Isolation and characterization of extragenic suppressor strains of Corynebacterium diphtheriae.

The isolation and characterization of two different nonsense suppressor strains of Corynebacterium diphtheriae C7 sup+(-)tox- are described. Appropriate lysogens of these strains with corynephage beta, carrying known class II tox premature polypeptide chain termination mutations [C7sup-1(betatox-30) and C7sup-2(betatox-45)], each produce a 62,000-dalton polypeptide with nicotinamide adenine dinucleotide: elongation factor-2 adenosine diphosphate ribosyltransferase activity in addition to a chain-terminated polypeptide of 30,000 or 45,000 daltons, respectively. In addition, purified protein of 62,000 daltons, resulting from the suppression of the nonsense mutations tox-30 and tox-45, will react with antisera purified against the terminal 17,000 daltons of the toxin molecule and are immunologically identical to toxin by radial immunodiffusion. The suppression pattern of lysogenic derivatives of C7sup-1(-)tox- and C7sup-2(-)tox- with other class II and III mutants of corynephage beta was determined.


Catabolite-repression-like phenomenon in Rhizobium meliloti.

We report a phenomenon similar to catabolite repression in Rhizobium meliloti. Succinate, which allows the highest observed rate of growth of R. meliloti, caused an immediate reduction of beta-galactosidase activity when added to cells growing in lactose. A Lac- mutant was unaltered in nodulation and nitrogen fixation capacities, but a pleiotropic mutant deficient in several catabolic properties was unable to produce effective nitrogen-fixing nodules.


Effects of altered rho gene product on the expression of the Escherichia coli histidine operon.

An altered rho gene product affects expression of the his operon of Escherichia coli. The effect is greater for the operator proximal portion of the his operon than for the operator distal portion. This "rho effect" appears to be independent of the site of action of hisT-altered histidyl-tRNA.


Cell fractions and enzymatic activities of Ureaplasma urealyticum.

The localization of some enzymic activities in cell fractions of Ureaplasma urealyticum was studied. A quantitative evaluation of the effectiveness of several cell lysis procedures was obtained by using labeled membranes and sucrose density gradient centrifugation. Ultrasonic treatment was found to be the most effective procedure for lysing the cells, whereas digitonin and osmotic shock caused the lysis of only 70 and 50% of the cells, respectively. The localization of selected enzymes in Ureaplasma cells resembled that found in other Mycoplasma species. Adenosine triphosphatase, ribonuclease, deoxyribonuclease, and p-nitrophenylphosphatase activities were located exclusively in the membrane fraction, whereas urease and L-histidine ammonia-lyase were located in the cytoplasm.


Co-induction of beta-galactosidase and the lactose-P-enolpyruvate phosphotransferase system in Streptococcus salivarius and Streptococcus mutans.

The addition of lactose, galactose, or isopropyl-beta-D-thiogalactoside (IPTG) to glucose-grown cells of Streptococcus salivarius 25975 resulted in the co-induction of both the lactose-P-enolpyruvate phosphotransferase system (lactose-PTS) and beta-galactosidase, with the latter the predominant metabolic system. With various strains of Streptococcus mutans and Streptococcus sanguis 10556, on the other hand, the lactose-PTS was the major metabolic pathway with beta-galactosidase induced either to low or negligible levels. In all cases, induction of the lactose-PTS resulted in the concomitant induction of 6-P-beta-galactosidase. The induction by lactose of both the lactose-PTS and beta-galactosidase in all strains was repressed by glucose and other catabolites, notably, fructose. Induction of beta-galactosidase in S. salivarius 25975 by IPTG was, however, relatively resistant to glucose repression. Induction experiments with IPTG and lactose suggested that a cellular metabolite of lactose metabolism was a repressor of enzyme activity. Exogenous cAMP was shown to reverse the transient repression by glucose of beta-galactosidase induction in cells of S. salivarius 25975 receiving lactose, provided the cells were grown with small amounts of toluene to overcome the permeability barrier to this nucleotide, cAMP, was however, unable to overcome the permanent repression of beta-galactosidase activity to a significant extent under these conditions.


Regulation of lac operon expression: reappraisal of the theory of catabolite repression.

The physiological state of Escherichia coli with respect to (permanent) catabolite repression was assessed by measuring the steady-state level of beta-galactosidase in induced or in constitutive cells under a variety of growth conditions. Four results were obtained. (i) Catabolite repression had a major effect on fully induced or constitutive expression of the lac gene, and the magnitude of this effect was found to be dependent on the promoter structure; cells with a wild-type lac promoter showed an 18-fold variation in lac expression, and cells with the lacP37 (formerly lac-L37) promoter exhibited several hundred-fold variation. (ii) Exogenous adenosine cyclic 3',5'-monophosphoric acid (cAMP) could not abolish catabolite repression, even though several controls demonstrated that cAMP was entering the cells in significant amounts. (Rapid intracellular degradation of cAMP could not be ruled out.) (iii) Neither the growth rate nor the presence of biosynthetic products altered the degree of catabolite repression; all variation could be related to the catabolites present in the growth medium. (iv) Slowing by imposing an amino acid restriction decreased the differential rate of beta-galactosidase synthesis from the wild-type lac promoter when bacteria were cultured in either the absence or presence of cAMP; this decreased lac expression also occurred when the bacteria harbored the catabolite-insensitive lacP5 (formerly lacUV5) promoter mutation. These findings support the idea that (permanent) catabolite repression is set by the catabolites in the growth medium and may not be related to an imbalance between catabolism and anabolism.


Stoichiometric studies on the oxidation of tetrahydropterin with ferri-cytochrome c.

The oxidation of tetrahydropterin with ferri-cytochrome c was studied using a tetrahydropterin-generating system composed of dihydropteridine reductase [EC 1.6.99.7] and NADH. Under aerobic conditions, 1.5 to 1.8 mol of cytochrome c was reduced per mol of NADH, whereas 2 mol of cytochrome c was reduced under anaerobic conditions. When superoxide dismutase [EC 1.15.1.1] was added to the system under aerobic conditions, only 1 mol of cytochrome c was reduced per mol of NADH, while the pterin oxidation was scarcely affected. Based on these results, we propose that the oxidation of tetrahydropterin to quinonoid dihydropterin proceeds via two steps: tetrahydropterin is first oxidized by ferri-cytochrome c to give a pterin intermediate, which has lost one electron, then in turn this reduces O2 to form O2-.


Diffusion-potential-induced oxidation and reduction of cytochromes in chromatophores from Rhodopseudomonas sphaeroides.

A membrane potential jump was induced by the addition of valinomycin in the presence of a KCl concentration gradient across the membrane of Rhodopseudomonas sphaeroides chromatophores. As well as a carotenoid band shift, which is known to be an indicator of membrane potential, absorbance changes due to the oxidation-reduction reactions of cytochromes accompanied the jump. Under aerobic conditions with no reductant added, a part of cytochrome c2 was reduced by an inside-positive potential jump of about 100 mV in the time range of tens of seconds. This can be explained by the location of the cytochrome on the inner side of the chromatophore membrane and electrophoretic flow of electrons across the membrane. On the other hand, in the presence of 1 mM ascorbate, a similar jump of membrane potential induced a rapid oxidation of cytochrome c2 and a subsequent reduction. A rapid reduction of b-type cytochrome was also observed. Antimycin A inhibited the c2 oxidation, but did not inhibit the b reduction. The oxidation of cytochrome c2 may be explained by a diffusion-potential-induced electron flow to cytochrome b and a simultaneous electron donation by cytochrome b and cytochrome c2 to a common electron acceptor, possibly a quinone.


A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function.

The biosynthesis of a secretory protein and a transmembrane viral glycoprotein are compared by two different experimental approaches. (a) NH2-terminal sequence analysis has been performed on various forms of the transmembrane glycoprotein of vesicular stomatitis virus synthesized in cell-free systems. The sequence data presented demonstrate that the nascent precursor of the glycoprotein contains a "signal sequence" of 16 amino acids at the NH2 terminus, whose sequence is Met-Lys-Cys-Leu-Leu-Tyr-Leu-Ala-Phe-Leu-Phe-Ile-(His-Val-Asn)-Cys. This signal sequence is proteolytically cleaved during the process of insertion into microsomal membranes prior to chain completion. The new NH2 terminus of the inserted, cleaved, and glycosylated membrane protein is located within the lumen of the microsomal vesicles and is identical to that of the authentic glycoprotein from virions. (b) Nascent chain competition experiments were performed between this glycoprotein, bovine pituitary prolactin (a secretory protein), and rabbit globin (a cytosolic protein). It was found that the nascent membrane glycoprotein, but not nascent globin, competed with nascent prolactin for membrane sites involved in the early biosynthetic event of transfer across membranes. These data suggest that an initially common pathway is involved in the biogenesis of secretory proteins and at least one class of integral membrane proteins.


Calcium-dependent 3':5'-cyclic nucleotide phosphodiesterase. Inhibition of basal activity at physiological levels of potassium ions.

A calcium-dependent cyclic nucleotide phosphodiesterase from rat cerebrum was, in the absence of activator protein, inhibited by various monovalent cations. The inhibition was rapid, readily reversible, and concentration-dependent, with 100 mM cesium, rubidium, or potassium ion inhibiting essentially all basal enzyme activity, while 100 mM sodium or lithium ions produced only moderate inhibition. The potency of the cations in inhibiting the enzyme was Cs greater than or equal to Rb greater than K greater than Na greater than or equal to Li. Potassium ions increased the apparent Km for cyclic GMP and cyclic AMP by 3- and 5-fold, respectively. At 100 mM, the monovalent cations inhibited enzyme activated by the calcium-dependent activator by only 15 to 30%, while at 55 mM no inhibition pertained. Potassium and sodium ions at 55 mM had no effect on the calcium-independent phosphodiesterase from rat cerebrum. The results indicate that at normal intracellular concentrations of potassium ions the activity of the calcium-dependent phosphodiesterase is virtually completely dependent on the presence of calcium plus activator protein.


Substrate inhibition of the mitochondrial and cytoplasmic malate dehydrogenases.

The mechanism that leads to an inhibition of enzyme activity in the presence of high concentrations of substrate was investigated with the two malate dehydrogenase isoenzymes obtained from pig heart. The inhibition is promoted by an abortive binary complex formed by the enzymes and the enol form of of oxalacelate. Neither the oxidized coenzyme nor the reduced coenzyme appears to be involved in the formation of this complex. These results suggest that the mechanism of substrate inhibition that occurs with the pig heart malate dehydrogenases is different from that observed with the lactate dehydrogenases from chicken hearts. The inhibition constants for oxalacetate are 2.0 mM with the mitochondrial enzyme and 4.5 mM with the cytoplasmic enzyme. Since the in vivo concentration of oxalacetate is reported to be about 10 micrometer, these data suggest that the substrate inhibition that is exhibited by the malate dehydrogenases may not be of any significance in vivo.


Characterization of NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system from Pseudomonas arvilla c-1.

NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system, was purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis and ultracentrifugation, from benzoate-induced cells of Pseudomonas arvilla. The molecular weight of the enzyme was determined to be 38,300 by sedimentation equilibrium analysis, 37,000 by Sephadex G-100 gel filtration, and 37,500 by sodium dodecyl sulfate disc gel electrophoresis, respectively, indicating that the enzyme consisted of a single polypeptide chain. The sedimentation coefficient was calculated to be 3.3 S. The Stokes radius for the enzyme was calculated to be 27 A. The isoelectric point of the enzyme was estimated to be pH 4.2. The enzyme contained 1 mol of FAD, 2 mol of iron, and 2 mol of labile sulfide/mol of enzyme. It exhibited absorption spectrum with maxima at 273, 340, 402, and 467 nm. Amino acid analysis of the enzyme revealed that it was devoid of tryptophan. The enzyme contained 9 mol of cysteine/mol of enzyme but no disulfide linkage. The turnover number of the enzyme for the NADH-dependent reduction of cytochrome c was 17,100 at 24 degrees C. Although NADPH also acted as an electron donor, NADH was highly superior to NADPH. Ferricyanide and 2,6-dichlorophenolindophenol served as electron acceptors. Certain other properties of the enzyme are also presented.


Heterogeneity of amino acid sequence in hippopotamus cytochrome c.

The amino acid sequences of chymotryptic and tryptic peptides of Hippopotamus amphibius cytochrome c were determined by a recent modification of the manual Edman sequential degradation procedure. They were ordered by comparison with the structure of the hog protein. The hippopotamus protein differs in three positions: serine, alanine, and glutamine replace alanine, glutamic acid, and lysine in positions 43, 92, and 100, respectively. Since the artiodactyl suborders diverged in the mid-Eocene some 50 million years ago, the fact that representatives of some of them show no differences in their cytochromes c (cow, sheep, and hog), while another exhibits as many as three such differences, verifies that even in relatively closely related lines of descent the rate at which cytochrome c changes in the course of evolution is not constant. Furthermore, 10.6% of the hippopotamus cytochrome c preparation was shown to contain isoleucine instead of valine at position 3, indicating that one of the four animals from which the protein was obtained was heterozygous in the cytochrome c gene. Such heterogeneity is a necessary condition of evolutionary variation and has not been previously observed in the cytochrome c of a wild mammalian population.


Inhibition of cyclic AMP-dependent protein kinase by analogues of a synthetic peptide substrate.

Analogues of the synthetic substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in which the serine is replaced by other amino acids inhibited the activity of the catalytic subunit of cyclic AMP-dependent protein kinase from beef skeletal muscle (Peak I). All of the analogues were competitive with respect to peptide substrate but apparent Ki values varied depending on the particular amino acid that was substituted for serine. Inhibition was also competitive with respect to mixed histone as determined in experiments utilizing one of the analogues. Acetylation of the terminal amino group of Leu-Arg-Arg-Ala-Ser-Leu-Gly lowered the Km for this substrate from 16 micrometer to 3 micrometer, but a similar modification of the inhibitory analogue Leu-Arg-Arg-Ala-Ala-Leu-Gly resulted in no major change in the Ki value. An amount of inhibitory peptide sufficient to inhibit the cyclic AMP-dependent protein kinase by 90% caused less than 10% inhibition of several cyclic AMP-independent protein kinases indicating a high degree of specificity of inhibition by the peptide analogues. The experiments show that synthetic peptide analogues could be useful in identifying phosphorylation reactions catalyzed by cyclic AMP-dependent protein kinase as distinguished from other protein kinase reactions.


Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts.

The low density lipoprotein (LDL) cell surface receptors on human fibroblasts grown in culture bind specific plasma lipoproteins, initiating a series of events which regulate intracellular cholesterol metabolism. Specificity for the interaction with the receptors resides with the protein moieties of the lipoproteins, specifically with the B and E apoproteins of LDL and certain high density lipoproteins (HDLc HDLl), respectively. It was previously established that the amino acid arginine is a functionally significant residue in or near the recognition sites on the B and E apoproteins and that modification of this residue abolishes the ability of these apolipoproteins to bind to the receptor. The present study indicates that lysine residues are also involved in the lipoprotein-receptor interaction. Chemical modification of 15% of the lysine residues of LDL by carbamylation with cyanate or 20% by acetoacetylation with diketene prevents the LDL from competitively displacing unmodified 125I-LDL from the high affinity receptor sites or from binding directly to the receptor. Moreover, quantitative reversal of the aceto-acetylation of the lysine residues of LDL by hydroxylamine treatment regenerates the lysyl residues and reestablishes greater than 90% of the original binding activity of the LDL. The reversibility of this reaction establishes that the loss of binding activity which follows lysine modification is not due to an irreversible alteration of the LDL or HDLc but is probably due to an alteration of a property of the recognition site associated with specific lysine residues. While acetoacetylation and carbamylation neutralize the positive charge on the epsilon-amino group of lysine, reductive methylation selectively modifies lysine residues of LDL and HDLc without altering the positive charge, yet abolishes their ability to bind to the receptor. Preservation of the charge but loss of binding activity following reductive methylation of the lipoproteins suggests that the specificity of the recognition site does not reside simply with the presence of positive charges but depends on other more specific properties of the site determined by the presence of a limited number of the lysine (and arginine) residues. The precise role of lysine remains to be defined, but its function may be to establish and maintain the conformation of the recognition site or the alignment of reactive residues, or both, or to chemically react, through its epsilon-amino group, with the receptor (hydrogen bond formation would be such a possibility).


A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella.

Treatment of demembranated sea urchin sperm axonemes with an extraction solution containing 0.6 M NaCl, pH 7.0 for 10 min at 4 degrees C yields a solution of dynein 1 having a low, latent specific ATPase activity of about 0.25 mumol of Pi mg(-1) min(-1). Exposure of this dynein solution to 0.1% Triton-X-100 for 10 min at 25 degrees C causes an increase in its ATPase activity to about 3 mumol of Pi mg(-1) min(-1). A similar activation can be obtained by treating at 42 degrees C or by reacting with 60 mol of p-chloromercuribenzene sulfonate/10(6) g of protein. The effects of these activating procedures are not additive, suggesting that they lead to a common activated state. Purification of the latent activity dynein 1 by sucrose density gradient centrifugation yields a monodisperse preparation sedimenting at 21 S, and having a molecular weight of 1,250,000 as determined by sedimentation diffusion and sedimentation equilibrium. Activation of the latent dynein 1 with Triton X-100 converts it to a form sedimenting at 10 to 14 S. The 21 S dynein is also converted to a 10 S form by dialysis against 5 mM imidazole/NaOH buffer, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, pH 7, although in this case, the ATPase activity is increased only about 3-fold, with another 3-fold activation being obtainable upon subsequent treatment with Triton X-100. The 21 S latent form of dynein 1 may represent the intact dynein arms that form moving cross-bridges and generate active sliding between adjacent doublet tubules of the flagellar axoneme. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate suggests a model in which the 21 S dynein 1 particle is composed of three subunits of about 330,000 daltons and one of each of three medium weight subunits of 126,000, 95,000, and 77,000 daltons. When latent dynein 1 is added back to NaCl-extracted axonemes in the presence of 0.15 M NaCl, it recombines stoichiometrically and restores the arms on the doublet tubules with a 6-fold activation of its ATPase activity measured in the absence of KCl.


Induction of metabolic changes and down regulation of bovine parathyroid hormone-responsive adenylate cyclase are dissociable in isolated osteoclastic and osteoblastic bone cells.

Bovine parathyroid hormone (PTH), dibutyryl cAMP, and calcium each induce similar metabolic changes in isolated bone cells. PTH and calcium, but not dibutyryl cAMP, result in desensitization of osteoclastic and osteoblastic bone cells to PTH. In osteoblastic cells, calcium effects are specific for PTH receptor.adenylate cyclase complexes and responsiveness to other hormones is not reduced while in osteoclastic cells, small effects of high calcium on prostaglandin E1- and epinephrine-inducible cAMP accompany the large decreases seen in cAMP response to PTH. The membrane effects of calcium and of PTH appear to be independently regulated as PTH-induced desensitization can be initiated in the absence of calcium. In addition, calcium effects on PTH-sensitive adenylate cyclase follow a different calcium dose-response than PTH-like metabolic changes. These results suggest that the effect of calcium on the membrane is not directly related to its induction of PTH-like metabolic changes. A possible role of calcium as an in vivo regulator of bone cell sensitivity to PTH is discussed.


Isoproterenol-induced desensitization of adenylate cyclase in human astrocytoma cells. Relation of loss of hormonal responsiveness and decrement in beta-adrenergic receptors.

Incubation of human astrocytoma cells (1321N1) with low concentrations of isoproterenol results in a specific loss of responsiveness to catecholamines as evidenced by a decreased accumulation of cAMP in intact cells, a reduction in isoproterenol-stimulated adenylate cyclase activity, and a decrease in beta-adrenergic receptor density, as measured by the specific binding of 125I-hydroxybenzylpindolol. The kinetics of desensitization suggest the involvement of two different reactions. The initial reaction involves a rapid loss of adenylate cyclase activity with little loss of beta-adrenergic receptors. Subsequently, a slower reaction results in the loss of measurable beta-adrenergic receptors. The degree of loss of both parameters was similar after 24 h of desensitization. It is concluded that the loss of beta-adrenergic receptors is an event that occurs as a result of the initial uncoupling of the beta-receptor-linked adenylate cyclase.


Evidence in intact cells for an involvement of GTP in the activation of adenylate cyclase.

Treatment of cultured normal rat kidney cells with virazole or mycophenolic acid which are inhibitors of IMP dehydrogenase decreases by 50 to 70% the ability of prostaglandin E1 or isoproterenol to elevate cAMP levels. Inhibition is maximal by 2 h. The response to cholera toxin is not significantly decreased. Basal cAMP is not affected. Under these conditions, GTP is decreased by 80%, ATP is only 10 to 15% decreased, and UTP and CTP are slightly increased. Normal GTP levels and the responses to prostaglandin E1 and isoproterenol are restored if guanosine, but not inosine, is added with the inhibitor. The response to isoproterenol is recovered within 5 min after removal of mycophenolic acid. Desensitization to prostaglandin E1 or isoproterenol stimulation occurs under conditions where GTP is 80% decreased. These results in intact cells provide direct evidence for a role for GTP in the activation of adenylate cyclase and support previous conclusions from studies with cell homogenates.


Radiologic findings in nasopharyngeal angiofibromas.

Juvenile nasopharyngeal angiofibromas produce radiographic changes which are sufficiently characteristic to render a diagnosis without resorting to biopsy. Computed tomography is also of value, particularly in following the response of these tumors to therapeutic irreadiation.


Tracheal histiocytoma: an inflammatory pseudotumor.

A tracheal histiocytoma produced airway obstruction in a child. These lesions contain histiocytic, fibrous, and xanthomatous elements and represent benign post-inflammatory reactions without predisposition to malignancy. Treatment is conservative surgical excision.


Endoplasmic reticulum marker enzymes in Golgi fractions--what does this mean?

NADPH cytochrome c (cyt c) reductase and glucose-6-phosphatase, two enzymes thought to be restricted to the endoplasmic reticulum (ER) and widely used as ER markers, are present in isolated Golgi fractions assayed immediately after their isolation. Both enzymes are rapidly inactivated in fractions stored at 0 degrees C in 0.25 M sucrose, conditions which do not affect the activity of other enzymes in the same preparation. The inactivation process was shown to be dependent on time and protein concentration and could be prevented by EDTA and catalase. Morphological evidence shows that extensive membrane damage occurs parallel with the inactivation. Taken together with the immunological data in the companion paper, the findings indicate that the enzymes NADPH cyt c reductase and probably glucose-6-phosphate are indigenous components of Golgi membranes.


Presence of NADPH-cytochrome P-450 reductase in rat liver Golgi membranes. Evidence obtained by immunoadsorption method.

Light Golgi fractions (GF(1+2)) prepared from rat liver homogenates by a modification of the Ehrenreich et al. procedure (J. Cell Biol. 59:45) had significant NADPH-cytochrome P(450) reductase (NADPH-cyt c reductase) activity if assayed immediately after their isolation. An antibody raised in rabbits against purified microsomal and Golgi fractions. To find out whether this activity is located in bona fide Golgi elements or in contaminating microsomal vesicles, we used the following 3-step immunoadsorption procedure: (a) antirabbit IgG (raised in goats) was conjugated to small (2-5 mum) polycrylamide (PA) beads; (b) rabbit anti NADPH-cyt c reductase was immunoadsorbed to the antibody-coated beads; and (c) GF(1+2) was reacted with the beads carrying the two successive layers of antibodies. The beads were then recovered by centrifugation, and were washed, fixed, embedded in agarose, and processed for transmission electromicroscopy. Antireductase- coated beads absorbed 60 percent of the NADPH-cyt c reductase (and comparable fractions of NADH-cyt c reductase and glucose-6-phosphatase) but only 20 percent of the galactosyltransferase activity of the input GF(1+2). Differential vesicle counts showed that approximately 72 percent of the immunoadsorbed vesicles were morphologically recognizable Golgi elements (vesicles with very low density lipoprotein [VLDL] clusters or Golgi cisternae); vesicles with single VLDL and smooth surfaced microsome-like vesicles were too few (approximately 25 percent) to account for the activity. It is concluded that NADPH-cytochrome P(450) reductase is a Golgi membrane enzyme of probably uneven distribution among the elements of the Golgi complex.


Computer simulation of aggregation in Dictyostelium discoideum.

The aggregation phase of development of the cellular slime mould Dictyostelium discoideum is simulated on a computer. The simulation is performed in 2 dimensions, and produces animated graphical output similar to time-lapse films. It is based on observations of cell behaviour as expressed by a set of rules for each cell, involving cell movement and release of and response to chemical signals. Following these rules, the simulation has reproduced many observed aggregation patterns: propagating waves of cell movement; formation of branching streams; entrainment of slower centres; and spiral centre formation. The simulation has proved to be an important adjunct to experimental work.


SV40-transformed hamster cells resistant to 100--250 microgram/ml of ethidium bromide.

SV40-transformed hamster cells were selected for resistance to ethidium bromide (EB). Several cell lines were established, which grew in the presence of up to 250 microgram/ml EB. The EB resistance is genetically stable. The cloned resistant cells show no difference in morphology, with the exception of the mitochondrial ultrastructure, which exhibits condensed cristae formation. The tumorigenicity of these cells in Syrian gold hamsters is considerably reduced. Incorporation of radioactive labelled thymidine into mitochondrial DNA is not influenced by the presence of the drug. Gel electrophoresis with mitochondrial proteins from wild-type and resistant cells reveals significantly different patterns. The mechanism of EB resistance is discussed.


Stimulation of dendritogenesis in the epidermal melanocytes of newborn mice by melanocyte-stimulating hormone.

The number of dendrites and the total length of dendrites in the epidermal melanocytes positive for the dopa reaction were shown to increase when newborn mice were injected with alpha-MSH or DBc-AMP. Moreover, both indices of the degree of dendritogenesis increased when skin explants of newborn mice were cultured in medium containing alpha-MSH or DBc-AMP. Electron-microscopic observation showed that the number of melanosomes was also increased by alpha-MSH treatment. Many mature melanosomes were observed in the dendrites of the epidermal melanocytes of alpha-MSH-injected mice. Highly dendritic melanocytes seem to be the cells stimulated by MSH to form melanosomes and translocate them to dendrites. Dendritogenesis stimulated by the hormone was suppressed by actinomycin D or cycloheximide, suggesting that the dendritogenesis in the epidermal melanocytes requires de novo transcription and translation.


Contact behaviour and pattern formation of BHK and polyoma virus-transformed BHK fibroblasts in culture.

Certain behavioural and morphological aspects of cellular transformation have been studied, using baby hamster kidney cells (BHK21/13) and polyoma virus-transformed BHK cells. BHK cellsgrow to monolayer arranged in parallel arrays, whereas the transformed cells show a much greater incidence of crisscrossing and multilayering. Time-lapse cinemicrography and scanning electron microscopy were used to examine the behaviour producing these striking differences in cellular pattern. It was found, contrary to previous thought, that in both cell lines, when contact is made ruffle to ruffle, ruffling is inhibited. When BHK cell contact each other ruffle to side, strong adhesions always occur, as evidence by a large deformation of the contacted cell margin, with accompanying paralysis of ruffling. Then, the contacting cell either changes direction, usually spreading along the side of the contacted cell, or occasionally continues to protrude and underlap the other cell, although the original adhesions are seen to remain. Transformed cells never form strong ruffle-to-side adhesions, and usually underlap each other totally. When the cells were filmed, fixed and the same cells relocated in the scanning electron microscope, neither cell type was seen to move over the surface of another (overlap). Rather, all cells crisscross by underlapping (moving under the other cell). SEM also reveals PyBHK cells to have many fewer side-to-substratum adhesions than BHK cells. The smaller number of these attachments could explain the ease with which PyBHK cells underlap.


Plastic multiwell plates to assay avian infectious bronchitis virus in organ cultures of chicken embryo trachea.

Simple assay systems for infectivity titrations of avian infectious bronchitis virus (IBV) in chicken embryo trachea organ cultures (OC) were developed using plastic multiplate wells with one tracheal ring per well; these assays appeared to be much more satisfactory than the conventional rolled-tube method. The medium, 0.05 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered Eagle minimal essential medium was not changed during observation. A medium containing 0.4% bovine serum albumin did not influence the virus yield, but did stabilize virus viability during storage. Reproducibility of results obtained in the OC system was confirmed by performing replicate titrations of the Beaudette strain with three different passage histories. The mean virus titers in the OC were lower than those in chicken embryos, depending on the IBV passage histories. The time required for ciliostasis was related not only to the concentration of virus, but also to the IBV passage history. Application of OC techniques for the constant serum-variable virus neutralization test gave low neutralization indexes with excellent reproducibility as compared with those obtained in the chicken embryo assay system. Also, the slopes of neutralization curves obtained by assays in OC were less steep than those seen in the chicken embryo system.


Center for Disease Control Diagnostic Immunology Proficiency Testing Program results for 1977.

A summary of the yearly data accumulated and analyzed in the diagnostic immunology portion of the Center for Disease Control 1977 Proficiency Testing Program provides information on the trends in testing protocols from over 900 participating laboratories. One hundred and fourteen specimens prepared by the Center for the Disease Control were distributed quarterly or in special surveys. The specimens chosen provided feedback from a broad range of tests commonly performed in diagnostic immunology laboratories and included those for rubella antibodies, hepatitis B surface antigen, bacterial antibodies, rheumatoid factor, immunoglobulins and other serum-specific proteins, syphilis serology, and carcinoembryonic antigen. This summary provides an overview of the trends observed since last year's report.


Role of rotavirus (reo-like) in weanling diarrhea of pigs.

Piglets weaned abruptly and precociously at 3 weeks of age and placed in a crowded nursery commenced diarrhea 3 to 5 days later. Death losses were low (approximately 6%), but weight gain ceased for 2 weeks. Large numbers of rotavirus (reo-like) particles were seen by electron microscopy in diarrhetic fluids. Sections of intestines showed a loss of adsorptive surface in that villi were shortened and fused with adjacent villi. Immunofluorescence revealed rotaviral antigens within damaged enterocytes. Rotavirus-containing gut fluid was harvested from sick, weaned piglets. This fluid, filtered free of bacteria, was used to inoculate per os colostrum-deprived piglets. These infected piglets developed diarrhea and dehydration, and large numbers of rotaviral particles were seen in their diarrhetic fluid. Also, rotaviral antigens were present in aberrant enterocytes, and the intestinal villi were shortened. Since the weaned piglets (3 weeks old) came from sows that were providing their piglet's intestine with passive antibody protection via milk, we concluded that the abrupt removal of the piglet from the gut-bathing antibody combined with the stress of weaning produced a neonate vulnerable to the ubiquitous rotavirus. Similar circumstances may prevail and operate in exacerbating rotaviral diarrhea in neonates of other species of mammals.


Essential role of GTP in the expression of adenylate cyclase activity after cholera toxin treatment.

Expression of activation of rat liver adenylate cyclase by the A1 peptide of cholera toxin and NAD is dependent on GTP. The nucleotide is effective either when added to the assay medium or during toxin (and NAD) treatment. Toxin treatment increases the Vmax for activation by GTP and the effect of GTP persists in toxin-treated membranes, a property seen in control membranes only with non-hydrolyzable analogs of GTP such as Gpp(NH)p. These observations could be explained by a recent report that cholera toxin acts to inhibit a GTPase associated with denylate cyclase. However, we have observed that one of the major effects of the toxin is to decrease the affinity of guanine nucleotides for the processes involved in the activation of adenylate cyclase and in the regulation of the binding of glucagon to its receptor. Moreover, the absence of lag time in the activation of adenylate cyclase by GTP, in contrast to by Gpp(NH)p, and the markedly reduced fluoride action after toxin treatment suggest that GTPase inhibition may not be the only action of cholera toxin on the adenylate cyclase system. We believe that the multiple effects of toxin action is a reflection of the recently revealed complexity of the regulation of adenylate cyclase by guanine nucleotides.


Growth arrest of Chinese hamster ovary cells in serum-free medium: changes in cell morphology and the intracellular and prostaglandin-induced levels of cyclic AMP.

When Chinese hamster ovary (CHO) cells are shifted from medium which contains serum into serum-free medium, they complete one cell doubling and arrest in the G1 phase of the cell cycle. During the first 72 hr of arrest, there is little change in intracellular adenosine 3':5'-phosphate (cAMP) level, and the cells retain their usual epithelial-like morphology. After 96 hr, the cAMP level doubles, the magnitude of the prostaglandin E1-induced changes in cAMP increases threefold, and the cells convert from a rounded, epithelial-like shape to an elongated, fibroblast-like form. The fact that these biochemical and cellular transitions are subsequent to the growth arrest shows that the cAMP increase is not the cause of the growth arrest but is consistent with a role for cAMP in the control of cell morphology. In addition, these changes point to the importance of the G1 phase for initiating cAMP-related events.


S49 lymphoma wild type and variant clones contain normal calcium dependent regulator.

Calcium dependent regulator is present in wild-type S49 lymphoma cells, in the variant that is deficient in adenylate cyclase activity (AC-), and in the uncoupled variant (UNC). The electrophoretic mobility and the ability to stimulate cyclic nucleotide phosphodiesterase of the calcium dependent regulator from each of these three clones are indistinguishable from those of the modulator protein isolated from bovine brain. Calcium dependent regulator does not appear to be involved in the defect responsible for the UNC or AC- variants.


Effect of lin-benzoadenosine and lin-benzoadenosine 3':5'-monophosphate on cyclic AMP-dependent protein kinase activity in vitro.

We have shown that the fluorescent "stretched-out" analog of cyclic AMP, namely linear-benzo-cyclic AMP, maximally activates brain protein kinase and protein kinase from skeletal muscle. The corresponding linear-benzoadenosine inhibits kinase activity slightly less than does adenosine. Thus, the 2.4A-wider versions of cyclic AMP and of adenosine interact with protein kinase in a manner similar to that of the natural compounds.


Possible role of cyclic nucleotides in the mechanism of the protective effect of methylprednisolone on the hypoxic rat heart.

The isolated isovolumic rat heart was used as a model of cardiac hypoxia. Force of cardiac contraction and cardiac cyclic nucleotide levels (cyclic GMP and cyclic AMP) were monitored in hearts subjected to hypoxia for 5 min and allowed to recover by reoxygenation. Hearts were obtained from both control animals and animals pretreated with methylprednisolone at 18 hr and 1 hr prior to sacrifice. Myocardial levels of cyclic GMP which were significantly (p less than 0.05) elevated above control during all periods of hypoxia were found to be lower when hearts were pretreated with methylprednisolone prior to hypoxic exposure. Hearts of animals pretreated with methylprednisolone also demonstrated better recovery during reoxygenation than did control hearts. These studies suggest that methylprednisolone may be beneficial in the prevention of myocardial failure following hypoxia via a modulation in myocardial cyclid GMP content.


Membrane-bound forms of Ca2+-dependent protein modulator: Ca2+-dependent and independent binding of modulator protein to the particulate fraction from brain.

Ca2+-dependent binding of modulator protein to the particulate fraction was studied. The particulate fraction from one gram of rat brain bound in a Ca2+-dependent fashion 144 microgram of modulator protein, representing more than one third of the total soluble modulator protein in this tissue. The binding site was present in both the mitochondrial and microsomal fractions, the specific activity of the microsomes being the higher. The binding was reversible with a physiological concentration of Ca2+, and was temperature-dependent, and the site can be saturated with modulator protein (4.5 microgram modulator protein per mg of microsomal protein). Tryptic digestion of the membranes caused complete disappearance of the binding activity, but heat-treatment for 5 min at 70 degrees C caused only 40% loss of activity. The binding site may be a known or unknown enzyme(s), the activity of which is regulated by Ca2+ and modulator. Alternatively, this binding site may be a nonenzymic protein that regulates the concentration of free modulator protein in the cell.


A semi quantitative method for cyclic nucleotide localization by immunocytochemistry and its application in determining the distribution of cyclic AMP in lungs of normal and pertussis-vaccinated mice following histamine or epinephrine challenge.

A method is described which allows quantitative comparison of cAMP content determined by immunocytochemical procedures. This technique was then employed to localize cAMP in lungs of normal and pertussis-vaccinated mice following saline, histamine, or epinephrine challenge. All primary pulmonary compartments were shown to contain some immunoreactive (cAMP) material. However, epinephrine and histamine challenge selectively increased the cAMP content of the vasculature. No effect of epinephrine or histamine was detected in bronchial smooth muscle or interstitial tissue. This increased cAMP accumulation was observed in both normal and pertussis-vaccinated mice following epinephrine challenge but only in pertussis-vaccinated mice after histamine administration. These results demonstrate that histamine and epinephrine stimulate cAMP accumulation in the same pulmonary compartments supporting earlier speculation that histamine acts indirectly through epinephrine release. Further, primary involvement of the vasculature supports a more prominent role for this tissue in pertussis mediated histamine hypersensitivity.


Positive inotropic but no relaxant effects of phenylephrine in cat papillary muscle.

The effect of phenylephrine (30 micron) in the presence of propranolol (1 micron) on electrically induced isometric twitches and on KCl-contractures was studied in papillary muscles from reserpine-pretreated cats at 25 degrees C. Under these conditions phenylephrine has previously been shown to act solely via alpha-adrenoceptors and not to increase c-AMP. Phenylephrine increased force of contraction, time to peak tension and relaxation time. Contracture tension remained unaffected. These data indicate that the stimulation of cardiac alpha-adrenoceptors, in contrast to beta-adrenergic stimulation, does not lead to so-called relaxant effects. This qualitative difference between the two responses is probably due to the different capacity of both stimuli to increase c-AMP.


Stimulation of hamster adipocyte cyclic 3':5'-nucleotide phosphodiesterase activity by ionophore A23187 and calcium.

Incubation of hamster isolated fat cells with the ionophore A23187 and calcium for 20 minutes caused 30-40% increases in the cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) activity of adipocyte homogenates when either 0.6 micron cyclic AMP or 0.6 micron cyclic GMP was the enzyme substrate. The stimulation of adipocyte cyclic AMP phosphodiesterase activity by A23187 and calcium was not antagonized by the adrenergic receptor blocking agents phentolamine and propranolol. The changes in enzyme activity produced by the ionophore and calcium were not associated with elevated intracellular cyclic AMP levels. Furthermore, A23187 and calcium acted to enhance adipocyte phosphodiesterase activity before, but not after, homogenization of the fat cells. These data suggest that the phosphodiesterase activity of hamster isolated fat cells may, at least in part, be regulated by fluctuations in intracellular calcium concentrations.


Role of cyclic AMP and protein kinase on the steroidogenic action of ACTH, prostaglandin E1 and dibutyryl cyclic AMP in normal adrenal cells and adrenal tumor cells from humans.

The role of the cyclic AMP-protein kinase system in mediating the steroidogenic effect of ACTH, prostaglandin E1 and dibutyryl cyclic AMP, induced similar stimulations of protein kinase activity, cyclic AMP was studied using human adrenal cells isolated from normal and adrenocortical secreting tumors. At high concentrations of ACTH, complete activation of protein kinase of normal adrenal cells was observed within 3 min, at the time when cyclic AMP production was slightly increased and there was still no stimulation of steroidogenesis. At supramaximal concentrations, ACTH, PGE1 and dibutyryl cyclic AMP and cortisol productions in adrenal cells isolated from normal and from one adrenocortical tumor. In one tumor in which the adenylate cyclase activity was insensitive to ACTH, the hormone was unable to stimulate protein kinase or steroidogenesis, but the cells responded to both PGE1 and dibutyryl cyclic AMP. In another tumor in which the adenylate cyclase was insensitive to PGE1, this compound also did not increase protein kinase activity or steroidogenesis, but both parameters were stimulated by ACTH and dibutyryl cyclic AMP. After incubation of normal adrenal cells with increasing concentrations of ACTH (0.01-100 nM) marked differences were found between cyclic AMP formation and cortisol production. However at the lowest concentrations of ACTH exerting an effect on steroid production a close linked correlation was found between protein kinase activation and cortisol production, but half-maximal and maximal cortisol production occurs at lower concentration of ACTH than was necessary to induce the same stimulation of protein kinase. Similar findings were found after incubating the adrenal cells with dibutyryl cyclic AMP (0.01-10 mM). The results implicate an important role of the cyclic AMP-protein kinase system during activation of adrenal cell steroidogenesis by low concentrations of steroidogenic compounds.


The concentration of cyclic AMP and the activity of cyclic AMP dependent protein kinase and an inhibitor in the adipose tissue of rats fed lard or glucose diets.

Measurements of tissue cyclic AMP (cAMP) concentration, the activity of cAMP-dependent protein kinase and the level of the enzyme's thermostable, macromolecular inhibitor were made on preparations of rat epididymal fat pad from animals fed high fat or high carbohydrate diets. The cAMP concentration from rats adapted to a high lard diet for 14-15 days was 153 +/- 17.8 pmoles/mg protein as opposed to 76 +/- 6.0 found with high glucose diet. No significant difference in total cAMP-dependent protein kinase activity was observed among rats fed high glucose, high lard or laboratory chow, although the enzyme's activity ratio (-cAMP)(+cAMP) was significantly elevated with lard feeding (0.49 +/- 0.02) as opposed to glucose feeding (0.43 +/- 0.01). Crude preparations from lard and glucose fed animals were equivalent in inhibitory activity when tested with enzyme from chow fed animals. Agarose column chromatography separated holoenzyme and C subunit forms of the protein kinase when 500 mM NaCl was present in the elution buffer. Absence of the salt allowed subunit reassociation to occur. Direct addition of NaCl greater than or equal to 75 mM significantly inhibited protein kinase activity. The results indicate that the adipose tissue of rats fed a high lard diet has a higher concentration of cAMP and an increased protein kinase activity ratio than tissue from rats fed a fat free, high glucose diet. Total cAMP-dependent protein kinase activity and the level of a thermostable macromolecular inhibitor remained unchanged.


Inhibition of plasminogen activator secretion by cyclic AMP in a macrophage-like cell line.

The continuous cell line, J774.2, exhibits many macrophage-like functions such as latex and Fc-mediated phagocytosis, antibody mediated phagocytosis, antibody mediated cytotoxicity, chemotaxis, and lysozyme secretion. Cyclic AMP stimulates Fc-mediated phagocytosis and inhibits the growth of J774.2. To further evaluate the relationship between cyclic AMP and the specialized functions exhibited by these cells. Variants deficient in phagocytosis, adenylate cyclase and cyclic AMP-dependent protein kinase were derived. We have now shown that J774.2 also secretes plasminogen activator and that this secretion is rapidly and specifically inhibited by 8-bromoadenosine 3':5'-cyclic monophosphoric acid (8 Br--cAMP) or cholera toxin under conditions where lysozyme secretion is unaltered. Utilizing protein kinase-deficient variants, the ability of cyclic AMP to inhibit plasminogen activator secretion was shown to be mediated by a cyclic AMP-dependent protein kinase. We conclude that cyclic AMP has diametrically opposing effects on two macrophage-like functions: Fc-mediated phagocytosis and plasminogen activator secretion.


The correlation of cyclic AMP and protein kinase activity in adipocytes with lipolysis stimulated by ACTH: the effect of adenosine deaminase and actinomycin D.

ACTH at levels as low as 0.05 mU/ml stimulated lipolysis, protein kinase and cyclic AMP accumulation in isolated fat cells from fed and fasted rats. Changes in cyclic AMP levels and in the protein kinase activity ratio were well correlated temporally. The protein kinase activity ratio was potentiated by adenosine deaminase. A sudden increase or decrease in either ACTH or dibutyryl cyclic AMP concentration was associated with a rapid and corresponding change in the rate of glycerol production. With ACTH, the changes in glycerol production were accompanied by appropriate changes in cyclic AMP levels. Actinomycin-D (10 UM) did not affect lipolysis or cyclic AMP accumulation activated by ACTH in fat cells.


Cyclic AMP-dependent regulation of ornithine decarboxylase activity in Chinese hamster ovary cells maintained with a salts/glucose medium.

Ornithine decarboxylase activity (ODC) increased about 7 fold 6--8 h following 10mM asparagine (ASN) addition to confluent cultures that had been previously serum deprived and then placed in a salts/glucose medium. Optimal concentrations of dibutyryl cAMP (dB cAMP) when incubated with the ASN caused up to a 50 fold increase in the activity of this enzyme after 7--8 h. The enhancement of ODC activity by ASN and dB cAMP was not sensitive to continuous (0--7 h) treatment with actinomycin D but similar treatment with cycloheximide depressed enzyme activity 40--60%. The synergistic stimulation of ODC activity by dB cAMP added with ASN was dose dependent and the dB cAMP stimulation of ODC activity displayed an absolute requirement for ASN when cells were maintained in the salts/glucose medium. The addition of dB cAMP always further enhanced ODC activity above the levels produced by addition of various levels of ASN (1 to 40mM) to the salts/glucose medium. Other agents which elevated cAMP levels such as 1-methyl-3-isobutylxanthine (IBMX) also enhanced ODC activity when administered with ASN. Additionally, treatment with sodium butyrate at concentrations ranging from 0.001mM to 5.0mM did not elevate ODC activity above the activity obtained with ASN alone. Addition of dB cAMP at various times after placing cells in salts/glucose medium with ASN further stimulated ODC activity only when added during the first 3-4 h. These results demonstrate the involvement of cAMP in the ASN mediated stimulation of ODC activity using cells maintained in a salts/glucose medium.


Inhibition of corrosion during autoclave sterilization of carbon steel dental instruments.

Comparisons were made of several dip-applied corrosion inhibitors for protection of carbon steel dental instruments during autoclaving. Although none were totally effective, several provided significant corrosion inhibition. Sodium nitrite and one proprietary inhibitor were about equally effective and notably superior to the other inhibitors investigated. Because sodium nitrite is a food preservative that is relatively harmless when ingested in small quantities, whereas commercial inhibitors are of proprietary composition and unspecified toxicity, we prefer to use sodium nitrite for corrosion inhibition during autoclaving. Sodium nitritie is readily available from chemical suppliers and is generally much less expensive than most commercial inhibitors. Although the autoclave used in this investigation is typical of many used in clinical situations, minor variations in design, steam impurities, and other unknown factors might effect the results. Further research is needed to determine the corrosive effects of impurities in the steam supply, residual detergents, method of postautoclave drying, steam supply deoxygenation, pH, and possibly the use of oxygen scavengers within the autoclave.


Role of cGMP in isolated rat parotid acinar cells.

The role of cGMP as the intracellular mediator of the activation of cholinergic receptors in the secretory cells of the rat parotid gland was investigated in vitro using isolated rat parotid acinar cells. It was demonstrated that this cyclic nucleotide is involved in the intracellular translation of activation of muscarinic cholinergic receptors in these cells.


Tendon transfers for radial nerve palsy: use of superficialis tendons for digital extension.

Since 1959, 22 patients have had wrist extension restored by transfer of the pronator teres to the extensor carpi radialis longus and brevis, common finger extension by transfer of the superficialis of the long finger, independent thumb and index finger extension by transfer of the superficialis of the ring finger, and abduction of the thumb by transfer of the flexor carpi radialis at the wrist joint level. Twenty-one of 22 patients have been evaluated from 8 months to 15 years after operation, with an average follow-up of 4.5 years. By our new system of evaluation, there were 10 excellent results, six good results, five fair results, and all patients improved. Sixteen patients obtained full, independent thumb-index finger extension, three had fair function, and two obtained thumb-index extension by tenodesis of the transfer. This procedure allows full metacarpophalangeal extension independent of wrist position, provides thumb-index finger extension independent of the ulnar three digits, and maintains the dorsal-radial-to-volar-ulnar plane of functional motion of the wrist by retaining the flexor carpi ulnaris.


Histochemical and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog submandibular glands.

p-Nitrophenyl phosphatase (p-NPPase) activity of (Na+-K+)-activated adenosine triphosphatase ((Na+-K+)-ATPase) on the acinar cells of dog submandibular gland was demonstrated by using light microscopy. The reaction products of p-NPPase of fresh frozen sections were seen to be localized on the basal parts of acini, and disappeared when the sections were incubated in medium containing 10(-3) Mouabain or in a K-free medium. Under the electron microscope, the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells. On the microvilli of the luminal plasma membrane of the acinar cell, a small quantity of the reaction products was also present. This localization of ATPase reaction products on the serous and mucous cells seemed to coincide well with that of p-NPPase activity observed on the acini under light microscopy. Possible explanations are given regarding distribution of the above mentioned enzymes in relation to the cation transport of the plasma membrane. Structural and functional asymmetrical properties of acinar cells of the dog submandibular gland are also discussed.


Preparation of crude Clostridium histolyticum collagenase for therapeutic purposes.

The production of a freeze-dried enzymatic preparation from the category of crude collagenases has been described. The method is based on the utilization of a highly proteolytic Clostridium histolyticum strain whose products have more advantageous properties for therapeutic purposes than the products of the strain commonly used as yet.


A comparative study of functional activity and cytochemical characteristics of human and rabbit microphages.

Substantial differences were found in the cytochemical indices of human and rabbit microphages. Regular changes in the enzymic activity and the levels of some biopolymers were observed in the microphages in the process of phagocytosis. The extent and (in some cases) tendency of these changes in human and rabbit microphages are not the same. Cytochemical indices provide significant information on the functional activity of the microphage system within one species. At the interspecies level, such information proves to be inadequate for the characteristic of the phagocytic ability of microphages unless humoral non-specific means of protection are taken into account.


Characterization of a specific adenosine receptor on human lymphocytes.

We have examined the mechanism of action of adenosine, a naturally occurring nucleoside that has profound effects on lymphocyte function. Adenosine (0.01 micrometer to 10 micrometer) increased lymphocytes cAMP levels in a dose-dependent fashion with a maximal (10 micrometer) increase of about 4-fold, whereas adenine, guanosine, and inosine had no effect on lymphocyte cAMP levels at concentrations of 100 micrometer. Adenosine appears to act on the cell surface since 1) 2-chloroadenosine, a poorly metabolized adenosine analogue, was as active as adenosine and 2) dipyridamole, which markedly inhibited [3H]-adenosine uptake by human lymphocytes, did not affect adenosine-induced accumulation of cAMP. The specificity of the adenosine effect was established by showing that the methylxanthine derivatives, theophylline and 3-isobutyl-1-methylxanthine (IBMX), specifically block the accumulation of cAMP in lymphocytes induced by adenosine. Theophylline is a competitive inhibitor of the effect of adenosine, with an estimated dissociation constant of theophylline-receptor complex of about 6.3 X 10(-7) M. The results suggest that adenosine increases the intracellular cAMP content of lymphocytes as a result of its interaction with a specific membrane receptor which results in the activation of adenylate cyclase.


Cell surface glycoproteins of rat lymphocytes. I. Correlation of mitogenic stimulation by periodate or neuraminidase and galactose oxidase with the presence of papain-sensitive glycoproteins.

Oxidation of viable rat lymph node lymphocytes with either periodate or a combination of neuraminidase and galactose oxidase (NGO), followed by reduction with tritiated sodium borohydride, labels similar sets of cell-surface molecules as assessed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Periodate and NGO induce blast transformation of lymph node lymphocytes (oxidative mitogenesis), and borohydride reduction inhibits the proliferative response. Thus, it is inferred that some or all of the glycoproteins that are labeled with tritiated borohydride may be involved in mediating the stimulation caused by the oxidizing agents. Treatment of lymph node lymphocytes with 5 units/ml papain abolishes the response to periodate or NGO but does not significantly affect the response to Con A. At the same time, papain treatment eliminates the labeled bands representing six high m.w. glycoproteins (175,000, 170,000, 160,000, 155,000, 100,000, and 70,000 daltons). No significant effect is seen on the labeling of the other components visualized in the slab gels. The results implicate the subset of six high m.w. papain-sensitive sialoglycoproteins in mediating oxidative mitogenesis of rat lymph node lymphocytes.


Role of the murine major histocompatibility complex in macrophage-mediated cytolysis.

Peritoneal cells from congeneic resistant mice infected with BCG displayed differential cytotoxicity toward tumor cells destroying more allogeneic tumor cells than syngeneic tumor cells. This observation was made regardless of the tumor cells used or the effector cell source. The responsible effector cell remained in a doubly adherent population, was sensitive to carrageenan and silica, insensitive to anti-thymocyte sera, and is probably a macrophage. Activated macrophages were capable of reacting against tumor cells as well as histoincompatible embryonic cells. These observations may indicate that macrophages are capable of discriminating cell surface components linked to the major histocompatibility complex.


Comparative toxicity of polychlorinated biphenyl and polybrominated biphenyl in the rat liver: light and electron microscopic alterations after subacute dietary exposure.

The comparative toxicity of polychlorinated biphenyl (PCB) and polybrominated biphenyl (PBB) in livers was studied in male Holtzman rats. Four-week-old animals were fed at dietary levels of 0, 5, 50, or 500 ppm for 5 weeks and then sacrificed. The mean liver-body weight ratios of the 50 and 500 ppm groups were increased. Histopathologic examination of the livers revealed fatty degenerative change associated with both compounds. This change was more marked at 500 than at 50 ppm. Various sized lamellar cytoplasmic inclusions were detected in livers of animals fed 500 ppm of either compound. However, the inclusions were more numerous in the PBB-treated rats. Several animals fed 50 ppm PBB had a few inclusions. In rats that received 500 ppm PBB, hypertrophic degenerative hepatocytes were present around the central veins. On the periphery of this change there were occasionally multinucleated hepatocytes. Electron microscopic examination at a dose level of 5 ppm in both the PCB and PBB groups showed a slight proliferation of smooth endoplasmic reticulum (SER), a moderate increase of lipid droplets and some liposomes, and a marked proliferation of Golgi condensing vesicles containing lipoprotein particles. A decreased number of mitochondria and lysosomes was also observed. At 50 ppm, similar but more marked ultrastructural alterations were seen. In addition, an increased number of branched and cup-shaped profiles of mitochondria and a decreased number of Golgi condensing vesicles containing lipoprotein particles were observed. Concentric membranous cytoplasmic whorls were encountered only in the 50 ppm PBB-treated rats. At 500 ppm the number of mitochondria decreased in both groups. There was also a marked increase in the number of SER and liposomes concomitant with a decreased number of Golgi condensing vesicles containing lipoprotein granules. Membranous whorls were also present in the 500 ppm groups.


T-cell lymphoma induction by radiation leukemia virus in athymic nude mice.

We report the development of extrathymic lymphoblastic lymphomas in RadLV-inoculated congenitally athymic nude mice. Thus, a leukemogenic virus which appears to require the presence of a thymus for its replication in normothymic mice can infect and transform target cells in the absence of this organ in the athymic host. The cells of one of these lymphomas have been established in vitro as a permanent cell line, BALB/Nu1. This cell line as well as a lymphoma induced in NIH/Swiss nude mice exhibit several T-cell markers, including terminal deoxynucleotidyl transferase activity, Thy-1.2, and Ly-2.2, but not Ly-1.2 nor TL. Ig determinants were not detected. The characteristics of the tumor cells support the view that cells with T-cell markers may normally exist in nude mice and undergo neoplastic transformation and clonal expansion after infection with a leukemogenic virus. The alternative possibility that virus-induced differentiation of prothymocytes may lead to the expression of Thy-1.2 and Ly-2.2 antigens is also considered. BALB/Nu1 cells release large numbers of type C viral particles. The virus, designated radiation leukemia virus (RadLV)/Nu1, has RTase activity and the protein profile characteristic of murine leukemia virus (MuLV). In radioimmunoassays, it cross-reacts completely with RadLV/VL3, a virus obtained from RadLV-induced C57BL/Ka thymic lymphoma cells in culture, and slightly with a xenotropic virus (BALB:virus-2) and with AKR MuLV. On inoculation into C57BL/Ka mice it has thymotropic and leukemogenic activity. In vitro it is B-tropic, poorly fibrotropic, and has limited xenotropic activity. Thus, RadLV/Nu1 appears to be biologically and serologically similar or identical to its parent virus, RadLV.


Comparative toxicity of polychlorinated biphenyl and polybrominated biphenylin the rat thyroid gland: light and electron microscopic alterations after subacute dietary exposure.

The comparative toxicity of polychlorinated biphenyl (PCB) and polybrominated biphenyl (PBB) in thyroid glands was studied in male Holtzman rats. Four-week-old animals were fed at dietary levels of 0, 5, 50, and 500 ppm for 5 weeks and then sacrificed. PCB and PBB produced similar dose-dependent ultrastructural lesions in thyroid follicular cells of rats. The daily administration of 5 ppm of either PCB or PBB resulted in the accumulation of large membrane-limited colloid droplets and electron-dense lysosomal bodies within the cytoplasm of follicular cells. Microvilli were short and abnormally branched, and unique cytoplasmic processes extended from the apical surface of follicular cells into the luminal colloid. Similar but more severe ultrastructural changes were observed in thyroid glands of rats administered 50 and 500 ppm of either PCB or PBB. Many follicular cells were distended with large abnormal lysosomal bodies and colloid droplets. Mitochondria were often vacuolated with disrupted cristae. Microvilli were blunt with abnormal branching or absent from areas of the luminal surface of follicular cells. Processes of apical cytoplasm often extended into the follicular lumen in areas devoid of microvilli. Follicular cells remained responsive after the feeding of either PCB or PBB and underwent moderate compensatory hypertrophy and hyperplasia. Thyroid follicles were smaller than in controls and were lined by more columnar cells that occasionally formed papillary projections into the follicular lumens. PCB and PBB produced similar ultrastructural lesions in thyroid follicular cells which appeared to interfere with the synthesis and secretion of thyroxine.


Recessive dystrophic epidermolysis bullosa. Evidence for increased collagenase as a genetic characteristic in cell culture.

Fibroblast cultures from patients with recessive dystrophic epidermolysis bullosa (RDEB) demonstrated an increased capacity to synthesize and secrete collagenase. This phenotypic trait appeared to distinguish RDEB from other genetically distinct forms of epidermolysis bullosa. The finding of increased collagenase may be a specific manifestation of these cells in that prototypic lysosomal and cytoplasmic enzymes were present in approximately normal concentrations. In addition, this trait persisted through many cell passages, suggesting that the property was genetically determined. The elevated concentrations of immunoreactive collagenase in fibroblast cultures of patients with RDEB reflected those previously observed in vivo (4) and support the concept of a pathogenetic role for the enzyme in the blistering phenomenon. In three of the cell lines, the increase in enzyme protein occurred in association with a structurally defective enzyme. The data suggest that this may be a characteristic of all RDEB cells.


In vitro assay for tumor promoters and anti-promoters.

Tumor promoters and anti-promoters have been shown to modify the induction of ornithine decarboxylase, the production of plasminogen activator, and the recovery of induced mutations. Data were presented to show that the recovery of mutagen-induced ouabain-resistant mutations in cultured Chinese hamster cells is increased with a tumor-promoter treatment and reduced by anti-promoter treatments. The results suggest that many induced mutations can either be repressed or derepressed by promoters or anti-promoters. The results also support the hypothesis that tumor initiation is due to a mutagenic event, while tumor promotion is the result of an epigenetic process involving cyclic nucleotide modulation of gene expression.


Preparation of functional smooth muscle cells from the rabbit aorta.

A procedure for dissociating the rabbit aorta into single, functional smooth muscle cells is described. After removal of adventitia and intima, slices of media were incubated with purified collagenase, elastase, and soybean trypsin inhibitor in a Krebs-Ringer buffer modified with Hepes, amino acids, and a [Ca2+] of 0.2 mM. After enzymatic digestion and mechanical shear, the yield of dispersed cells was approximately 25% based on DNA recovered. Greater than 95% of the cells excluded trypan blue and approximately 80-90% adhered to tissue culture dishes. By phase contrast microscopy, most of the cells were elongate and approximately 10 micron X 30 micron in size. The remainder were either spherical or highly crenated and contracted. Electron microscopy of the cells showed that immediately after dissociation greater than 95% could be identified as smooth muscle, though most had undergone some degree of structural change compared to cells in situ. Depending on the preparation, from 5 to 50% of these cells contracted in response to agonists. Cells shortened by 10-15% and developed numerous evaginations when stimulated by angiotensin II norepinephrine, or carbamylcholine. Cells relaxed after washout of agonists and could subsequently be restimulated. Specific inhibitors of each of the agonists blocked the contractile response. Dispersed cells cultured for 1-5 days contracted in even higher numbers than the freshly prepared cells, suggesting restoration of hormone binding and/or contractile function in culture. This preparation provides a system in which the physiology of individual vascular smooth muscle cells may be studied.


The biologic significance of alloreactivity. The ontogeny of T-cell sets specific for alloantigens or modified self antigens.

We have analyzed the cellular basis of T-cell reactivity against lymphocytes expressing major histocompatibility complex (MHC) products that are foreign by virtue of polymorphism (alloantigens) or because of modification by chemicals or viruses. We find that early in ontogeny, prekiller activity against both trinitrophenyl (TNP)-coupled autologous MHC products and allogeneic MHC products resides in the same (Ly123(+)) T-cell pool; later in ontogeny alloreactivity is invested in Ly23 cells which, when activated, lyse TNP-coupled autologous cells as well as appropriate allogeneic target cells. We demonstrate that stimulation of Ly123(+) T cells in vitro by autologous cells coated with chemically-inactivated Sendai virus results in the formation of Ly23(+) cytolytic T lymphocytes (CTL) that specifically lyse both virus modified autologous target cells and unmodified allogeneic target cells. These results suggest the following model to account for the presence of large numbers of alloreactive T-cell clones in adult animals: continuous stimulation of Ly123 cells by autologous MHC antigens associated with foreign materials such as a virus results in the formation of Ly23 memory progeny carrying receptors that recognize MHC products that are foreign due to genetic polymorphism (alloantigens). In general, these studies indicate that alloaggression (as manifest by Ly23 cells in the CTL response) reflects a high degree of cross stimulation between physiologically relevant antigens, e.g., viral determinants associated with self MHC products, and biologically irrelevant allelic variants of the MHC.


In vitro effects of Epstein-Barr virus on peripheral blood mononuclear cells from patients with rheumatoid arthritis and normal subjects.

Peripheral blood mononuclear cells from 10 patients with rheumatoid arthritis and 9 control subjects were cultured in vitro for 30 days with and without infection by Epstein-Barr virus. All cultures showed polyclonal stimulation of B cells as indicated by rising levels of IgM in the culture supernates, reaching maximal at 18-24 days, and with no quantitative or kinetic difference between the RA and control cells. IgM anti-IgG was also produced in both groups and maximally at 18-24 days, but in greater quantity by the RA lymphocytes. The anti-IgG made by the RA lymphocytes was more easily absorbed by solid phase IgG than was the anti-IgG made by the normal lymphocytes and thus was judged to be of higher affinity. RA lymphocytes uninfected with EBV had higher transformation scores than did the normal controls and developed spontaneously into permanent cell lines in six instances.


The induction of virus-specific cytotoxic T lymphocytes with solubilized viral and membrane proteins.

Reconstituted membranes were prepared from detergent solubilized P815 (H-2d) tumor cell membranes and solubilized Sendai virus protein. These reconstituted membranes stimulated a virus-specific H-2-restricted secondary CTL response. Stimulating activity was dependent upon the presence of both viral and P815 protein in the same membrane and was restricted to the H-2 specificity present in the reconstituted membrane. Liposomes prepared from solubilized Sendai virus proteins and partially purified H-2 alloantigen also had activity for CTL induction. The results demonstrate the feasibility of using detergent solubilized membrane proteins to study antigen recognition by virus-specific, H-2 restricted cytolytic T lymphocytes.


Specific adsorption of H-2-restricted cytotoxi T cells to macrophage monolayers.

These experiments tested whether Tc cells specific for foreign antigen (X) plus self H-2 adsorbed to macrophage monolayers displaying (a) X with allogeneic H-2; (B) self H-2 alone; (c) X plus self H-2. Specific adsorption occurred only in case (c), a result compatible with altered self and requiring further operational assumptions in dual recognition models.


Genomic RNA of the murine coronavirus JHM.

Genomic RNA extracted from the purified murine coronavirus JHM sediments between 52S and 54S in aqueous sucrose gradients. The RNA is single-stranded and has an apparent mol. wt. of 5.4 to 6.5 X 10(6), as determined by electrophoresis in polyacrylamide agarose gels of different concentrations. The presence of polyadenylate sequences in the RNA is demonstrated by binding to oligo-)dT) cellulose and digestion with ribonucleases A and T1. The purified RNA does not dissociate into subunits at high temperatures or in high concentrations of DMSO and is infectious.


Early events in the interaction between foot-and mouth disease virus and primary pig kidney cells.

Foot-and-mouth disease virus (FMDV) attached to pig kidney cells at 0 degrees C and could only be recovered in a form with a sedimentation coefficient and buoyant density lower than that of the native virus. Incubation of the virus-cell complex at 37 degrees C caused disruption of about 80% of the particles into a 12S protein sub-unit that had the same polypeptide composition as that produced by reducing the pH of the virus below pH7. The remaining 20% had the same polypeptide and RNA composition as the native virus but it had a lower sedimentation coefficient, buoyant density and specific infectivity. These lower values are probably due to the association of the virus with cell membrane components. The 12S subunits were shown to be located inside the cell, indicating that disruption of the virus had occurred within the cell. The results are discussed in relation to the different cell mediated alteration of other picornaviruses.


Mastomys natalensis papilloma virus (MnPV), the causative agent of epithelial proliferations: characterization of the virus particle.

A virus (MnPV) with the structural characteristics of papilloma viruses was isolated from benign and malignant proliferations of adult animals of the inbred line 'GRA Giessen' of Mastomys natalensis. The particles can be banded in CsCl gradients at densities of 1.34 g/ml (full particles) and 1.29 g/ml (empty particles). The virus DNA has a buoyant density of 1.7104 g/ml and can exist in three different conformations (supercoiled circular, nicked circular and linear), the sedimentation values of which have been determined as 23 to 24S, 16 to 17S and 14 to 15S, respectively. Although the mol. wt. of MnPV DNA is similar to that of HPV 1 DNA, the size of the fragments obtained after cleavage of MnPV DNA with the restriction endonuclease Hae III is quite different from the pattern seen with human papilloma virus. The virion contains 12 different polypeptides; the major structural protein has a mol. wt. of 56 000. MnPV is shown to be the causative agent of the skin proliferations, because tumours can be induced by inoculation of purified virus, whereas no cutaneous alterations are observed when the particles are inoculated in the presence of anti-MnPV serum. MnPV can be re-isolated from the experimentally induced tumours.


Efficient transfer of interferon-induced virus resistance between human cells.

The rate of development of interferon-induced virus resistance in a mixture of two human cell types (U and WISH) is determined by the cell type (WISH) in the mixture which responds first. This phenomenon has been shown with two types of interferon assay procedure, and with both vesicular stomatitis virus and Sindbis virus. The transfer of virus resistance from one human cell (WISH) to another (U) (homospecific transfer) is much more efficient than the transfer from mouse L cells to WISH cells (heterospecific transfer), as shown by a much lower ratio of donor to recipient cells required for maximum transfer as well as a more rapid transfer. Thus, virus protection afforded by the interferon system is amplified more efficiently in mixtures of different human cells than in mixtures of mouse and human cells. These results suggest that, in a mixed population of cells such as occurs in vivo, more slowly responding cells might be influenced by cells which respond more rapidly to interferon. A defensive role is suggested for this mechanism which amplifies protection due to interferon.


Virus mutation during 'slow infection': temporal development and characterization of mutants of visna virus recovered from sheep.

Visna virus could be recovered from peripheral blood leukocytes of sheep for years after intracerebral inoculation. Viruses recovered from sheep prior to and several months after development of antibody were antigenically identical to the parental strain used for inoculation. Subsequently, mutant viruses which were not neutralized by the animals' sera were obtained. Longitudinal studies of leukocyte viruses collected from two infected sheep showed that more than one strain of virus could co-exist in the animal. Virus neutralization tests using sequentially collected sera and the viruses recovered from leukocytes revealed a sequential development of antibody to parental and then to each strain of mutant virus. Characterization of two of the mutant viruses showed that they were antigenically stable, virulent in cell culture and when inoculated into new sheep, elicited antibodies which cross reacted with the parental virus from which they were derived. This continuous mutation of Visna virus in persistently infected sheep may be a mechanism for the production of chronic disease.


A re-appraisal of the biochemical map of foot-and-mouth disease virus RNA.

The proteins induced by infection of BHK 21 cells with foot-and-mouth disease virus have been compared by tryptic peptide analysis. The results indicate that there are three primary products 5'--P88, P52, P100--3'. The polypeptide P56, which we considered previously to be a primary product, is derived from the region of the genome that codes for P100. The results indicate that there are alternative cleavage pathways of P100, the polypeptide coded for by the 3' end of the genome.


Effect of interferon on transcription and translation of vesicular stomatitis virus in human and simian cell cultures.

The effect of interferons on vesicular stomatitis virus (VSV) primary transcription, amplified RNA synthesis [i.e. the sum of primary transcription RNA replication (leading to [ - ] RNA) and secondary transcription (leading to [ + ] RNA) and virus protein synthesis were studied. In a human cell line, both human and simian interferons inhibited the initiation of primary transcription and amplified RNA synthesis. In contrast, in a simian cell line tested similarly, the initiation of these activities was not affected, though they decreased as the infection progressed. Nevertheless, virus protein synthesis was completely inhibited. These results demonstrate that the action of interferon on virus transcription and/or translation may depend more on the host cell than on the particular interferon used.


Synthesis of herpes simplex virus DNA in preparations of chromatin from infected cell nuclei.

Chromatin prepared from cells infected with Herpes simplex virus type 1 or type 2 can synthesize both virus and cell DNA in vitro. The rate of synthesis is comparable to that of isolated whole nuclei. Incorporation is limited, and both cell and virus DNA synthesis are sensitive to the presence of virus-specific antiserum and phosphonoacetate. In chromatin from cells infected with a phosphonoacetate resistant virus mutant, both types of DNA synthesis are resistant to the presence of the inhibitor.


The intracellular organization of actin and tubulin in cultured C-1300 mouse neuroblastoma cells (clone NB41A3).

Cells of clone NB41A3 of the C-1300 mouse neuroblastoma were grown to a critical density at which many of the cells flatten, assume a variety of shapes and sizes and some sprout processes resembling neurites. We have studied the distribution of actin and tubulin in these cells using fluorescence microscopy and antibodies against actin or tubulin under these conditions. Actin-containing structures are variably arranged and predominantly associated with motile areas of the cell periphery including the growth cone. Microtubules appear to run radially from the perinuclear area towards the cell periphery. When neurites are present, microtubules converge into them and run to the growth cone but rarely contact its edge.


Correlations of Tc-99m pyrophosphate myocardial scintigraphy and the results of coronary-artery bypass surgery.

This study indicates that abnormal myocardial scintigrams with Tc-99m pyrophosphate tend to improve after coronary-artery bypass surgery, frequently changing from positive to normal. The significance of this change is uncertain. It does not correlate well with the clinical state but may simply reflect the natural course of myocardial scintigraphy in response to hospitalization, medical management, and presumably improved myocardial oxygenation. Postoperative myocardial scintigrams are useful in detecting perioperative infarction. ECG interpretation may be difficult in the immediate postoperative period and, in three cases, there were scintigraphic criteria of perioperative infarction without diagnostic changes on ECG. Patients with positive preoperative scintigrams, especially in association with Functional Class IV angina, are at increased risk during coronary-artery bypass surgery.


The specificity of pyrophosphate myocardial scintigrams in patients with prior myocardial infarction: concise communication.

Fifty-five patients with old (9 days to 10 yr) transmural infarcts but with no evidence of recent infarction, were imaged with Tc-99m pyrophosphate. Discrete uptake was rare in the setting of an old infarct. Diffuse uptake was neither sensitive to, nor specific for, acute infarction. Prior infarction will rarely cause diagnostic error if the discrete pattern is required for a positive diagnosis.


Ultrastructure of the lung in the rat following exposure to crocidolite asbestos and quartz.

Lung tissue from rats that had inhaled U.I.C.C. crocidolite asbestos or quartz particles showed thickening of the interstitial tissue and the presence of collagen fibres. Aggregates of macrophages in the alveolar spaces were a dominant feature of all the sections examined from asbestos exposed rats. According to the ultrastructural studies described here, all the macrophages were mature cells, indicating that the ingested crocidolite asbestos was non-toxic. Lung sections of rats exposed to quartz particles were significantly different. Single cells were found in the alveolar spaces and many macrophages showed a characteristic pattern of vacuole formation. Other cells contained intracellular membranous lamellated bodies, similar to those found in Type II pneumocytes. Cells containing lamellated bodies were also found in the interstitial tissue. These findings suggest that the two mineral dusts have quite different biological effects on the macrophage and that the development of pulmonary fibrosis may, to some extent, be caused by a different mechanism in each instance.


Malignant synovioma: electron microscopical findings in three patients and review of the literature.

Ultrastructural findings in three malignant synoviomas are described. Two typical "biphasic" tumours contained "epithelial" cells possessing filopodia or microvilli, specialised cell attachment and a basal lamina, and smaller "stromal" cells showing transitions to fibroblasts. In one case, the microvilli included fibrils resembling those in epithelial cells of the intestine and renal tubules. The third tumour was mainly spindle-celled with little epithelial differentiation and no clear division into cell types, but intracytoplasmic microfibrils were conspicuous, forming ovoid masses. The tumour cells differ a good deal from normal human synovial cells but some of the features of the neoplasm are found in inflamed human synovium and in normal synovial membranes of other species. The cytoplasmic fibrils in the third case are similar to those reported by others in epithelioid sarcoma, a tumour that may be of related origin to synovioma; however, the phenomenon may be merely degenerative.